David M. Holtzman

The National Institute on Aging and the Alzheimer's Association charged a workgroup with the task of developing criteria for the symptomatic predementia phase of Alzheimer's disease (AD), referred to in this article as mild cognitive impairment due to AD. The workgroup developed the following two sets of criteria: (1) core clinical criteria that could be used by healthcare providers without access to advanced imaging techniques or cerebrospinal fluid analysis, and (2) research criteria that could be used in clinical research settings, including clinical trials. The second set of criteria incorporate the use of biomarkers based on imaging and cerebrospinal fluid measures. The final set of criteria for mild cognitive impairment due to AD has four levels of certainty, depending on the presence and nature of the biomarker findings. Considerable work is needed to validate the criteria that use biomarkers and to standardize biomarker analysis for use in community settings.

In 2011, the National Institute on Aging and Alzheimer's Association created separate diagnostic recommendations for the preclinical, mild cognitive impairment, and dementia stages of Alzheimer's disease. Scientific progress in the interim led to an initiative by the National Institute on Aging and Alzheimer's Association to update and unify the 2011 guidelines. This unifying update is labeled a "research framework" because its intended use is for observational and interventional research, not routine clinical care. In the National Institute on Aging and Alzheimer's Association Research Framework, Alzheimer's disease (AD) is defined by its underlying pathologic processes that can be documented by postmortem examination or in vivo by biomarkers. The diagnosis is not based on the clinical consequences of the disease (i.e., symptoms/signs) in this research framework, which shifts the definition of AD in living people from a syndromal to a biological construct. The research framework focuses on the diagnosis of AD with biomarkers in living persons. Biomarkers are grouped into those of β amyloid deposition, pathologic tau, and neurodegeneration [AT(N)]. This ATN classification system groups different biomarkers (imaging and biofluids) by the pathologic process each measures. The AT(N) system is flexible in that new biomarkers can be added to the three existing AT(N) groups, and new biomarker groups beyond AT(N) can be added when they become available. We focus on AD as a continuum, and cognitive staging may be accomplished using continuous measures. However, we also outline two different categorical cognitive schemes for staging the severity of cognitive impairment: a scheme using three traditional syndromal categories and a six-stage numeric scheme. It is important to stress that this framework seeks to create a common language with which investigators can generate and test hypotheses about the interactions among different pathologic processes (denoted by biomarkers) and cognitive symptoms. We appreciate the concern that this biomarker-based research framework has the potential to be misused. Therefore, we emphasize, first, it is premature and inappropriate to use this research framework in general medical practice. Second, this research framework should not be used to restrict alternative approaches to hypothesis testing that do not use biomarkers. There will be situations where biomarkers are not available or requiring them would be counterproductive to the specific research goals (discussed in more detail later in the document). Thus, biomarker-based research should not be considered a template for all research into age-related cognitive impairment and dementia; rather, it should be applied when it is fit for the purpose of the specific research goals of a study. Importantly, this framework should be examined in diverse populations. Although it is possible that β-amyloid plaques and neurofibrillary tau deposits are not causal in AD pathogenesis, it is these abnormal protein deposits that define AD as a unique neurodegenerative disease among different disorders that can lead to dementia. We envision that defining AD as a biological construct will enable a more accurate characterization and understanding of the sequence of events that lead to cognitive impairment that is associated with AD, as well as the multifactorial etiology of dementia. This approach also will enable a more precise approach to interventional trials where specific pathways can be targeted in the disease process and in the appropriate people.

The order and magnitude of pathologic processes in Alzheimer's disease are not well understood, partly because the disease develops over many years. Autosomal dominant Alzheimer's disease has a predictable age at onset and provides an opportunity to determine the sequence and magnitude of pathologic changes that culminate in symptomatic disease.In this prospective, longitudinal study, we analyzed data from 128 participants who underwent baseline clinical and cognitive assessments, brain imaging, and cerebrospinal fluid (CSF) and blood tests. We used the participant's age at baseline assessment and the parent's age at the onset of symptoms of Alzheimer's disease to calculate the estimated years from expected symptom onset (age of the participant minus parent's age at symptom onset). We conducted cross-sectional analyses of baseline data in relation to estimated years from expected symptom onset in order to determine the relative order and magnitude of pathophysiological changes.Concentrations of amyloid-beta (Aβ)(42) in the CSF appeared to decline 25 years before expected symptom onset. Aβ deposition, as measured by positron-emission tomography with the use of Pittsburgh compound B, was detected 15 years before expected symptom onset. Increased concentrations of tau protein in the CSF and an increase in brain atrophy were detected 15 years before expected symptom onset. Cerebral hypometabolism and impaired episodic memory were observed 10 years before expected symptom onset. Global cognitive impairment, as measured by the Mini-Mental State Examination and the Clinical Dementia Rating scale, was detected 5 years before expected symptom onset, and patients met diagnostic criteria for dementia at an average of 3 years after expected symptom onset.We found that autosomal dominant Alzheimer's disease was associated with a series of pathophysiological changes over decades in CSF biochemical markers of Alzheimer's disease, brain amyloid deposition, and brain metabolism as well as progressive cognitive impairment. Our results require confirmation with the use of longitudinal data and may not apply to patients with sporadic Alzheimer's disease. (Funded by the National Institute on Aging and others; DIAN ClinicalTrials.gov number, NCT00869817.).

Microglia play a pivotal role in the maintenance of brain homeostasis but lose homeostatic function during neurodegenerative disorders. We identified a specific apolipoprotein E (APOE)-dependent molecular signature in microglia from models of amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), and Alzheimer's disease (AD) and in microglia surrounding neuritic β-amyloid (Aβ)-plaques in the brains of people with AD. The APOE pathway mediated a switch from a homeostatic to a neurodegenerative microglia phenotype after phagocytosis of apoptotic neurons. TREM2 (triggering receptor expressed on myeloid cells 2) induced APOE signaling, and targeting the TREM2-APOE pathway restored the homeostatic signature of microglia in ALS and AD mouse models and prevented neuronal loss in an acute model of neurodegeneration. APOE-mediated neurodegenerative microglia had lost their tolerogenic function. Our work identifies the TREM2-APOE pathway as a major regulator of microglial functional phenotype in neurodegenerative diseases and serves as a novel target that could aid in the restoration of homeostatic microglia.

Alzheimer disease (AD) is a heterogeneous disease with a complex pathobiology. The presence of extracellular β-amyloid deposition as neuritic plaques and intracellular accumulation of hyperphosphorylated tau as neurofibrillary tangles remains the primary neuropathologic criteria for AD diagnosis. However, a number of recent fundamental discoveries highlight important pathological roles for other critical cellular and molecular processes. Despite this, no disease-modifying treatment currently exists, and numerous phase 3 clinical trials have failed to demonstrate benefits. Here, we review recent advances in our understanding of AD pathobiology and discuss current treatment strategies, highlighting recent clinical trials and opportunities for developing future disease-modifying therapies.

One in 3 individuals will experience a stroke, dementia or both. Moreover, twice as many individuals will have cognitive impairment short of dementia as either stroke or dementia. The commonly used stroke scales do not measure cognition, while dementia criteria focus on the late stages of cognitive impairment, and are heavily biased toward the diagnosis of Alzheimer disease. No commonly agreed standards exist for identifying and describing individuals with cognitive impairment, particularly in the early stages, and especially with cognitive impairment related to vascular factors, or vascular cognitive impairment.The National Institute for Neurological Disorders and Stroke (NINDS) and the Canadian Stroke Network (CSN) convened researchers in clinical diagnosis, epidemiology, neuropsychology, brain imaging, neuropathology, experimental models, biomarkers, genetics, and clinical trials to recommend minimum, common, clinical and research standards for the description and study of vascular cognitive impairment.The results of these discussions are reported herein.The development of common standards represents a first step in a process of use, validation and refinement. Using the same standards will help identify individuals in the early stages of cognitive impairment, will make studies comparable, and by integrating knowledge, will accelerate the pace of progress.

Abstract During the past decade, a conceptual shift occurred in the field of Alzheimer's disease (AD) considering the disease as a continuum. Thanks to evolving biomarker research and substantial discoveries, it is now possible to identify the disease even at the preclinical stage before the occurrence of the first clinical symptoms. This preclinical stage of AD has become a major research focus as the field postulates that early intervention may offer the best chance of therapeutic success. To date, very little evidence is established on this “silent” stage of the disease. A clarification is needed about the definitions and lexicon, the limits, the natural history, the markers of progression, and the ethical consequence of detecting the disease at this asymptomatic stage. This article is aimed at addressing all the different issues by providing for each of them an updated review of the literature and evidence, with practical recommendations.

The epsilon4 allele of apolipoprotein E (APOE) is the major genetic risk factor for Alzheimer's disease (AD). Although there have been numerous studies attempting to elucidate the underlying mechanism for this increased risk, how apoE4 influences AD onset and progression has yet to be proven. However, prevailing evidence suggests that the differential effects of apoE isoforms on Abeta aggregation and clearance play the major role in AD pathogenesis. Other potential mechanisms, such as the differential modulation of neurotoxicity and tau phosphorylation by apoE isoforms as well as its role in synaptic plasticity and neuroinflammation, have not been ruled out. Inconsistent results among studies have made it difficult to define whether the APOE epsilon4 allele represents a gain of toxic function, a loss of neuroprotective function, or both. Therapeutic strategies based on apoE propose to reduce the toxic effects of apoE4 or to restore the physiological, protective functions of apoE.

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2) </= 25 minutes), mainly by vascular transport across the blood-brain barrier (BBB). The efflux transport system for Ass(1-40) at the BBB was half saturated at 15.3 nM, and the maximal transport capacity was reached between 70 nM and 100 nM. Ass(1-40) clearance was substantially inhibited by the receptor-associated protein, and by antibodies against LDL receptor-related protein-1 (LRP-1) and alpha(2)-macroglobulin (alpha(2)M). As compared to adult wild-type mice, clearance was significantly reduced in young and old apolipoprotein E (apoE) knockout mice, and in old wild-type mice. There was no evidence that Ass was metabolized in brain interstitial fluid and degraded to smaller peptide fragments and amino acids before its transport across the BBB into the circulation. LRP-1, although abundant in brain microvessels in young mice, was downregulated in older animals, and this downregulation correlated with regional Ass accumulation in brains of Alzheimer's disease (AD) patients. We conclude that the BBB removes Ass from the brain largely via age-dependent, LRP-1-mediated transport that is influenced by alpha(2)M and/or apoE, and may be impaired in AD.

Amyloid-beta (Abeta) accumulation in the brain extracellular space is a hallmark of Alzheimer's disease. The factors regulating this process are only partly understood. Abeta aggregation is a concentration-dependent process that is likely responsive to changes in brain interstitial fluid (ISF) levels of Abeta. Using in vivo microdialysis in mice, we found that the amount of ISF Abeta correlated with wakefulness. The amount of ISF Abeta also significantly increased during acute sleep deprivation and during orexin infusion, but decreased with infusion of a dual orexin receptor antagonist. Chronic sleep restriction significantly increased, and a dual orexin receptor antagonist decreased, Abeta plaque formation in amyloid precursor protein transgenic mice. Thus, the sleep-wake cycle and orexin may play a role in the pathogenesis of Alzheimer's disease.

Active immunization with the amyloid beta (A beta) peptide has been shown to decrease brain A beta deposition in transgenic mouse models of Alzheimer's disease and certain peripherally administered anti-A beta antibodies were shown to mimic this effect. In exploring factors that alter A beta metabolism and clearance, we found that a monoclonal antibody (m266) directed against the central domain of A beta was able to bind and completely sequester plasma A beta. Peripheral administration of m266 to PDAPP transgenic mice, in which A beta is generated specifically within the central nervous system (CNS), results in a rapid 1,000-fold increase in plasma A beta, due, in part, to a change in A beta equilibrium between the CNS and plasma. Although peripheral administration of m266 to PDAPP mice markedly reduces A beta deposition, m266 did not bind to A beta deposits in the brain. Thus, m266 appears to reduce brain A beta burden by altering CNS and plasma A beta clearance.

Amyloid-beta(42) (Abeta(42)) appears central to Alzheimer's disease (AD) pathogenesis and is a major component of amyloid plaques. Mean cerebrospinal fluid (CSF) Abeta(42) is decreased in dementia of the Alzheimer's type. This decrease may reflect plaques acting as an Abeta(42) "sink," hindering transport of soluble Abeta(42) between brain and CSF. We investigated this hypothesis.We compared the in vivo brain amyloid load (via positron emission tomography imaging of the amyloid-binding agent, Pittsburgh Compound-B [PIB]) with CSF Abeta(42) and other measures (via enzyme-linked immunosorbent assay) in clinically characterized research subjects.Subjects fell into two nonoverlapping groups: those with positive PIB binding had the lowest CSF Abeta(42) level, and those with negative PIB binding had the highest CSF Abeta(42) level. No relation was observed between PIB binding and CSF Abeta(40), tau, phospho-tau(181), plasma Abeta(40), or plasma Abeta(42). Importantly, PIB binding and CSF Abeta(42) did not consistently correspond with clinical diagnosis; three cognitively normal subjects were PIB-positive with low CSF Abeta(42), suggesting the presence of amyloid in the absence of cognitive impairment (ie, preclinical AD).These observations suggest that brain amyloid deposition results in low CSF Abeta(42), and that amyloid imaging and CSF Abeta(42) may potentially serve as antecedent biomarkers of (preclinical) AD.

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is central to Alzheimer's disease pathogenesis. Abeta aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Abeta levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Abeta levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Abeta metabolism and ultimately region-specific Abeta deposition. The findings also have important implications for treatment development.

In the first of our State of the Art Review series, David M. Holtzman, John C. Morris, and Alison M. Goate explore the rapid pace of Alzheimer’s disease research and the challenges to translating research breakthroughs into clinical treatments.

Human apolipoprotein E has three isoforms: APOE2, APOE3 and APOE4. APOE4 is a major genetic risk factor for Alzheimer's disease and is associated with Down's syndrome dementia and poor neurological outcome after traumatic brain injury and haemorrhage. Neurovascular dysfunction is present in normal APOE4 carriers and individuals with APOE4-associated disorders. In mice, lack of Apoe leads to blood-brain barrier (BBB) breakdown, whereas APOE4 increases BBB susceptibility to injury. How APOE genotype affects brain microcirculation remains elusive. Using different APOE transgenic mice, including mice with ablation and/or inhibition of cyclophilin A (CypA), here we show that expression of APOE4 and lack of murine Apoe, but not APOE2 and APOE3, leads to BBB breakdown by activating a proinflammatory CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes. This, in turn, leads to neuronal uptake of multiple blood-derived neurotoxic proteins, and microvascular and cerebral blood flow reductions. We show that the vascular defects in Apoe-deficient and APOE4-expressing mice precede neuronal dysfunction and can initiate neurodegenerative changes. Astrocyte-secreted APOE3, but not APOE4, suppressed the CypA-nuclear factor-κB-matrix-metalloproteinase-9 pathway in pericytes through a lipoprotein receptor. Our data suggest that CypA is a key target for treating APOE4-mediated neurovascular injury and the resulting neuronal dysfunction and degeneration.

Human apoE4 increases the concentration of soluble Aβ in the brain by impairing its clearance.

Considerable overlap has been identified in the risk factors, comorbidities and putative pathophysiological mechanisms of Alzheimer disease and related dementias (ADRDs) and type 2 diabetes mellitus (T2DM), two of the most pressing epidemics of our time. Much is known about the biology of each condition, but whether T2DM and ADRDs are parallel phenomena arising from coincidental roots in ageing or synergistic diseases linked by vicious pathophysiological cycles remains unclear. Insulin resistance is a core feature of T2DM and is emerging as a potentially important feature of ADRDs. Here, we review key observations and experimental data on insulin signalling in the brain, highlighting its actions in neurons and glia. In addition, we define the concept of 'brain insulin resistance' and review the growing, although still inconsistent, literature concerning cognitive impairment and neuropathological abnormalities in T2DM, obesity and insulin resistance. Lastly, we review evidence of intrinsic brain insulin resistance in ADRDs. By expanding our understanding of the overlapping mechanisms of these conditions, we hope to accelerate the rational development of preventive, disease-modifying and symptomatic treatments for cognitive dysfunction in T2DM and ADRDs alike.

Senile plaques accumulate over the course of decades in the brains of patients with Alzheimer's disease. A fundamental tenet of the amyloid hypothesis of Alzheimer's disease is that the deposition of amyloid-beta precedes and induces the neuronal abnormalities that underlie dementia. This idea has been challenged, however, by the suggestion that alterations in axonal trafficking and morphological abnormalities precede and lead to senile plaques. The role of microglia in accelerating or retarding these processes has been uncertain. To investigate the temporal relation between plaque formation and the changes in local neuritic architecture, we used longitudinal in vivo multiphoton microscopy to sequentially image young APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice. Here we show that plaques form extraordinarily quickly, over 24 h. Within 1-2 days of a new plaque's appearance, microglia are activated and recruited to the site. Progressive neuritic changes ensue, leading to increasingly dysmorphic neurites over the next days to weeks. These data establish plaques as a critical mediator of neuritic pathology.

Alzheimer disease (AD) is biologically defined by the presence of β-amyloid-containing plaques and tau-containing neurofibrillary tangles. AD is a genetic and sporadic neurodegenerative disease that causes an amnestic cognitive impairment in its prototypical presentation and non-amnestic cognitive impairment in its less common variants. AD is a common cause of cognitive impairment acquired in midlife and late-life but its clinical impact is modified by other neurodegenerative and cerebrovascular conditions. This Primer conceives of AD biology as the brain disorder that results from a complex interplay of loss of synaptic homeostasis and dysfunction in the highly interrelated endosomal/lysosomal clearance pathways in which the precursors, aggregated species and post-translationally modified products of Aβ and tau play important roles. Therapeutic endeavours are still struggling to find targets within this framework that substantially change the clinical course in persons with AD. Alzheimer disease is a neurodegenerative disorder that causes cognitive impairment. This Primer by Knopman et al. reviews the epidemiology of cognitive manifestations and risk factors, summarizes the pathophysiology from a synaptic disorder perspective, and reviews advances in the diagnosis, management and quality of life of persons living with Alzheimer disease.

APOE4 is the strongest genetic risk factor for late-onset Alzheimer disease. ApoE4 increases brain amyloid-β pathology relative to other ApoE isoforms. However, whether APOE independently influences tau pathology, the other major proteinopathy of Alzheimer disease and other tauopathies, or tau-mediated neurodegeneration, is not clear. By generating P301S tau transgenic mice on either a human ApoE knock-in (KI) or ApoE knockout (KO) background, here we show that P301S/E4 mice have significantly higher tau levels in the brain and a greater extent of somatodendritic tau redistribution by three months of age compared with P301S/E2, P301S/E3, and P301S/EKO mice. By nine months of age, P301S mice with different ApoE genotypes display distinct phosphorylated tau protein (p-tau) staining patterns. P301S/E4 mice develop markedly more brain atrophy and neuroinflammation than P301S/E2 and P301S/E3 mice, whereas P301S/EKO mice are largely protected from these changes. In vitro, E4-expressing microglia exhibit higher innate immune reactivity after lipopolysaccharide treatment. Co-culturing P301S tau-expressing neurons with E4-expressing mixed glia results in a significantly higher level of tumour-necrosis factor-α (TNF-α) secretion and markedly reduced neuronal viability compared with neuron/E2 and neuron/E3 co-cultures. Neurons co-cultured with EKO glia showed the greatest viability with the lowest level of secreted TNF-α. Treatment of P301S neurons with recombinant ApoE (E2, E3, E4) also leads to some neuronal damage and death compared with the absence of ApoE, with ApoE4 exacerbating the effect. In individuals with a sporadic primary tauopathy, the presence of an ε4 allele is associated with more severe regional neurodegeneration. In individuals who are positive for amyloid-β pathology with symptomatic Alzheimer disease who usually have tau pathology, ε4-carriers demonstrate greater rates of disease progression. Our results demonstrate that ApoE affects tau pathogenesis, neuroinflammation, and tau-mediated neurodegeneration independently of amyloid-β pathology. ApoE4 exerts a 'toxic' gain of function whereas the absence of ApoE is protective.

To investigate the ability of cerebrospinal fluid (CSF) and plasma measures to discriminate early-stage Alzheimer disease (AD) (defined by clinical criteria and presence/absence of brain amyloid) from nondemented aging and to assess whether these biomarkers can predict future dementia in cognitively normal individuals.Evaluation of CSF beta-amyloid(40) (Abeta(40)), Abeta(42), tau, phosphorylated tau(181), and plasma Abeta(40) and Abeta(42) and longitudinal clinical follow-up (from 1 to 8 years).Longitudinal studies of healthy aging and dementia through an AD research center.Community-dwelling volunteers (n = 139) aged 60 to 91 years and clinically judged as cognitively normal (Clinical Dementia Rating [CDR], 0) or having very mild (CDR, 0.5) or mild (CDR, 1) AD dementia.Individuals with very mild or mild AD have reduced mean levels of CSF Abeta(42) and increased levels of CSF tau and phosphorylated tau(181). Cerebrospinal fluid Abeta(42) level completely corresponds with the presence or absence of brain amyloid (imaged with Pittsburgh Compound B) in demented and nondemented individuals. The CSF tau/Abeta(42) ratio (adjusted hazard ratio, 5.21; 95% confidence interval, 1.58-17.22) and phosphorylated tau(181)/Abeta(42) ratio (adjusted hazard ratio, 4.39; 95% confidence interval, 1.62-11.86) predict conversion from a CDR of 0 to a CDR greater than 0.The very mildest symptomatic stage of AD exhibits the same CSF biomarker phenotype as more advanced AD. In addition, levels of CSF Abeta(42), when combined with amyloid imaging, augment clinical methods for identifying in individuals with brain amyloid deposits whether dementia is present or not. Importantly, CSF tau/Abeta(42) ratios show strong promise as antecedent (preclinical) biomarkers that predict future dementia in cognitively normal older adults.

Apolipoprotein E (apoE) alleles determine the age-adjusted relative risk (epsilon4 > epsilon3) for Alzheimer's disease (AD). ApoE may affect AD pathogenesis by promoting deposition of the amyloid-beta (Abeta) peptide and its conversion to a fibrillar form. To determine the effect of apoE on Abeta deposition and AD pathology, we compared APP(V717F) transgenic (TG) mice expressing mouse, human, or no apoE (apoE(-/-)). A severe, plaque-associated neuritic dystrophy developed in APP(V717F) TG mice expressing mouse or human apoE. Though significant levels of Abeta deposition also occurred in APP(V717F) TG, apoE(-/-) mice, neuritic degeneration was virtually absent. Expression of apoE3 and apoE4 in APP(V717F) TG, apoE(-/-) mice resulted in fibrillar Abeta deposits and neuritic plaques by 15 months of age and substantially (>10-fold) more fibrillar deposits were observed in apoE4-expressing APP(V717F) TG mice. Our data demonstrate a critical and isoform-specific role for apoE in neuritic plaque formation, a pathological hallmark of AD.

Apolipoprotein E is associated with age-related risk for Alzheimer's disease and plays critical roles in Aβ homeostasis. We report that ApoE plays a role in facilitating the proteolytic clearance of soluble Aβ from the brain. The endolytic degradation of Aβ peptides within microglia by neprilysin and related enzymes is dramatically enhanced by ApoE. Similarly, Aβ degradation extracellularly by insulin-degrading enzyme is facilitated by ApoE. The capacity of ApoE to promote Aβ degradation is dependent upon the ApoE isoform and its lipidation status. The enhanced expression of lipidated ApoE, through the activation of liver X receptors, stimulates Aβ degradation. Indeed, aged Tg2576 mice treated with the LXR agonist GW3965 exhibited a dramatic reduction in brain Aβ load. GW3965 treatment also reversed contextual memory deficits. These data demonstrate a mechanism through which ApoE facilitates the clearance of Aβ from the brain and suggest that LXR agonists may represent a novel therapy for AD.

Alzheimer's disease (AD) is a slowly progressing disorder in which pathophysiological abnormalities, detectable in vivo by biomarkers, precede overt clinical symptoms by many years to decades. Five AD biomarkers are sufficiently validated to have been incorporated into clinical diagnostic criteria and commonly used in therapeutic trials. Current AD biomarkers fall into two categories: biomarkers of amyloid-β plaques and of tau-related neurodegeneration. Three of the five are imaging measures and two are cerebrospinal fluid analytes. AD biomarkers do not evolve in an identical manner but rather in a sequential but temporally overlapping manner. Models of the temporal evolution of AD biomarkers can take the form of plots of biomarker severity (degree of abnormality) versus time. In this Review, we discuss several time-dependent models of AD that take into consideration varying age of onset (early versus late) and the influence of aging and co-occurring brain pathologies that commonly arise in the elderly.

Using a mouse model of Alzheimer's disease and in vivo microdialysis, this study shows that neuronal activity drives the level of interstitial fluid amyloid-β and subsequent amyloid-β plaque deposition. Amyloid-β (Aβ) plaque deposition in specific brain regions is a pathological hallmark of Alzheimer's disease. However, the mechanism underlying the regional vulnerability to Aβ deposition in Alzheimer's disease is unknown. Herein, we provide evidence that endogenous neuronal activity regulates the regional concentration of interstitial fluid (ISF) Aβ, which drives local Aβ aggregation. Using in vivo microdialysis, we show that ISF Aβ concentrations in several brain regions of APP transgenic mice before plaque deposition were commensurate with the degree of subsequent plaque deposition and with the concentration of lactate, a marker of neuronal activity. Furthermore, unilateral vibrissal stimulation increased ISF Aβ, and unilateral vibrissal deprivation decreased ISF Aβ and lactate, in contralateral barrel cortex. Long-term unilateral vibrissal deprivation decreased amyloid plaque formation and growth. Our results suggest a mechanism to account for the vulnerability of specific brain regions to Aβ deposition in Alzheimer's disease.

Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.

To examine interactions of apolipoprotein E (APOE) genotype with age and with in vivo measures of preclinical Alzheimer disease (AD) in cognitively normal aging.Two hundred forty-one cognitively normal individuals, aged 45-88 years, had cerebral amyloid imaging studies with Pittsburgh Compound-B (PIB). Of the 241 individuals, 168 (70%) also had cerebrospinal fluid (CSF) assays of amyloid-beta(42) (Abeta(42)), tau, and phosphorylated tau (ptau(181)). All individuals were genotyped for APOE.The frequency of individuals with elevated mean cortical binding potential (MCBP) for PIB rose in an age-dependent manner from 0% at ages 45-49 years to 30.3% at 80-88 years. Reduced levels of CSF Abeta(42) appeared to begin earlier (18.2% of those aged 45-49 years) and increase with age in higher frequencies (50% at age 80-88 years) than elevations of MCBP. There was a gene dose effect for the APOE4 genotype, with greater MCBP increases and greater reductions in CSF Abeta(42) with increased numbers of APOE4 alleles. Individuals with an APOE2 allele had no increase in MCBP with age and had higher CSF Abeta(42) levels than individuals without an APOE2 allele. There was no APOE4 or APOE2 effect on CSF tau or ptau(181).Increasing cerebral Abeta deposition with age is the pathobiological phenotype of APOE4. The biomarker sequence that detects Abeta deposition may first be lowered CSF Abeta(42), followed by elevated MCBP for PIB. A substantial proportion of cognitively normal individuals have presumptive preclinical AD.

Apolipoprotein E (APOE) is a 299-aminoacid protein encoded by the APOE gene. Three common polymorphisms in the APOE gene, ɛ2, ɛ3, and ɛ4, result in a single aminoacid change in the APOE protein. APOE ɛ2, ɛ3, and ɛ4 alleles strongly alter, in a dose-dependent manner, the likelihood of developing Alzheimer's disease and cerebral amyloid angiopathy. In particular, APOE ɛ4 is associated with increased risk for Alzheimer's disease whereas APOE ɛ2 is associated with decreased risk. The effects of APOE genotype on risk of these diseases are likely to be mediated by differential effects of APOE on amyloid-β accumulation in the brain and its vasculature. Response to treatment for Alzheimer's disease might differ according to APOE genotype. Because convincing evidence ties the APOE genotype to risk of Alzheimer's disease and cerebral amyloid angiopathy, APOE has been studied in other neurological diseases. APOE ɛ4 is associated with poor outcome after traumatic brain injury and brain haemorrhage, although the mechanisms underlying these associations are unclear. The possibility that APOE has a role in these and other neurological diseases has been of great interest, but convincing associations have not yet emerged.

In the premature infant, hypoxic-ischemic damage to the cerebral white matter [periventricular leukomalacia (PVL)] is a common and leading cause of brain injury that often results in chronic neurologic disability from cerebral palsy. The cellular basis for the propensity of white matter injury to occur in the developing brain and the greater resistance of the adult white matter to similar injury remains unknown. By using a neonatal rat model of hypoxic-ischemic injury, we found that the mechanism of perinatal white matter injury involved maturation-dependent vulnerability in the oligodendroctye (OL) lineage. The timing of appearance of late OL progenitors was the major developmental factor that accounted for the susceptibility of the neonatal white matter to injury. Late OL progenitors were the major OL lineage stage killed by apoptosis, whereas early OL progenitors and more mature OLs were highly resistant. The density of pyknotic late OL progenitors was significantly increased in the ischemic hemisphere (67 ± 31 cells/mm²) versus the control hemisphere (2.2 ± 0.4 cells/mm²; mean ± SEM; <i>p</i> = 0.05), which resulted in the death of 72 ± 6% of this OL stage. Surviving late OL progenitors displayed a reactive response in which an increase in cell density was accompanied by accelerated maturation to a P27/kip1-positive oligodendrocyte. Because we showed recently that late OL progenitors populate human cerebral white matter during the high risk period for PVL (Back et al., 2001), maturation-dependent vulnerability of OL progenitors to hypoxia–ischemia may underlie the selective vulnerability to PVL of the white matter in the premature infant.

Factors other than age and genetics may increase the risk of developing Alzheimer disease (AD). Accumulation of the amyloid-β (Aβ) peptide in the brain seems to initiate a cascade of key events in the pathogenesis of AD. Moreover, evidence is emerging that the sleep-wake cycle directly influences levels of Aβ in the brain. In experimental models, sleep deprivation increases the concentration of soluble Aβ and results in chronic accumulation of Aβ, whereas sleep extension has the opposite effect. Furthermore, once Aβ accumulates, increased wakefulness and altered sleep patterns develop. Individuals with early Aβ deposition who still have normal cognitive function report sleep abnormalities, as do individuals with very mild dementia due to AD. Thus, sleep and neurodegenerative disease may influence each other in many ways that have important implications for the diagnosis and treatment of AD.

Glia have been implicated in Alzheimer's disease (AD) pathogenesis. Variants of the microglia receptor triggering receptor expressed on myeloid cells 2 (TREM2) increase AD risk, and activation of disease-associated microglia (DAM) is dependent on TREM2 in mouse models of AD. We surveyed gene-expression changes associated with AD pathology and TREM2 in 5XFAD mice and in human AD by single-nucleus RNA sequencing. We confirmed the presence of Trem2-dependent DAM and identified a previously undiscovered Serpina3n+C4b+ reactive oligodendrocyte population in mice. Interestingly, remarkably different glial phenotypes were evident in human AD. Microglia signature was reminiscent of IRF8-driven reactive microglia in peripheral-nerve injury. Oligodendrocyte signatures suggested impaired axonal myelination and metabolic adaptation to neuronal degeneration. Astrocyte profiles indicated weakened metabolic coordination with neurons. Notably, the reactive phenotype of microglia was less evident in TREM2-R47H and TREM2-R62H carriers than in non-carriers, demonstrating a TREM2 requirement in both mouse and human AD, despite the marked species-specific differences.

Apolipoprotein E (APOE) genotype is the major genetic risk factor for Alzheimer disease (AD); the 14 allele increases risk and the 12 allele is protective.In the central nervous system (CNS), apoE is produced by glial cells, is present in high-density-like lipoproteins, interacts with several receptors that are members of the low-density lipoprotein receptor (LDLR) family, and is a protein that binds to the amyloid-b (Ab) peptide.There are a variety of mechanisms by which apoE isoform may influence risk for AD.There is substantial evidence that differential effects of apoE isoform on AD risk are influenced by the ability of apoE to affect Ab aggregation and clearance in the brain.Other mechanisms are also likely to play a role in the ability of apoE to influence CNS function as well as AD, including effects on synaptic plasticity, cell signaling, lipid transport and metabolism, and neuroinflammation.ApoE receptors, including LDLRs, Apoer2, very low-density lipoprotein receptors (VLDLRs), and lipoprotein receptor-related protein 1 (LRP1) appear to influence both the CNS effects of apoE as well as Ab metabolism and toxicity.Therapeutic strategies based on apoE and apoE receptors may include influencing apoE/Ab interactions, apoE structure, apoE lipidation, LDLR receptor family member function, and signaling.Understanding the normal and disease-related biology connecting apoE, apoE receptors, and AD is likely to provide novel insights into AD pathogenesis and treatment.A lzheimer disease (AD), specifically the late- onset form of AD (LOAD), is the most common cause of dementia in individuals older than 60 years of age.Although mutations in the genes PS1, PS2, and APP cause less common forms of early-onset, autosomal dominant familial AD (FAD), these cases represent ,1% of AD.In addition to the genes that cause FAD, LOAD also has a strong genetic component.Although several susceptibility genes for AD have been reported, by far the strongest genetic risk factor for LOAD is apolipoprotein

Neurotoxic amyloid beta peptide (Abeta) accumulates in the brains of individuals with Alzheimer disease (AD). The APOE4 allele is a major risk factor for sporadic AD and has been associated with increased brain parenchymal and vascular amyloid burden. How apoE isoforms influence Abeta accumulation in the brain has, however, remained unclear. Here, we have shown that apoE disrupts Abeta clearance across the mouse blood-brain barrier (BBB) in an isoform-specific manner (specifically, apoE4 had a greater disruptive effect than either apoE3 or apoE2). Abeta binding to apoE4 redirected the rapid clearance of free Abeta40/42 from the LDL receptor-related protein 1 (LRP1) to the VLDL receptor (VLDLR), which internalized apoE4 and Abeta-apoE4 complexes at the BBB more slowly than LRP1. In contrast, apoE2 and apoE3 as well as Abeta-apoE2 and Abeta-apoE3 complexes were cleared at the BBB via both VLDLR and LRP1 at a substantially faster rate than Abeta-apoE4 complexes. Astrocyte-secreted lipo-apoE2, lipo-apoE3, and lipo-apoE4 as well as their complexes with Abeta were cleared at the BBB by mechanisms similar to those of their respective lipid-poor isoforms but at 2- to 3-fold slower rates. Thus, apoE isoforms differentially regulate Abeta clearance from the brain, and this might contribute to the effects of APOE genotype on the disease process in both individuals with AD and animal models of AD.

Alzheimer's disease affects millions of people around the world. Currently, there are no treatments that prevent or slow the disease. Like other neurodegenerative diseases, Alzheimer's disease is characterized by protein misfolding in the brain. This process and the associated brain damage begin years before the substantial neurodegeneration that accompanies dementia. Studies using new neuroimaging techniques and fluid biomarkers suggest that Alzheimer's disease pathology can be detected preclinically. These advances should allow the design of new clinical trials and early mechanism-based therapeutic intervention.

The amyloid cascade is perhaps the most dominant hypothesis in the field of Alzheimer's disease pathogenesis but it is also one of the most controversial. Here, we present two Perspective articles which argue both for and against the amyloid hypothesis. In this piece, Drs. Musiek and Holtzman argue that, despite sometimes conflicting data, there is ample evidence to suggest that Aβ accumulation is a key initiator of AD-related pathology and may act as a trigger of downstream effects such as tau aggregation. The amyloid hypothesis, which has been the predominant framework for research in Alzheimer's disease (AD), has been the source of considerable controversy. The amyloid hypothesis postulates that amyloid-β peptide (Aβ) is the causative agent in AD. It is strongly supported by data from rare autosomal dominant forms of AD. However, the evidence that Aβ causes or contributes to age-associated sporadic AD is more complex and less clear, prompting criticism of the hypothesis. We provide an overview of the major arguments for and against the amyloid hypothesis. We conclude that Aβ likely is the key initiator of a complex pathogenic cascade that causes AD. However, we argue that Aβ acts primarily as a trigger of other downstream processes, particularly tau aggregation, which mediate neurodegeneration. Aβ appears to be necessary, but not sufficient, to cause AD. Its major pathogenic effects may occur very early in the disease process.

Accumulation of amyloid-β (Aβ) within extracellular spaces of the brain is a hallmark of Alzheimer disease (AD).In sporadic, late-onset AD, there is little evidence for increased Aβ production, suggesting that decreased elimination from the brain may contribute to elevated levels of Aβ and plaque formation.Efflux transport of Aβ across the blood-brain barrier (BBB) contributes to Aβ removal from the brain.P-glycoprotein (Pgp) is highly expressed on the luminal surface of brain capillary endothelial cells and contributes to the BBB.In Pgp-null mice, we show that [ 125 I]Aβ 40 and [ 125 I]Aβ 42 microinjected into the CNS clear at half the rate that they do in WT mice.When amyloid precursor protein-transgenic (APP-transgenic) mice were administered a Pgp inhibitor, Aβ levels within the brain interstitial fluid significantly increased within hours of treatment.Furthermore, APP-transgenic, Pgp-null mice had increased levels of brain Aβ and enhanced Aβ deposition compared with APP-transgenic, Pgp WT mice.These data establish a direct link between Pgp and Aβ metabolism in vivo and suggest that Pgp activity at the BBB could affect risk for developing AD as well as provide a novel diagnostic and therapeutic target.

Amyloid β-peptide (Aβ) clearance from the central nervous system (CNS) maintains its low levels in brain. In Alzheimer's disease, Aβ accumulates in brain possibly because of its faulty CNS clearance and a deficient efflux across the blood—brain barrier (BBB). By using human-specific enzyme-linked immunosorbent assays, we measured a rapid 30 mins efflux at the BBB and transport via the interstitial fluid (ISF) bulk flow of human-unlabeled Aβ and of Aβ transport proteins, apolipoprotein E (apoE) and apoJ in mice. We show (i) Aβ40 is cleared rapidly across the BBB via low-density lipoprotein receptor-related protein (LRP)1 at a rate of 0.21 pmol/min g ISF or 6-fold faster than via the ISF flow; (ii) Aβ42 is removed across the BBB at a rate 1.9-fold slower compared with Aβ40; (iii) apoE, lipid-poor isoform 3, is cleared slowly via the ISF flow and across the BBB (0.03–0.04 pmol/min g ISF), and after lipidation its transport at the BBB becomes barely detectable within 30 mins; (iv) apoJ is eliminated rapidly across the BBB (0.16 pmol/ming ISF) via LRP2. Clearance rates of unlabeled and corresponding 125 I-labeled Aβ and apolipoproteins were almost identical, but could not be measured at low physiologic levels by mass spectrometry. Amyloid β-peptide 40 binding to apoE3 reduced its efflux rate at the BBB by 5.7-fold, whereas Aβ42 binding to apoJ enhanced Aβ42 BBB clearance rate by 83%. Thus, Aβ, apoE, and apoJ are cleared from brain by different transport pathways, and apoE and apoJ may critically modify Aβ clearance at the BBB.

Sleep and circadian problems are very common in Alzheimer disease (AD). Recent animal studies suggest a bidirectional relationship between sleep and β-amyloid (Aβ), a key molecule involved in AD pathogenesis.To test whether Aβ deposition in preclinical AD, prior to the appearance of cognitive impairment, is associated with changes in quality or quantity of sleep.Cross-sectional study conducted from October 2010 to June 2012.General community volunteers at the Washington University Knight Alzheimer's Disease Research Center.Cognitively normal individuals (n = 145) 45 years and older were recruited from longitudinal studies of memory and aging at the Washington University Knight Alzheimer's Disease Research Center. Valid actigraphy data were recorded in 142. The majority (124 of 142) were recruited from the Adult Children Study, in which all were aged 45 to 75 years at baseline and 50% have a parental history of late-onset AD. The rest were recruited from a community volunteer cohort in which all were older than 60 years and healthy at baseline.Sleep was objectively measured using actigraphy for 2 weeks. Sleep efficiency, which is the percentage of time in bed spent asleep, was the primary measure of sleep quality. Total sleep time was the primary measure of sleep quantity. Cerebrospinal fluid Aβ42 levels were used to determine whether amyloid deposition was present or absent. Concurrent sleep diaries provided nap information.Amyloid deposition, as assessed by Aβ42 levels, was present in 32 participants (22.5%). This group had worse sleep quality, as measured by sleep efficiency (80.4% vs 83.7%), compared with those without amyloid deposition, after correction for age, sex, and APOEε4 allele carrier status (P = .04). In contrast, quantity of sleep was not significantly different between groups, as measured by total sleep time. Frequent napping, 3 or more days per week, was associated with amyloid deposition (31.2% vs 14.7%; P = .03).Amyloid deposition in the preclinical stage of AD appears to be associated with worse sleep quality but not with changes in sleep quantity.

PET imaging of pathological tau correlates more closely with Alzheimer’s disease–related cognitive impairment than does imaging of β-amyloid.

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial receptor that recognizes changes in the lipid microenvironment, which may occur during amyloid β (Aβ) accumulation and neuronal degeneration in Alzheimer’s disease (AD). Rare TREM2 variants that affect TREM2 function lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents microglial clustering around Aβ deposits. However, the origin of myeloid cells surrounding amyloid and the impact of TREM2 on Aβ accumulation are a matter of debate. Using parabiosis, we found that amyloid-associated myeloid cells derive from brain-resident microglia rather than from recruitment of peripheral blood monocytes. To determine the impact of TREM2 deficiency on Aβ accumulation, we examined Aβ plaques in the 5XFAD model of AD at the onset of Aβ-related pathology. At this early time point, Aβ accumulation was similar in TREM2-deficient and -sufficient 5XFAD mice. However, in the absence of TREM2, Aβ plaques were not fully enclosed by microglia; they were more diffuse, less dense, and were associated with significantly greater neuritic damage. Thus, TREM2 protects from AD by enabling microglia to surround and alter Aβ plaque structure, thereby limiting neuritic damage.

Disruptions of normal circadian rhythms and sleep cycles are consequences of aging and can profoundly affect health. Accumulating evidence indicates that circadian and sleep disturbances, which have long been considered symptoms of many neurodegenerative conditions, may actually drive pathogenesis early in the course of these diseases. In this Review, we explore potential cellular and molecular mechanisms linking circadian dysfunction and sleep loss to neurodegenerative diseases, with a focus on Alzheimer’s disease. We examine the interplay between central and peripheral circadian rhythms, circadian clock gene function, and sleep in maintaining brain homeostasis, and discuss therapeutic implications. The circadian clock and sleep can influence a number of key processes involved in neurodegeneration, suggesting that these systems might be manipulated to promote healthy brain aging.

In addition to pathology in the gray matter, there are also abnormalities in the white matter in Alzheimer's disease (AD). Sulfatide species are a class of myelin-specific sphingolipids and are involved in certain diseases of the central nervous system. To assess whether sulfatide content in gray and white matter in human subjects is associated with both the presence of Alzheimer's disease (AD) pathology as well as the stage of dementia, we analyzed the sulfatide content of brain tissue lipid extracts by electrospray ionization mass spectrometry from 22 subjects whose cognitive status at time of death varied from no dementia to very severe dementia. All subjects with dementia had AD pathology. The results demonstrate that: (i) sulfatides were depleted up to 93% in gray matter and up to 58% in white matter from all examined brain regions from AD subjects with very mild dementia, whereas all other major classes of lipid (except plasmalogen) in these subjects were not altered in comparison to those from age-matched subjects with no dementia; (ii) there was no apparent deficiency in the biosynthesis of sulfatides in very mild AD subjects as characterized by the examination of galactocerebroside sulfotransferase activities in post-mortem brain tissues; (iii) the content of ceramides (a class of potential degradation products of sulfatides) was elevated more than three-fold in white matter and peaked at the stage of very mild dementia. The findings demonstrate that a marked decrease in sulfatides is associated with AD pathology even in subjects with very mild dementia and that these changes may be linked with early events in the pathological process of AD.

The deposition of amyloid-beta (Abeta) peptides into amyloid plaques precedes the cognitive dysfunction of Alzheimer's disease (AD) by years. Biomarkers indicative of brain amyloid burden could be useful for identifying individuals at high risk for developing AD. As in AD in humans, baseline plasma Abeta levels in a transgenic mouse model of AD did not correlate with brain amyloid burden. However, after peripheral administration of a monoclonal antibody to Abeta (m266), we observed a rapid increase in plasma Abeta and the magnitude of this increase was highly correlated with amyloid burden in the hippocampus and cortex. This method may be useful for quantifying brain amyloid burden in patients at risk for or those who have been diagnosed with AD.

<h3>Objective</h3> We examined whether plasma β-amyloid (Aβ)42/Aβ40, as measured by a high-precision assay, accurately diagnosed brain amyloidosis using amyloid PET or CSF p-tau181/Aβ42 as reference standards. <h3>Methods</h3> Using an immunoprecipitation and liquid chromatography–mass spectrometry assay, we measured Aβ42/Aβ40 in plasma and CSF samples from 158 mostly cognitively normal individuals that were collected within 18 months of an amyloid PET scan. <h3>Results</h3> Plasma Aβ42/Aβ40 had a high correspondence with amyloid PET status (receiver operating characteristic area under the curve [AUC] 0.88, 95% confidence interval [CI] 0.82–0.93) and CSF p-tau181/Aβ42 (AUC 0.85, 95% CI 0.79–0.92). The combination of plasma Aβ42/Aβ40, age, and <i>APOE</i> ε4 status had a very high correspondence with amyloid PET (AUC 0.94, 95% CI 0.90–0.97). Individuals with a negative amyloid PET scan at baseline and a positive plasma Aβ42/Aβ40 (&lt;0.1218) had a 15-fold greater risk of conversion to amyloid PET-positive compared to individuals with a negative plasma Aβ42/Aβ40 (<i>p</i> = 0.01). <h3>Conclusions</h3> Plasma Aβ42/Aβ40, especially when combined with age and <i>APOE</i> ε4 status, accurately diagnoses brain amyloidosis and can be used to screen cognitively normal individuals for brain amyloidosis. Individuals with a negative amyloid PET scan and positive plasma Aβ42/Aβ40 are at increased risk for converting to amyloid PET-positive. Plasma Aβ42/Aβ40 could be used in prevention trials to screen for individuals likely to be amyloid PET-positive and at risk for Alzheimer disease dementia. <h3>Classification of evidence</h3> This study provides Class II evidence that plasma Aβ42/Aβ40 levels accurately determine amyloid PET status in cognitively normal research participants.

Certain disease states are characterized by disturbances in production, accumulation or clearance of protein. In Alzheimer disease, accumulation of amyloid-beta (Abeta) in the brain and disease-causing mutations in amyloid precursor protein or in enzymes that produce Abeta indicate dysregulation of production or clearance of Abeta. Whether dysregulation of Abeta synthesis or clearance causes the most common form of Alzheimer disease (sporadic, >99% of cases), however, is not known. Here, we describe a method to determine the production and clearance rates of proteins within the human central nervous system (CNS). We report the first measurements of the fractional production and clearance rates of Abeta in vivo in the human CNS to be 7.6% per hour and 8.3% per hour, respectively. This method may be used to search for novel biomarkers of disease, to assess underlying differences in protein metabolism that contribute to disease and to evaluate treatments in terms of their pharmacodynamic effects on proposed disease-causing pathways.

Alzheimer's disease (AD) is the most common cause of dementia. Much is known concerning AD pathophysiology but our understanding of the disease at the systems level remains incomplete. Previous AD research has used resting-state functional connectivity magnetic resonance imaging (rs-fcMRI) to assess the integrity of functional networks within the brain. Most studies have focused on the default-mode network (DMN), a primary locus of AD pathology. However, other brain regions are inevitably affected with disease progression. We studied rs-fcMRI in five functionally defined brain networks within a large cohort of human participants of either gender (n = 510) that ranged in AD severity from unaffected [clinical dementia rating (CDR) 0] to very mild (CDR 0.5) to mild (CDR 1). We observed loss of correlations within not only the DMN but other networks at CDR 0.5. Within the salience network (SAL), increases were seen between CDR 0 and CDR 0.5. However, at CDR 1, all networks, including SAL, exhibited reduced correlations. Specific networks were preferentially affected at certain CDR stages. In addition, cross-network relations were consistently lost with increasing AD severity. Our results demonstrate that AD is associated with widespread loss of both intranetwork and internetwork correlations. These results provide insight into AD pathophysiology and reinforce an integrative view of the brain's functional organization.

Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼ 300 fM) and synuclein (∼ 300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.

To explore the hypothesis that alterations in ethanolamine plasmalogen may be directly related to the severity of dementia in Alzheimer's disease (AD), we performed a systematic examination of plasmalogen content in cellular membranes of gray and white matter from different regions of human subjects with a spectrum of AD clinical dementia ratings (CDR) using electrospray ionization mass spectrometry (ESI/MS). The results demonstrate: (1) a dramatic decrease in plasmalogen content (up to 40 mol% of total plasmalogen) in white matter at a very early stage of AD (i.e. CDR 0.5); (2) a correlation of the deficiency in gray matter plasmalogen content with the AD CDR (i.e. approximately 10 mol% of deficiency at CDR 0.5 (very mild dementia) to approximately 30 mol% of deficiency at CDR 3 (severe dementia); (3) an absence of alterations of plasmalogen content and molecular species in cerebellar gray matter at any CDR despite dramatic alterations of plasmalogen content in cerebellar white matter. Alterations of ethanolamine plasmalogen content in two mouse models of AD, APP(V717F) and APPsw, were also examined by ESI/MS. A plasmalogen deficiency was present (up to 10 mol% of total plasmalogen at the age of 18 months) in cerebral cortices, but was absent in cerebella from both animal models. These results suggest plasmalogen deficiency may play an important role in the AD pathogenesis, particularly in the white matter, and suggest that altered plasmalogen content may contribute to neurodegeneration, synapse loss and synaptic dysfunction in AD.

Background: Trans-cellular propagation of aggregation may be important in neurodegeneration, but mechanisms are unknown.Results: Tau fibrils are secreted into the extracellular space, where they directly trigger aggregation in recipient cells by contacting native protein. Conclusion:Trans-cellular movement of Tau fibrils seeds subsequent aggregation.Significance: Therapies that block trans-cellular movement, including antibodies, may have an important role in neurodegenerative diseases. Aggregation of the microtubule associated proteinTau is associated with several neurodegenerative disorders, including Alzheimer disease and frontotemporal dementia.In Alzheimer disease, Tau pathology spreads progressively throughout the brain, possibly along existing neural networks.However, it is still unclear how the propagation of Tau misfolding occurs.Intriguingly, in animal models, vaccine-based therapies have reduced Tau and synuclein pathology by uncertain mechanisms, given that these proteins are intracellular.We have previously speculated that trans-cellular propagation of misfolding could be mediated by a process similar to prion pathogenesis, in which fibrillar Tau aggregates spread pathology from cell to cell.However, there has been little evidence to demonstrate true transcellular propagation of Tau misfolding, in which Tau aggregates from one cell directly contact Tau protein in the recipient cell to trigger further aggregation.Here we have observed that intracellular Tau fibrils are directly released into the medium and then taken up by co-cultured cells.Internalized Tau aggregates induce fibrillization of intracellular Tau in these naive recipient cells via direct protein-protein contact that we demonstrate using FRET.Tau aggregation can be amplified across several generations of cells.An anti-Tau monoclonal antibody blocks Tau aggregate propagation by trapping fibrils in the extracellular space and preventing their uptake.Thus, propagation of Tau protein misfolding among cells can be mediated by release and subsequent uptake of fibrils that directly contact native protein in recipient cells.These results support the model of aggregate propagation by templated conformational change and suggest a mechanism for vaccine-based therapies in neurodegenerative diseases.

Tau aggregation occurs in neurodegenerative diseases including Alzheimer’s disease and many other disorders collectively termed tauopathies. trans-cellular propagation of tau pathology, mediated by extracellular tau aggregates, may underlie pathogenesis of these conditions. P301S tau transgenic mice express mutant human tau protein and develop progressive tau pathology. Using a cell-based biosensor assay, we screened anti-tau monoclonal antibodies for their ability to block seeding activity present in P301S brain lysates. We infused three effective antibodies or controls into the lateral ventricle of P301S mice for 3 months. The antibodies markedly reduced hyperphosphorylated, aggregated, and insoluble tau. They also blocked development of tau seeding activity detected in brain lysates using the biosensor assay, reduced microglial activation, and improved cognitive deficits. These data imply a central role for extracellular tau aggregates in the development of pathology. They also suggest that immunotherapy specifically designed to block trans-cellular aggregate propagation will be a productive treatment strategy.

Programmed cell death (apoptosis) is a normal process in the developing nervous system. Recent data suggest that certain features seen in the process of programmed cell death may be favored in the developing versus the adult brain in response to different brain injuries. In a well characterized model of neonatal hypoxia-ischemia, we demonstrate marked but delayed cell death in which there is prominent DNA laddering, TUNEL-labeling, and nuclei with condensed chromatin. Caspase activation, which is required in many cases of apoptotic cell death, also followed a delayed time course after hypoxia-ischemia. Administration of boc-aspartyl(OMe)-fluoromethylketone, a pan-caspase inhibitor, was significantly neuroprotective when given by intracerebroventricular injection 3 h after cerebral hypoxia-ischemia. In addition, systemic injections of boc-aspartyl(OMe)-fluoromethylketone also given in a delayed fashion, resulted in significant neuroprotection. These findings suggest that caspase inhibitors may be able to provide benefit over a prolonged therapeutic window after hypoxic-ischemic events in the developing brain, a major contributor to static encephalopathy and cerebral palsy.

Severe amyloidosis and plaque-localized neuro-inflammation are key pathological features of Alzheimer's disease (AD). In addition to astrocyte and microglial reactivity, emerging evidence suggests a role of gut microbiota in regulating innate immunity and influencing brain function. Here, we examine the role of the host microbiome in regulating amyloidosis in the APPSWE/PS1ΔE9 mouse model of AD. We show that prolonged shifts in gut microbial composition and diversity induced by long-term broad-spectrum combinatorial antibiotic treatment regime decreases Aβ plaque deposition. We also show that levels of soluble Aβ are elevated and that levels of circulating cytokine and chemokine signatures are altered in this setting. Finally, we observe attenuated plaque-localised glial reactivity in these mice and significantly altered microglial morphology. These findings suggest the gut microbiota community diversity can regulate host innate immunity mechanisms that impact Aβ amyloidosis.

The sleep-wake cycle regulates interstitial fluid (ISF) and cerebrospinal fluid (CSF) levels of β-amyloid (Aβ) that accumulates in Alzheimer's disease (AD). Furthermore, chronic sleep deprivation (SD) increases Aβ plaques. However, tau, not Aβ, accumulation appears to drive AD neurodegeneration. We tested whether ISF/CSF tau and tau seeding and spreading were influenced by the sleep-wake cycle and SD. Mouse ISF tau was increased ~90% during normal wakefulness versus sleep and ~100% during SD. Human CSF tau also increased more than 50% during SD. In a tau seeding-and-spreading model, chronic SD increased tau pathology spreading. Chemogenetically driven wakefulness in mice also significantly increased both ISF Aβ and tau. Thus, the sleep-wake cycle regulates ISF tau, and SD increases ISF and CSF tau as well as tau pathology spreading.

New research criteria for preclinical Alzheimer's disease have been proposed, which include stages for cognitively normal individuals with abnormal amyloid markers (stage 1), abnormal amyloid and neuronal injury markers (stage 2), or abnormal amyloid and neuronal injury markers and subtle cognitive changes (stage 3). We aimed to investigate the prevalence and long-term outcome of preclinical Alzheimer's disease according to these criteria.Participants were cognitively normal (clinical dementia rating [CDR]=0) community-dwelling volunteers aged at least 65 years who were enrolled between 1998 and 2011 at the Washington University School of Medicine (MO, USA). CSF amyloid-β1-42 and tau concentrations and a memory composite score were used to classify participants as normal (both markers normal), preclinical Alzheimer's disease stage 1-3, or suspected non-Alzheimer pathophysiology (SNAP, abnormal injury marker without abnormal amyloid marker). The primary outcome was the proportion of participants in each preclinical AD stage. Secondary outcomes included progression to CDR at least 0·5, symptomatic Alzheimer's disease (score of at least 0·5 for memory and at least one other domain and cognitive impairments deemed to be due to Alzheimer's disease), and mortality. We undertook survival analyses using subdistribution and standard Cox hazards models and linear mixed models.Of 311 participants, 129 (41%) were classed as normal, 47 (15%) as stage 1, 36 (12%) as stage 2, 13 (4%) as stage 3, 72 (23%) as SNAP, and 14 (5%) remained unclassified. The 5-year progression rate to CDR at least 0·5, symptomatic Alzheimer's disease was 2% for participants classed as normal, 11% for stage 1, 26% for stage 2, 56% for stage 3, and 5% for SNAP. Compared with individuals classed as normal, participants with preclinical Alzheimer's disease had an increased risk of death after adjusting for covariates (hazard ratio 6·2, 95% CI 1·1-35·0; p=0·040).Preclinical Alzheimer's disease is common in cognitively normal elderly people and is associated with future cognitive decline and mortality. Thus, preclinical Alzheimer's disease could be an important target for therapeutic intervention.National Institute of Aging of the National Institutes of Health (P01-AG003991, P50-AG05681, P01-AG02676), Internationale Stichting Alzheimer Onderzoek, the Center for Translational Molecular Medicine project LeARN, the EU/EFPIA Innovative Medicines Initiative Joint Undertaking, and the Charles and Joanne Knight Alzheimer Research Initiative.

Objective: To determine whether preclinical Alzheimer disease (AD), as detected by the amyloid-imaging agent Pittsburgh Compound B (PiB) in cognitively normal older adults, is associated with risk of symptomatic AD.Design: A longitudinal cohort study of cognitively normal older adults assessed with positron emission tomography (PET) to determine the mean cortical binding potential for PiB and followed up with annual clinical and cognitive assessments for progression to very mild dementia of the Alzheimer type (DAT).

Aggregation of β-amyloid (Aβ) in the brain begins to occur years before the clinical onset of Alzheimer's disease (AD). Before Aβ aggregation, concentrations of extracellular soluble Aβ in the interstitial fluid (ISF) space of the brain, which are regulated by neuronal activity and the sleep-wake cycle, correlate with the amount of Aβ deposition in the brain seen later. The amount and quality of sleep decline with normal aging and to a greater extent in AD patients. How sleep quality as well as the diurnal fluctuation in Aβ change with age and Aβ aggregation is not well understood. We report a normal sleep-wake cycle and diurnal fluctuation in ISF Aβ in the brain of the APPswe/PS1δE9 mouse model of AD before Aβ plaque formation. After plaque formation, the sleep-wake cycle markedly deteriorated and diurnal fluctuation of ISF Aβ dissipated. As in mice, diurnal fluctuation of cerebrospinal fluid Aβ in young adult humans with presenilin mutations was also markedly attenuated after Aβ plaque formation. Virtual elimination of Aβ deposits in the mouse brain by active immunization with Aβ(42) normalized the sleep-wake cycle and the diurnal fluctuation of ISF Aβ. These data suggest that Aβ aggregation disrupts the sleep-wake cycle and diurnal fluctuation of Aβ. Sleep-wake behavior and diurnal fluctuation of Aβ in the central nervous system may be functional and biochemical indicators, respectively, of Aβ-associated pathology.

Tau is primarily a cytoplasmic protein that stabilizes microtubules. However, it is also found in the extracellular space of the brain at appreciable concentrations. Although its presence there may be relevant to the intercellular spread of tau pathology, the cellular mechanisms regulating tau release into the extracellular space are not well understood. To test this in the context of neuronal networks in vivo, we used in vivo microdialysis. Increasing neuronal activity rapidly increased the steady-state levels of extracellular tau in vivo. Importantly, presynaptic glutamate release is sufficient to drive tau release. Although tau release occurred within hours in response to neuronal activity, the elimination rate of tau from the extracellular compartment and the brain is slow (half-life of ∼11 d). The in vivo results provide one mechanism underlying neuronal tau release and may link trans-synaptic spread of tau pathology with synaptic activity itself.

Significance It has been proposed that differential physical interactions of apolipoprotein E (apoE) isoforms with soluble amyloid-β (Aβ) in brain fluids influence the metabolism of Aβ, providing a major mechanism to account for how APOE influences Alzheimer’s disease risk. The current study challenges this proposal and clearly shows that lipoproteins containing apoE isoforms are unlikely to play a significant role in Aβ metabolism by binding directly to Aβ in physiological fluids such as cerebrospinal fluid or interstitial fluid. Our in vitro and in vivo results suggest that apoE isoforms influence Aβ metabolism by competing for the same clearance pathways within the brain.

Neurotrophins activate several different intracellular signaling pathways that in some way exert neuroprotective effects. In vitro studies of sympathetic and cerebellar granule neurons have demonstrated that the survival effects of neurotrophins can be mediated via activation of the phosphatidylinositol 3-kinase (PI3-kinase) pathway. Neurotrophin-mediated protection of other neuronal types in vitro can be mediated via the extracellular signal-related protein kinase (ERK) pathway. Whether either of these pathways contributes to the neuroprotective effects of neurotrophins in the brain in vivo has not been determined. Brain-derived neurotrophic factor (BDNF) is markedly neuroprotective against neonatal hypoxic-ischemic (H-I) brain injury in vivo . We assessed the role of the ERK and PI3-kinase pathways in neonatal H-I brain injury in the presence and absence of BDNF. Intracerebroventricular administration of BDNF to postnatal day 7 rats resulted in phosphorylation of ERK1/2 and the PI3-kinase substrate AKT within minutes. Effects were greater on ERK activation and occurred in neurons. Pharmacological inhibition of ERK, but not the PI3-kinase pathway, inhibited the ability of BDNF to block H-I-induced caspase-3 activation and tissue loss. These findings suggest that neuronal ERK activation in the neonatal brain mediates neuroprotection against H-I brain injury, a model of cerebral palsy.

To study the pathogenesis of central nervous system abnormalities in Down syndrome (DS), we have analyzed a new genetic model of DS, the partial trisomy 16 (Ts65Dn) mouse. Ts65Dn mice have an extra copy of the distal aspect of mouse chromosome 16, a segment homologous to human chromosome 21 that contains much of the genetic material responsible for the DS phenotype. Ts65Dn mice show developmental delay during the postnatal period as well as abnormal behaviors in both young and adult animals that may be analogous to mental retardation. Though the Ts65Dn brain is normal on gross examination, there is age-related degeneration of septohippocampal cholinergic neurons and astrocytic hypertrophy, markers of the Alzheimer disease pathology that is present in elderly DS individuals. These findings suggest that Ts65Dn mice may be used to study certain developmental and degenerative abnormalities in the DS brain.

Abstract Introduction Cerebrospinal fluid analysis and other measurements of amyloidosis, such as amyloid‐binding positron emission tomography studies, are limited by cost and availability. There is a need for a more practical amyloid β (Aβ) biomarker for central nervous system amyloid deposition. Methods We adapted our previously reported stable isotope labeling kinetics protocol to analyze the turnover kinetics and concentrations of Aβ38, Aβ40, and Aβ42 in human plasma. Results Aβ isoforms have a half‐life of approximately 3 hours in plasma. Aβ38 demonstrated faster turnover kinetics compared with Aβ40 and Aβ42. Faster fractional turnover of Aβ42 relative to Aβ40 and lower Aβ42 and Aβ42/Aβ40 concentrations in amyloid‐positive participants were observed. Discussion Blood plasma Aβ42 shows similar amyloid‐associated alterations as we have previously reported in cerebrospinal fluid, suggesting a blood‐brain transportation mechanism of Aβ. The stability and sensitivity of plasma Aβ measurements suggest this may be a useful screening test for central nervous system amyloidosis.

See Mander et al. (doi:10.1093/awx174) for a scientific commentary on this article. Sleep deprivation increases amyloid-β, suggesting that chronically disrupted sleep may promote amyloid plaques and other downstream Alzheimer’s disease pathologies including tauopathy or inflammation. To date, studies have not examined which aspect of sleep modulates amyloid-β or other Alzheimer’s disease biomarkers. Seventeen healthy adults (age 35–65 years) without sleep disorders underwent 5–14 days of actigraphy, followed by slow wave activity disruption during polysomnogram, and cerebrospinal fluid collection the following morning for measurement of amyloid-β, tau, total protein, YKL-40, and hypocretin. Data were compared to an identical protocol, with a sham condition during polysomnogram. Specific disruption of slow wave activity correlated with an increase in amyloid-β40 (r = 0.610, P = 0.009). This effect was specific for slow wave activity, and not for sleep duration or efficiency. This effect was also specific to amyloid-β, and not total protein, tau, YKL-40, or hypocretin. Additionally, worse home sleep quality, as measured by sleep efficiency by actigraphy in the six nights preceding lumbar punctures, was associated with higher tau (r = 0.543, P = 0.045). Slow wave activity disruption increases amyloid-β levels acutely, and poorer sleep quality over several days increases tau. These effects are specific to neuronally-derived proteins, which suggests they are likely driven by changes in neuronal activity during disrupted sleep.

Traumatic axonal injury (TAI) may contribute greatly to neurological impairments after traumatic brain injury, but it is difficult to assess with conventional imaging. We quantitatively compared diffusion tensor imaging (DTI) signal abnormalities with histological and electron microscopic characteristics of pericontusional TAI in a mouse model. Two DTI parameters, relative anisotropy and axial diffusivity, were significantly reduced 6 h to 4 d after trauma, corresponding to relatively isolated axonal injury. One to 4 weeks after trauma, relative anisotropy remained decreased, whereas axial diffusivity “pseudo-normalized” and radial diffusivity increased. These changes corresponded to demyelination, edema, and persistent axonal injury. At every time point, DTI was more sensitive to injury than conventional magnetic resonance imaging, and relative anisotropy distinguished injured from control mice with no overlap between groups. Remarkably, DTI changes strongly predicted the approximate time since trauma. These results provide an important validation of DTI for pericontusional TAI and suggest novel clinical and forensic applications.

Apolipoprotein E (apoE) and clusterin can influence structure, toxicity, and accumulation of the amyloid-beta (Abeta) peptide in brain. Both molecules may also be involved in Abeta metabolism prior to its deposition. To assess this possibility, we compared PDAPP transgenic mice that develop age-dependent Abeta accumulation in the absence of apoE or clusterin as well as in the absence of both proteins. apoE(-/-) and clusterin(-/-) mice accumulated similar Abeta levels but much less fibrillar Abeta. In contrast, apoE(-/-)/clusterin(-/-) mice had both earlier onset and markedly increased Abeta and amyloid deposition. Both apoE(-/-) and apoE(-/-)/clusterin(-/-) mice had elevated CSF and brain interstitial fluid Abeta, as well as significant differences in the elimination half-life of interstitial fluid Abeta measured by in vivo microdialysis. These findings demonstrate additive effects of apoE and clusterin on influencing Abeta deposition and that apoE plays an important role in regulating extracellular CNS Abeta metabolism independent of Abeta synthesis.

Brain aging is associated with diminished circadian clock output and decreased expression of the core clock proteins, which regulate many aspects of cellular biochemistry and metabolism. The genes encoding clock proteins are expressed throughout the brain, though it is unknown whether these proteins modulate brain homeostasis. We observed that deletion of circadian clock transcriptional activators aryl hydrocarbon receptor nuclear translocator-like (Bmal1) alone, or circadian locomotor output cycles kaput (Clock) in combination with neuronal PAS domain protein 2 (Npas2), induced severe age-dependent astrogliosis in the cortex and hippocampus. Mice lacking the clock gene repressors period circadian clock 1 (Per1) and period circadian clock 2 (Per2) had no observed astrogliosis. Bmal1 deletion caused the degeneration of synaptic terminals and impaired cortical functional connectivity, as well as neuronal oxidative damage and impaired expression of several redox defense genes. Targeted deletion of Bmal1 in neurons and glia caused similar neuropathology, despite the retention of intact circadian behavioral and sleep-wake rhythms. Reduction of Bmal1 expression promoted neuronal death in primary cultures and in mice treated with a chemical inducer of oxidative injury and striatal neurodegeneration. Our findings indicate that BMAL1 in a complex with CLOCK or NPAS2 regulates cerebral redox homeostasis and connects impaired clock gene function to neurodegeneration.

Alzheimer disease is more than a pure proteopathy. Chronic neuroinflammation stands out during the pathogenesis of the disease and in turn modulates disease progression. The central nervous system (CNS) is separated from the blood circulation by the blood–brain barrier. In Alzheimer disease, neuroinflammation heavily relies on innate immune responses that are primarily mediated by CNS-resident microglia. APOE (which encodes apolipoprotein E) is the strongest genetic risk factor for Alzheimer disease, and APOE was recently shown to affect the disease in part through its immunomodulatory function. This function of APOE is likely linked to triggering receptor expressed on myeloid cells 2 (TREM2), which is expressed by microglia in the CNS. Here, we review the rapidly growing literature on the role of disease-associated microglia, TREM2 and APOE in the pathogenesis of Alzheimer disease and present an integrated view of innate immune function in Alzheimer disease. In this Review, Shi and Holtzman highlight our growing understanding of the innate immune mechanisms that contribute to Alzheimer disease with a specific focus on microglial cells, apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2).

Models of Alzheimer's disease propose a sequence of amyloid β (Aβ) accumulation, hypometabolism, and structural decline that precedes the onset of clinical dementia. These pathological features evolve both temporally and spatially in the brain. In this study, we aimed to characterise where in the brain and when in the course of the disease neuroimaging biomarkers become abnormal.Between Jan 1, 2009, and Dec 31, 2015, we analysed data from mutation non-carriers, asymptomatic carriers, and symptomatic carriers from families carrying gene mutations in presenilin 1 (PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) enrolled in the Dominantly Inherited Alzheimer's Network. We analysed 11C-Pittsburgh Compound B (11C-PiB) PET, 18F-Fluorodeoxyglucose (18F-FDG) PET, and structural MRI data using regions of interest to assess change throughout the brain. We estimated rates of biomarker change as a function of estimated years to symptom onset at baseline using linear mixed-effects models and determined the earliest point at which biomarker trajectories differed between mutation carriers and non-carriers. This study is registered at ClinicalTrials.gov (number NCT00869817) FINDINGS: 11C-PiB PET was available for 346 individuals (162 with longitudinal imaging), 18F-FDG PET was available for 352 individuals (175 with longitudinal imaging), and MRI data were available for 377 individuals (201 with longitudinal imaging). We found a sequence to pathological changes, with rates of Aβ deposition in mutation carriers being significantly different from those in non-carriers first (across regions that showed a significant difference, at a mean of 18·9 years [SD 3·3] before expected onset), followed by hypometabolism (14·1 years [5·1] before expected onset), and lastly structural decline (4·7 years [4·2] before expected onset). This biomarker ordering was preserved in most, but not all, regions. The temporal emergence within a biomarker varied across the brain, with the precuneus being the first cortical region for each method to show divergence between groups (22·2 years before expected onset for Aβ accumulation, 18·8 years before expected onset for hypometabolism, and 13·0 years before expected onset for cortical thinning).Mutation carriers had elevations in Aβ deposition, reduced glucose metabolism, and cortical thinning compared with non-carriers which preceded the expected onset of dementia. Accrual of these pathologies varied throughout the brain, suggesting differential regional and temporal vulnerabilities to Aβ, metabolic decline, and structural atrophy, which should be taken into account when using biomarkers in a clinical setting as well as designing and evaluating clinical trials.US National Institutes of Health, the German Center for Neurodegenerative Diseases, and the Medical Research Council Dementias Platform UK.

White matter hyperintensities (WMHs) are areas of increased signal on T2-weighted magnetic resonance imaging (MRI) scans that most commonly reflect small vessel cerebrovascular disease. Increased WMH volume is associated with risk and progression of Alzheimer's disease (AD). These observations are typically interpreted as evidence that vascular abnormalities play an additive, independent role contributing to symptom presentation, but not core features of AD. We examined the severity and distribution of WMH in presymptomatic PSEN1, PSEN2, and APP mutation carriers to determine the extent to which WMH manifest in individuals genetically determined to develop AD.The study comprised participants (n = 299; age = 39.03 ± 10.13) from the Dominantly Inherited Alzheimer Network, including 184 (61.5%) with a mutation that results in AD and 115 (38.5%) first-degree relatives who were noncarrier controls. We calculated the estimated years from expected symptom onset (EYO) by subtracting the affected parent's symptom onset age from the participant's age. Baseline MRI data were analyzed for total and regional WMH. Mixed-effects piece-wise linear regression was used to examine WMH differences between carriers and noncarriers with respect to EYO.Mutation carriers had greater total WMH volumes, which appeared to increase approximately 6 years before expected symptom onset. Effects were most prominent for the parietal and occipital lobe, which showed divergent effects as early as 22 years before estimated onset.Autosomal-dominant AD is associated with increased WMH well before expected symptom onset. The findings suggest the possibility that WMHs are a core feature of AD, a potential therapeutic target, and a factor that should be integrated into pathogenic models of the disease. Ann Neurol 2016;79:929-939.

ABCA1 is an ATP-binding cassette protein that transports cellular cholesterol and phospholipids onto high density lipoproteins (HDL) in plasma. Lack of ABCA1 in humans and mice causes abnormal lipidation and increased catabolism of HDL, resulting in very low plasma apoA-I, apoA-II, and HDL. Herein, we have used <i>Abca1</i>^-/- mice to ask whether ABCA1 is involved in lipidation of HDL in the central nervous system (CNS). ApoE is the most abundant CNS apolipoprotein and is present in HDL-like lipoproteins in CSF. We found that <i>Abca1</i>^-/- mice have greatly decreased apoE levels in both the cortex (80% reduction) and the CSF (98% reduction). CSF from <i>Abca1</i>^-/- mice had significantly reduced cholesterol as well as small apoE-containing lipoproteins, suggesting abnormal lipidation of apoE. Astrocytes, the primary producer of CNS apoE, were cultured from <i>Abca1</i>^+/+, ^+/-, and ^-/- mice, and nascent lipoprotein particles were collected. <i>Abca1</i>^-/- astrocytes secreted lipoprotein particles that had markedly decreased cholesterol and apoE and had smaller apoE-containing particles than particles from <i>Abca1</i>^+/+ astrocytes. These findings demonstrate that ABCA1 plays a critical role in CNS apoE metabolism. Since apoE isoforms and levels strongly influence Alzheimer's disease pathology and risk, these data suggest that ABCA1 may be a novel therapeutic target.

We have applied in situ atomic force microscopy to directly observe the aggregation of Alzheimer's beta-amyloid peptide (Abeta) in contact with two model solid surfaces: hydrophilic mica and hydrophobic graphite. The time course of aggregation was followed by continuous imaging of surfaces remaining in contact with 10-500 microM solutions of Abeta in PBS (pH 7.4). Visualization of fragile nanoscale aggregates of Abeta was made possible by the application of a tapping mode of imaging, which minimizes the lateral forces between the probe tip and the sample. The size and the shape of Abeta aggregates, as well as the kinetics of their formation, exhibited pronounced dependence on the physicochemical nature of the surface. On hydrophilic mica, Abeta formed particulate, pseudomicellar aggregates, which at higher Abeta concentration had the tendency to form linear assemblies, reminiscent of protofibrillar species described recently in the literature. In contrast, on hydrophobic graphite Abeta formed uniform, elongated sheets. The dimensions of those sheets were consistent with the dimensions of beta-sheets with extended peptide chains perpendicular to the long axis of the aggregate. The sheets of Abeta were oriented along three directions at 120 degrees to each other, resembling the crystallographic symmetry of a graphite surface. Such substrate-templated self-assembly may be the distinguishing feature of beta-sheets in comparison with alpha-helices. These studies show that in situ atomic force microscopy enables direct assessment of amyloid aggregation in physiological fluids and suggest that Abeta fibril formation may be driven by interactions at the interface of aqueous solutions and hydrophobic substrates, as occurs in membranes and lipoprotein particles in vivo.

Background Disease-modifying therapies for Alzheimer's disease (AD) would be most effective during the preclinical stage (pathology present, cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design. Methods AD-associated changes in cerebrospinal fluid (CSF) were measured using two-dimensional difference gel electrophoresis and liquid chromatography tandem mass spectrometry. Subsequently, CSF YKL-40 was measured by enzyme-linked immunosorbent assay in the discovery cohort (n = 47), validation cohort (n = 292) with paired plasma samples (n = 237), frontotemporal lobar degeneration (PSP; n = 9), and progressive supranuclear palsy (PSP; n = 6). Immunohistochemistry was performed to identify source(s) of YKL-40 in human AD brain. Results Discovery and validation cohorts, showed higher mean CSF YKL-40 in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) versus control subjects (CDR 0) and PSP subjects. Importantly, CSF YKL-40/Aβ42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR > 0 conversion), as well as the best CSF biomarkers identified to date, tau/Aβ42 and p-tau 181/Aβ42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 versus CDR 0, and correlated with CSF levels. YKL-40 immunoreactivity labeled astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aβ deposition. Conclusions These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD and, together with Aβ42, has potential prognostic utility as a biomarker for preclinical AD. Disease-modifying therapies for Alzheimer's disease (AD) would be most effective during the preclinical stage (pathology present, cognition intact) before significant neuronal loss occurs. Therefore, biomarkers that detect AD pathology in its early stages and predict dementia onset and progression will be invaluable for patient care and efficient clinical trial design. AD-associated changes in cerebrospinal fluid (CSF) were measured using two-dimensional difference gel electrophoresis and liquid chromatography tandem mass spectrometry. Subsequently, CSF YKL-40 was measured by enzyme-linked immunosorbent assay in the discovery cohort (n = 47), validation cohort (n = 292) with paired plasma samples (n = 237), frontotemporal lobar degeneration (PSP; n = 9), and progressive supranuclear palsy (PSP; n = 6). Immunohistochemistry was performed to identify source(s) of YKL-40 in human AD brain. Discovery and validation cohorts, showed higher mean CSF YKL-40 in very mild and mild AD-type dementia (Clinical Dementia Rating [CDR] 0.5 and 1) versus control subjects (CDR 0) and PSP subjects. Importantly, CSF YKL-40/Aβ42 ratio predicted risk of developing cognitive impairment (CDR 0 to CDR > 0 conversion), as well as the best CSF biomarkers identified to date, tau/Aβ42 and p-tau 181/Aβ42. Mean plasma YKL-40 was higher in CDR 0.5 and 1 versus CDR 0, and correlated with CSF levels. YKL-40 immunoreactivity labeled astrocytes near a subset of amyloid plaques, implicating YKL-40 in the neuroinflammatory response to Aβ deposition. These data demonstrate that YKL-40, a putative indicator of neuroinflammation, is elevated in AD and, together with Aβ42, has potential prognostic utility as a biomarker for preclinical AD.

Disturbances in the sleep-wake cycle and circadian rhythms are common symptoms of Alzheimer Disease (AD), and they have generally been considered as late consequences of the neurodegenerative processes. Recent evidence demonstrates that sleep-wake and circadian disruption often occur early in the course of the disease and may even precede the development of cognitive symptoms. Furthermore, the sleep-wake cycle appears to regulate levels of the pathogenic amyloid-beta peptide in the brain, and manipulating sleep can influence AD-related pathology in mouse models via multiple mechanisms. Finally, the circadian clock system, which controls the sleep-wake cycle and other diurnal oscillations in mice and humans, may also have a role in the neurodegenerative process. In this review, we examine the current literature related to the mechanisms by which sleep and circadian rhythms might impact AD pathogenesis, and we discuss potential therapeutic strategies targeting these systems for the prevention of AD.

Coding variants in the triggering receptor expressed on myeloid cells 2 (TREM2) are associated with late-onset Alzheimer's disease (AD). We demonstrate that amyloid plaque seeding is increased in the absence of functional Trem2. Increased seeding is accompanied by decreased microglial clustering around newly seeded plaques and reduced plaque-associated apolipoprotein E (ApoE). Reduced ApoE deposition in plaques is also observed in brains of AD patients carrying TREM2 coding variants. Proteomic analyses and microglia depletion experiments revealed microglia as one origin of plaque-associated ApoE. Longitudinal amyloid small animal positron emission tomography demonstrates accelerated amyloidogenesis in Trem2 loss-of-function mutants at early stages, which progressed at a lower rate with aging. These findings suggest that in the absence of functional Trem2, early amyloidogenesis is accelerated due to reduced phagocytic clearance of amyloid seeds despite reduced plaque-associated ApoE.

The cerebrospinal fluid (CSF) biomarkers amyloid β (Aβ)-42, total-tau (T-tau), and phosphorylated-tau (P-tau) demonstrate good diagnostic accuracy for Alzheimer's disease (AD). However, there are large variations in biomarker measurements between studies, and between and within laboratories. The Alzheimer's Association has initiated a global quality control program to estimate and monitor variability of measurements, quantify batch-to-batch assay variations, and identify sources of variability. In this article, we present the results from the first two rounds of the program.The program is open for laboratories using commercially available kits for Aβ, T-tau, or P-tau. CSF samples (aliquots of pooled CSF) are sent for analysis several times a year from the Clinical Neurochemistry Laboratory at the Mölndal campus of the University of Gothenburg, Sweden. Each round consists of three quality control samples.Forty laboratories participated. Twenty-six used INNOTEST enzyme-linked immunosorbent assay kits, 14 used Luminex xMAP with the INNO-BIA AlzBio3 kit (both measure Aβ-(1-42), P-tau(181P), and T-tau), and 5 used Meso Scale Discovery with the Aβ triplex (AβN-42, AβN-40, and AβN-38) or T-tau kits. The total coefficients of variation between the laboratories were 13% to 36%. Five laboratories analyzed the samples six times on different occasions. Within-laboratory precisions differed considerably between biomarkers within individual laboratories.Measurements of CSF AD biomarkers show large between-laboratory variability, likely caused by factors related to analytical procedures and the analytical kits. Standardization of laboratory procedures and efforts by kit vendors to increase kit performance might lower variability, and will likely increase the usefulness of CSF AD biomarkers.

Nerve growth factor (NGF) appears to act as a neurotrophic factor for basal forebrain and caudate-putamen cholinergic neurons. The mechanism by which NGF transduces its signal in these neurons is yet to be defined. Recent data indicate that the product of the trk gene, p140trk, is a critical component of the NGF receptor. Herein, we show that p140trk mRNA is highly restricted in its distribution in the adult rat forebrain, that it is present in cholinergic neurons, and that most if not all cholinergic neurons contain p140trk mRNA. Furthermore, induction of trk expression by NGF suggests that neurotrophin-mediated up-regulation of their receptor tyrosine kinases is an important feature of their actions and that neurotrophins may regulate the activity of responsive neurons through increasing the level of their receptors.

Alzheimer's disease is characterized by the progressive deposition of beta-amyloid (Abeta) within the brain parenchyma and its subsequent accumulation into senile plaques. Pathogenesis of the disease is associated with perturbations in Abeta homeostasis and the inefficient clearance of these soluble and insoluble peptides from the brain. Microglia have been reported to mediate the clearance of fibrillar Abeta (fAbeta) through receptor-mediated phagocytosis; however, their participation in clearance of soluble Abeta peptides (sAbeta) is largely unknown. We report that microglia internalize sAbeta from the extracellular milieu through a nonsaturable, fluid phase macropinocytic mechanism that is distinct from phagocytosis and receptor-mediated endocytosis both in vitro and in vivo. The uptake of sAbeta is dependent on both actin and tubulin dynamics and does not involve clathrin assembly, coated vesicles or membrane cholesterol. Upon internalization, fluorescently labeled sAbeta colocalizes to pinocytic vesicles. Microglia rapidly traffic these soluble peptides into late endolysosomal compartments where they are subject to degradation. Additionally, we demonstrate that the uptake of sAbeta and fAbeta occurs largely through distinct mechanisms and upon internalization are segregated into separate subcellular vesicular compartments. Significantly, we found that upon proteolytic degradation of fluorescently labeled sAbeta, the fluorescent chromophore is retained by the microglial cell. These studies identify an important mechanism through which microglial cells participate in the maintenance of Abeta homeostasis, through their capacity to constitutively clear sAbeta peptides from the brain.

Soluble amyloid-β (Aβ) peptide converts to structures with high β-sheet content in Alzheimer's disease (AD). Soluble Aβ is released by neurons into the brain interstitial fluid (ISF), in which it can convert into toxic aggregates. Because assessment of ISF Aβ levels may provide unique insights into Aβ metabolism and AD, an in vivo microdialysis technique was developed to measure it. Our Aβ microdialysis technique was validated ex vivo with human CSF and then in vivo in awake, freely moving mice. Using human amyloid precursor protein (APP) transgenic mice, we found that, before the onset of AD-like pathology, ISF Aβ in hippocampus and cortex correlated with levels of APP in those tissues. After the onset of Aβ deposition, significant changes in the ISF Aβ 40 /Aβ 42 ratio developed without changes in Aβ 1-x . These changes differed from changes seen in tissue lysates from the same animals. By rapidly inhibiting Aβ production, we found that ISF Aβ half-life was short (∼2 hr) in young mice but was twofold longer in mice with Aβ deposits. This increase in half-life, without an increase in steady-state levels, suggests that inhibition of Aβ synthesis reveals a portion of the insoluble Aβ pool that is in dynamic equilibrium with ISF Aβ. This now measurable in vivo pool is a likely target for new diagnostic and therapeutic strategies.

The cerebrospinal fluid (CSF) biomarkers amyloid beta 1-42, total tau, and phosphorylated tau are used increasingly for Alzheimer's disease (AD) research and patient management. However, there are large variations in biomarker measurements among and within laboratories.Data from the first nine rounds of the Alzheimer's Association quality control program was used to define the extent and sources of analytical variability. In each round, three CSF samples prepared at the Clinical Neurochemistry Laboratory (Mölndal, Sweden) were analyzed by single-analyte enzyme-linked immunosorbent assay (ELISA), a multiplexing xMAP assay, or an immunoassay with electrochemoluminescence detection.A total of 84 laboratories participated. Coefficients of variation (CVs) between laboratories were around 20% to 30%; within-run CVs, less than 5% to 10%; and longitudinal within-laboratory CVs, 5% to 19%. Interestingly, longitudinal within-laboratory CV differed between biomarkers at individual laboratories, suggesting that a component of it was assay dependent. Variability between kit lots and between laboratories both had a major influence on amyloid beta 1-42 measurements, but for total tau and phosphorylated tau, between-kit lot effects were much less than between-laboratory effects. Despite the measurement variability, the between-laboratory consistency in classification of samples (using prehoc-derived cutoffs for AD) was high (>90% in 15 of 18 samples for ELISA and in 12 of 18 samples for xMAP).The overall variability remains too high to allow assignment of universal biomarker cutoff values for a specific intended use. Each laboratory must ensure longitudinal stability in its measurements and use internally qualified cutoff levels. Further standardization of laboratory procedures and improvement of kit performance will likely increase the usefulness of CSF AD biomarkers for researchers and clinicians.

The epsilon 4 allele of apolipoprotein E (apoE) is a major risk factor for Alzheimer disease, suggesting that apoE may directly influence neurons in the aging brain. Recent data suggest that apoE-containing lipoproteins can influence neurite outgrowth in an isoform-specific fashion. The neuronal mediators of apoE effects have not been clarified. We show here that in a central nervous system-derived neuronal cell line, apoE3 but not apoE4 increases neurite extension. The effect of apoE3 was blocked at low nanomolar concentrations by purified 39-kDa protein that regulates ligand binding to the low density lipoprotein receptor-related protein (LRP). Anti-LRP antibody also completely abolished the neurite-promoting effect of apoE3. Understanding isoform-specific cell biological processes mediated by apoE-LRP interactions in central nervous system neurons may provide insight into Alzheimer disease pathogenesis.

Cerebrospinal fluid (CSF) tau, tau phosphorylated at threonine 181 (ptau), and Aβ₄₂ are established biomarkers for Alzheimer's disease (AD) and have been used as quantitative traits for genetic analyses. We performed the largest genome-wide association study for cerebrospinal fluid (CSF) tau/ptau levels published to date (n = 1,269), identifying three genome-wide significant loci for CSF tau and ptau: rs9877502 (p = 4.89 × 10⁻⁹ for tau) located at 3q28 between GEMC1 and OSTN, rs514716 (p = 1.07 × 10⁻⁸ and p = 3.22 × 10⁻⁹ for tau and ptau, respectively), located at 9p24.2 within GLIS3 and rs6922617 (p = 3.58 × 10⁻⁸ for CSF ptau) at 6p21.1 within the TREM gene cluster, a region recently reported to harbor rare variants that increase AD risk. In independent data sets, rs9877502 showed a strong association with risk for AD, tangle pathology, and global cognitive decline (p = 2.67 × 10⁻⁴, 0.039, 4.86 × 10⁻⁵, respectively) illustrating how this endophenotype-based approach can be used to identify new AD risk loci.

Longitudinal cerebrospinal fluid biomarker analyses reveal decreases in neuronal injury markers in later stages of autosomal-dominant Alzheimer’s disease.

Variants in the gene encoding the triggering receptor expressed on myeloid cells 2 (TREM2) were recently found to increase the risk for developing Alzheimer's disease (AD). In the brain, TREM2 is predominately expressed on microglia, and its association with AD adds to increasing evidence implicating a role for the innate immune system in AD initiation and progression. Thus far, studies have found TREM2 is protective in the response to amyloid pathology while variants leading to a loss of TREM2 function impair microglial signaling and are deleterious. However, the potential role of TREM2 in the context of tau pathology has not yet been characterized. In this study, we crossed Trem2+/+ (T2+/+) and Trem2-/- (T2-/-) mice to the PS19 human tau transgenic line (PS) to investigate whether loss of TREM2 function affected tau pathology, the microglial response to tau pathology, or neurodegeneration. Strikingly, by 9 mo of age, T2-/-PS mice exhibited significantly less brain atrophy as quantified by ventricular enlargement and preserved cortical volume in the entorhinal and piriform regions compared with T2+/+PS mice. However, no TREM2-dependent differences were observed for phosphorylated tau staining or insoluble tau levels. Rather, T2-/-PS mice exhibited significantly reduced microgliosis in the hippocampus and piriform cortex compared with T2+/+PS mice. Gene expression analyses and immunostaining revealed microglial activation was significantly attenuated in T2-/-PS mice, and there were lower levels of inflammatory cytokines and astrogliosis. These unexpected findings suggest that impairing microglial TREM2 signaling reduces neuroinflammation and is protective against neurodegeneration in the setting of pure tauopathy.

The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-β fibrils (fAβ), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Aβ (sAβ) could degrade fAβ as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAβ and that this ability is not shared by other sAβ-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAβ was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAβ breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAβ were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Aβ that are cleaved during its degradation. Only MMP-9 digests contained fragments (Aβ1-20 and Aβ1-30) from fAβ1-42 substrate; the corresponding cleavage sites are thought to be important for β-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAβ and may contribute to ongoing clearance of plaques from amyloid-laden brains. The pathological hallmark of Alzheimer disease is the senile plaque principally composed of tightly aggregated amyloid-β fibrils (fAβ), which are thought to be resistant to degradation and clearance. In this study, we explored whether proteases capable of degrading soluble Aβ (sAβ) could degrade fAβ as well. We demonstrate that matrix metalloproteinase-9 (MMP-9) can degrade fAβ and that this ability is not shared by other sAβ-degrading enzymes examined, including endothelin-converting enzyme, insulin-degrading enzyme, and neprilysin. fAβ was decreased in samples incubated with MMP-9 compared with other proteases, assessed using thioflavin-T. Furthermore, fAβ breakdown with MMP-9 but not with other proteases was demonstrated by transmission electron microscopy. Proteolytic digests of purified fAβ were analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify sites of Aβ that are cleaved during its degradation. Only MMP-9 digests contained fragments (Aβ1-20 and Aβ1-30) from fAβ1-42 substrate; the corresponding cleavage sites are thought to be important for β-pleated sheet formation. To determine whether MMP-9 can degrade plaques formed in vivo, fresh brain slices from aged APP/PS1 mice were incubated with proteases. MMP-9 digestion resulted in a decrease in thioflavin-S (ThS) staining. Consistent with a role for endogenous MMP-9 in this process in vivo, MMP-9 immunoreactivity was detected in astrocytes surrounding amyloid plaques in the brains of aged APP/PS1 and APPsw mice, and increased MMP activity was selectively observed in compact ThS-positive plaques. These findings suggest that MMP-9 can degrade fAβ and may contribute to ongoing clearance of plaques from amyloid-laden brains. One of the key pathological features of Alzheimer disease (AD) 3The abbreviations used are: AD, Alzheimer disease; ECE, endothelin-converting enzyme; Aβ, amyloid-β peptide; fAβ, amyloid-β fibrils; sAβ, soluble Aβ; IDE, insulin-degrading enzyme; MMP-9, matrix metalloproteinase-9; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NEP, neprilysin; TEM, transmission electron microscopy; ThS, thioflavin-S; ThT, thioflavin-T; TIMP, tissue inhibitors of MMP; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MES, 4-morpholineethanesulfonic acid; PBS, phosphate-buffered saline. is the senile plaque, extracellular deposits found throughout the brains of AD patients, composed primarily of the amyloid-β peptide (Aβ). Aggregated Aβ in senile plaques can be found in two conformations: non-β-pleated-sheet (nonfibrillar) conformation, known as “diffuse plaques” or β-pleated sheet (fibrillar) conformation, known as “compact plaques” (1Lorenzo A. Yankner B.A. Ann. N. Y. Acad. Sci. 1996; 777: 89-95Crossref PubMed Scopus (138) Google Scholar). The 42-amino acid peptide (Aβ1-42), the predominant peptide length found in senile plaques, has a remarkable propensity to aggregate at high concentrations to form a β-pleated sheet structure (2McLaurin J. Yang D. Yip C.M. Fraser P.E. J. Struct. Biol. 2000; 130: 259-270Crossref PubMed Scopus (214) Google Scholar, 3Serpell L.C. Biochim. Biophys. Acta. 2000; 1502: 16-30Crossref PubMed Scopus (833) Google Scholar). This is generally viewed as an irreversible process resulting in the formation of fibrillar Aβ (fAβ), which is insoluble and resistant to proteolysis, endowing compact plaques with a resistance to degradation and clearance. Given the purported irreversibility of Aβ fibril formation and the resistance to degradation, one might expect that senile plaques would continue to grow throughout disease progression; however, careful observational studies indicate that plaque size remains relatively constant over a wide range of disease durations (4Hyman B.T. Marzloff K. Arriagada P.V. J. Neuropathol. Exp. Neurol. 1993; 52: 594-600Crossref PubMed Scopus (245) Google Scholar). In addition, in vivo imaging in the APPsw (Tg2576) transgenic mouse model of Alzheimer's disease (using multiphoton microscopy) demonstrated that plaques remain constant in size over a period of many months (5Christie R.H. Bacskai B.J. Zipfel W.R. Williams R.M. Kajdasz S.T. Webb W.W. Hyman B.T. J. Neurosci. 2001; 21: 858-864Crossref PubMed Google Scholar). These observations have led some to believe that plaques, once formed, are in dynamic equilibrium with their environment, balancing formation with degradation (6Cruz L. Urbanc B. Buldyrev S.V. Christie R. Gomez-Isla T. Havlin S. McNamara M. Stanley H.E. Hyman B.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7612-7616Crossref PubMed Scopus (89) Google Scholar). In support of this idea, a few isolated plaques in APPsw mice were found to decrease in size (5Christie R.H. Bacskai B.J. Zipfel W.R. Williams R.M. Kajdasz S.T. Webb W.W. Hyman B.T. J. Neurosci. 2001; 21: 858-864Crossref PubMed Google Scholar), raising the possibility that endogenous mechanisms for plaque clearance may exist. Moreover, a semiquantitative analysis of plaque burden in Alzheimer disease cases revealed that the most advanced cases (by Braak staging) actually had a slightly lower frequency of plaques than less advanced cases (7Thal D.R. Arendt T. Waldmann G. Holzer M. Zedlick D. Rub U. Schober R. Neurobiol. Aging. 1998; 19: 517-525Crossref PubMed Scopus (42) Google Scholar). While plaques and amyloid fibrils have been viewed by some as resistant to proteolytic degradation, it is possible that certain (as yet unidentified) proteases may contribute to endogenous mechanisms leading to plaque clearance. A growing list of proteases are known to degrade soluble Aβ (sAβ) in vitro, including NEP (8Howell S. Nalbantoglu J. Crine P. Peptides. 1995; 16: 647-652Crossref PubMed Scopus (186) Google Scholar), IDE (9Kurochkin I.V. Goto S. FEBS Lett. 1994; 345: 33-37Crossref PubMed Scopus (346) Google Scholar), ECE (10Eckman E.A. Reed D.K. Eckman C.B. J. Biol. Chem. 2001; 276: 24540-24548Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), angiotensin-converting enzyme (11Hu J. Igarashi A. Kamata M. Nakagawa H. J. Biol. Chem. 2001; 276: 47863-47868Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar), the plasmin system (12Tucker H.M. Kihiko M. Caldwell J.N. Wright S. Kawarabayashi T. Price D. Walker D. Scheff S. McGillis J.P. Rydel R.E. Estus S. J. Neurosci. 2000; 20: 3937-3946Crossref PubMed Google Scholar), and MMP-9 (13Backstrom J.R. Lim G.P. Cullen M.J. Tokes Z.A. J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar). Each of these proteases appears to have distinguishing Aβ cleavage sites, generating characteristic fragments. However, experimental evidence that these proteases degrade sAβ in vivo is available only for a few: gene deletion of NEP (14Iwata N. Tsubuki S. Takaki Y. Shirotani K. Lu B. Gerard N.P. Gerard C. Hama E. Lee H.J. Saido T.C. Science. 2001; 292: 1550-1552Crossref PubMed Scopus (848) Google Scholar), ECE (15Eckman E.A. Watson M. Marlow L. Sambamurti K. Eckman C.B. J. Biol. Chem. 2003; 278: 2081-2084Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), or IDE (16Farris W. Mansourian S. Chang Y. Lindsley L. Eckman E.A. Frosch M.P. Eckman C.B. Tanzi R.E. Selkoe D.J. Guenette S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4162-4167Crossref PubMed Scopus (1226) Google Scholar) in mice resulted in increased steady-state levels of Aβ in brain, suggesting a role in regulating endogenous basal levels of Aβ in vivo. Although all of these proteases demonstrate the ability to degrade sAβ, none has been convincingly shown to degrade Aβ fibrils or compact plaques. MMP-9, a zinc-dependent metalloprotease, has not been well studied with regards to its potential role in amyloid clearance. Initially synthesized as an inactive proenzyme, pro-MMP-9 is cleaved into an active form upon cellular release by other proteases (17Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3903) Google Scholar); this property puts MMP-9 in a unique position to regulate extracellular Aβ levels. Once activated, MMP-9 can be regulated by interacting with endogenous tissue inhibitors of MMPs (TIMPs) (17Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3903) Google Scholar). Although expressed at low basal levels in normal brain tissue, MMP-9 expression is induced in neurons, astrocytes, microglia, and vascular cells under a variety of pathological conditions (18Gu Z. Cui J. Brown S. Fridman R. Mobashery S. Strongin A.Y. Lipton S.A. J. Neurosci. 2005; 25: 6401-6408Crossref PubMed Scopus (373) Google Scholar, 19Rosenberg G.A. Cunningham L.A. Wallace J. Alexander S. Estrada E.Y. Grossetete M. Razhagi A. Miller K. Gearing A. Brain Res. 2001; 893: 104-112Crossref PubMed Scopus (355) Google Scholar, 20Zhang X. Cheng M. Chintala S.K. Neurosci. Lett. 2004; 356: 140-144Crossref PubMed Scopus (35) Google Scholar, 21Anthony D.C. Ferguson B. Matyzak M.K. Miller K.M. Esiri M.M. Perry V.H. Neuropathol. Appl. Neurobiol. 1997; 23: 406-415Crossref PubMed Scopus (240) Google Scholar, 22Maier C.M. Hsieh L. Yu F. Bracci P. Chan P.H. Stroke. 2004; 35: 1169-1174Crossref PubMed Scopus (84) Google Scholar, 23Berman N.E. Marcario J.K. Yong C. Raghavan R. Raymond L.A. Joag S.V. Narayan O. Cheney P.D. Neurobiol. Dis. 1999; 6: 486-498Crossref PubMed Scopus (58) Google Scholar). Its expression is increased in the brains of AD patients (13Backstrom J.R. Lim G.P. Cullen M.J. Tokes Z.A. J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar, 24Asahina M. Yoshiyama Y. Hattori T. Clin. Neuropathol. 2001; 20: 60-63PubMed Google Scholar). Moreover MMP-9 expression in astrocytes is induced in the presence of the Aβ peptide (25Deb S. Wenjun Zhang J. Gottschall P.E. Brain Res. 2003; 970: 205-213Crossref PubMed Scopus (75) Google Scholar). In this study, we investigated the possibility that certain sAβ-degrading proteases were capable of degrading fAβ and amyloid plaques. Specifically, we examined the well studied sAβ-degrading proteases, ECE, IDE, and NEP, as well as MMP-9. We further examined the expression and activity of fAβ-degrading proteases in the brains of transgenic mouse models of AD. APP Transgenic Mice—Tg2576 (APPsw) and APP/PS1 mice, models of AD, were used in this study. The production, genotyping, and background strains of these mice have been described previously (26Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3712) Google Scholar). Transgenic mice were compared with age-matched littermate wild-type controls. All experimental protocols were approved by the Animal Studies Committee at Washington University. Aβ Preparation and Analysis—Synthetic human Aβ1-42 (Bachem) or Aβ1-40 (American Peptide), dissolved in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5 mm, was diluted in MQ water to a final concentration of 25 μm immediately prior to use. Analysis with Tris-Tricine gels indicate that the vast majority of Aβ in this preparation (referred to as sAβ) was in the monomer form (data not shown). To prepare Aβ fibrils (fAβ), 5 mm Aβ1-42 or Aβ1-40 in Me2SO was diluted in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortexed for 30 s, and incubated at 37 °C for 5 days (27Stine Jr., W.B. Dahlgren K.N. Krafft G.A. LaDu M.J. J. Biol. Chem. 2003; 278: 11612-11622Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar). For mass spectrometry (MS) experiments, fAβ was further purified by centrifugation at 14,000 × g for 30 min. Fibrils were confirmed by thioflavin-T (ThT) fluorescence and electron microscopy (see below). Some experiments were repeated using Aβ1-42 from a different manufacturer (American Peptide). ThT assay: 10 μl of sample was added to 0.2 ml of 10 μm ThT in 50 mm glycine buffer (pH 10.5). Fluorescence intensity was monitored at λex of 440 nm and λem of 530 nm (28LeVine III, H. Arch. Biochem. Biophys. 1997; 342: 306-316Crossref PubMed Scopus (107) Google Scholar). Proteolytic Digestions—Human recombinant ECE, ICE, NEP, and MMP-9 (R&D Systems) were used for digestion reactions using the following buffers: ECE, 0.1 m MES, 0.1 m NaCl (pH 6.0); IDE, 50 mm Tris, 1 m NaCl (pH 7.5); NEP, 0.1 m MES (pH 6.5); MMP-9, 50 mm Tris-HCl (pH 7.5), 10 mm CaCl2, 150 mm NaCl, 0.05% Brij 35. Pro-MMP-9 was activated with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. For sAβ digestions, 100 nm protease was incubated with 2.5 μm sAβ at 37 °C for varying periods of time (4 h to 5 days), then analyzed by MS. For fAβ digestions (employing ThT or TEM), 200 nm protease was added to 10 μl of fAβ in reaction buffer and incubated at 37 °C for 4 h to 5 days. After digestion, the reaction was analyzed by ThT assay or TEM. For fAβ digestions (employing MS), 10 μl of fAβ was applied to a Microcon 50-kDa centrifugal ultrafiltration unit (YM-50, Millipore) and washed with 500 μl of 10 mm HCl three times, then equilibrated with 500 μl of MQ water. Thirty μl of reaction buffer was added to the fAβ (retentate) and centrifuged; the filtrate was analyzed by MS (see below) to ensure that any contaminating Aβ monomer or fragments were washed away. Ten μl of 300nm protease and 20 μl of reaction buffer was added to the fAβ (retentate) and incubated at 37 °C for 24 h, then centrifuged and analyzed by MS to detect liberated fragments of fAβ. Mass Spectrometry—Samples were first passed through a reverse phase C18 Ziptip (Millipore), according to the manufacturer's instructions, then diluted 1:1 with matrix solution (a saturated solution of 3,5-dimethoxy-4-hydroxycinnamic acid in 50% acetonitrile with 0.1% trifluoroacetic acid in water), loaded onto a plate, and allowed to dry. The sample was then analyzed on an Applied Biosystesms (Foster City, CA) Voyager DE-STR matrix-assisted laser desorption-ionization time-of-flight mass spectrometer (MALDI-TOF MS) operated in linear mode. Insulin was used as an internal standard, and a calibration line constructed from its [M + H]+ and [M + 2H]2+ ions was used for quantification (29Wang R. Sweeney D. Gandy S.E. Sisodia S.S. J. Biol. Chem. 1996; 271: 31894-31902Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Electron Microscopy—Small volumes (5 μl) of fAβ samples were absorbed onto glow-discharged, carbon-coated, formvar/carbon-filmed 150-mesh copper grids. Grids were stained with 0.5% uranyl acetate and dried in a light-protected environment overnight before being viewed in a TEM (H-7500) operated at 80 kV (30Yang F. Lim G.P. Begum A.N. Ubeda O.J. Simmons M.R. Ambegaokar S.S. Chen P.P. Kayed R. Glabe C.G. Frautschy S.A. Cole G.M. J. Biol. Chem. 2005; 280: 5892-5901Abstract Full Text Full Text PDF PubMed Scopus (2014) Google Scholar). In Situ Plaque Degradation—Brains were removed from anesthetized 9-month-old APP/PS1 mice after perfusion with cold saline and snap-frozen on dry ice. Five-μm cryostat sections were collected on slides. Every other section was flipped 180° so that identical faces of adjacent sections were exposed. Paired adjacent sections (one incubated with buffer, the other with 70 nm protease) were incubated at 37 °C for 5 days, stained with thioflavin-S (ThS) (31Sun A. Nguyen X.V. Bing G. J. Histochem. Cytochem. 2002; 50: 463-472Crossref PubMed Scopus (105) Google Scholar), then imaged with fluorescence microscopy (Olympus BX60). Using a 20× objective lens, identical fields from paired sections were photographed (precise alignment was confirmed by superimposing plaque staining patterns). The area of ThS fluorescence was determined using image analysis software (Sigma Scan) and expressed as a fraction of total area. Fractional area was compared between paired sections. The specific activities (Aβ degrading activity) of all proteases were approximately equivalent (Fig. 2C). Immunohistochemistry—APPsw mice (>16 months), APP/PS1 (>6 months), and age-matched wild-type mice were deeply anesthetized and perfused transcardially with 4% paraformaldehyde in 0.1 m phosphate buffer (pH 7.4). Brain cryostat sections (16 μm) were processed for immunofluorescence double labeling, a mixture of rabbit anti-MMP-9 (1:800, gift from Dr. Robert Senior (32Betsuyaku T. Fukuda Y. Parks W.C. Shipley J.M. Senior R.M. Am. J. Pathol. 2000; 157: 525-535Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and mouse anti-GFAP (1:2000; Sigma) or pan-Aβ antibody (1:1000; Biosource International) were applied to the sections overnight at 4 °C, then Cy3-conjugated donkey anti-rabbit IgG antibody (1:800; Jackson ImmunoResearch) and Alexa Fluoro 488-conjugated donkey anti-mouse IgG antibody (1:400; Molecular Probes) (33Yan P. Xu J. Li Q. Chen S. Kim G.M. Hsu C.Y. Xu X.M. J. Neurosci. 1999; 19: 9355-9363Crossref PubMed Google Scholar). Sections were coverslipped and examined using confocal microscopy (Zeiss LSM). In Situ Zymography—Fresh frozen sections (10μ m) were incubated with DQ-gelatin (EnzCheck; Molecular Probes) at a concentration of 0.1 mg/ml in 1% low gel temperature agarose (Sigma) in PBS containing 4′,6-diamidino-2-phenylindole (1.0 μg/ml). Sections were incubated for 24 h at room temperature. For further immunostaining, sections were washed and fixed with ice-cold acetone and alcohol (1:1), then subjected to immunofluorescent staining as described above. Specificity of MMP activity was confirmed by the addition of 20 mm EDTA, which inhibits activity (34Goussev S. Hsu J.Y. Lin Y. Tjoa T. Maida N. Werb Z. Noble-Haeusslein L.J. J. Neurosurg. 2003; 99: 188-197Crossref PubMed Scopus (25) Google Scholar). Statistics—Student's t test was employed to determine differences between two groups (ThT comparison ± protease). Paired comparisons (in situ plaque degradation assay) were analyzed using the paired t test. p values <0.05 were considered statistically significant. Cleavage Sites for Aβ-degrading Proteases—Of the candidate proteases capable of degrading sAβ in vitro, only ECE, IDE, and NEP have evidence supporting a role in vivo (14Iwata N. Tsubuki S. Takaki Y. Shirotani K. Lu B. Gerard N.P. Gerard C. Hama E. Lee H.J. Saido T.C. Science. 2001; 292: 1550-1552Crossref PubMed Scopus (848) Google Scholar, 15Eckman E.A. Watson M. Marlow L. Sambamurti K. Eckman C.B. J. Biol. Chem. 2003; 278: 2081-2084Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 16Farris W. Mansourian S. Chang Y. Lindsley L. Eckman E.A. Frosch M.P. Eckman C.B. Tanzi R.E. Selkoe D.J. Guenette S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4162-4167Crossref PubMed Scopus (1226) Google Scholar). To directly compare Aβ-degrading activity and specific peptide fragments generated by digestion between these proteases and MMP-9, freshly prepared synthetic human Aβ1-42 or Aβ1-40 was incubated with each protease for 4 h prior to analysis with MALDI-TOF MS. Incubation of Aβ1-42 with each recombinant human protease (ECE, IDE, NEP, and MMP-9) resulted in relatively specific profiles of Aβ fragments (Fig. 1, A-F). The putative Aβ1-42 cleavage sites for each protease, based on the fragments generated, are shown in Fig. 1G. The major fragments generated from proteolytic cleavage of Aβ1-40 were similar to those generated from Aβ1-42 (supplemental Fig. 1). In general, the Aβ cleavage sites are in good agreement with previous studies (10Eckman E.A. Reed D.K. Eckman C.B. J. Biol. Chem. 2001; 276: 24540-24548Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 13Backstrom J.R. Lim G.P. Cullen M.J. Tokes Z.A. J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar, 24Asahina M. Yoshiyama Y. Hattori T. Clin. Neuropathol. 2001; 20: 60-63PubMed Google Scholar, 35Morelli L. Llovera R. Gonzalez S.A. Affranchino J.L. Prelli F. Frangione B. Ghiso J. Castano E.M. J. Biol. Chem. 2003; 278: 23221-23226Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 36Iwata N. Tsubuki S. Takaki Y. Watanabe K. Sekiguchi M. Hosoki E. Kawashima-Morishima M. Lee H.J. Hama E. Sekine-Aizawa Y. Saido T.C. Nat. Med. 2000; 6: 143-150Crossref PubMed Scopus (0) Google Scholar). However, a few differences were noted, possibly reflecting differences in the sources of the enzymes used (human versus rat), in the nature of Aβ substrate, in the digestion conditions (e.g. different buffers), or in detection and purification methods. MMP-9 generated six fragments that, in contrast to other proteases, were mostly in the hydrophobic C-terminal region of the Aβ peptide. All proteases examined exhibited a specific activity for sAβ degradation that was remarkably similar (see below). MMP-9 Degrades fAβ in Vitro—To determine whether sAβ-degrading proteases could degrade fAβ, human recombinant proteases (ECE, IDE, NEP, and MMP-9) or buffer alone were incubated with preformed Aβ fibrils. These fibrils were formed by incubating Aβ1-42 or Aβ1-40 at 37 °C for 5 days (see “Experimental Procedures”). For Aβ1-42, ThT fluorescence reached maximal values within 1 day, while Aβ1-40 reached maximal fluorescence within 3 days. Preformed Aβ fibrils were then incubated with proteases for 1-5 days at 37 °C and examined using ThT fluorescence. Of the four proteases examined, only MMP-9 reduced ThT fluorescence compared with buffer controls (fAβ1-42, Fig. 2A, fAβ1-40; supplemental Fig. 2), suggesting that fAβ was degraded by this protease. This decrease in ThT fluorescence was time-dependent and declined to 70% of initial values by 96 h (Fig. 2B). To ensure that similar results could be obtained with Aβ preparations from different batches or manufacturers, we repeated the digestions using fAβ1-42 from two different sources (American Peptide and Bachem) and at least two different lots and obtained similar results in each case (data not shown). Because our experiments directly compared the proteolytic activity of four different enzymes, we determined the specific activity for sAβ-degradation using MALDI-TOF MS data after 0 and 5 days of incubating proteases alone (without substrate) at 37 °C. Specific sAβ degrading activity was remarkably similar for all four proteases at 0 days, declining to ∼50% after 5 days (Fig. 2C). Therefore, the observed differences in fAβ degrading activity did not reflect different specific activities for sAβ degradation among the proteases. To visualize ultrastructural changes in Aβ fibrils after incubation with proteases, we performed TEM on preformed fAβ incubated in buffer with or without the proteases. TEM of aggregated synthetic human Aβ1-42 demonstrated fibrils with an approximate diameter of 10 nm, consistent with prior reports (37Shirahama T. Cohen A.S. J. Cell Biol. 1967; 33: 679-708Crossref PubMed Scopus (284) Google Scholar, 38Serpell L.C. Blake C.C. Fraser P.E. Biochemistry. 2000; 39: 13269-13275Crossref PubMed Scopus (161) Google Scholar). In addition, thicker aggregates of fibers (80-100 nm diameter) were occasionally observed. Incubation of fAβ with ECE, IDE, or NEP, pro-MMP-9 produced no changes compared with PBS alone (Fig. 3, A-D), but MMP-9 incubation mixtures contained fewer fibrils. In addition, amorphous structures suggestive of decomposed fibrils were observed in the MMP-9 incubation mixtures (Fig. 3, F and G). These amorphous structures were very similar to that observed for α-helical structures (39Nilsson M.R. Methods (Amst.). 2004; 34: 151-160Google Scholar) or after Aβ fibril deaggregation with curcumin (30Yang F. Lim G.P. Begum A.N. Ubeda O.J. Simmons M.R. Ambegaokar S.S. Chen P.P. Kayed R. Glabe C.G. Frautschy S.A. Cole G.M. J. Biol. Chem. 2005; 280: 5892-5901Abstract Full Text Full Text PDF PubMed Scopus (2014) Google Scholar). To explore the potential mechanisms of fibril disruption by MMP-9, we determined whether Aβ fragments were released by MMP-9 digestion of fAβ. Aβ fragments generated by incubating purified fAβ with ECE, IDE, NEP, or MMP-9 were isolated and analyzed by MALDI-TOF MS. MMP-9 was the only protease examined that produced Aβ fragments (Fig. 4C), with molecular masses of 2461.68 and 3390.59 daltons corresponding to Aβ1-20 and Aβ1-30. Inhibition of MMP-9 activity with EDTA prevented the generation of Aβ fragments (Fig. 4B), suggesting that proteolytic cleavage at Phe20-Ala21 or Ala30-Ile31 may be important for fibril degradation. Impurities in the MMP-9 preparation are indicated by * and are also seen in the MS of the protease alone (supplemental Fig. 3). Masses of the impurities did not coincide with Aβ fragments. MMP-9 Degrades Compact Plaques in Situ—Although amyloid plaques are primarily composed of the Aβ peptide, numerous other components are known to colocalize in senile plaques, including proteoglycans, serum constituents, metal ions, and others. Furthermore, posttranslational modifications of Aβ may further stabilize amyloid deposits in plaques (40Atwood C.S. Martins R.N. Smith M.A. Perry G. Peptides. 2002; 23: 1343-1350Crossref PubMed Scopus (115) Google Scholar) Thus, the ability of an enzyme to degrade Aβ fibrils need not imply an ability to degrade amyloid plaques. To determine whether MMP-9 degrades amyloid plaques, we developed a sensitive method to compare the load of compact plaques in adjacent brain sections from aged APP/PS1 mice. Adjacent brain sections were incubated with proteases or buffer alone and then stained with ThS, and the area of fluorescence was compared between matching high power fields from adjacent sections. The area of ThS fluorescence (thioflavin load) in brain sections incubated with ECE, IDE, or NEP did not differ from those incubated with buffer alone, but incubation with MMP-9 resulted in a significant decrease in thioflavin load (Fig. 5A-D), suggesting that MMP-9 could degrade compact amyloid plaques. Fig. 5E illustrates a representative comparison between two corresponding high power fields from adjacent sections (upper panels) and the same fields with contrast thresholding to illustrate plaque load (lower panels). Note the relative decrease in plaque sizes in the section incubated with MMP-9 (arrows, +MMP-9) compared with that incubated with buffer alone (-MMP-9). MMP-9 Expression and Activity in Brains of APPsw Mice— Unlike other MMPs, MMP-9 is expressed in specific cells at low basal levels, and its expression can be induced by a variety of stimuli, including growth factors, cytokines, reactive oxygen species, or stressors (41Van den Steen P.E. Dubois B. Nelissen I. Rudd P.M. Dwek R.A. Opdenakker G. Crit. Rev. Biochem. Mol. Biol. 2002; 37: 375-536Crossref PubMed Scopus (749) Google Scholar). We examined MMP-9 expression in the brains of aged APPsw, APP/PS1, and wild-type littermate mice. Aged wild-type mice demonstrated a few isolated cells with MMP-9 immunoreactivity localized primarily in the corpus callosum (Fig. 6A), while APPsw mice had many more cells with prominent MMP-9 immunostaining throughout the brain (including cortex, corpus callosum, and hippocampus, Fig. 6, B and C). Double staining with ThS revealed that many of the MMP-9 immunoreactive cells appeared to surround ThS-positive compact plaques (Fig. 6C). Moreover, the cells had the appearance of activated astrocytes, based on their hypertrophic cell bodies and immunoreactivity with anti-GFAP antibodies (as illustrated by the double labeling experiment in Fig. 6, D-F). Similar expression profiles were found in 9-month-old APP/PS1 mice (data not shown). MMP-9 protein expression does not necessarily imply proteolytic activity. MMP-9 activity, like that of other MMPs, is stringently controlled through multiple mechanisms including transcriptional regulation, release from cells, activation through propeptide cleavage, and interaction with TIMPs. Thus, to examine net MMP-9 (gelatinase) activity in the brains of APPsw mice, in situ zymography was performed by incubating brain sections with fluorescently labeled gelatin. Gelatinase activity was detected within many but not all amyloid plaques (Fig. 6, G-I) in aged APPsw mice. Double labeling with ThS and anti-MMP-9 antibodies revealed that the vast majority of plaques with gelatinase activity were ThS-positive compact plaques surrounded by astrocytes expressing MMP-9 (data

Studies of subjects with dementia of the Alzheimer type have reported correlations between increases in activity of the hypothalamic-pituitary-adrenal (HPA) axis and hippocampal degeneration. In this study, the authors sought to determine whether increases in plasma cortisol, a marker of HPA activity, were associated with clinical and cognitive measures of the rate of disease progression in subjects with Alzheimer-type dementia.Thirty-three subjects with very mild and mild Alzheimer-type dementia and 21 subjects without dementia were assessed annually for up to 4 years with the Clinical Dementia Rating scale and a battery of neuropsychological tests. Plasma was obtained at 8 a.m. on a single day and assayed for cortisol. Rates of change over time in the clinical and cognitive measures were derived from growth curve models.In the subjects with dementia, but not in those without dementia, higher plasma cortisol levels were associated with more rapidly increasing symptoms of dementia and more rapidly decreasing performance on neuropsychological tests associated with temporal lobe function. No associations were observed between plasma cortisol levels and clinical and cognitive assessments obtained at the single assessment closest in time to the plasma collection.Higher HPA activity, as reflected by increased plasma cortisol levels, is associated with more rapid disease progression in subjects with Alzheimer-type dementia.

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

Alzheimer disease (AD) is the most common cause of dementia in the elderly. Clinicopathological studies support the presence of a long preclinical phase of the disease, with the initial deposition of AD pathology estimated to begin approximately 10-15 years prior to the onset of clinical symptoms. The hallmark clinical phenotype of AD is a gradual and progressive decline in two or more cognitive domains, most commonly involving episodic memory and executive functions, that is sufficient to cause social or occupational impairment. Current diagnostic criteria can accurately identify AD in the majority of cases. As disease-modifying therapies are being developed, there is growing interest in the identification of individuals in the earliest symptomatic, as well as presymptomatic, stages of disease, because it is in this population that such therapies may have the greatest chance of success. The use of informant-based methods to establish cognitive and functional decline of an individual from previously attained levels of performance best allows for the identification of individuals in the very mildest stages of cognitive impairment.

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-beta peptide (Abeta). Although familial AD appears to be caused by Abeta overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Abeta. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Abeta degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Abeta degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Abeta, lowering levels and producing several fragments after incubation with synthetic human Abeta(1-40) and Abeta(1-42). This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Abeta were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Abeta levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Abeta clearance by promoting Abeta catabolism.

For therapies for Alzheimer's disease (AD) to have the greatest impact, it will likely be necessary to treat individuals in the "preclinical" (presymptomatic) stage. Fluid and neuroimaging measures are being explored as possible biomarkers of AD pathology that could aid in identifying individuals in this stage to target them for clinical trials and to direct and monitor therapy. The objective of this study was to determine whether cerebrospinal fluid (CSF) biomarkers for AD suggest the presence of brain damage in the preclinical stage of AD.We investigated the relation between structural neuroimaging measures (whole-brain volume) and levels of CSF amyloid-beta (Abeta)(40), Abeta(42), tau, and phosphorylated tau(181) (ptau(181)), and plasma Abeta(40) and Abeta(42) in well-characterized research subjects with very mild and mild dementia of the Alzheimer type (n = 29) and age-matched, cognitively normal control subjects (n = 69).Levels of CSF tau and ptau(181), but not Abeta(42), correlated inversely with whole-brain volume in very mild and mild dementia of the Alzheimer type, whereas levels of CSF Abeta(42), but not tau or ptau(181), were positively correlated with whole-brain volume in nondemented control subjects.Reduction in CSF Abeta(42), likely reflecting Abeta aggregation in the brain, is associated with brain atrophy in the preclinical phase of AD. This suggests that there is toxicity associated with Abeta aggregation before the onset of clinically detectable disease. Increases in CSF tau (and ptau(181)) are later events that correlate with further structural damage and occur with clinical onset and progression.

Alzheimer's disease (AD) has a long preclinical phase in which amyloid and tau cerebral pathology accumulate without producing cognitive symptoms. Resting state functional connectivity magnetic resonance imaging has demonstrated that brain networks degrade during symptomatic AD. It is unclear to what extent these degradations exist before symptomatic onset. In this study, we investigated graph theory metrics of functional integration (path length), functional segregation (clustering coefficient), and functional distinctness (modularity) as a function of disease severity. Further, we assessed whether these graph metrics were affected in cognitively normal participants with cerebrospinal fluid evidence of preclinical AD. Clustering coefficient and modularity, but not path length, were reduced in AD. Cognitively normal participants who harbored AD biomarker pathology also showed reduced values in these graph measures, demonstrating brain changes similar to, but smaller than, symptomatic AD. Only modularity was significantly affected by age. We also demonstrate that AD has a particular effect on hub-like regions in the brain. We conclude that AD causes large-scale disconnection that is present before onset of symptoms.

Major imaging biomarkers of Alzheimer's disease include amyloid deposition [imaged with [(11)C]Pittsburgh compound B (PiB) PET], altered glucose metabolism (imaged with [(18)F]fluro-deoxyglucose PET), and structural atrophy (imaged by MRI). Recently we published the initial subset of imaging findings for specific regions in a cohort of individuals with autosomal dominant Alzheimer's disease. We now extend this work to include a larger cohort, whole-brain analyses integrating all three imaging modalities, and longitudinal data to examine regional differences in imaging biomarker dynamics. The anatomical distribution of imaging biomarkers is described in relation to estimated years from symptom onset. Autosomal dominant Alzheimer's disease mutation carrier individuals have elevated PiB levels in nearly every cortical region 15 y before the estimated age of onset. Reduced cortical glucose metabolism and cortical thinning in the medial and lateral parietal lobe appeared 10 and 5 y, respectively, before estimated age of onset. Importantly, however, a divergent pattern was observed subcortically. All subcortical gray-matter regions exhibited elevated PiB uptake, but despite this, only the hippocampus showed reduced glucose metabolism. Similarly, atrophy was not observed in the caudate and pallidum despite marked amyloid accumulation. Finally, before hypometabolism, a hypermetabolic phase was identified for some cortical regions, including the precuneus and posterior cingulate. Additional analyses of individuals in which longitudinal data were available suggested that an accelerated appearance of volumetric declines approximately coincides with the onset of the symptomatic phase of the disease.

Although there are no proven ways to delay onset or slow progression of Alzheimer's disease (AD), studies suggest that diet can affect risk. Pomegranates contain very high levels of antioxidant polyphenolic substances as compared to other fruits and vegetables. Polyphenols have been shown to be neuroprotective in different model systems. We asked whether dietary supplementation with pomegranate juice (PJ) would influence behavior and AD-like pathology in a transgenic mouse model. Transgenic mice (APPsw/Tg2576) received either PJ or sugar water control from 6 to 12.5 months of age. PJ-treated mice learned water maze tasks more quickly and swam faster than controls. Mice treated with PJ had significantly less (∼ 50%) accumulation of soluble Aβ42 and amyloid deposition in the hippocampus as compared to control mice. These results suggest that further studies to validate and determine the mechanism of these effects, as well as whether substances in PJ may be useful in AD, should be considered.

By comparing the amino acid sequences of 11 mammalian and 1 avian prion proteins (PrP), structural analyses predicted four alpha-helical regions. Peptides corresponding to these regions of Syrian hamster PrP were synthesized, and, contrary to predictions, three of the four spontaneously formed amyloids as shown by electron microscopy and Congo red staining. By IR spectroscopy, these amyloid peptides exhibited secondary structures composed largely of beta-sheets. The first of the predicted helices is the 14-amino acid peptide corresponding to residues 109-122; this peptide and the overlapping 15-residue sequence 113-127 both form amyloid. The most highly amyloidogenic peptide is AGAAAAGA, which corresponds to Syrian hamster PrP residues 113-120 and is conserved across all species for which the PrP sequence has been determined. Two other predicted alpha-helices corresponding to residues 178-191 and 202-218 form amyloids and exhibit considerable beta-sheet structure when synthesized as peptides. These findings suggest the possibility that the conversion of the cellular isoform of PrP to the scrapie isoform of PrP involves the transition of one or more putative PrP alpha-helices into beta-sheets and that prion diseases are disorders of protein conformation.

Report26 November 2009Open Access Cerebrospinal fluid tau and ptau181 increase with cortical amyloid deposition in cognitively normal individuals: Implications for future clinical trials of Alzheimer's disease Anne M. Fagan Corresponding Author Anne M. Fagan [email protected] Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mark A. Mintun Mark A. Mintun Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Aarti R. Shah Aarti R. Shah Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Patricia Aldea Patricia Aldea Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Catherine M. Roe Catherine M. Roe Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Robert H. Mach Robert H. Mach Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daniel Marcus Daniel Marcus Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author John C. Morris John C. Morris Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author David M. Holtzman David M. Holtzman Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Anne M. Fagan Corresponding Author Anne M. Fagan [email protected] Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Mark A. Mintun Mark A. Mintun Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Aarti R. Shah Aarti R. Shah Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Patricia Aldea Patricia Aldea Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Catherine M. Roe Catherine M. Roe Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Robert H. Mach Robert H. Mach Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Daniel Marcus Daniel Marcus Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author John C. Morris John C. Morris Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author David M. Holtzman David M. Holtzman Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA Search for more papers by this author Author Information Anne M. Fagan *,1,2,3, Mark A. Mintun2,4, Aarti R. Shah1,3, Patricia Aldea4, Catherine M. Roe1,2, Robert H. Mach2,4, Daniel Marcus4, John C. Morris1,2,5 and David M. Holtzman1,2,3,6 1Department of Neurology, Washington University School of Medicine, St. Louis, MO, USA 2Alzheimer's Disease Research Center, Washington University School of Medicine, St. Louis, MO, USA 3Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, MO, USA 4Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA 5Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA 6Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO, USA *Tel: +1 341 362 3453; Fax: +1 341 362 2244 EMBO Mol Med (2009)1:371-380https://doi.org/10.1002/emmm.200900048 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Alzheimer's disease (AD) pathology is estimated to develop many years before detectable cognitive decline. Fluid and imaging biomarkers may identify people in early symptomatic and even preclinical stages, possibly when potential treatments can best preserve cognitive function. We previously reported that cerebrospinal fluid (CSF) levels of amyloid-β42 (Aβ42) serve as an excellent marker for brain amyloid as detected by the amyloid tracer, Pittsburgh compound B (PIB). Using data from 189 cognitively normal participants, we now report a positive linear relationship between CSF tau/ptau181 (primary constituents of neurofibrillary tangles) with the amount of cortical amyloid. We observe a strong inverse relationship of cortical PIB binding with CSF Aβ42 but not for plasma Aβ species. Some individuals have low CSF Aβ42 but no cortical PIB binding. Together, these data suggest that changes in brain Aβ42 metabolism and amyloid formation are early pathogenic events in AD, and that significant disruptions in CSF tau metabolism likely occur after Aβ42 initially aggregates and increases as amyloid accumulates. These findings have important implications for preclinical AD diagnosis and treatment. The paper explained PROBLEM: AD pathology is estimated to develop many years before detectable cognitive decline. Once symptoms are apparent, the brain has already experienced substantial neuronal and synaptic loss. Thus there is a great need to develop biomarkers that can identify people in the very earliest symptomatic and even ‘preclinical’ stages, prior to any cognitive impairment, when potential treatments will have the best opportunity to preserve cognitive function. RESULTS: We analysed CSF samples and in parallel determined the amount of cortical amyloid as evidenced by retention of the in vivo amyloid binding agent, PIB, in 189 cognitively normal research participants (age 43–89 years). We observed a positive linear relationship between the levels of CSF tau and ptau181 (primary constituents of neurofibrillary tangles) with the amount of cortical amyloid. We also observed a strong inverse relationship between cortical PIB binding and CSF Aβ42 (the primary constituent of amyloid plaques), but not plasma Aβ species, demonstrating that a low level of CSF Aβ42 is an excellent marker of brain amyloid even in the absence of cognitive symptoms. IMPACT: The data obtained shed light on the potential utility of PIB amyloid imaging and CSF Aβ42, tau and ptau181 as antecedent (‘preclinical’) biomarkers of AD and also provide insight into the normal time course of the pathophysiology of the disease as reflected in the CSF. These findings have important implications for preclinical AD diagnosis and treatment, and should aid in the design and evaluation of secondary prevention trials in AD. INTRODUCTION Alzheimer's disease (AD) is a progressive and fatal neurodegenerative disorder that currently affects ∼10.6 million people in the US and Europe, with projected estimates reaching 15.4 million by the year 2030 (Alzheimer's Association). Clinicopathological studies support the notion of a long ‘preclinical’ stage of the disease, with brain pathology (amyloid plaques and neurofibrillary tangles) estimated to begin ∼10–20 years prior to significant neuronal cell death and the consequent appearance of any behavioural signs or symptoms (Braak & Braak, 1997; Gomez-Isla et al, 1996; Hulette et al, 1998; Markesbery et al, 2006; Morris & Price, 2001; Price et al, 2001). Fluid and imaging biomarkers of this pathology are currently being sought in order to confirm early diagnoses and, importantly, to identify individuals in the preclinical stage so emerging therapies ultimately have a chance to preserve normal brain function (Craig-Schapiro et al, 2008). We recently reported an inverse relationship between cortical amyloid deposits, as viewed by positron emission tomography (PET) imaging with the amyloid binding agent, Pittsburgh compound B (PIB) and the amount of cerebrospinal fluid (CSF) amyloid-β42 (Aβ42), the primary constituent of brain amyloid plaques (Fagan et al, 2006, 2007). Individuals with cortical amyloid (as detected by PET PIB) had low CSF Aβ42 whereas those without cortical amyloid had high CSF Aβ42. This relationship was observed independent of clinical status; several cognitively normal individuals had a CSF Aβ42/PIB profile indistinguishable from that of other individuals diagnosed clinically with early stage dementia of the Alzheimer type (DAT). These observations are consistent with the idea of preclinical AD and suggest that these measures may have clinical utility as antecedent biomarkers of the disease. We have now obtained CSF and PIB data from 189 cognitively normal individuals ranging in age from 43 to 89 years. We explored the relationship between in vivo brain amyloid and CSF markers of proteins present in neurons and constituents of neurofibrillary tangles (tau and ptau181), as well as Aβ species in plasma, and investigated whether the CSF Aβ42/PIB relationship remains robust in this large cohort of non-demented individuals. The data we obtained shed further light on the potential utility of these measures as antecedent biomarkers of AD, and also provide insight into the normal time course of the pathophysiology of the disease as reflected in CSF, information that should aid in the design and evaluation of secondary prevention trials. RESULTS One hundred and eighty-nine research participants with a clinical dementia rating of 0 (CDR 0, indicating cognitively normal) (Morris, 1993) met the selection criterion of having a PIB scan within 2 years of CSF collection by lumbar puncture (LP). Combined PIB and biomarker data from 25 of these participants have been reported by us in previous studies (Fagan et al, 2006, 2009), whereas the remaining 164 are unique to the present study. The demographic characteristics of the present cohort are similar to what we have previously published with the exception of age (Table 1). By design, we have included a wide range of ages in the present study (43–89 years, normally distributed) so as to better capture the various biomarker correlations during the potential preclinical stage of AD (which is estimated to begin 10–20 years prior to cognitive symptoms). Therefore, the mean age of our cohort is younger (64.7 ± 10.4) than what we have reported on previously (71.41 ± 8.62; Fagan et al, 2009). In keeping with this age difference, CSF Aβ42 levels in the present study (652 ± 235 pg/ml) are higher than that reported by us in our studies of older individuals (572 ± 208 pg/ml) and, as expected, CSF tau (285 ± 151 pg/ml vs. 334 ± 180 pg/ml), and ptau181 (52 ± 23 pg/ml vs. 61 ± 27 pg/ml) levels are lower (Fagan et al, 2009). The absolute plasma Aβ values cannot be compared between our various studies because different methods were used to measure these analytes. Table 1. Participant demographic characteristics, psychometric performance and biomarker values CDR 0 participants N 189 Age at LP (year) 64.7 (10.4) Age range (year) 43–89 M/F (%F) 62/127 (67%) APOE ε4–/ε4+ (% ε4+) 125/64 (34%) Selective remembering (possible score, 0–48) 31.2 (6.3) Animal naming 21.6 (5.3) Trailmaking A (# of connections/s) 0.916 (0.303) Trailmaking B (# of connections/s) 0.384 (0.151) CSF Aβ38 1228 (514) CSF Aβ40 8958 (4464) CSF Aβ42 652 (235) CSF tau 285 (151) CSF ptau181 52 (23) Plasma Aβ1–40 217 (60) Plasma Aβx–40 37 (11) Plasma Aβ1–42 193 (44) Plasma Aβx–42 25 (9) PIB MCBP 0.0931 (0.213) Fluid psychometric and PET PIB (MCBP) values are represented as means (standard deviations). CSF and plasma values are in pg/ml. PIB MCBP values are in arbitrary units (generated by Logan graphical analyses). APOE, apolipoprotein E; CDR, clinical dementia rating; CSF, cerebrospinal fluid; LP, lumbar puncture; MCBP, mean cortical binding potential; PIB, Pittsburgh compound B. Given the wide range of ages in the present cohort, we first investigated whether any of the biomarker measures correlated with age at the time of LP. As shown in Fig. 1, positive correlations were observed between age and cortical amyloid (represented by mean cortical PIB binding potential (MCBP); Mintun et al, 2006), CSF tau and ptau181 and plasma Aβ1–40. An inverse correlation was observed between age and CSF Aβ42, and no relationships between age and CSF Aβ38 or Aβ40 were found. Due to these age effects, all subsequent analyses were corrected for age. Figure 1. Cortical amyloid as detected by PET PIB and fluid biomarkers in CDR 0 participants (n = 189) as a function of age. Levels of A.. cortical amyloid are positively correlated with age in this CDR 0 cohort, B.. The level of CSF Aβ38 is not correlated with age, C.. nor is CSF Aβ40. D.. CSF Aβ42 is negatively correlated with age. E.. Positive correlations with age are observed for CSF tau, F.. CSF ptau181 and G.. plasma Aβ1–40. H.. Plasma Aβ1–42 is not correlated with age in this cohort. Download figure Download PowerPoint We next investigated whether the inverse relationship between CSF Aβ42 and cortical PIB binding that we had reported previously in a mixed cohort of mildly demented and non-demented individuals was observed in this cohort of cognitively normal individuals. Overall, 29 participants in this cohort had MCBP values greater than or equal to 0.18 whereas 160 participants had MCBPs below 0.18 (Fig 2A). In individuals with MCBP values greater than 0.18, PIB retention is visualized in the neocortex and appears qualitatively greater than background levels. We continued to observe a robust and linear relationship between CSF Aβ42 and cortical amyloid in this group of cognitively normal individuals (Fig 2B). Every participant with high PIB binding had CSF Aβ42 values <582 pg/ml; 86% had CSF Aβ42 values <500 pg/ml. A large majority (84%) of participants with low PIB binding had CSF Aβ42 values >500 pg/ml (Fig 2A). Consistent with our previous findings (Fagan et al, 2006, 2007), many of the CDR 0 participants within this broad age range had little or no cortical amyloid and high mean CSF Aβ42 levels (≥500 pg/ml) (Fig 2B). Twenty-five of the 189 CDR 0 participants displayed the typical AD biomarker phenotype in relation to Aβ, with high PIB binding and low CSF Aβ42 (Fig 2B). In many cases their PET PIB scans were indistinguishable from demented individuals with DAT (CDR > 0) (Fig 2C). In contrast to CSF Aβ42, CSF Aβ40 was not related to the presence or amount of cortical amyloid in these individuals (Fig 2D). Similarly, levels of CSF Aβ38 were not correlated with cortical amyloid load (Fig 2E), but the ratio of CSF Aβ38/Aβ42 was positively correlated with amyloid load (Fig 2F), likely due to the drop in CSF Aβ42 with amyloid deposition. Figure 2. Cortical amyloid as detected by PET PIB and its relationship to CSF Aβ in CDR 0 participants (n = 189). A.. A high percentage (84%) of participants with low PIB values (MCBP < 0.18) had high CSF Aβ42 levels (mean (SD) = 705 pg/ml (211)) whereas the vast majority of participants (86%) in the cohort who had high PIB binding (MCBP ≥ 0.18) had low CSF Aβ42 (mean (SD) = 362 pg/ml (115)). Horizontal lines represent the group means, and these means are statistically different from each other (asterisk, p < 0.0001). B.. Relationship between CSF Aβ42 levels and cortical amyloid. Most participants had low MCBP values. The vast majority (86%) of participants with MCBPs ≥ 0.18 had low CSF Aβ42 levels. These CDR 0 participants are hypothesized to have preclinical AD. The box outlined by dashed lines identifies the 28 individuals who have low cortical PIB binding (MCBP < 0.18) with low CSF Aβ42. There is a linear relationship between CSF Aβ42 and the amount of cortical amyloid although CSF Aβ42 appears to drop and then stay low as the amyloid load increases. C.. MRI (left) and PET PIB (right) images of a representative low PIB (MCBP = 0.0270) CDR 0 participant (top panel), a high PIB (MCBP = 0.7790) CDR 0 participant (middle panel), and a high PIB (MCBP = 0.7812) CDR > 0 participant (bottom panel). The amount of cortical PIB binding (yellow-red corresponds to high binding) in the high PIB CDR 0 participant and the high PIB CDR > 0 participant is comparable, whereas there is only background PIB binding (green) in white matter tracks in the low PIB CDR 0 participant. D,E.. No relationship between CSF Aβ40 (D) and CSF Aβ38 (E) levels and cortical amyloid was observed in this cognitively normal cohort (r = −0.0287, p = 0.6963; r = 0.06851, p = 0.3515, respectively). F.. A negative correlation was found between cortical amyloid and the CSF Aβ38/Aβ42 ratio. All Pearson correlation coefficients are corrected for age. n.s., not significant. Download figure Download PowerPoint Twenty-eight CDR 0 individuals showed a mismatch, appearing in the ‘lower quadrant’ of Fig 2B (whose boundaries are indicated by the square outlined in a dashed line in Fig 2B) in that they had little or no cortical PIB binding (MCPB < 0.18) but had low CSF Aβ42 (<500 pg/ml). The mean interval between LP and PIB scans for individuals in this quadrant did not differ statistically from the mean intervals of those participants in the other quadrants (i.e. low PIB/high CSF Aβ42 and high PIB/low CSF Aβ42) (p = 0.4693). In addition, this low PIB/low CSF Aβ42 group (‘lower quadrant’, n = 28) did not differ from the low PIB/high CSF Aβ42 group (‘upper quadrant’, n = 132) in the frequency of the ε4 allele of APOE (39% vs. 27%, respectively, p = 0.2049), nor did it differ from the high PIB/low CSF Aβ42 (‘PIB-positive quadrant’) group (39% vs. 62%, respectively), but it did approach statistical significance (p = 0.0854). The ε4+ frequency in the high PIB/low CSF Aβ42 (‘PIB-positive quadrant’) group did, however, differ significantly from that of the low PIB/high CSF Aβ42 (‘upper quadrant’) group (p = 0.0003). We did not observe any significant associations between quadrant membership and performance on any of the psychometric tests when adjusted for age (Selective Reminding, p = 0.2486; Animal Naming, p = 0.1209; Trailmaking A, p = 0.8561; Trailmaking B, p = 0.2817). The groups also did not differ in the percentage of self-reported presence or absence of heart disease, diabetes, history of stroke and/or TIAs, prior head trauma (with loss of consciousness) or NSAID use (all p > 0.05, Fisher's exact test). However, the low PIB/low CSF Aβ42 group (‘lower quadrant’) had a greater frequency of reported hypertension than the low PIB/high CSF Aβ42 group (‘upper quadrant’) (50% vs. 29.6%, respectively, p = 0.0469) and a greater frequency of arthritis than the low PIB/high CSF Aβ42 group (14.3% vs. 3.03%, respectively, p = 0.0323). The biological significance of these findings, if any, remains unclear but warrants further investigation. Overall, longitudinal PIB follow-up of the participants in this lower quadrant will be required to understand whether their low CSF Aβ42 represents Aβ aggregation in diffuse (PIB-negative) plaques, oligomeric forms prior to substantial fibrillar (PIB-positive) Aβ deposition or simply reflects the low end of the normal spectrum of CSF Aβ42 levels. It is interesting to note that one of the individuals in this quadrant (having no cortical PIB binding but low CSF Aβ42) has come to autopsy 2 years after LP and PIB testing. This participant was CDR 0 at the time of LP and PIB (6 months apart) but received a CDR rating of 0.5 (very mild dementia) just prior to death. Subsequent histological analysis of the brain revealed abundant diffuse but few neuritic (amyloid) plaques, (Cairns et al, 2009) consistent with the first proposed hypothesis. Despite the strong relationship between PIB binding and CSF Aβ42, we observed no relationship between cortical amyloid load and plasma levels of Aβ42, Aβx–42, Aβ40 or Aβx–40 (Fig. 3). Our previous study reported the same results in a much smaller, clinically mixed cohort. Furthermore, for the present study we used the xMAP plasma kit (Inno-Bia Plasma Aβ Forms Multiplex Assay) which generates reliable values in the lower pg/ml range required for plasma measures (Blennow et al, 2009; Lachno et al, 2009). We obtained reliable values (with low coefficients of variability) for all but five samples; these five had very low levels of Aβx–42 that were below the level of detection so they were assigned a value of 0 pg/ml. Figure 3. Cortical amyloid as detected by PET PIB and its relationship to plasma Aβ42 and Aβ40 species in CDR 0 participants (n = 189). No relationship was observed between mean cortical PIB binding and plasma A.. Aβ1–40 (r = −0.0724, p = 0.3234), B.. Aβ x–40 (r = 0.04583, p = 0.5323), C.. Aβ1–42 (r = −0.1015, p = 0.1658) or D.. Aβx–42 (r = −0.03869, p = 0.5981). Five participants had levels of plasma Aβx–42 below the level of detection so they are represented as having 0 pg/ml. All Pearson correlation coefficients are corrected for age. n.s., not significant. Download figure Download PowerPoint Importantly, analysis of this CDR 0 cohort revealed a novel pattern of increases in CSF tau (and ptau181) with increasing cortical amyloid deposition (Fig 4A,B). Elevations in CSF tau in general did not appear to occur substantially in participants with an MCBP less than 0.5 but did increase in many, but not all, participants with binding potentials 0.5 and greater. Regression analyses correcting for age revealed a linear relationship between CSF tau (and ptau181) and PIB binding (Fig 4A,B). In addition, the ratios of CSF tau/Aβ42 and ptau181/Aβ42 also increased linearly with amyloid deposition and the correlations were particularly robust (Fig 4C,D). Similar to what we observed for CSF tau and ptau181, the ratios of tau and ptau181 to CSF Aβ42 were generally not elevated until substantial PIB binding values were reached. Figure 4. Cortical amyloid as detected by PET PIB and its relationship to CSF tau and ptau181 and the ratios of CSF tau/Aβ42 and ptau181/Aβ42 in CDR 0 participants (n = 189). A linear relationship is observed between the amount of cortical amyloid and A.. the levels of CSF tau B.. the levels of CSF ptau181 C.. the ratios of CSF tau/Aβ42 and D.. the ratios of the ptau181/Aβ42. The correlations between the CSF tau(s)/Aβ42 ratios and MCBP remain significant even when the statistical outlier (high PIB, high ratio) is omitted from the analysis (tau/Aβ42, r = 0.74227, p < 0.0001; ptau181/Aβ42, r = 0.73510, p < 0.0001). All Pearson correlation coefficients are corrected for age. Download figure Download PowerPoint All participants in this cognitively normal cohort were administered a common battery of psychometric tests, including Selective Reminding (a measure of episodic memory) (Grober et al, 1988), Animal Naming (assesses semantic memory) (Goodglass & Kaplan, 1983) and a speeded visuospatial test with two parts: Trailmakings A and B (Armitage, 1946). None of the biomarker measures showed significant associations with performance on any of the psychometric tests, with the exception of negative correlations between Trailmaking A and CSF Aβ38 (r = −0.14621, p = 0.0464) and plasma Aβ1–40 (r = −0.24069, p = 0.0009). Due to the number of statistical tests conducted overall, however, some statistically significant differences could be due to chance. DISCUSSION The data presented here are part of an ongoing longitudinal study investigating fluid and imaging measures as possible antecedent (preclinical) biomarkers of AD. Importantly, they shed light on what may be the pathophysiology of the earliest events in the disease process and their relationship with CSF biomarkers. Consistent with our previous, smaller studies which included both non-demented and demented individuals (Fagan et al, 2006, 2007), we observed a robust relationship between cortical PIB binding and levels of CSF Aβ42 but not CSF Aβ40 (or Aβ38) in this large cohort of cognitively normal participants. While this relationship is linear, visual inspection of the graphs gives the impression of CSF Aβ42 levels dropping early in the disease process and staying low as the amount of cortical amyloid increases. The lack of correlation we observed between CSF Aβ38 and the amount of cortical amyloid is consistent with other studies suggesting this Aβ species does not change with dementia status (Mehta & Pirttila, 2005; Welge et al, 2009). However, these studies suggested that the ratio of Aβ38 to Aβ42, as opposed to Aβ38 alone, may have better specificity for distinguishing AD from controls or other non-AD dementias, although not all studies have reported this (Schoonenboom et al, 2005). The positive correlation we observed between cortical amyloid and the ratio of Aβ38 to Aβ42 is likely driven by the drop in Aβ42, as suggested by others (Mehta & Pirttila, 2005). We were also now able to measure, with great sensitivity and reliability, a number of Aβ species in plasma, including Aβ1–40, Aβx–40, Aβ1–42 and Aβx–42. However, the level of these species did not in any way relate to the presence or amount of amyloid in the brain. Previous studies investigating the utility of plasma Aβ species have reported mixed results. While plasma Aβ42 has been reported to be neither specific nor sensitive for a clinical diagnosis of mild cognitive impairment (MCI) or AD (Fukumoto et al, 2003), nor in predicting the probability of progression from MCI to AD (Hansson et al, 2008), other studies have reported that the ratio of plasma Aβ42/Aβ40 may be useful as an antecedent marker for identifying risk for developing cognitive impairment in cognitively normal elders (Graff-Radford et al, 2007). Others have reported alterations in the direction of change in plasma Aβ species over the course of the disease (Schupf et al, 2008). Using the same protocol as we used in the present study, a very recent study reported significant decreases in the levels of plasma Aβ42 and the Aβ42/Aβ40 ratio in those who were classified as having AD or MCI that would progress to AD (with subjects preselected based on clinical and CSF biomarker profiles) compared to those who did not have the ‘abnormal’ profile (Lewczuk et al, 2009). However, this decrease was not particularly robust, on the order of 10–15%, with great overlap between the groups. Thus, while the mechanism(s) underlying potential changes in Aβ metabolism in plasma is still unclear, our cross-sectional data indicate that Aβ42 levels in plasma do not reflect the amount of amyloid in the brain in cognitively normal individuals (nor are they related to the level of Aβ42 in the CSF, data not shown). In this large cohort we now observed a new grouping of participants; those who had low CSF Aβ42 levels in the absence of cortical PIB binding. It is unlikely that this is simply an APOE effect (Sunderland et al, 2004) since the frequency of the ε4 allele did not differ between the low PIB groups with low versus high CSF Aβ42. Instead, these data suggest that CSF Aβ42 may drop prior to amyloid becoming detectable by PIB, or this drop may reflect the presence of diffuse plaques and/or oligomeric Aβ species, consistent with the participant in the lower (low PIB/low CSF Aβ42) quadrant who has come to autopsy with abundant diffuse, but few amyloid (neuritic), plaques (Cairns et al, 2009). However, we cannot rule out the possib

Accumulation of amyloid-beta (Abeta) by overproduction or underclearance in the central nervous system (CNS) is hypothesized to be a necessary event in the pathogenesis of Alzheimer's disease. However, previously, there has not been a method to determine drug effects on Abeta production or clearance in the human CNS. The objective of this study was to determine the effects of a gamma-secretase inhibitor on the production of Abeta in the human CNS.We utilized a recently developed method of stable-isotope labeling combined with cerebrospinal fluid sampling to directly measure Abeta production during treatment of a gamma-secretase inhibitor, LY450139. We assessed whether this drug could decrease CNS Abeta production in healthy men (age range, 21-50 years) at single oral doses of 100, 140, or 280mg (n = 5 per group).LY450139 significantly decreased the production of CNS Abeta in a dose-dependent fashion, with inhibition of Abeta generation of 47, 52, and 84% over a 12-hour period with doses of 100, 140, and 280mg, respectively. There was no difference in Abeta clearance.Stable isotope labeling of CNS proteins can be utilized to assess the effects of drugs on the production and clearance rates of proteins targeted as potential disease-modifying treatments for Alzheimer's disease and other CNS disorders. Results from this approach can assist in making decisions about drug dosing and frequency in the design of larger and longer clinical trials for diseases such as Alzheimer's disease, and may accelerate effective drug validation. Ann Neurol 2009.

Huntington's disease is a neurodegenerative disease caused by the accumulation of mutant htt protein. Now, two groups led by Dimitri Krainc and Wenzhen Duan report that mutant htt binds and inactivates the deacetylase enzyme SIRT1 and that SIRT1 overexpression is protective in Huntington's disease mouse models. Huntington's disease is a fatal neurodegenerative disorder caused by an expanded polyglutamine repeat in huntingtin (HTT) protein. We previously showed that calorie restriction ameliorated Huntington's disease pathogenesis and slowed disease progression in mice that model Huntington's disease (Huntington's disease mice)1. We now report that overexpression of sirtuin 1 (Sirt1), a mediator of the beneficial metabolic effects of calorie restriction, protects neurons against mutant HTT toxicity, whereas reduction of Sirt1 exacerbates mutant HTT toxicity. Overexpression of Sirt1 improves motor function, reduces brain atrophy and attenuates mutant-HTT–mediated metabolic abnormalities in Huntington's disease mice. Further mechanistic studies suggested that Sirt1 prevents the mutant-HTT–induced decline in brain-derived neurotrophic factor (BDNF) concentrations and the signaling of its receptor, TrkB, and restores dopamine- and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) concentrations in the striatum. Sirt1 deacetylase activity is required for Sirt1-mediated neuroprotection in Huntington's disease cell models. Notably, we show that mutant HTT interacts with Sirt1 and inhibits Sirt1 deacetylase activity, which results in hyperacetylation of Sirt1 substrates such as forkhead box O3A (Foxo3a), thereby inhibiting its pro-survival function. Overexpression of Sirt1 counteracts the mutant-HTT–induced deacetylase deficit, enhances the deacetylation of Foxo3a and facilitates cell survival. These findings show a neuroprotective role for Sirt1 in mammalian Huntington's disease models and open new avenues for the development of neuroprotective strategies in Huntington's disease.

LY450139 dihydrate, a γ-secretase inhibitor, was studied in a randomized, controlled trial of 70 patients with Alzheimer disease. Subjects were given 30 mg for 1 week followed by 40 mg for 5 weeks. Treatment was well tolerated. Aβ_1-40 in plasma decreased by 38.2%; in CSF, Aβ_1-40 decreased by 4.42 ± 9.55% (<i>p</i> = not significant). Higher drug doses may result in additional decreases in plasma Aβ concentrations and a measurable decrease in CSF Aβ.

As we age, we experience changes in our nighttime sleep and daytime wakefulness. Individuals afflicted with Alzheimer’s disease (AD) can develop sleep problems even before memory and other cognitive deficits are reported. As the disease progresses and cognitive changes ensue, sleep disturbances become even more debilitating. Thus, it is imperative to gain a better understanding of the relationship between sleep and AD pathogenesis. We postulate a bidirectional relationship between sleep and the neuropathological hallmarks of AD; in particular, the accumulation of amyloid-β (Aβ) and tau. Our research group has shown that extracellular levels of both Aβ and tau fluctuate during the normal sleep−wake cycle. Disturbed sleep and increased wakefulness acutely lead to increased Aβ production and decreased Aβ clearance, whereas Aβ aggregation and deposition is enhanced by chronic increased wakefulness in animal models. Once Aβ accumulates, there is evidence in both mice and humans that this results in disturbed sleep. New findings from our group reveal that acute sleep deprivation increases levels of tau in mouse brain interstitial fluid (ISF) and human cerebrospinal fluid (CSF) and chronic sleep deprivation accelerates the spread of tau protein aggregates in neural networks. Finally, recent evidence also suggests that accumulation of tau aggregates in the brain correlates with decreased nonrapid eye movement (NREM) sleep slow wave activity. In this review, we first provide a brief overview of the AD and sleep literature and then highlight recent advances in the understanding of the relationship between sleep and AD pathogenesis. Importantly, the effects of the bidirectional relationship between the sleep−wake cycle and tau have not been previously discussed in other reviews on this topic. Lastly, we provide possible directions for future studies on the role of sleep in AD.

Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid β-peptide (Aβ). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Aβ, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Aβ production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Aβ. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo. Apolipoprotein E (apoE) genotype has a major influence on the risk for Alzheimer disease (AD). Different apoE isoforms may alter AD pathogenesis via their interactions with the amyloid β-peptide (Aβ). Mice lacking the lipid transporter ABCA1 were found to have markedly decreased levels and lipidation of apoE in the central nervous system. We hypothesized that if Abca1-/- mice were bred to the PDAPP mouse model of AD, PDAPP Abca1-/ mice would have a phenotype similar to that of PDAPP Apoe+/- and PDAPP Apoe-/- mice, which develop less amyloid deposition than PDAPP Apoe+/+ mice. In contrast to this prediction, 12-month-old PDAPP Abca -/- mice had significantly higher levels of hippocampal Aβ, and cerebral amyloid angiopathy was significantly more common compared with PDAPP Abca1+/+ mice. Amyloid precursor protein (APP) C-terminal fragments were not different between Abca1 genotypes prior to plaque deposition in 3-month-old PDAPP mice, suggesting that deletion of Abca1 did not affect APP processing or Aβ production. As expected, 3-month-old PDAPP Abca1-/- mice had decreased apoE levels, but they also had a higher percentage of carbonate-insoluble apoE, suggesting that poorly lipidated apoE is less soluble in vivo. We also found that 12-month-old PDAPP Abca1-/- mice had a higher percentage of carbonate-insoluble apoE and that apoE deposits co-localize with amyloid plaques, demonstrating that poorly lipidated apoE co-deposits with insoluble Aβ. Together, these data suggest that despite substantially lower apoE levels, poorly lipidated apoE produced in the absence of ABCA1 is strongly amyloidogenic in vivo. Apolipoprotein E (apoE) 2The abbreviations used are: apoEapolipoprotein EAβamyloid β-peptideADAlzheimer diseaseAPPamyloid precursor proteinCAAcerebral amyloid angiopathyCSFcerebrospinal fluidCTFC-terminal fragmentELISAenzyme-linked immunosorbent assayHDLhigh density lipoproteinLDLRlow density lipoprotein receptorLXRliver X receptorBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolMES4-morpholineethanesulfonic acidLRPlow density lipoprotein receptors. genotype is a strong determinant of risk for Alzheimer disease (AD) and cerebral amyloid angiopathy (CAA) (1Greenberg S.M. Rebeck G.W. Vonsattel J.P. Gomez-Isla T. Hyman B.T. Ann. Neurol. 1995; 38: 254-259Crossref PubMed Scopus (468) Google Scholar, 2Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3751) Google Scholar, 3Wisniewski T. Ghiso J. Frangione B. Neurobiol. 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Studies have also shown a profound effect of apoE on Aβ deposition and conformation in vivo. The PDAPP and Tg2576 transgenic mouse models of AD, which overexpress the human amyloid precursor protein (APP) containing AD-causing mutations, develop several aspects of AD-like pathology beginning at 6–9 months of age, including diffuse and fibrillar Aβ deposits, neuritic plaques, gliosis, and CAA (12Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Guido T. Hagopian S. Johnson-Wood K. Masliah E. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2254) Google Scholar, 13Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3724) Google Scholar). 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Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). In vitro studies revealed that the primary cultures of astrocytes, the major producers of apoE in the central nervous system, secrete apoE in small, very poorly lipidated lipoprotein particles if they are derived from Abca1-/- mice (28Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). Particles derived from Abca1-/- mice contained only 0.69 μg of total cholesterol/μg of apoE, whereas particles from Abca1+/+ mice contained 2.3 μg of total cholesterol/μg of apoE. The decreased central nervous system apoE levels seen in the Abca1-/- mice likely result from rapid catabolism of the poorly lipidated apoE-containing HDL particles. The dramatic alterations in central nervous system of apoE produced by Abca1 deletion provide an opportunity to determine how changes in apoE levels and lipidation influence Aβ metabolism in vivo. In the present study, we bred Abca1-/- mice to a well characterized APP transgenic mouse model of AD (PDAPP) that develops age- and region-dependent AD-like pathology (15Bales K.R. Verina T. Dodel R.C. Du Y. Altstiel L. Bender M. Hyslop P. Johnstone E.M. Little S.P. Cummins D.J. Piccardo P. Ghetti B. Paul S.M. Nat. Genet. 1997; 17: 263-264Crossref PubMed Scopus (711) Google Scholar). Because Abca1-/- mice have a greatly decreased apoE, we hypothesized that PDAPP Abca1-/- mice would develop similar levels of pathology as aged PDAPP mice with either no apoE or 50% less apoE (PDAPP Apoe-/- or Apoe+/- mice), both of which have significantly lower levels of nonfibrillar and fibrillar Aβ deposits as well as less CAA. Most interestingly and contrary to our hypothesis, 12-month-old PDAPP Abca1-/- mice had increased parenchymal Aβ levels, amyloid deposition, and CAA. These results suggest that the poorly lipidated apoE formed in the absence of ABCA1 facilitates amyloidogenesis, even when present at low levels. Animals and Tissue Collection—Mice heterozygous for an Abca1 deletion gene on a DBA background were obtained from The Jackson Laboratory, Bar Harbor, ME (strain name, DBA/1-Abca1tm1Jdm). Transgenic mice overexpressing human APP containing the V717F familial Alzheimer disease mutation on a C57Bl/6 background, referred to as PDAPP mice (12Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Guido T. Hagopian S. Johnson-Wood K. Masliah E. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2254) Google Scholar), were obtained from Lilly. The Abca1+/- and PDAPP mice were bred to one another for three generations to produce mice of all Abca1 genotypes that were hemizygous for the PDAPP transgene. All mice were genotyped by PCR. Animals used for experiments were either 3 or 12 months old and were of the same generation. At the appropriate ages, the mice were anesthetized with pentobarbital, and CSF was collected from the cisterna magna as described (30DeMattos R.B. Bales K.R. Parsadanian M. O'Dell M.A. Foss E.M. Paul S.M. Holtzman D.M. J. Neurochem. 2002; 81: 229-236Crossref PubMed Scopus (225) Google Scholar), and the animals were perfused with phosphate-buffered saline/heparin (3 units/ml). The hippocampi and cortices were dissected from the brains and frozen on dry ice. Aβ and ApoE ELISAs—Hippocampi were subjected to a serial extraction method using carbonate and guanidine buffers as described previously (31DeMattos R.B. Cirrito J.R. Parsadanian M. May P.C. O'Dell M.A. Taylor J.W. Harmony J.A. Aronow B.J. Bales K.R. Paul S.M. Holtzman D.M. Neuron. 2004; 41: 193-202Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). Briefly, hippocampi were homogenized in 10 μl/mg carbonate buffer (100 mm sodium carbonate, 50 mm NaCl, protease inhibitors, pH 11.5) and centrifuged at 20,000 × g for 25 min. The carbonate-soluble supernatant was collected, and the pellet was re-homogenized with 700 μl of guanidine buffer (5 m guanidine, 50 mm Tris, protease inhibitors, pH 8.0). The homogenate was centrifuged at 20,000 × g for 25 min, and the guanidine extract was collected. Aβ and apoE quantification was performed by ELISAs that have been described previously (28Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 32Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Lieberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1550-1555Crossref PubMed Scopus (585) Google Scholar). Levels of Aβ and apoE in all hippocampal samples were normalized to total protein, which was determined by BCA assay (Pierce). Histology—Frozen hemibrains from the 12-month-old mice were cut in 50-μm coronal sections from the genu of the corpus collosum to the caudal end of the hippocampus by using a sliding microtome. Sections were incubated with 3D6, an anti-Aβ monoclonal antibody, to detect Aβ deposits, and immunohistochemistry was performed as described previously (33Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Crossref PubMed Scopus (737) Google Scholar). Thioflavine S was used to stain sections for the sub-set of Aβ that was in a β-pleated sheet (amyloid) conformation as described previously (15Bales K.R. Verina T. Dodel R.C. Du Y. Altstiel L. Bender M. Hyslop P. Johnstone E.M. Little S.P. Cummins D.J. Piccardo P. Ghetti B. Paul S.M. Nat. Genet. 1997; 17: 263-264Crossref PubMed Scopus (711) Google Scholar, 33Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Crossref PubMed Scopus (737) Google Scholar). To examine whether apoE and amyloid were co-localized, slides were incubated with an anti-apoE antibody (Calbiochem) and then stained with thioflavine S. The area of the cingulate cortex and hippocampus covered by Aβ immunoreactivity and thioflavine S staining in sections 19, 25, and 31 (from rostral to caudal) were quantified by using stereological techniques (area fraction fractionator) as described previously (33Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Crossref PubMed Scopus (737) Google Scholar). Western Blots—Cortices were sonicated in 10 μl/mg RIPA buffer with protease inhibitors, and the homogenate was spun at 20,000 × g for 25 min. The supernatant was collected, and total protein levels were measured by BCA assay (Pierce). 15 μg of total protein was loaded per lane. Samples were run on 4–12% BisTris gels with MES running buffer (Invitrogen). Following electrophoresis, proteins were transferred to nitrocellulose membranes, which were then blocked in 4% milk in phosphate-buffered saline and probed with an antibody to the C-terminal 22 amino acids of APP (Invitrogen). For a loading control, the blots were stripped and re-probed with an anti-tubulin antibody (Sigma). Densitometric analyses used the Kodak 1D Image Analysis software. Statistical Analysis—All analyses were performed using PRISM version 3.00 (Graphpad, San Diego). Error bars in figures represent the means ± S.E. For all tests of significance between genotypes, an analysis of variance was performed followed by Tukey's post hoc repeated measures testing between all groups. The p values listed are the Tukey's post hoc result. Values not listed are not significant. For testing the significance of CAA frequency in the three genotypes, a 2 degrees of freedom χ2 test was performed. Aβ Levels in CSF and Hippocampus—Levels of Aβ40 and Aβ42 in the CSF and hippocampi of 3- and 12-month-old PDAPP Abca1+/+, Abca1+/-, and Abca1-/- mice were measured using a highly sensitive ELISA. Levels of Aβ in the CSF of the mice did not vary significantly by Abca1 genotype in either 3- or 12-month-old PDAPP mice (data not shown). Previous studies have shown that multiple pools of Aβ exist in the brain that can be differentiated via serial extraction of the tissue in various buffers. We chose to perform carbonate extraction of the brain tissue followed by re-extraction with 5 m guanidine because similar methods have been used in publications relevant to the current study (31DeMattos R.B. Cirrito J.R. Parsadanian M. May P.C. O'Dell M.A. Taylor J.W. Harmony J.A. Aronow B.J. Bales K.R. Paul S.M. Holtzman D.M. Neuron. 2004; 41: 193-202Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 32Johnson-Wood K. Lee M. Motter R. Hu K. Gordon G. Barbour R. Khan K. Gordon M. Tan H. Games D. Lieberburg I. Schenk D. Seubert P. McConlogue L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1550-1555Crossref PubMed Scopus (585) Google Scholar, 33Holtzman D.M. Bales K.R. Tenkova T. Fagan A.M. Parsadanian M. Sartorius L.J. Mackey B. Olney J. McKeel D. Wozniak D. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2892-2897Crossref PubMed Scopus (737) Google Scholar, 34Fagan A.M. Watson M. Parsadanian M. Bales K.R. Paul S.M. Holtzman D.M. Neurobiol. Dis. 2002; 9: 305-318Crossref PubMed Scopus (222) Google Scholar). The carbonate-soluble Aβ probably represents Aβ that is normally soluble in vivo or is loosely associated with membranes. Aβ that requires 5 m guanidine for extraction is likely more strongly bound to membranes or, in the case of the 12-month-old PDAPP mice, deposited into relatively insoluble amyloid plaques. At 3 months of age, PDAPP Abca1-/- mice had significantly higher levels of carbonate-soluble Aβ40 than PDAPP Abca1+/+ mice (Fig. 1A), but levels of carbonate-soluble Aβ42 and carbonate-insoluble Aβ40 and Aβ42 did not vary by Abca1 genotype (Fig. 1, A and B). To investigate whether this increase of carbonate-soluble Aβ40 in PDAPP Abca1-/- mice was a result of increased Aβ generation, we examined levels of APP and C-terminal fragments of APP (APP-CTFs) that are produced during the process of Aβ generation. We found that levels of APP and APP-CTFs, including CTF-γ, were not significantly different in 3-month-old PDAPP Abca1+/+ and PDAPP Abca1-/- mice (Fig. 1E and data not shown). This suggests that the Aβ generation is not affected by the Abca1 genotype. Instead, the increase of carbonate-soluble Aβ40 in 3-month-old PDAPP Abca1-/- mice may be because of decreased clearance of Aβ40 by poorly lipidated apoE. In fact, recent in vivo data suggest that apoE plays a role in Aβ40 transport and clearance (31DeMattos R.B. Cirrito J.R. Parsadanian M. May P.C. O'Dell M.A. Taylor J.W. Harmony J.A. Aronow B.J. Bales K.R. Paul S.M. Holtzman D.M. Neuron. 2004; 41: 193-202Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar, 35Fryer J.D. Simmons K. Parsadanian M. Bales K.R. Paul S.M. Sullivan P.M. Holtzman D.M. J. Neurosci. 2005; 25: 2803-2810Crossref PubMed Scopus (233) Google Scholar). At 12 months of age, Aβ deposition had begun to occur in PDAPP mice of all genotypes. The amount of carbonate-soluble Aβ increased ∼10-fold in 12-month-old mice as compared with 3-month-old mice but did not vary by Abca1 genotype (Fig. 1C). Moreover, there was a >100-fold increase in carbonate-insoluble Aβ42 levels that was because of the deposition of large amounts of Aβ42 in amyloid plaques, which require extraction in 5 m guanidine (Fig. 1D). Most interestingly, at this time point PDAPP Abca1-/- mice had >3-fold higher levels of Aβ40 and Aβ42 than PDAPP Abca1+/+ mice (Fig. 1D, note changes in units and scales). Additionally, the average percentage of total Aβ that was carbonate-insoluble was significantly higher in PDAPP Abca1-/- mice (92% in Abca1+/+ mice and 98% in Abca1-/- mice, p < 0.001). This suggests that more of the Aβ deposits in Abca1-/- mice were contained within insoluble plaques, potentially as a result of increased Aβ fibrillogenesis caused by poorly lipidated apoE. Histological Analysis of Brains—Brain sections from the 12-month-old mice were immunostained for total Aβ and stained with thioflavine S for detection of fibrillar Aβ in amyloid plaques. PDAPP Abca1-/- mice had a higher average percentage of their cortex and hippocampus covered by Aβ immunoreactive and thioflavine S-positive deposits (Fig. 2A). However, stereological quantification of the Aβ and thioflavine S-positive plaque load did not show a significant difference between genotypes (Fig. 3). Brains were also examined for the presence of CAA with thioflavine S staining (Fig. 2B). Of 12 PDAPP Abca1+/+ mice, none had observable CAA, whereas 1 of 9 PDAPP Abca1-/- and 4 of 11 PDAPP Abca1-/- mice had observable CAA (p = 0.05).FIGURE 3Stereological analysis of Aβ immunoreactivity and thioflavine S-positive amyloid in 12-month-old PDAPP Abca1 mice. A–D, the graphs represent the area of cortex and hippocampus covered by Aβ immunoreactivity and thioflavine S-positive amyloid, referred to as Aβ and thioflavine S load. In the 12-month-old group, n = 12 for PDAPP Abca1+/+, n = 9 for PDAPP Abca1+/-, and n = 11 for PDAPP Abca1-/- mice. N.S., not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) ApoE Levels in Brain—Levels of apoE in the hippocampi of the mice were measured by ELISA. As expected, the 3-month-old PDAPP Abca1-/- mice had markedly decreased apoE levels, which were ∼25% the level found in PDAPP Abca1+/+ mice (Fig. 4A). Most interestingly, even prior to plaque formation and despite the fact that Abca1-/- mice have much lower levels of total tissue apoE, Abca1-/- mice had a higher percentage of apoE in the carbonate-insoluble fraction from the hippocampus (Fig. 4B). This suggests that poorly lipidated apoE is less soluble in vivo. After plaque deposition began, tissue-associated apoE increased in PDAPP mice of all Abca1 genotypes and became less soluble. In 12-month-old PDAPP mice, ∼40% of the apoE was not soluble in carbonate buffer and required 5 m guanidine for extraction (Fig. 4B), which is characteristic of proteins in amyloid plaques. More importantly, 12-month-old PDAPP mice of all Abca1 genotypes had approximately equal levels of total apoE, which indicates that PDAPP Abca1-/- mice accumulate large amounts of apoE between 3 and 12 months of age. Additionally, the apoE accumulated by the 12-month-old PDAPP Abca1-/- mice contained a higher percentage of carbonate-insoluble apoE than found in the PDAPP Abca1+/+ and PDAPP Abca1+/- mice. In vivo, apoE normally co-deposits with Aβ into plaques. To confirm that apoE from both PDAPP Abca1+/+ and PDAPP Abca1-/- mice was present in plaques, sections of brain were double-stained with anti-apoE and thioflavine S. The apoE deposits co-localized with the thioflavine S staining in both the PDAPP Abca1+/+ and PDAPP Abca1-/- mice, showing that apoE was associated with amyloid plaques (Fig. 4C). These findings show that despite the fact that PDAPP Abca1-/- mice initially have much lower levels of apoE, the apoE that is present efficiently binds to and becomes associated with the deposited Aβ. Together, our findings suggest that the poorly lipidated apoE in PDAPP Abca1-/- mice promotes Aβ fibrillogenesis to a greater extent than normally lipidated apoE-containing HDL in the brain. Despite initially having much lower levels of apoE in the brain, PDAPP Abca1-/- mice developed increased Aβ levels and CAA in the brain. Further supporting our findings, two other groups have observed a similar phenotype using completely independent lines of APP transgenic mice with different APP mutations and different promoters (48Hirsch-Reinshagen V. Maia L.F. Burgess B.L. Blain J.-F. Naus K.E. McIsaac S.A. Parkinson P.F. Chan J.Y. Tansley G.H. Hayden M.R. Poirier J. Van Nostrand W. Wellington C.L. J. Biol. Chem. 2005; 280: 43243-43256Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 49Koldamova R. Staufenbiel M. Lefterov I. J. Biol. Chem. 2005; 280: 43236-43242Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Although the increases in Aβ levels and CAA were significant but not dramatic in PDAPP Abca1-/- versus PDAPP Abca1+/+ mice at 12 months of age, these results were counter to what we expected because previous studies showed an ∼50% decrease in apoE levels in Apoe+/- mice resulted in a >50% decrease in Aβ levels and amyloid deposition in both PDAPP Apoe+/- and Tg2576 Apoe+/- mice (14Bales K.R. Verina T. Cummins D.J. Du Y. Dodel R.C. Saura J. Fishman C.E. DeLong C.A. Piccardo P. Petegnief V. Ghetti B. Paul S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15233-15238Crossref PubMed Scopus (416) Google Scholar, 17Holtzman D.M. Fagan A.M. Mackey B. Tenkova T. Sartorius L. Paul S.M. Bales K. Ashe K.H. Irizarry M.C. Hyman B.T. Ann. Neurol. 2000; 47: 739-747Crossref PubMed Scopus (269) Google Scholar). The demonstration that PDAPP Abca1-/- mice, which have ∼25% of normal apoE levels at 3 months of age, develop increased Aβ deposition by 12 months of age suggests that poorly lipidated apoE formed in the absence of ABCA1 strongly promotes Aβ fibrillogenesis in an age-dependent manner relative to normally lipidated murine apoE. We considered three possible mechanisms by which Abca1 deletion could affect Aβ levels. First, we hypothesized that because ABCA1 exports cholesterol and phospholipids from cells and alterations of cellular lipids have been shown to modulate APP processing (36Wolozin B. Neuron. 2004; 41: 7-10Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar), it was possible that Abca1 deletion could have modified brain Aβ levels via the effects on production of Aβ from APP. However, we found that Abca1 deletion had no effect on levels of APP or APP-CTFs prior to Aβ deposition, which suggests that ABCA1 is not influencing Aβ production. Additionally, in our previous work we found no differences in brain total cholesterol or brain neutral lipid distribution between Abca1+/+ and-/- mice (28Wahrle S.E. Jiang H. Parsadanian M. Legleiter J. Han X. Fryer J.D. Kowalewski T. Holtzman D.M. J. Biol. Chem. 2004; 279: 40987-40993Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). It is conceivable that lipid levels could be altered in certain subpopulations of cells (astrocytes or microglia) within the brain that could influence Aβ metabolism. This needs to be assessed in future experiments. A second possible mechanism by which Abca1 deletion could increase Aβ deposition is that the lipid-poor apoE in Abca1 Abca1-/- mice may impair receptor-mediated clearance of Aβ. This hypothesis is based on data showing that lipid-poor apoE is a poor ligand for the low density lipoprotein receptor (LDLR) and LDLR-related protein, the major apoE receptors in brain (37Ruiz J. Kouiavskaia D. Migliorini M. Robinson S. Saenko E.L. Gorlatova N. Li D. Lawrence D. Hyman B.T. Weisgraber K.H. Strickland D.K. J. Lipid Res. 2005; 46: 1721-1731Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 38Narita M. Holtzman D.M. Fagan A.M. LaDu M.J. Yu L. Han X. Gross R.W. Bu G. Schwartz A.L. J. Biochem. (Tokyo). 2002; 132: 743-749Crossref PubMed Scopus (40) Google Scholar, 39Fryer J.D. Demattos R.B. McCormick L.M. O'Dell M.A. Spinner M.L. Bales K.R. Paul S.M. Sullivan P.M. Parsadanian M. Bu G. Holtzman D.M. J. Biol. Chem. 2005; 280: 25754-25759Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Previous data from our laboratory have shown increased soluble Aβ in the brains of 3-month-old PDAPP mice lacking apoE, which is possibly due to a lack of receptor-mediated clearance of apoE-Aβ complexes (31DeMattos R.B. Cirrito J.R. Parsadanian M. May P.C. O'Dell M.A. Taylor J.W. Harmony J.A. Aronow B.J. Bales K.R. Paul S.M. Holtzman D.M. Neuron. 2004; 41: 193-202Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar). In the current study, we found increased soluble Aβ40 in the brains of PDAPP Abca1-/- mice, also possibly a result of impaired receptor-mediated clearance of apoE-Aβ complexes. However, PDAPP mice lacking apoE, and therefore lacking receptor-mediated clearance of apoE-Aβ complexes, have decreased Aβ deposition, whereas PDAPP Abca1-/- mice have increased Aβ deposition. This strongly suggests that receptor-mediated clearance of Aβ is not the main reason for the increase in Aβ we observed in 12-month-old PDAPP Abca1-/- mice. Finally, we think the most likely mechanism by which Abca1 deletion increases Aβ deposition is by affecting the lipidation state of apoE. Experiments have shown that apoE lipidation affects interactions with Aβ in vitro that is likely to influence the probability that Aβ will aggregate. De-lipidated apoE3 and apoE4 form similar amounts of SDS-stable complexes with Aβ (2Strittmatter W.J. Saunders A.M. Schmechel D. Pericak-Vance M. Enghild J. Salvesen G.S. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1977-1981Crossref PubMed Scopus (3751) Google Scholar, 40Strittmatter W.J. Weisgraber K.H. Huang D.Y. Dong L.M. Salvesen G.S. Pericak-Vance M. Schmechel D. Saunders A.M. Goldgaber D. Roses A.D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8098-8102Crossref PubMed Scopus (1249) Google Scholar). In contrast, cell-secreted, lipidated apoE2 and apoE3 interact with Aβ and form a much greater amount of SDS-stable complex than apoE4 (41LaDu M.J. Pederson T.M. Frail D.E. Reardon C.A. Getz G.S. 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LXR dimerizes with retinoid X receptor to transcriptionally induce a group of lipid-related genes including Abca1. It was recently shown that the LXR agonist T0901317 decreased brain Aβ40 and Aβ42 levels when given over several days to 3-month-old APP transgenic mice (APP23) prior to plaque deposition (46Koldamova R.P. Lefterov I.M. Staufenbiel M. Wolfe D. Huang S. Glorioso J.C. Walter M. Roth M.G. Lazo J.S. J. Biol. Chem. 2005; 280: 4079-4088Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). One possible mechanism for this effect is that the LXR agonist modulated cellular cholesterol levels and directly affected APP processing into Aβ (36Wolozin B. Neuron. 2004; 41: 7-10Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Although the authors found a difference in secreted APP fragments, they did not see any differences in APP-CTFs that would support this mechanism. An alternative hypothesis to explain these data is that the induction of ABCA1 increases lipidation of apoE, which could affect Aβ levels and ultimately Aβ deposition. Because a decrease in ABCA1 results in more amyloid deposition, increasing ABCA1 protein or function might be predicted to decrease amyloid deposition via increasing apoE lipidation. This hypothesis needs to be tested directly. If ABCA1 influences amyloid deposition by altering the level and lipidation state of apoE, it will be important to assess the effects of ABCA1 on both murine and human apoE. This is because murine apoE appears to increase amyloid deposition, whereas human apoE appears to delay and decrease amyloid deposition (34Fagan A.M. Watson M. Parsadanian M. Bales K.R. Paul S.M. Holtzman D.M. Neurobiol. Dis. 2002; 9: 305-318Crossref PubMed Scopus (222) Google Scholar, 35Fryer J.D. Simmons K. Parsadanian M. Bales K.R. Paul S.M. Sullivan P.M. Holtzman D.M. J. Neurosci. 2005; 25: 2803-2810Crossref PubMed Scopus (233) Google Scholar, 47Holtzman D.M. J. Mol. Neurosci. 2004; 23: 247-254Crossref PubMed Scopus (121) Google Scholar). In sum, the absence of ABCA1 resulted in an increase in amyloid deposition and CAA in PDAPP mice. This effect appears likely because of promotion of Aβ fibrillogenesis by the poorly lipidated apoE particles produced in the brains of Abca1-/- mice. These results emphasize the potential importance of ABCA1 not only in regulating apoE levels and lipidation but also the consequences of its absence on Aβ deposition and conformation. As such, ABCA1 can be hypothesized to be a potential therapeutic target in AD, and this hypothesis can be tested in future studies. We thank Dr. John Cirrito and Mary Beth Finn for helpful comments. We also thank Cheryl Wellington and Iliya Lefterov for sharing their results prior to submission of this manuscript.