Matthew P. Frosch

The neuropathological hallmarks of Alzheimer disease (AD) include "positive" lesions such as amyloid plaques and cerebral amyloid angiopathy, neurofibrillary tangles, and glial responses, and "negative" lesions such as neuronal and synaptic loss.Despite their inherently cross-sectional nature, postmortem studies have enabled the staging of the progression of both amyloid and tangle pathologies, and, consequently, the development of diagnostic criteria that are now used worldwide.In addition, clinicopathological correlation studies have been crucial to generate hypotheses about the pathophysiology of the disease, by establishing that there is a continuum between "normal" aging and AD dementia, and that the amyloid plaque build-up occurs primarily before the onset of cognitive deficits, while neurofibrillary tangles, neuron loss, and particularly synaptic loss, parallel the progression of cognitive decline.Importantly, these cross-sectional neuropathological data have been largely validated by longitudinal in vivo studies using modern imaging biomarkers such as amyloid PET and volumetric MRI.

We present a practical guide for the implementation of recently revised National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease (AD). Major revisions from previous consensus criteria are: (1) recognition that AD neuropathologic changes may occur in the apparent absence of cognitive impairment, (2) an “ABC” score for AD neuropathologic change that incorporates histopathologic assessments of amyloid β deposits (A), staging of neurofibrillary tangles (B), and scoring of neuritic plaques (C), and (3) more detailed approaches for assessing commonly co-morbid conditions such as Lewy body disease, vascular brain injury, hippocampal sclerosis, and TAR DNA binding protein (TDP)-43 immunoreactive inclusions. Recommendations also are made for the minimum sampling of brain, preferred staining methods with acceptable alternatives, reporting of results, and clinico-pathologic correlations.

A consensus panel from the United States and Europe was convened recently to update and revise the 1997 consensus guidelines for the neuropathologic evaluation of Alzheimer's disease (AD) and other diseases of brain that are common in the elderly. The new guidelines recognize the pre-clinical stage of AD, enhance the assessment of AD to include amyloid accumulation as well as neurofibrillary change and neuritic plaques, establish protocols for the neuropathologic assessment of Lewy body disease, vascular brain injury, hippocampal sclerosis, and TDP-43 inclusions, and recommend standard approaches for the workup of cases and their clinico-pathologic correlation.

The Alzheimer Disease Genetics Consortium (ADGC) performed a genome-wide association study of late-onset Alzheimer disease using a three-stage design consisting of a discovery stage (stage 1) and two replication stages (stages 2 and 3). Both joint analysis and meta-analysis approaches were used. We obtained genome-wide significant results at MS4A4A (rs4938933; stages 1 and 2, meta-analysis P (P(M)) = 1.7 × 10(-9), joint analysis P (P(J)) = 1.7 × 10(-9); stages 1, 2 and 3, P(M) = 8.2 × 10(-12)), CD2AP (rs9349407; stages 1, 2 and 3, P(M) = 8.6 × 10(-9)), EPHA1 (rs11767557; stages 1, 2 and 3, P(M) = 6.0 × 10(-10)) and CD33 (rs3865444; stages 1, 2 and 3, P(M) = 1.6 × 10(-9)). We also replicated previous associations at CR1 (rs6701713; P(M) = 4.6 × 10(-10), P(J) = 5.2 × 10(-11)), CLU (rs1532278; P(M) = 8.3 × 10(-8), P(J) = 1.9 × 10(-8)), BIN1 (rs7561528; P(M) = 4.0 × 10(-14), P(J) = 5.2 × 10(-14)) and PICALM (rs561655; P(M) = 7.0 × 10(-11), P(J) = 1.0 × 10(-10)), but not at EXOC3L2, to late-onset Alzheimer's disease susceptibility.

Clinicopathologic correlation studies are critically important for the field of Alzheimer disease (AD) research. Studies on human subjects with autopsy confirmation entail numerous potential biases that affect both their general applicability and the validity of the correlations. Many sources of data variability can weaken the apparent correlation between cognitive status and AD neuropathologic changes. Indeed, most persons in advanced old age have significant non-AD brain lesions that may alter cognition independently of AD. Worldwide research efforts have evaluated thousands of human subjects to assess the causes of cognitive impairment in the elderly, and these studies have been interpreted in different ways. We review the literature focusing on the correlation of AD neuropathologic changes (i.e. β-amyloid plaques and neurofibrillary tangles) with cognitive impairment. We discuss the various patterns of brain changes that have been observed in elderly individuals to provide a perspective for understanding AD clinicopathologic correlation and conclude that evidence from many independent research centers strongly supports the existence of a specific disease, as defined by the presence of Aβ plaques and neurofibrillary tangles. Although Aβ plaques may play a key role in AD pathogenesis, the severity of cognitive impairment correlates best with the burden of neocortical neurofibrillary tangles.

<h2>Summary</h2> Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the <i>CDK4</i>, <i>EGFR</i>, and <i>PDGFRA</i> loci and by mutations in the <i>NF1</i> locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE --). IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE -- mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive accumulation in selected neurons of protein inclusions containing alpha-synuclein and ubiquitin. Rare inherited forms of PD are caused by autosomal dominant mutations in alpha-synuclein or by autosomal recessive mutations in parkin, an E3 ubiquitin ligase. We hypothesized that these two gene products interact functionally, namely, that parkin ubiquitinates alpha-synuclein normally and that this process is altered in autosomal recessive PD. We have now identified a protein complex in normal human brain that includes parkin as the E3 ubiquitin ligase, UbcH7 as its associated E2 ubiquitin conjugating enzyme, and a new 22-kilodalton glycosylated form of alpha-synuclein (alphaSp22) as its substrate. In contrast to normal parkin, mutant parkin associated with autosomal recessive PD failed to bind alphaSp22. In an in vitro ubiquitination assay, alphaSp22 was modified by normal but not mutant parkin into polyubiquitinated, high molecular weight species. Accordingly, alphaSp22 accumulated in a non-ubiquitinated form in parkin-deficient PD brains. We conclude that alphaSp22 is a substrate for parkin's ubiquitin ligase activity in normal human brain and that loss of parkin function causes pathological alphaSp22 accumulation. These findings demonstrate a critical biochemical reaction between the two PD-linked gene products and suggest that this reaction underlies the accumulation of ubiquitinated alpha-synuclein in conventional PD.

Automated analysis of MRI data of the subregions of the hippocampus requires computational atlases built at a higher resolution than those that are typically used in current neuroimaging studies. Here we describe the construction of a statistical atlas of the hippocampal formation at the subregion level using ultra-high resolution, ex vivo MRI. Fifteen autopsy samples were scanned at 0.13 mm isotropic resolution (on average) using customized hardware. The images were manually segmented into 13 different hippocampal substructures using a protocol specifically designed for this study; precise delineations were made possible by the extraordinary resolution of the scans. In addition to the subregions, manual annotations for neighboring structures (e.g., amygdala, cortex) were obtained from a separate dataset of in vivo, T1-weighted MRI scans of the whole brain (1mm resolution). The manual labels from the in vivo and ex vivo data were combined into a single computational atlas of the hippocampal formation with a novel atlas building algorithm based on Bayesian inference. The resulting atlas can be used to automatically segment the hippocampal subregions in structural MRI images, using an algorithm that can analyze multimodal data and adapt to variations in MRI contrast due to differences in acquisition hardware or pulse sequences. The applicability of the atlas, which we are releasing as part of FreeSurfer (version 6.0), is demonstrated with experiments on three different publicly available datasets with different types of MRI contrast. The results show that the atlas and companion segmentation method: 1) can segment T1 and T2 images, as well as their combination, 2) replicate findings on mild cognitive impairment based on high-resolution T2 data, and 3) can discriminate between Alzheimer's disease subjects and elderly controls with 88% accuracy in standard resolution (1mm) T1 data, significantly outperforming the atlas in FreeSurfer version 5.3 (86% accuracy) and classification based on whole hippocampal volume (82% accuracy).

Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.

Tumor subclasses differ according to the genotypes and phenotypes of malignant cells as well as the composition of the tumor microenvironment (TME). We dissected these influences in isocitrate dehydrogenase (IDH)–mutant gliomas by combining 14,226 single-cell RNA sequencing (RNA-seq) profiles from 16 patient samples with bulk RNA-seq profiles from 165 patient samples. Differences in bulk profiles between IDH-mutant astrocytoma and oligodendroglioma can be primarily explained by distinct TME and signature genetic events, whereas both tumor types share similar developmental hierarchies and lineages of glial differentiation. As tumor grade increases, we find enhanced proliferation of malignant cells, larger pools of undifferentiated glioma cells, and an increase in macrophage over microglia expression programs in TME. Our work provides a unifying model for IDH-mutant gliomas and a general framework for dissecting the differences among human tumor subclasses.

Converging evidence suggests that the accumulation of cerebral amyloid beta-protein (Abeta) in Alzheimer's disease (AD) reflects an imbalance between the production and degradation of this self-aggregating peptide. Upregulation of proteases that degrade Abeta thus represents a novel therapeutic approach to lowering steady-state Abeta levels, but the consequences of sustained upregulation in vivo have not been studied. Here we show that transgenic overexpression of insulin-degrading enzyme (IDE) or neprilysin (NEP) in neurons significantly reduces brain Abeta levels, retards or completely prevents amyloid plaque formation and its associated cytopathology, and rescues the premature lethality present in amyloid precursor protein (APP) transgenic mice. Our findings demonstrate that chronic upregulation of Abeta-degrading proteases represents an efficacious therapeutic approach to combating Alzheimer-type pathology in vivo.

Transgenic mice carrying disease-linked forms of genes associated with Alzheimer disease often demonstrate deposition of the beta-amyloid as senile plaques and cerebral amyloid angiopathy. We have characterized the natural history of beta-amyloid deposition in APPswe/PS1dE9 mice, a particularly aggressive transgenic mouse model generated with mutant transgenes for APP (APPswe: KM594/5NL) and PS1 (dE9: deletion of exon 9). Ex vivo histochemistry showed Abeta deposition by 4 months with a progressive increase in plaque number up to 12 months and a similar increase of Abeta levels. In vivo multiphoton microscopy at weekly intervals showed increasing beta-amyloid deposition as CAA and plaques. Although first appearing at an early age, CAA progressed at a significantly slower rate than in the Tg2576 mice. The consistent and early onset of beta-amyloid accumulation in the APPswe/PS1dE9 model confirms its utility for studies of biochemical and pathological mechanisms underlying beta-amyloid deposition, as well as exploring new therapeutic treatments.

Could similar changes in SOD1 underlie both familial and sporadic ALS? Here, Bosco et al. find that wild-type SOD1 from sporadic ALS tissues shows conformational changes similar to those seen in familial ALS and that aberrant wild-type SOD1 can be pathogenic, potentially as a result of the same SOD1-dependent mechanism seen in familial ALS. Many mutations confer one or more toxic function(s) on copper/zinc superoxide dismutase 1 (SOD1) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we found that oxidized wild-type SOD1 and mutant SOD1 share a conformational epitope that is not present in normal wild-type SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord were markedly C4F6 immunoreactive, indicating that an aberrant wild-type SOD1 species was present. Recombinant, oxidized wild-type SOD1 and wild-type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to that of FALS-linked mutant SOD1. Our findings suggest that wild-type SOD1 can be pathogenic in SALS and identify an SOD1-dependent pathogenic mechanism common to FALS and SALS.

<b><i>Background:</i></b> Pathologic changes in the Alzheimer disease (AD) brain occur in a hierarchical neuroanatomical pattern affecting cortical, subcortical, and limbic regions. <b><i>Objective:</i></b> To define the time course of pathologic and biochemical changes—amyloid deposition, amyloid β-peptide (Aβ) accumulation, neurofibrillary tangle (NFT) formation, synaptic loss, and gliosis—within the temporal association cortex of AD cases of varying disease duration, relative to control brains. <b><i>Methods:</i></b> Stereologic assessments of amyloid burden and tangle density as well as ELISA-based measurements of Aβ, synaptophysin, and glial fibrillary acidic protein (GFAP) were performed in the superior temporal sulcus from a cohort of 83 AD and 26 nondemented control brains. <b><i>Results:</i></b> Relative to control cases, AD brains were characterized by accumulation of NFT and amyloid plaques, increase of tris- and formic acid–extractable Aβ species, reduced levels of synaptophysin, and elevated levels of GFAP. In AD cases, the duration of dementia correlated with the degree of tangle formation, gliosis, and synaptic loss but not with any Aβ measures. Accumulation of Aβ, measured both neuropathologically and biochemically, was markedly increased in AD brains independent of disease duration, even in cases of short duration. <b><i>Conclusions:</i></b> These data support distinct processes in the initiation and progression of AD pathology within the temporal cortex: Deposition of Aβ reaches a “ceiling” early in the disease process, whereas NFT formation, synaptic loss, and gliosis continue throughout the course of the illness.

Amyloid imaging related abnormalities (ARIA) have now been reported in clinical trials with multiple therapeutic avenues to lower amyloid-β burden in Alzheimer's disease (AD). In response to concerns raised by the Food and Drug Administration, the Alzheimer's Association Research Roundtable convened a working group to review the publicly available trial data, attempts at developing animal models, and the literature on the natural history and pathology of related conditions. The spectrum of ARIA includes signal hyperintensities on fluid attenuation inversion recoverysequences thought to represent "vasogenic edema" and/or sulcal effusion (ARIA-E), as well as signal hypointensities on GRE/T2* thought to represent hemosiderin deposits (ARIA-H), including microhemorrhage and superficial siderosis. The etiology of ARIA remains unclear but the prevailing data support vascular amyloid as a common pathophysiological mechanism leading to increased vascular permeability. The workgroup proposes recommendations for the detection and monitoring of ARIA in ongoing AD clinical trials, as well as directions for future research.

Objective To examine region‐ and substrate‐specific autoradiographic and in vitro binding patterns of positron emission tomography tracer [F‐18]‐AV‐1451 (previously known as T807), tailored to allow in vivo detection of paired helical filament‐tau–containing lesions, and to determine whether there is off‐target binding to other amyloid/non‐amyloid proteins. Methods We applied [F‐18]‐AV‐1451 phosphor screen autoradiography, [F‐18]‐AV‐1451 nuclear emulsion autoradiography, and [H‐3]‐AV‐1451 in vitro binding assays to the study of postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration–tau, frontotemporal lobar degeneration–transactive response DNA binding protein 43 (TDP‐43), progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, cerebral amyloid angiopathy and elderly controls free of pathology. Results Our data suggest that [F‐18]‐AV‐1451 strongly binds to tau lesions primarily made of paired helical filaments in Alzheimer brains (eg, intraneuronal and extraneuronal tangles and dystrophic neurites), but does not seem to bind to a significant extent to neuronal and glial inclusions mainly composed of straight tau filaments in non‐Alzheimer tauopathy brains or to lesions containing β‐amyloid, α‐synuclein, or TDP‐43. [F‐18]‐AV‐1451 off‐target binding to neuromelanin‐ and melanin‐containing cells and, to a lesser extent, to brain hemorrhagic lesions was identified. Interpretation Our data suggest that [F‐18]‐AV‐1451 holds promise as a surrogate marker for the detection of brain tau pathology in the form of tangles and paired helical filament‐tau–containing neurites in Alzheimer brains but also point to its relatively lower affinity for lesions primarily made of straight tau filaments in non‐Alzheimer tauopathy cases and to the existence of some [F‐18]‐AV‐1451 off‐target binding. These findings provide important insights for interpreting in vivo patterns of [F‐18]‐AV‐1451 retention. Ann Neurol 2015 Ann Neurol 2015;78:Ann Neurol 2015;78:679–696

<h2>Abstract</h2> Accumulation of misfolded proteins as insoluble aggregates occurs in several neurodegenerative diseases. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein (αS) accumulates in insoluble inclusions. To identify soluble αS oligomers that precede insoluble aggregates, we probed the cytosols of mesencephalic neuronal (MES) cells, normal and αS-transgenic mouse brains, and normal, PD, and DLB human brains. All contained highly soluble oligomers of αS whose detection was enhanced by delipidation. Exposure of living MES neurons to polyunsaturated fatty acids (PUFAs) increased αS oligomer levels, whereas saturated FAs decreased them. PUFAs directly promoted oligomerization of recombinant αS. Transgenic mice accumulated soluble oligomers with age. PD and DLB brains had elevated amounts of the soluble, lipid-dependent oligomers. We conclude that αS interacts with PUFAs in vivo to promote the formation of highly soluble oligomers that precede the insoluble αS aggregates associated with neurodegeneration.

Frontotemporal lobar degeneration (FTLD) is the second most common cause of presenile dementia. The predominant neuropathology is FTLD with TAR DNA-binding protein (TDP-43) inclusions (FTLD-TDP). FTLD-TDP is frequently familial, resulting from mutations in GRN (which encodes progranulin). We assembled an international collaboration to identify susceptibility loci for FTLD-TDP through a genome-wide association study of 515 individuals with FTLD-TDP. We found that FTLD-TDP associates with multiple SNPs mapping to a single linkage disequilibrium block on 7p21 that contains TMEM106B. Three SNPs retained genome-wide significance following Bonferroni correction (top SNP rs1990622, P = 1.08 x 10(-11); odds ratio, minor allele (C) 0.61, 95% CI 0.53-0.71). The association replicated in 89 FTLD-TDP cases (rs1990622; P = 2 x 10(-4)). TMEM106B variants may confer risk of FTLD-TDP by increasing TMEM106B expression. TMEM106B variants also contribute to genetic risk for FTLD-TDP in individuals with mutations in GRN. Our data implicate variants in TMEM106B as a strong risk factor for FTLD-TDP, suggesting an underlying pathogenic mechanism.

The cellular composition of H3K27M gliomas Diffuse midline gliomas with histone H3 lysine27-to-methionine mutations (H3K27M-glioma) are an aggressive type of childhood cancer with few options for treatment. Filbin et al. used a single-cell sequencing approach to study the oncogenic programs, genetics, and cellular hierarchies of H3K27M-glioma. Tumors were mainly composed of cells resembling oligodendrocyte precursor cells, whereas differentiated malignant cells were a smaller fraction. In comparison with other gliomas, these cancers had distinct oncogenic programs and stem cell–like profiles that contributed to their stable tumor-propagating potential. The analysis also identified a lineage-specific marker that may be useful in developing therapies. Science , this issue p. 331

This study creates a compendium of gene expression in normal human tissues suitable as a reference for defining basic organ systems biology. Using oligonucleotide microarrays, we analyze 59 samples representing 19 distinct tissue types. Of approximately 7,000 genes analyzed, 451 genes are expressed in all tissue types and designated as housekeeping genes. These genes display significant variation in expression levels among tissues and are sufficient for discerning tissue-specific expression signatures, indicative of fundamental differences in biochemical processes. In addition, subsets of tissue-selective genes are identified that define key biological processes characterizing each organ. This compendium highlights similarities and differences among organ systems and different individuals and also provides a publicly available resource (Human Gene Expression Index, the HuGE Index, http://www.hugeindex.org) for future studies of pathophysiology.

Dissecting hyperactivation in AD Progressive accumulation of amyloid β (Aβ) in the brain is a defining feature of Alzheimer's disease (AD). At late stages of AD, pathological Aβ accumulations cause neurodegeneration and cell death. However, neuronal dysfunction, consisting of an excessively increased activity in a fraction of brain neurons, already occurs in early stages of the disease. Zott et al. explored the cellular basis of this hyperactivity in mouse models of AD (see the Perspective by Selkoe). Aβ-mediated hyperactivation was linked to a defect in synaptic transmission exclusively in active neurons, with the most-active neurons having the highest risk of hyperactivation. Aβ-containing brain extracts from human AD patients sustained this vicious cycle, underscoring the potential relevance of this pathological mechanism in humans. Science , this issue p. 559 ; see also p. 540

Platelet-derived growth factors (PDGFs) are growth-regulatory molecules that stimulate chemotaxis, proliferation, and increased metabolism of primarily connective tissue cells. In a survey of normal tissues, we found specific immunostaining for PDGF B-chain in neurons, principal dendrites, some axons, and probable terminals throughout the brain, in the dorsal horn of the spinal cord, and in the posterior pituitary of a nonhuman primate (Macaca nemestrina). PDGF activity was extracted from brain cortex and posterior pituitary, and ubiquitous expression of transcripts for the two chains of PDGF and both PDGF receptors was detected throughout the brain and posterior pituitary. A transgenic model was also evaluated in which the chloramphenicol acetyltransferase gene was placed under transcriptional control of the PDGF B-chain promoter. The transgene was preferentially expressed within neural cell bodies in the cortex, hippocampus, and cerebellum. PDGF may act as a neuronal regulatory agent. Neuronal release of PDGF could contribute to nerve regeneration and to glial proliferation that leads to gliosis and scarring.

Combined measurement of diverse molecular and anatomical traits that span multiple levels remains a major challenge in biology. Here, we introduce a simple method that enables proteomic imaging for scalable, integrated, high-dimensional phenotyping of both animal tissues and human clinical samples. This method, termed SWITCH, uniformly secures tissue architecture, native biomolecules, and antigenicity across an entire system by synchronizing the tissue preservation reaction. The heat- and chemical-resistant nature of the resulting framework permits multiple rounds (>20) of relabeling. We have performed 22 rounds of labeling of a single tissue with precise co-registration of multiple datasets. Furthermore, SWITCH synchronizes labeling reactions to improve probe penetration depth and uniformity of staining. With SWITCH, we performed combinatorial protein expression profiling of the human cortex and also interrogated the geometric structure of the fiber pathways in mouse brains. Such integrated high-dimensional information may accelerate our understanding of biological systems at multiple levels.

Senile plaques are a prominent pathological feature of Alzheimer's disease (AD), but little is understood about the association of glial cells with plaques or about the dynamics of glial responses through the disease course. We investigated the progression of reactive glial cells and their relationship with AD pathological hallmarks to test whether glial cells are linked only to amyloid deposits or also to tangle deposition, thus integrating both lesions as a marker of disease severity. We conducted a quantitative stereology-based post-mortem study on the temporal neocortex of 15 control subjects without dementia and 91 patients with AD, including measures of amyloid load, neurofibrillary tangles, reactive astrocytes, and activated microglia. We also addressed the progression of glial responses in the vicinity (≤50 μm) of dense-core plaques and tangles. Although the amyloid load reached a plateau early after symptom onset, astrocytosis and microgliosis increased linearly throughout the disease course. Moreover, glial responses correlated positively with tangle burden, whereas astrocytosis correlated negatively with cortical thickness. However, neither correlated with amyloid load. Glial responses increased linearly around existing plaques and in the vicinity of tangles. These results indicate that the progression of astrocytosis and microgliosis diverges from that of amyloid deposition, arguing against a straightforward relationship between glial cells and plaques. They also suggest that reactive glia might contribute to the ongoing neurodegeneration. Senile plaques are a prominent pathological feature of Alzheimer's disease (AD), but little is understood about the association of glial cells with plaques or about the dynamics of glial responses through the disease course. We investigated the progression of reactive glial cells and their relationship with AD pathological hallmarks to test whether glial cells are linked only to amyloid deposits or also to tangle deposition, thus integrating both lesions as a marker of disease severity. We conducted a quantitative stereology-based post-mortem study on the temporal neocortex of 15 control subjects without dementia and 91 patients with AD, including measures of amyloid load, neurofibrillary tangles, reactive astrocytes, and activated microglia. We also addressed the progression of glial responses in the vicinity (≤50 μm) of dense-core plaques and tangles. Although the amyloid load reached a plateau early after symptom onset, astrocytosis and microgliosis increased linearly throughout the disease course. Moreover, glial responses correlated positively with tangle burden, whereas astrocytosis correlated negatively with cortical thickness. However, neither correlated with amyloid load. Glial responses increased linearly around existing plaques and in the vicinity of tangles. These results indicate that the progression of astrocytosis and microgliosis diverges from that of amyloid deposition, arguing against a straightforward relationship between glial cells and plaques. They also suggest that reactive glia might contribute to the ongoing neurodegeneration. Activated glia is a prominent feature of Alzheimer's disease (AD) neuropathological features, with both reactive astrocytes and activated microglia clustering around and within dense-core amyloid plaques (ie, thioflavin-S–positive plaques).1Itagaki S. McGeer P.L. Akiyama H. Zhu S. Selkoe D. Relationship of microglia and astrocytes to amyloid deposits of Alzheimer disease.J Neuroimmunol. 1989; 24: 173-182Abstract Full Text PDF PubMed Scopus (765) Google Scholar A better understanding of how these reactive glial cells accrue during the disease course and how they relate to the classic AD pathological hallmarks [ie, amyloid plaques and neurofibrillary tangles (NFTs)] is crucial for the following reasons: i) a body of preclinical evidence implicates these glial cells in AD pathophysiological features; ii) new positron emission tomographic (PET) radiotracers for amyloid plaques, NFTs, and, particularly, activated glial cells are being developed as diagnostic and progression biomarkers; and iii) clinical trials with anti-inflammatory therapies, ranging from nonsteroidal anti-inflammatory drugs (NSAIDs) to i.v. Ig, are under development. In a previous quantitative neuropathological study,2Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Early Aβ accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain.Neurology. 2004; 62: 925-931Crossref PubMed Scopus (523) Google Scholar we observed a positive linear correlation between astrocytosis in the temporal neocortex, as measured with a glial fibrillary acidic protein (GFAP) enzyme-linked immunosorbent assay, and the duration of the disease from the onset of cognitive symptoms, despite the plaque burden remaining stable throughout the course of the disease. We hypothesized that a certain threshold of amyloid burden might be needed to trigger glial responses within a particular region of the cortex and that, once triggered, glial responses would reflect a pathogenic cascade increasingly independent of plaques. In the present study, we sought to extend that observation and test the hypothesis that, although initially linked to plaques, glial responses increasingly reflect the widespread ongoing neurodegenerative process. We quantified the number of reactive astrocytes and activated microglial cells in the temporal neocortex of a large cohort of controls without dementia and subjects with AD at different stages of the disease and investigated both their apparent progression throughout the disease course and their relation to the local burden of amyloid plaques and NFTs. Although glial association with amyloid plaques has long been assumed, we found a dissociation between these pathological features, with a linear increase of reactive glia despite a relatively stable plaque burden. The magnitude of these glial changes correlated with the burden of NFTs. A closer analysis in a subset of subjects with AD revealed that reactive glial cells increased both in the proximity of dense-core plaques and near NFTs, thus supporting a previously not described association between glial responses and neurofibrillary degeneration. Formalin-fixed, paraffin-embedded tissue specimens from the temporal association isocortex (Brodmann area 38) of 91 patients with AD and 15 controls without dementia were obtained from the Massachusetts Alzheimer Disease Research Center Brain Bank. They were consecutively selected by tissue availability. All of the study subjects or their next of kin gave written informed consent for the brain donation, and the Massachusetts General Hospital Institutional Review Board approved the study protocol. The demographic characteristics of both groups are depicted in Table 1. All of the patients with AD fulfilled the National Institute of Neurological and Communicative Disorders and Stroke–Alzheimer's Disease and Related Disorders Associations criteria for probable AD3McKhann G. Drachman D. Folstein M. Katzman R. Price D. Stadlan E.M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of the Department of Health and Human Services Task Force on Alzheimer's Disease.Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar and the National Institute on Aging–Reagan criteria for high likelihood of AD.4The National Institute on Aging, and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease Consensus recommendations for the postmortem diagnosis of Alzheimer's disease.Neurobiol Aging. 1997; 18: S1-S2PubMed Google Scholar Cases with cerebrovascular disease considered severe enough to contribute to the dementia syndrome were excluded because cerebrovascular disease is a major cause of focal gliosis. Cases with Lewy body pathological features were also excluded. Sections (8-μm thick) were deparaffinized for immunohistochemistry by standard methods. Primary and secondary antibodies, pretreatments for antigen retrieval, and visualization strategies are summarized in Table 2. For stereological quantitative studies, immunostained sections were developed with 3,3′-diaminobenzidine (Vector Laboratories, Burlingame, CA), lightly counterstained with Mayer's hematoxylin, dehydrated with increasing concentrations of ethanol, cleared with xylene, and cover-slipped with Permount mounting media (Fisher Scientific, Fair Lawn, NJ). Nearly adjacent temporal sections from a subset of 40 subjects with AD and six controls without dementia were immunostained using fluorescently labeled secondary antibodies, counterstained with 0.05% thioflavin-S (Sigma, St Louis, MO) in 50% ethanol for 8 minutes, and cover-slipped with Vectashield mounting media with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).Table 1Demographic Characteristics of the Cohorts without Dementia and with AD and Their Corresponding Subsets Included in the Quantitative Neuropathological StudyCharacteristicsControl cohort (n = 15)AD cohort (n = 91)P valueControl subset (n = 6)AD subset (n = 40)P valueAge at death (years)⁎Data are given as mean ± SD. P values were obtained using the two-tailed Mann-Whitney U-test.79.9 ± 13.379.0 ± 7.8NS83.7 ± 14.077.6 ± 8.60.0429Female sex†Data are given as number (percentage) of each group. P values were obtained using the two-tailed χ2 test with Fisher's exact test.10 (66.7)58 (63.7)NS4 (66.7)26 (65.0)NSDisease duration (years)‡Data are given as median (interquartile range).NA9.8 (6.8–13.7)NANA9.9 (5.4–15.5)NAAPOE genotype†Data are given as number (percentage) of each group. P values were obtained using the two-tailed χ2 test with Fisher's exact test. APOEε4 carriers4 (26.7)59 (64.8)0.00902 (33.3)21 (52.5)NS APOEε4 alleles§To obtain percentages, the denominators for this row were doubled.4 (13.3)70 (38.5)0.00702 (16.7)25 (31.2)NSPost-mortem interval (hours)⁎Data are given as mean ± SD. P values were obtained using the two-tailed Mann-Whitney U-test.22.3 ± 12.813.9 ± 9.10.008521.0 ± 11.114.1 ± 6.2NSInformation about cause of death was available in only 44 of the 91 subjects with AD because nursing homes are the main source of our brain donation program. Patients with protracted death (mostly aspirative pneumonia and cancer, n = 31) did not differ from patients with sudden death (mostly pulmonary emboli and myocardial infarction, n = 13) regarding their age at death (P = 0.2261), disease duration (P = 0.9898), or amount of astrocytosis (P = 0.1870) and microgliosis (P = 0.9180). Statistically significant P values are boldfaced.NA, not applicable; NS, not significant. Data are given as mean ± SD. P values were obtained using the two-tailed Mann-Whitney U-test.† Data are given as number (percentage) of each group. P values were obtained using the two-tailed χ2 test with Fisher's exact test.‡ Data are given as median (interquartile range).§ To obtain percentages, the denominators for this row were doubled. Open table in a new tab Table 2Antibodies, Antigen Retrieval Protocols, and Visualization Strategies Used in the IHC StudiesPrimary antibodyHostDilutionAntigen retrieval⁎Citrate buffer + MW indicates 0.01 mol/L citrate buffer (pH 6.0) with 0.05% Tween 20 in a microwave oven at 95°C for 20 minutes.Secondary antibody†All secondary antibodies were obtained from Jackson ImmunoResearch Labs (West Grove, PA).Visualization strategy10D5 (Elan Pharmaceuticals, Inc.)Ms1:50Citrate buffer + MW and 90% formic acid for 5 minutesHRP anti-Ms (1:200)DAB (Vector Laboratories)PHF1 (gift from Dr. Peter Davies)Ms1:200Citrate buffer + MWBiotin anti-Ms (1:200)ABC kit + DAB (Vector Laboratories for both)GFAP (catalogue no. G9269; Sigma)Rb1:1000Citrate buffer + MWi) Biotin anti-Rb (1:200) and ii) Cy3 anti-Rb (1:200)i) ABC kit + DAB (Vector Laboratories for both) and ii) noneCD68 (catalogue no. M0814; Dako, Glostrup, Denmark)Ms1:100Citrate buffer + MWBiotin anti-Ms (1:200)ABC kit + DAB (Vector Laboratories for both)Iba1 (catalogue no. 019-19741; Wako)Rb1:250Citrate buffer + MWCy3 anti-Rb (1:200)NoneNAB61 (gift from Dr. Virginia Lee)Ms1:500NoneBiotin anti-Ms (1:200)ABC kit (Vector Laboratories) + streptavidin-Cy3 (1:200) (Invitrogen)ABC, avidin-biotin complex; DAB, 3,3′-diaminobenzidine; HRP, horseradish peroxidase; Ms, mouse; Rb, rabbit. Citrate buffer + MW indicates 0.01 mol/L citrate buffer (pH 6.0) with 0.05% Tween 20 in a microwave oven at 95°C for 20 minutes.† All secondary antibodies were obtained from Jackson ImmunoResearch Labs (West Grove, PA). Open table in a new tab Information about cause of death was available in only 44 of the 91 subjects with AD because nursing homes are the main source of our brain donation program. Patients with protracted death (mostly aspirative pneumonia and cancer, n = 31) did not differ from patients with sudden death (mostly pulmonary emboli and myocardial infarction, n = 13) regarding their age at death (P = 0.2261), disease duration (P = 0.9898), or amount of astrocytosis (P = 0.1870) and microgliosis (P = 0.9180). Statistically significant P values are boldfaced. NA, not applicable; NS, not significant. ABC, avidin-biotin complex; DAB, 3,3′-diaminobenzidine; HRP, horseradish peroxidase; Ms, mouse; Rb, rabbit. We took advantage of stereology tools to perform unbiased quantitative neuropathological studies in these brain specimens. All analyses were conducted blinded to disease status. Cortical thickness was measured in sections stained with Luxol fast blue H&E, as previously described.5Freeman S.H. Kandel R. Cruz L. Rozkalne A. Newell K. Frosch M.P. Hedley-Whyte E.T. Locascio J.J. Lipsitz L.A. Hyman B.T. Preservation of neuronal number despite age-related cortical brain atrophy in elderly subjects without Alzheimer disease.J Neuropathol Exp Neurol. 2008; 67: 1205-1212Crossref PubMed Scopus (144) Google Scholar Briefly, the image analysis software CAST (Olympus, Copenhagen, Denmark), mounted on an upright BX51 Olympus microscope (Olympus) and coupled with a motorized stage and a charge-coupled device camera, was used to randomly sample the cortex of the entire section and measure the thickness of the full cortex. The measurements of full cortical thickness in 20 random sites were averaged. Amyloid load and stereology-based studies on 3,3′-diaminobenzidine sections were conducted in an upright Leica DMRB microscope (Leica, Wetzlar, Germany) equipped with a motorized stage and a charge-coupled device camera (model DC330; DAGE-MTI, Inc., Michigan City, IN) and coupled with the software BIOQUANT NOVA PRIME, version 6.90.10 (MBSR, Nashville, TN). Amyloid load was measured as the percentage of total surface stained by the N-terminal–specific anti-amyloid β (Aβ) antibody 10D5 (Elan Pharmaceuticals, Inc., Dublin, Ireland) in a full-thickness strip of cortex (approximately 1-cm long) using the optical threshold application of the software. The total number of amyloid plaques in a 1-cm-long strip of cortex was calculated by dividing the total number of particles higher than the threshold by the area analyzed (both parameters provided by the software) and then correcting the resultant density by the cortical thickness. Paired helical filament 1–positive NFTs, GFAP-positive astrocytes, and CD68-positive microglial cells were counted with the optic dissector technique,6Hyman B.T. Gómez-Isla T. Irizarry M.C. Stereology: a practical primer for neuropathology.J Neuropathol Exp Neurol. 1998; 57: 305-310Crossref PubMed Scopus (77) Google Scholar using either 100 cells or 1000 optical dissectors as the end point. The objective/dissector size used in each case was 40/150 × 150 μm for paired helical filament 1–positive neurons, 40/50 × 50 μm for GFAP-positive astrocytes, and 100/20 × 20 μm for CD68-positive microglial cells. Intraneuronal and extracellular ghost tangles were not distinguished. Because different pathological features tend to accumulate in specific layers of the cortex (ie, reactive astrocytes in layer I and NFTs in layers II and V), care was taken to cover all of the six cortical layers in the systematic random sampling to avoid selection bias. As with the amyloid plaques, the densities of NFTs, astrocytes, and microglial cells were calculated by dividing the number of cells counted in single sections by the total area of the dissectors analyzed. To avoid any overestimation of densities because of disease-related cortical atrophy, these densities were then corrected by the cortical thickness to estimate the total number of cells within a full-thickness 1-cm-long strip of cortex. We performed additional quantitative studies in a subset of 40 AD cases selected from the original AD cohort on the basis of a wide range of disease duration and in a subset of six controls without dementia (ie, those with enough dense-core plaques). These subsets were representative of their corresponding cohorts in demographic characteristics, and the AD subset was also comparable to the entire AD cohort in neuropathological quantitative measures (Table 1; see also Supplemental Table S1 and Figure S1 at http://ajp.amjpathol.org). To study the progression of compact and oligomeric species of Aβ, we quantified the number of dense-core plaques and oligomeric Aβ-positive plaques in sections doubly stained with thioflavin-S and NAB61 antibody. The NAB61 antibody was provided by Dr. Virginia Lee (University of Pennsylvania, Philadelphia) and has been previously characterized. It is a conformation-specific anti-Aβ mouse monoclonal antibody that binds to Aβ dimers, small oligomers, and higher-order Aβ assemblies and stains a subset of mature dense-core plaques.7Lee E.B. Leng L.Z. Zhang B. Kwong L. Trojanowski J.Q. Abel T. Lee V.M. Targeting amyloid-β peptide (Aβ) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Aβ precursor protein (APP) transgenic mice.J Biol Chem. 2006; 281: 4292-4299Crossref PubMed Scopus (257) Google Scholar Virtually no thioflavin-S–negative plaque was immunoreactive for NAB61. In this study, 100 dense-core plaques per case were randomly sampled, as previously described, and their positivity for NAB61 was qualitatively assessed. The densities of dense-core plaques and NAB61-positive plaques obtained were corrected by the cortical thickness to calculate total numbers of plaques within a 1-cm-long full-thickness strip of cortex. Single sections from the subset of 40 AD cases were also doubly stained with thioflavin-S and GFAP or Iba1 to investigate the spatial relationship between glial responses and dense-core plaques and NFTs along the course of the disease. Optimal fluorescent immunolabeling of activated microglia was achieved with antibody Iba1 (Wako, Osaka, Japan), another marker widely used for activated microglia. Sections were placed on the motorized stage of an upright BX51 Olympus microscope equipped with CAST stereology software. One hundred GFAP-positive astrocytes or Iba1-positive microglial cells per section were randomly selected under the ×20 or the ×40 objective, respectively, and their distance with respect to the closest dense-core plaque or NFT was measured with the appropriate tool of the software. For consistency, only cells with a visible nucleus in the DAPI staining were considered. Astrocytes and microglial cells were classified into three categories: i) close to plaques, if located ≤50 μm from the edge of a plaque (regardless of the presence of an NFT within this boundary); ii) close to NFTs, if located ≤50 μm from an NFT but far (>50 μm) from dense-core plaques; and iii) far from plaques and NFTs, if the closest plaque and NFT to the glial cell were located >50 μm. Densities of glial cells in each of these categories were obtained as previously described. The APOE genotype was determined in all of the study subjects by restriction fragment length polymorphism analysis, as previously described.8Ingelsson M. Shin Y. Irizarry M.C. Hyman B.T. Genotyping of apolipoprotein E: comparative evaluation of different protocols.Curr Protoc Hum Gen. 2003; 38: 1-13Google Scholar Statistics were performed, and graphs were obtained with GraphPad Prism software for Mac, version 5.0. The normality of data sets was tested with the D'Agostino-Pearson omnibus test. For correlations of cortical thickness, amyloid load, and total number of astrocytes/microglia with disease duration, two different fit models were examined using the least-squares fitting method: linear regression versus one-phase exponential association (or decay in the case of cortical thickness). The first model assumes a linear increase of the pathological features over time, whereas the second model consists of an initial increase followed by a plateau. Next, these two fit models were compared using the Akaike's Informative Criteria method with no constraints, and the model most likely to have generated the data was selected based on the magnitude of the difference between both fit models, the probabilities of the models being correct (as calculated by the statistical software), and their goodness of fit (R2). When the straight-line model was preferred, a P value indicating whether the slope of the straight line is significantly different from 0 and both the correlation coefficient (r) and the P value of Spearman's rank correlation test were also reported. Because none of the data sets was normally distributed, cross correlations between these pathological quantitative measures were investigated with the Spearman's rank correlation test. The significance level was set at a two-sided P < 0.05 in all statistical analyses. We have previously used disease duration (defined from the onset of cognitive symptoms) as a proxy of disease severity to avoid the floor effects of neuropsychological tests in patients with advanced dementia, who are typically not testable. More important, the three major pathological correlates of cognitive decline (ie, NFT burden, neuron loss, and synaptic loss) also correlated with disease duration in our previous quantitative post-mortem studies2Ingelsson M. Fukumoto H. Newell K.L. Growdon J.H. Hedley-Whyte E.T. Frosch M.P. Albert M.S. Hyman B.T. Irizarry M.C. Early Aβ accumulation and progressive synaptic loss, gliosis, and tangle formation in AD brain.Neurology. 2004; 62: 925-931Crossref PubMed Scopus (523) Google Scholar, 9Arriagada P.V. Growdon J.H. Hedley-Whyte E.T. Hyman B.T. Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease.Neurology. 1992; 42: 631-639Crossref PubMed Google Scholar, 10Gómez-Isla T. Hollister R. West H. Mui S. Growdon J.H. Petersen R.C. Parisi J.E. Hyman B.T. Neuronal loss correlates with but exceeds neurofibrillary tangles in Alzheimer's disease.Ann Neurol. 1997; 41: 17-24Crossref PubMed Scopus (1119) Google Scholar on the temporal neocortex. Herein, we measured the cortical thickness of the temporal neocortex specimens from the AD cohort as an index of synaptic, dendritic, and neuronal integrity. We found a significant negative correlation between cortical thickness and symptomatic disease duration, further validating the use of disease duration as a surrogate of disease severity (r = −0.3977, P < 0.0001) (Figure 1A and Table 3).Table 3Summary of the Results from the AD and Control CohortsVariableAD cohort (n = 91)AD + CTRL with plaques (n = 101)AD + all CTRL (n = 106)LinearOne-phase exponentialLinearOne-phase exponentialLinearOne-phase exponentialCortical thickness (μm) ΔAICc1.804NANA Probability (%)71.1328.87100Not converged100Not converged Goodness (R2)0.17970.18310.2130NA0.2296NA Slope ≠ 0? (P)<0.0001NA<0.0001NA<0.0001NA Spearman's r−0.3977NA−0.4523NA−0.4649NA Spearman′s P<0.0001NA<0.0001NA<0.0001NAAmyloid burden (%) ΔAICc4.34710.8318.23 Probability (%)10.2289.780.4499.560.0199.99 Goodness (R2)0.06570.10930.23170.30990.31170.4205 Slope ≠ 0? (P)0.0142NA<0.0001NA<0.0001NATotal amyloid plaques ΔAICc1.76111.9621.72 Probability (%)29.3170.690.2599.75<0.01>99.99 Goodness (R2)0.03910.05750.21010.29830.29520.4258 Slope ≠ 0? (P)0.0602NA<0.0001NA<0.0001NATotal astrocytes ΔAICc0.75453.0053.953 Probability (%)59.3240.6881.8018.2087.8312.17 Goodness (R2)0.19510.18840.24330.22040.27050.2428 Slope ≠ 0? (P)<0.0001NA<0.0001NA<0.0001NA Spearman's r0.4070NA0.5037NA0.5471NA Spearman's P<0.0001NA<0.0001NA<0.0001NATotal microglia ΔAICc5.17133.1842.25 Probability (%)92.997.01>99.99<0.01>99.99<0.01 Goodness (R2)0.09600.04310.1728−0.14890.2109−0.1755 Slope ≠ 0? (P)0.0028NA<0.0001NA<0.0001NA Spearman's r0.3545NA0.4326NA0.4728NA Spearman's P0.0006NA<0.0001NA<0.0001NAThe probability of being correct and the goodness of fit (R2) of both the linear regression and the one-phase exponential association models (or decay, in the case of cortical thickness) are shown for the main neuropathological measures in the AD cohort alone, the AD cohort plus the controls without dementia and with plaques, and the AD cohort plus the entire control cohort. The best-fit model is boldfaced. In the linear regression model, P indicates whether the slope is significantly different from 0. When the linear regression model was the preferred-fit model, the correlation coefficient and the P value from the Spearman's rank correlation test are also shown. For the amyloid burden and the total number of amyloid plaques, the nonlinear model remains the best fit, despite the linear fit yielding a straight line with a slope significantly different from 0 (because of the anchoring effect of controls close to 0). Also, the R2 of the one-phase exponential association model is negative for some neuropathological measures, indicating that the best-fit curve fits the data even worse than a horizontal line. Statistics in Materials and Methods provides further details.ΔAICc, magnitude of the difference between both fit models; CTRL, control without dementia; NA, not applicable. Open table in a new tab The probability of being correct and the goodness of fit (R2) of both the linear regression and the one-phase exponential association models (or decay, in the case of cortical thickness) are shown for the main neuropathological measures in the AD cohort alone, the AD cohort plus the controls without dementia and with plaques, and the AD cohort plus the entire control cohort. The best-fit model is boldfaced. In the linear regression model, P indicates whether the slope is significantly different from 0. When the linear regression model was the preferred-fit model, the correlation coefficient and the P value from the Spearman's rank correlation test are also shown. For the amyloid burden and the total number of amyloid plaques, the nonlinear model remains the best fit, despite the linear fit yielding a straight line with a slope significantly different from 0 (because of the anchoring effect of controls close to 0). Also, the R2 of the one-phase exponential association model is negative for some neuropathological measures, indicating that the best-fit curve fits the data even worse than a horizontal line. Statistics in Materials and Methods provides further details. ΔAICc, magnitude of the difference between both fit models; CTRL, control without dementia; NA, not applicable. Next, we traced the progression of amyloid deposition and patterns of glial immunostaining throughout the clinical disease course. Amyloid burden, determined as the percentage of cortical surface immunoreactive for the anti-Aβ antibody 10D5, reached a plateau early after symptomatic onset and remained relatively stable thereafter (Figure 1B and Table 3). An analysis of total number of plaques yielded similar results (Figure 1C and Table 3). Like 10D5-immunoreactive plaques in the original AD cohort, the number of dense-core plaques determined in a subset of 40 AD cases remained relatively stable throughout the disease clinical course after an initial increase (Figure 2, A and B, and Table 4). Last, the amount of NAB61-positive oligomeric Aβ-enriched plaques also remained constant throughout the disease clinical course (Figure 2, C and D, and Table 4).Table 4Summary of the Results from the AD and Control Subsets Concerning Fibrillar and Oligomeric Aβ BurdenVariableAD subset (n = 40)AD + CTRL with dense-core plaques (n = 46)LinearOne-phase exponentialLinearOne-phase exponentialTotal dense-core plaques ΔAICc0.25251.833 Probability (%)46.8553.1528.5771.43 Goodness (R2)0.02480.03100.12970.1637 Slope ≠ 0? (P)0.3313NA0.0140NATotal NAB61 + plaques ΔAICcNANA Probability (%)100Not converged100Not converged Goodness (R2)0.00020.0361 Slope ≠ 0? (P)0.93130.2060 Spearman's r−0.0037NA0.2267NA Spearman's P0.9816NA0.1298NAThe ΔAICc represents the magnitude of the difference between the two fit models compared. The best-fit model is boldfaced. In the linear model, P indicates whether the slope is significantly different from 0. When the linear regression model was the preferred-fit model, the correlation coefficient and the P value from the Spearman's rank correlation test are also shown. The nonlinear model is the best fit for total dense-core plaques in the AD + CTRL analysis, despite the linear fit yielding a straight line with a slope significantly different from 0 (likely because of the anchoring effect caused by the controls). Statistics in Materials and Methods provides further details.ΔAICc, magnitude of the difference between both fit models; CTRL, control without dementia; NA, not applicable. Open table in a new tab The ΔAICc represents the magnitude of the difference between the two fit models compared. The best-fit model is boldfaced. In the linear model, P indicates

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD.

The identification of amyloid deposits in living Alzheimer disease (AD) patients is important for both early diagnosis and for monitoring the efficacy of newly developed anti-amyloid therapies. Methoxy-X04 is a derivative of Congo red and Chrysamine-G that contains no acid groups and is therefore smaller and much more lipophilic than Congo red or Chrysamine-G. Methoxy-X04 retains in vitro binding affinity for amyloid beta (Abeta) fibrils (Ki = 26.8 nM) very similar to that of Chrysamine-G (Ki = 25.3 nM). Methoxy-X04 is fluorescent and stains plaques, tangles, and cerebrovascular amyloid in postmortem sections of AD brain with good specificity. Using multiphoton microscopy to obtain high-resolution (1 microm) fluorescent images from the brains of living PSI/APP mice, individual plaques could be distinguished within 30 to 60 min after a single i.v. injection of 5 to 10 mg/kg methoxy-X04. A single i.p. injection of 10 mg/kg methoxy-X04 also produced high contrast images of plaques and cerebrovascular amyloid in PSI/APP mouse brain. Complementary quantitative studies using tracer doses of carbon- 11-labeled methoxy-X04 show that it enters rat brain in amounts that suggest it is a viable candidate as a positron emission tomography (PET) amyloid-imaging agent for in vivo human studies.

To explore the clinical effects of inflammation associated with vascular deposits of the amyloid beta peptide (A beta), we analyzed 42 consecutive patients with pathologically diagnosed cerebral amyloid angiopathy (CAA) for evidence of an inflammatory response. Inflammation with giant-cell reaction surrounding amyloid-laden vessels was identified in 7 of the 42 cases. The clinical symptoms in each of the seven were subacute cognitive decline or seizure rather than hemorrhagic stroke, the primary clinical presentation in 33 of 35 patients with noninflammatory CAA (p < 0.001). Inflammatory CAA also was associated with radiographic white matter abnormalities, significantly younger age at presentation, and a marked overrepresentation of the apolipoprotein E epsilon 4/epsilon 4 genotype (71% vs 4%, p < 0.001). Of the six inflammatory CAA patients with available follow-up information, five demonstrated clinical and radiographic improvement after immunosuppressive treatment. The syndrome of CAA-related perivascular inflammation appears to represent a subset of CAA with clinically distinct symptoms that may respond to immunosuppressive treatment. These data add to evidence that inflammation against A beta can cause vascular dysfunction, a potential mechanism for the toxic response recently observed in clinical trials of A beta immunization.

Sporadic cerebral amyloid angiopathy is a common, well-defined small vessel disease and a largely untreatable cause of intracerebral haemorrhage and contributor to age-related cognitive decline. The term ‘cerebral amyloid angiopathy’ now encompasses not only a specific cerebrovascular pathological finding, but also different clinical syndromes (both acute and progressive), brain parenchymal lesions seen on neuroimaging and a set of diagnostic criteria—the Boston criteria, which have resulted in increasingly detected disease during life. Over the past few years, it has become clear that, at the pathophysiological level, cerebral amyloid angiopathy appears to be in part a protein elimination failure angiopathy and that this dysfunction is a feed-forward process, which potentially leads to worsening vascular amyloid-β accumulation, activation of vascular injury pathways and impaired vascular physiology. From a clinical standpoint, cerebral amyloid angiopathy is characterized by individual focal lesions (microbleeds, cortical superficial siderosis, microinfarcts) and large-scale alterations (white matter hyperintensities, structural connectivity, cortical thickness), both cortical and subcortical. This review provides an interdisciplinary critical outlook on various emerging and changing concepts in the field, illustrating mechanisms associated with amyloid cerebrovascular pathology and neurological dysfunction.

To determine the correspondence between uptake of Pittsburgh Compound B (PiB) in life and measures of beta-amyloid (Abeta) in postmortem tissue analysis. Patient A 76-year-old man with a clinical diagnosis of dementia with Lewy bodies underwent fluorodeoxyglucose (18)F and PiB positron emission tomographic brain scans. Imaging revealed marked region specific binding of PiB and abnormal fluorodeoxyglucose uptake. Intervention Autopsy was performed 3 months after the PiB scan.Autopsy confirmed the clinical diagnosis; in addition, there was severe cerebral amyloid angiopathy and only moderate numbers of parenchymal Abeta plaques. Biochemical measures revealed a positive correlation between Abeta levels and regional PiB binding.This report confirms that PiB detects Abeta in the living patient and demonstrates that amyloid deposited as cerebral amyloid angiopathy can be the dominant source of signal.

The synthesis of a new lipophilic thioflavin-T analogue (2-[4′-(methylamino)phenyl]benzothiazole, 6) with high affinity for amyloid is reported. Intravenous injection of [11C]-labeled 6 in control mice resulted in high brain uptake. Amyloid deposits were imaged with multiphoton microscopy in the brains of living transgenic mice following the systemic injection of unlabeled 6. [11C]6 is a promising amyloid imaging agent for Alzheimer's disease.

Alzheimer's disease (AD) and related dementias are a major public health challenge and present a therapeutic imperative for which we need additional insight into molecular pathogenesis. We performed a genome-wide association study and analysis of known genetic risk loci for AD dementia using neuropathologic data from 4,914 brain autopsies. Neuropathologic data were used to define clinico-pathologic AD dementia or controls, assess core neuropathologic features of AD (neuritic plaques, NPs; neurofibrillary tangles, NFTs), and evaluate commonly co-morbid neuropathologic changes: cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), hippocampal sclerosis of the elderly (HS), and vascular brain injury (VBI). Genome-wide significance was observed for clinico-pathologic AD dementia, NPs, NFTs, CAA, and LBD with a number of variants in and around the apolipoprotein E gene (APOE). GalNAc transferase 7 (GALNT7), ATP-Binding Cassette, Sub-Family G (WHITE), Member 1 (ABCG1), and an intergenic region on chromosome 9 were associated with NP score; and Potassium Large Conductance Calcium-Activated Channel, Subfamily M, Beta Member 2 (KCNMB2) was strongly associated with HS. Twelve of the 21 non-APOE genetic risk loci for clinically-defined AD dementia were confirmed in our clinico-pathologic sample: CR1, BIN1, CLU, MS4A6A, PICALM, ABCA7, CD33, PTK2B, SORL1, MEF2C, ZCWPW1, and CASS4 with 9 of these 12 loci showing larger odds ratio in the clinico-pathologic sample. Correlation of effect sizes for risk of AD dementia with effect size for NFTs or NPs showed positive correlation, while those for risk of VBI showed a moderate negative correlation. The other co-morbid neuropathologic features showed only nominal association with the known AD loci. Our results discovered new genetic associations with specific neuropathologic features and aligned known genetic risk for AD dementia with specific neuropathologic changes in the largest brain autopsy study of AD and related dementias.

The amygdala is composed of multiple nuclei with unique functions and connections in the limbic system and to the rest of the brain. However, standard in vivo neuroimaging tools to automatically delineate the amygdala into its multiple nuclei are still rare. By scanning postmortem specimens at high resolution (100-150µm) at 7T field strength (n = 10), we were able to visualize and label nine amygdala nuclei (anterior amygdaloid, cortico-amygdaloid transition area; basal, lateral, accessory basal, central, cortical medial, paralaminar nuclei). We created an atlas from these labels using a recently developed atlas building algorithm based on Bayesian inference. This atlas, which will be released as part of FreeSurfer, can be used to automatically segment nine amygdala nuclei from a standard resolution structural MR image. We applied this atlas to two publicly available datasets (ADNI and ABIDE) with standard resolution T1 data, used individual volumetric data of the amygdala nuclei as the measure and found that our atlas i) discriminates between Alzheimer's disease participants and age-matched control participants with 84% accuracy (AUC=0.915), and ii) discriminates between individuals with autism and age-, sex- and IQ-matched neurotypically developed control participants with 59.5% accuracy (AUC=0.59). For both datasets, the new ex vivo atlas significantly outperformed (all p < .05) estimations of the whole amygdala derived from the segmentation in FreeSurfer 5.1 (ADNI: 75%, ABIDE: 54% accuracy), as well as classification based on whole amygdala volume (using the sum of all amygdala nuclei volumes; ADNI: 81%, ABIDE: 55% accuracy). This new atlas and the segmentation tools that utilize it will provide neuroimaging researchers with the ability to explore the function and connectivity of the human amygdala nuclei with unprecedented detail in healthy adults as well as those with neurodevelopmental and neurodegenerative disorders.

A subset of patients with cerebral amyloid angiopathy (CAA) present with cognitive symptoms, seizures, headaches, T2-hyperintense MRI lesions, and neuropathologic evidence of CAA-associated vascular inflammation.To analyze the risk factors, diagnostic characteristics, and long-term course of this disorder.We assessed 14 consecutive patients with pathologically diagnosed CAA-related inflammation, 12 with available neuroimaging and follow-up data. Patients were evaluated for MRI appearance, APOE genotype, and clinical course over a 46.8 +/- 29.1-month follow-up.Baseline MRI scans were characterized by asymmetric T2-hyperintense lesions extending to the subcortical white matter and occasionally the overlying gray matter, with signal properties suggesting vasogenic edema. Subjects could be divided into three groups based on response to immunosuppressive treatment: monophasic improvement (7/12), initial improvement followed by symptomatic relapse (3/12), and no evident response to treatment (2/12). The volume of MRI hyperintensities correlated with the severity of clinical symptoms. One patient experienced symptomatic intracerebral hemorrhage within a region of recurrent MRI hyperintensity. The APOE epsilon4/epsilon4 genotype was strongly associated with CAA-related inflammation, present in 76.9% (10/13) of subjects vs 5.1% (2/39) with symptomatic but noninflammatory CAA (p < 0.0001).Cerebral amyloid angiopathy-related inflammation represents a clinically, pathologically, radiographically, and genetically distinct disease subtype with implications for clinical practice and ongoing immunotherapeutic approaches to Alzheimer disease.

Objective To investigate miR‐155 in the SOD1 mouse model and human sporadic and familial amyotrophic lateral sclerosis (ALS). Methods NanoString microRNA, microglia and immune gene profiles, protein mass spectrometry, and RNA‐seq analyses were measured in spinal cord microglia, splenic monocytes, and spinal cord tissue from SOD1 mice and in spinal cord tissue of familial and sporadic ALS. miR‐155 was targeted by genetic ablation or by peripheral or centrally administered anti–miR‐155 inhibitor in SOD1 mice. Results In SOD1 mice, we found loss of the molecular signature that characterizes homeostatic microglia and increased expression of miR‐155. There was loss of the microglial molecules P2ry12 , Tmem119, Olfml3 , transcription factors Egr1 , Atf3 , Jun , Fos , and Mafb, and the upstream regulators Csf1r, Tgfb1, and Tgfbr1, which are essential for microglial survival. Microglia biological functions were suppressed including phagocytosis. Genetic ablation of miR‐155 increased survival in SOD1 mice by 51 days in females and 27 days in males and restored the abnormal microglia and monocyte molecular signatures. Disease severity in SOD1 males was associated with early upregulation of inflammatory genes, including Apoe in microglia. Treatment of adult microglia with apolipoprotein E suppressed the M0‐homeostatic unique microglia signature and induced an M1‐like phenotype. miR‐155 expression was increased in the spinal cord of both familial and sporadic ALS. Dysregulated proteins that we identified in human ALS spinal cord were restored in SOD1 G93A /miR‐155 −/− mice. Intraventricular anti–miR‐155 treatment derepressed microglial miR‐155 targeted genes, and peripheral anti–miR‐155 treatment prolonged survival. Interpretation We found overexpression of miR‐155 in the SOD1 mouse and in both sporadic and familial human ALS. Targeting miR‐155 in SOD1 mice restores dysfunctional microglia and ameliorates disease. These findings identify miR‐155 as a therapeutic target for the treatment of ALS. ANN NEUROL 2015;77:75–99

Tau pathology is known to spread in a hierarchical pattern in Alzheimer's disease (AD) brain during disease progression, likely by trans-synaptic tau transfer between neurons. However, the tau species involved in inter-neuron propagation remains unclear. To identify tau species responsible for propagation, we examined uptake and propagation properties of different tau species derived from postmortem cortical extracts and brain interstitial fluid of tau-transgenic mice, as well as human AD cortices. Here we show that PBS-soluble phosphorylated high-molecular-weight (HMW) tau, though very low in abundance, is taken up, axonally transported, and passed on to synaptically connected neurons. Our findings suggest that a rare species of soluble phosphorylated HMW tau is the endogenous form of tau involved in propagation and could be a target for therapeutic intervention and biomarker development.

An estimated 200 million patients worldwide have surgery each year. Anesthesia and surgery have been reported to facilitate emergence of Alzheimer's disease. The commonly used inhalation anesthetic isoflurane has previously been reported to induce apoptosis, and to increase levels and aggregation of Alzheimer's disease-associated amyloid beta-protein (Abeta) in cultured cells. However, the in vivo relevance has not been addressed.We therefore set out to determine effects of isoflurane on caspase activation and levels of beta-site amyloid precursor protein-cleaving enzyme (BACE) and Abeta in naive mice, using Western blot, immunohistochemistry, and reverse transcriptase polymerase chain reaction.Here we show for the first time that a clinically relevant isoflurane anesthesia (1.4% isoflurane for 2 hours) leads to caspase activation and modest increases in levels of BACE 6 hours after anesthesia in mouse brain. Isoflurane anesthesia induces caspase activation, and increases levels of BACE and Abeta up to 24 hours after anesthesia. Isoflurane may increase BACE levels by reducing BACE degradation. Moreover, the Abeta aggregation inhibitor, clioquinol, was able to attenuate isoflurane-induced caspase-3 activation in vivo.Given that transient insults to brain may lead to long-term brain damage, these findings suggest that isoflurane may promote Alzheimer's disease neuropathogenesis and, as such, have implications for use of isoflurane in humans, pending human study confirmation.

Patent foramen ovale and pulmonary arteriovenous shunts are associated with serious complications such as cerebral emboli, stroke, and migraine with aura. The pathophysiological mechanisms that link these conditions are unknown. We aimed to establish a mechanism linking microembolization to migraine aura in an experimental animal model.We introduced particulate or air microemboli into the carotid circulation in mice to determine whether transient microvascular occlusion, insufficient to cause infarcts, triggered cortical spreading depression (CSD), a propagating slow depolarization that underlies migraine aura.Air microemboli reliably triggered CSD without causing infarction. Polystyrene microspheres (10 microm) or cholesterol crystals (<70 microm) triggered CSD in 16 of 28 mice, with 60% of the mice (40% of those with CSD) showing no infarcts or inflammation on detailed histological analysis of serial brain sections. No evidence of injury was detected on magnetic resonance imaging examination (9.4T; T2 weighted) in 14 of 15 selected animals. The occurrence of CSD appeared to be related to the magnitude and duration of flow reduction, with a triggering mechanism that depended on decreased brain perfusion but not sustained tissue damage.In a mouse model, microemboli triggered CSD, often without causing microinfarction. Paradoxical embolization then may link cardiac and extracardiac right-to-left shunts to migraine aura. If translatable to humans, a subset of migraine auras may belong to a spectrum of hypoperfusion disorders along with transient ischemic attacks and silent infarcts.

The apolipoprotein E ε4 gene is the most important genetic risk factor for sporadic Alzheimer's disease, but the link between this gene and neurodegeneration remains unclear. Using array tomography, we analysed >50000 synapses in brains of 11 patients with Alzheimer's disease and five non-demented control subjects and found that synapse loss around senile plaques in Alzheimer's disease correlates with the burden of oligomeric amyloid-β in the neuropil and that this synaptotoxic oligomerized peptide is present at a subset of synapses. Further analysis reveals apolipoprotein E ε4 patients with Alzheimer's disease have significantly higher oligomeric amyloid-β burden and exacerbated synapse loss around plaques compared with apolipoprotein E ε3 patients. Apolipoprotein E4 protein colocalizes with oligomeric amyloid-β and enhances synaptic localization of oligomeric amyloid-β by >5-fold. Biochemical characterization shows that the amyloid-β enriched at synapses by apolipoprotein E4 includes sodium dodecyl sulphate-stable dimers and trimers. In mouse primary neuronal culture, lipidated apolipoprotein E4 enhances oligomeric amyloid-β association with synapses via a mechanism involving apolipoprotein E receptors. Together, these data suggest that apolipoprotein E4 is a co-factor that enhances the toxicity of oligomeric amyloid-β both by increasing its levels and directing it to synapses, providing a link between apolipoprotein E ε4 genotype and synapse loss, a major correlate of cognitive decline in Alzheimer's disease.

Controlled tissue fixation with polyepoxides protects protein fluorescence, antigenicity, nucleic acids and tissue architecture. Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra- and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.

Alzheimer's disease (AD) causes unrelenting, progressive cognitive impairments, but its course is heterogeneous, with a broad range of rates of cognitive decline1. The spread of tau aggregates (neurofibrillary tangles) across the cerebral cortex parallels symptom severity2,3. We hypothesized that the kinetics of tau spread may vary if the properties of the propagating tau proteins vary across individuals. We carried out biochemical, biophysical, MS and both cell- and animal-based-bioactivity assays to characterize tau in 32 patients with AD. We found striking patient-to-patient heterogeneity in the hyperphosphorylated species of soluble, oligomeric, seed-competent tau. Tau seeding activity correlates with the aggressiveness of the clinical disease, and some post-translational modification (PTM) sites appear to be associated with both enhanced seeding activity and worse clinical outcomes, whereas others are not. These data suggest that different individuals with 'typical' AD may have distinct biochemical features of tau. These data are consistent with the possibility that individuals with AD, much like people with cancer, may have multiple molecular drivers of an otherwise common phenotype, and emphasize the potential for personalized therapeutic approaches for slowing clinical progression of AD.

Each additional copy of the apolipoprotein E4 (APOE4) allele is associated with a higher risk of Alzheimer's dementia, while the APOE2 allele is associated with a lower risk of Alzheimer's dementia, it is not yet known whether APOE2 homozygotes have a particularly low risk. We generated Alzheimer's dementia odds ratios and other findings in more than 5,000 clinically characterized and neuropathologically characterized Alzheimer's dementia cases and controls. APOE2/2 was associated with a low Alzheimer's dementia odds ratios compared to APOE2/3 and 3/3, and an exceptionally low odds ratio compared to APOE4/4, and the impact of APOE2 and APOE4 gene dose was significantly greater in the neuropathologically confirmed group than in more than 24,000 neuropathologically unconfirmed cases and controls. Finding and targeting the factors by which APOE and its variants influence Alzheimer's disease could have a major impact on the understanding, treatment and prevention of the disease.

Paravascular drainage of solutes, including β-amyloid (Aβ), appears to be an important process in brain health and diseases such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). However, the major driving force for clearance remains largely unknown. Here we used in vivo two-photon microscopy in awake head-fixed mice to assess the role of spontaneous vasomotion in paravascular clearance. Vasomotion correlated with paravascular clearance of fluorescent dextran from the interstitial fluid. Increasing the amplitude of vasomotion by means of visually evoked vascular responses resulted in increased clearance rates in the visual cortex of awake mice. Evoked vascular reactivity was impaired in mice with CAA, which corresponded to slower clearance rates. Our findings suggest that low-frequency arteriolar oscillations drive drainage of solutes. Targeting naturally occurring vasomotion in patients with CAA or AD may be a promising early therapeutic option for prevention of Aβ accumulation in the brain.

The biophysical features of neurons shape information processing in the brain. Cortical neurons are larger in humans than in other species, but it is unclear how their size affects synaptic integration. Here, we perform direct electrical recordings from human dendrites and report enhanced electrical compartmentalization in layer 5 pyramidal neurons. Compared to rat dendrites, distal human dendrites provide limited excitation to the soma, even in the presence of dendritic spikes. Human somas also exhibit less bursting due to reduced recruitment of dendritic electrogenesis. Finally, we find that decreased ion channel densities result in higher input resistance and underlie the lower coupling of human dendrites. We conclude that the increased length of human neurons alters their input-output properties, which will impact cortical computation. VIDEO ABSTRACT.

Neurofibrillary tangles (NFT), intracellular inclusions of abnormal fibrillar forms of microtubule associated protein tau, accumulate in Alzheimer's disease (AD) and other tauopathies and are believed to cause neuronal dysfunction, but the mechanism of tau-mediated toxicity are uncertain. Tau overexpression in cell culture impairs localization and trafficking of organelles. Here we tested the hypothesis that, in the intact brain, changes in mitochondrial distribution occur secondary to pathological changes in tau. Array tomography, a high-resolution imaging technique, was used to examine mitochondria in the reversible transgenic (rTg)4510, a regulatable transgenic, mouse model and AD brain tissue. Mitochondrial distribution is progressively disrupted with age in rTg4510 brain, particularly in somata and neurites containing Alz50-positive tau aggregates. Suppression of soluble tau expression with doxycycline resulted in complete recovery of mitochondrial distribution, despite the continued presence of aggregated tau. The effect on mitochondrial distribution occurs without concomitant alterations in neuropil mitochondrial size, as assessed by both array tomography and electron microscopy. Similar mitochondrial localization alterations were also observed in human AD tissue in Alz50+ neurons, confirming the relevance of tau to mitochondrial trafficking observed in this animal model. Because abnormalities reverted to normal if soluble tau was suppressed in rTg4510 mice, even in the continued presence of fibrillar tau inclusions, we suggest that soluble tau plays an important role in mitochondrial abnormalities, which likely contribute to neuronal dysfunction in AD.

T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder causing dementia. Massive deposition of amyloid β peptide (Aβ) as senile plaques in the brain is the pathological hallmark of AD, but oligomeric, soluble forms of Aβ have been implicated as the synaptotoxic component. The apolipoprotein E ε 4 (apoE ε4) allele is known to be a genetic risk factor for developing AD. However, it is still unknown how apoE impacts the process of Aβ oligomerization. Here, we found that the level of Aβ oligomers in APOE ε4/ε4 AD patient brains is 2.7 times higher than those in APOE ε3/ε3 AD patient brains, matched for total plaque burden, suggesting that apoE4 impacts the metabolism of Aβ oligomers. To test this hypothesis, we examined the effect of apoE on Aβ oligomer formation. Using both synthetic Aβ and a split-luciferase method for monitoring Aβ oligomers, we observed that apoE increased the level of Aβ oligomers in an isoform-dependent manner (E2 &lt; E3 &lt; E4). This effect appears to be dependent on the ApoE C-terminal domain. Moreover, these results were confirmed using endogenous apoE isolated from the TBS-soluble fraction of human brain, which increased the formation of Aβ oligomers. Together, these data show that lipidated apoE, especially apoE4, increases Aβ oligomers in the brain. Higher levels of Aβ oligomers in the brains of APOE ε4/ε4 carriers compared with APOE ε3/ε3 carriers may increase the loss of dendritic spines and accelerate memory impairments, leading to earlier cognitive decline in AD.

<h3>Importance</h3> Cerebral amyloid angiopathy–related inflammation (CAA-ri) is an important diagnosis to reach in clinical practice because many patients with the disease respond to immunosuppressive therapy. Reliable noninvasive diagnostic criteria for CAA-ri would allow some patients to avoid the risk of brain biopsy. <h3>Objective</h3> To test the sensitivity and specificity of clinical and neuroimaging-based criteria for CAA-ri. <h3>Design, Setting, and Participants</h3> We modified the previously proposed clinicoradiological criteria and retrospectively analyzed clinical medical records and magnetic resonance imaging fluid-attenuated inversion recovery and gradient-echo scans obtained from individuals with CAA-ri and noninflammatory CAA. At 2 referral centers between October 1, 1995, and May 31, 2013, and between January 1, 2009, and December 31, 2011, participants included 17 individuals with pathologically confirmed CAA-ri and 37 control group members with pathologically confirmed noninflammatory CAA. The control group was further divided into those with past lobar intracerebral hemorrhage (ICH) (n = 21) and those with cerebral microbleeds only and no history of ICH (n = 16). The dates of our analysis were September 1, 2012, to August 31, 2015. <h3>Main Outcomes and Measures</h3> The sensitivity and specificity of prespecified criteria for probable CAA-ri (requiring asymmetric white matter hyperintensities extending to the subcortical white matter) and possible CAA-ri (not requiring the white matter hyperintensities to be asymmetric). <h3>Results</h3> The 17 patients in the CAA-ri group were a mean (SD) of 68 (8) years and 8 (47%) were women. In the CAA-ri group, 14 of 17 (82%) met the criteria for both probable and possible CAA-ri. In the control group having noninflammatory CAA with lobar ICH, 1 of 21 (5%) met the criteria for possible CAA-ri, and none met the criteria for probable CAA-ri. In the control group having noninflammatory CAA with no ICH, 11 of 16 (69%) met the criteria for possible CAA-ri, and 1 of 16 (6%) met the criteria for probable CAA-ri. These findings yielded a sensitivity and specificity of 82% and 97%, respectively, for the probable criteria and a sensitivity and specificity of 82% and 68%, respectively, for the possible criteria. <h3>Conclusions and Relevance</h3> Our data suggest that a reliable diagnosis of CAA-ri can be reached from basic clinical and magnetic resonance imaging information alone, with good sensitivity and excellent specificity.

The interstitial fluid (ISF) drainage pathway has been hypothesized to underlie the clearance of solutes and metabolites from the brain. Previous work has implicated the perivascular spaces along arteries as the likely route for ISF clearance; however, it has never been demonstrated directly. The accumulation of amyloid β (Aβ) peptides in brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD), and it is likely related to an imbalance between production and clearance of the peptide. Aβ drainage along perivascular spaces has been postulated to be one of the mechanisms that mediate the peptide clearance from the brain. We therefore devised a novel method to visualize solute clearance in real time in the living mouse brain using laser guided bolus dye injections and multiphoton imaging. This methodology allows high spatial and temporal resolution and revealed the kinetics of ISF clearance. We found that the ISF drains along perivascular spaces of arteries and capillaries but not veins, and its clearance exhibits a bi-exponential profile. ISF drainage requires a functional vasculature, as solute clearance decreased when perfusion was impaired. In addition, reduced solute clearance was observed in transgenic mice with significant vascular amyloid deposition; we suggest the existence of a feed-forward mechanism, by which amyloid deposition promotes further amyloid deposition. This important finding provides a mechanistic link between cerebrovascular disease and Alzheimer disease and suggests that facilitation of Aβ clearance along the perivascular pathway should be considered as a new target for therapeutic approaches to Alzheimer disease and cerebral amyloid angiopathy.

Alzheimer's disease (AD) is defined by the presence of intraneuronal neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau aggregates as well as extracellular amyloid-beta plaques. The presence and spread of tau pathology through the brain is classified by Braak stages and thought to correlate with the progression of AD. Several in vitro and in vivo studies have examined the ability of tau pathology to move from one neuron to the next, suggesting a "prion-like" spread of tau aggregates may be an underlying cause of Braak tau staging in AD. Using the HEK293 TauRD-P301S-CFP/YFP expressing biosensor cells as a highly sensitive and specific tool to identify the presence of seed competent aggregated tau in brain lysate-i.e., tau aggregates that are capable of recruiting and misfolding monomeric tau-, we detected substantial tau seeding levels in the entorhinal cortex from human cases with only very rare NFTs, suggesting that soluble tau aggregates can exist prior to the development of overt tau pathology. We next looked at tau seeding levels in human brains of varying Braak stages along six regions of the Braak Tau Pathway. Tau seeding levels were detected not only in the brain regions impacted by pathology, but also in the subsequent non-pathology containing region along the Braak pathway. These data imply that pathogenic tau aggregates precede overt tau pathology in a manner that is consistent with transneuronal spread of tau aggregates. We then detected tau seeding in frontal white matter tracts and the optic nerve, two brain regions comprised of axons that contain little to no neuronal cell bodies, implying that tau aggregates can indeed traverse along axons. Finally, we isolated cytosolic and synaptosome fractions along the Braak Tau Pathway from brains of varying Braak stages. Phosphorylated and seed competent tau was significantly enriched in the synaptic fraction of brain regions that did not have extensive cellular tau pathology, further suggesting that aggregated tau seeds move through the human brain along synaptically connected neurons. Together, these data provide further evidence that the spread of tau aggregates through the human brain along synaptically connected networks results in the pathogenesis of human Alzheimer's disease.

Rare mutations in the gene encoding for tau (MAPT, microtubule-associated protein tau) cause frontotemporal dementia-spectrum (FTD-s) disorders, including FTD, progressive supranuclear palsy (PSP) and corticobasal syndrome, and a common extended haplotype spanning across the MAPT locus is associated with increased risk of PSP and Parkinson's disease. We identified a rare tau variant (p.A152T) in a patient with a clinical diagnosis of PSP and assessed its frequency in multiple independent series of patients with neurodegenerative conditions and controls, in a total of 15 369 subjects. Tau p.A152T significantly increases the risk for both FTD-s (n = 2139, OR = 3.0, CI: 1.6-5.6, P = 0.0005) and Alzheimer's disease (AD) (n = 3345, OR = 2.3, CI: 1.3-4.2, P = 0.004) compared with 9047 controls. Functionally, p.A152T (i) decreases the binding of tau to microtubules and therefore promotes microtubule assembly less efficiently; and (ii) reduces the tendency to form abnormal fibers. However, there is a pronounced increase in the formation of tau oligomers. Importantly, these findings suggest that other regions of the tau protein may be crucial in regulating normal function, as the p.A152 residue is distal to the domains considered responsible for microtubule interactions or aggregation. These data provide both the first genetic evidence and functional studies supporting the role of MAPT p.A152T as a rare risk factor for both FTD-s and AD and the concept that rare variants can increase the risk for relatively common, complex neurodegenerative diseases, but since no clear significance threshold for rare genetic variation has been established, some caution is warranted until the findings are further replicated.

Cerebral amyloid angiopathy (CAA) is an age-related small vessel disease, characterised pathologically by progressive deposition of amyloid β in the cerebrovascular wall. The Boston criteria are used worldwide for the in-vivo diagnosis of CAA but have not been updated since 2010, before the emergence of additional MRI markers. We report an international collaborative study aiming to update and externally validate the Boston diagnostic criteria across the full spectrum of clinical CAA presentations.In this multicentre, hospital-based, retrospective, MRI and neuropathology diagnostic accuracy study, we did a retrospective analysis of clinical, radiological, and histopathological data available to sites participating in the International CAA Association to formulate updated Boston criteria and establish their diagnostic accuracy across different populations and clinical presentations. Ten North American and European academic medical centres identified patients aged 50 years and older with potential CAA-related clinical presentations (ie, spontaneous intracerebral haemorrhage, cognitive impairment, or transient focal neurological episodes), available brain MRI, and histopathological assessment for CAA diagnosis. MRI scans were centrally rated at Massachusetts General Hospital (Boston, MA, USA) for haemorrhagic and non-haemorrhagic CAA markers, and brain tissue samples were rated by neuropathologists at the contributing sites. We derived the Boston criteria version 2.0 (v2.0) by selecting MRI features to optimise diagnostic specificity and sensitivity in a prespecified derivation cohort (Boston cases 1994-2012, n=159), then externally validated the criteria in a prespecified temporal validation cohort (Boston cases 2012-18, n=59) and a geographical validation cohort (non-Boston cases 2004-18; n=123), comparing accuracy of the new criteria to the currently used modified Boston criteria with histopathological assessment of CAA as the diagnostic standard. We also assessed performance of the v2.0 criteria in patients across all cohorts who had the diagnostic gold standard of brain autopsy.The study protocol was finalised on Jan 15, 2017, patient identification was completed on Dec 31, 2018, and imaging analyses were completed on Sept 30, 2019. Of 401 potentially eligible patients presenting to Massachusetts General Hospital, 218 were eligible to be included in the analysis; of 160 patient datasets from other centres, 123 were included. Using the derivation cohort, we derived provisional criteria for probable CAA requiring the presence of at least two strictly lobar haemorrhagic lesions (ie, intracerebral haemorrhages, cerebral microbleeds, or foci of cortical superficial siderosis) or at least one strictly lobar haemorrhagic lesion and at least one white matter characteristic (ie, severe visible perivascular spaces in centrum semiovale or white matter hyperintensities in a multispot pattern). The sensitivity and specificity of these criteria were 74·8% (95% CI 65·4-82·7) and 84·6% (71·9-93·1) in the derivation cohort, 92·5% (79·6-98·4) and 89·5% (66·9-98·7) in the temporal validation cohort, 80·2% (70·8-87·6) and 81·5% (61·9-93·7) in the geographical validation cohort, and 74·5% (65·4-82·4) and 95·0% (83·1-99·4) in all patients who had autopsy as the diagnostic standard. The area under the receiver operating characteristic curve (AUC) was 0·797 (0·732-0·861) in the derivation cohort, 0·910 (0·828-0·992) in the temporal validation cohort, 0·808 (0·724-0·893) in the geographical validation cohort, and 0·848 (0·794-0·901) in patients who had autopsy as the diagnostic standard. The v2.0 Boston criteria for probable CAA had superior accuracy to the current Boston criteria (sensitivity 64·5% [54·9-73·4]; specificity 95·0% [83·1-99·4]; AUC 0·798 [0·741-0854]; p=0·0005 for comparison of AUC) across all individuals who had autopsy as the diagnostic standard.The Boston criteria v2.0 incorporate emerging MRI markers of CAA to enhance sensitivity without compromising their specificity in our cohorts of patients aged 50 years and older presenting with spontaneous intracerebral haemorrhage, cognitive impairment, or transient focal neurological episodes. Future studies will be needed to determine generalisability of the v.2.0 criteria across the full range of patients and clinical presentations.US National Institutes of Health (R01 AG26484).

Brain metastases from lung adenocarcinoma (BM-LUAD) frequently cause patient mortality. To identify genomic alterations that promote brain metastases, we performed whole-exome sequencing of 73 BM-LUAD cases. Using case-control analyses, we discovered candidate drivers of brain metastasis by identifying genes with more frequent copy-number aberrations in BM-LUAD compared to 503 primary LUADs. We identified three regions with significantly higher amplification frequencies in BM-LUAD, including MYC (12 versus 6%), YAP1 (7 versus 0.8%) and MMP13 (10 versus 0.6%), and significantly more frequent deletions in CDKN2A/B (27 versus 13%). We confirmed that the amplification frequencies of MYC, YAP1 and MMP13 were elevated in an independent cohort of 105 patients with BM-LUAD. Functional assessment in patient-derived xenograft mouse models validated the notion that MYC, YAP1 or MMP13 overexpression increased the incidence of brain metastasis. These results demonstrate that somatic alterations contribute to brain metastases and that genomic sequencing of a sufficient number of metastatic tumors can reveal previously unknown metastatic drivers.

Alzheimer's disease (AD) is a complex genetic disorder with no effective treatments. More than 20 common markers have been identified, which are associated with AD. Recently, several rare variants have been identified in Amyloid Precursor Protein (APP), Triggering Receptor Expressed On Myeloid Cells 2 (TREM2) and Unc-5 Netrin Receptor C (UNC5C) that affect risk for AD. Despite the many successes, the genetic architecture of AD remains unsolved. We used Genome-wide Complex Trait Analysis to (1) estimate phenotypic variance explained by genetics; (2) calculate genetic variance explained by known AD single nucleotide polymorphisms (SNPs); and (3) identify the genomic locations of variation that explain the remaining unexplained genetic variance. In total, 53.24% of phenotypic variance is explained by genetics, but known AD SNPs only explain 30.62% of the genetic variance. Of the unexplained genetic variance, approximately 41% is explained by unknown SNPs in regions adjacent to known AD SNPs, and the remaining unexplained genetic variance outside these regions.

Objective Recent studies have shown that positron emission tomography (PET) tracer AV‐1451 exhibits high binding affinity for paired helical filament (PHF)‐tau pathology in Alzheimer's brains. However, the ability of this ligand to bind to tau lesions in other tauopathies remains controversial. Our goal was to examine the correlation of in vivo and postmortem AV‐1451 binding patterns in three autopsy‐confirmed non‐Alzheimer tauopathy cases. Methods We quantified in vivo retention of [F‐18]‐AV‐1451 and performed autoradiography, [H‐3]‐AV‐1451 binding assays, and quantitative tau measurements in postmortem brain samples from two progressive supranuclear palsy (PSP) cases and a MAPT P301L mutation carrier. They all underwent [F‐18]‐AV‐1451 PET imaging before death. Results The three subjects exhibited [F‐18]‐AV‐1451 in vivo retention predominantly in basal ganglia and midbrain. Neuropathological examination confirmed the PSP diagnosis in the first two subjects; the MAPT P301L mutation carrier had an atypical tauopathy characterized by grain‐like tau‐containing neurites in gray and white matter with heaviest burden in basal ganglia. In all three cases, autoradiography failed to show detectable [F‐18]‐AV‐1451 binding in multiple brain regions examined, with the exception of entorhinal cortex (reflecting incidental age‐related neurofibrillary tangles) and neuromelanin‐containing neurons in the substantia nigra (off‐target binding). The lack of a consistent significant correlation between in vivo [F‐18]‐AV‐1541 retention and postmortem in vitro binding and tau measures in these cases suggests that this ligand has low affinity for tau lesions primarily made of straight tau filaments. Interpretation AV‐1451 may have limited utility for in vivo selective and reliable detection of tau aggregates in these non‐Alzheimer tauopathies. ANN NEUROL 2017;81:117–128

Amyloid plaques and neurofibrillary tangles co-occur in Alzheimer disease, but with different topological and temporal patterns. Whether these two lesions are independent or pathobiologically related is uncertain. For example, amyloid deposition in the neocortex precedes the spread of tau neurofibrillary tangles from the limbic areas to the cortex. We examined the aggregation properties of tau isolated from human cases with early tau pathology (Braak II) with and without plaques. Using a well-established HEK cell biosensor assay, we show that tau from cases with plaques has an enhanced ability to induce tau aggregates compared to tau from cases without plaques. To further explore this effect, we combined mice carrying the APP/PS1 transgene array that develop plaques with rTg4510 mice carrying the P301L mutant human tau transgene that develop extensive tau pathology with age. The resulting APP/PS1-rTg4510 mice had a threefold increase in tau seeding activity over the rTg4510 strain, without change in tau production or extracellular release. Surprisingly, this effect was observed before overt amyloid deposition. The enhancement of tau aggregation was also apparent by an increase in histological measures of tau pathology in young APP/PS1-rTg4510 mice and an increase in high-molecular-weight tau. Overall, these data provide evidence that amyloid β acts to enhance tau pathology by increasing the formation of tau species capable of seeding new aggregates.

Because APOE locus variants contribute to risk of late-onset Alzheimer disease (LOAD) and to differences in age at onset (AAO), it is important to know whether other established LOAD risk loci also affect AAO in affected participants.To investigate the effects of known Alzheimer disease risk loci in modifying AAO and to estimate their cumulative effect on AAO variation using data from genome-wide association studies in the Alzheimer Disease Genetics Consortium.The Alzheimer Disease Genetics Consortium comprises 14 case-control, prospective, and family-based data sets with data on 9162 participants of white race/ethnicity with Alzheimer disease occurring after age 60 years who also had complete AAO information, gathered between 1989 and 2011 at multiple sites by participating studies. Data on genotyped or imputed single-nucleotide polymorphisms most significantly associated with risk at 10 confirmed LOAD loci were examined in linear modeling of AAO, and individual data set results were combined using a random-effects, inverse variance-weighted meta-analysis approach to determine whether they contribute to variation in AAO. Aggregate effects of all risk loci on AAO were examined in a burden analysis using genotype scores weighted by risk effect sizes.Age at disease onset abstracted from medical records among participants with LOAD diagnosed per standard criteria.Analysis confirmed the association of APOE with earlier AAO (P = 3.3 × 10(-96)), with associations in CR1 (rs6701713, P = 7.2 × 10(-4)), BIN1 (rs7561528, P = 4.8 × 10(-4)), and PICALM (rs561655, P = 2.2 × 10(-3)) reaching statistical significance (P < .005). Risk alleles individually reduced AAO by 3 to 6 months. Burden analyses demonstrated that APOE contributes to 3.7% of the variation in AAO (R(2) = 0.256) over baseline (R(2) = 0.221), whereas the other 9 loci together contribute to 2.2% of the variation (R(2) = 0.242).We confirmed an association of APOE (OMIM 107741) variants with AAO among affected participants with LOAD and observed novel associations of CR1 (OMIM 120620), BIN1 (OMIM 601248), and PICALM (OMIM 603025) with AAO. In contrast to earlier hypothetical modeling, we show that the combined effects of Alzheimer disease risk variants on AAO are on the scale of, but do not exceed, the APOE effect. While the aggregate effects of risk loci on AAO may be significant, additional genetic contributions to AAO are individually likely to be small.

Genetic loci for Alzheimer's disease (AD) have been identified in whites of European ancestry, but the genetic architecture of AD among other populations is less understood.We conducted a transethnic genome-wide association study (GWAS) for late-onset AD in Stage 1 sample including whites of European Ancestry, African-Americans, Japanese, and Israeli-Arabs assembled by the Alzheimer's Disease Genetics Consortium. Suggestive results from Stage 1 from novel loci were followed up using summarized results in the International Genomics Alzheimer's Project GWAS dataset.Genome-wide significant (GWS) associations in single-nucleotide polymorphism (SNP)-based tests (P < 5 × 10-8) were identified for SNPs in PFDN1/HBEGF, USP6NL/ECHDC3, and BZRAP1-AS1 and for the interaction of the (apolipoprotein E) APOE ε4 allele with NFIC SNP. We also obtained GWS evidence (P < 2.7 × 10-6) for gene-based association in the total sample with a novel locus, TPBG (P = 1.8 × 10-6).Our findings highlight the value of transethnic studies for identifying novel AD susceptibility loci.

Reversible cerebral vasoconstriction syndromes (RCVS) and primary angiitis of the central nervous system (PACNS) are invariably considered in the differential diagnosis of new cerebral arteriopathies. However, prompt and accurate diagnosis remains challenging. Here we compared the features of 159 RCVS to 47 PACNS patients and developed criteria for prompt bedside diagnosis. Recurrent thunderclap headache (TCH), and single TCH combined with either normal neuroimaging, border zone infarcts, or vasogenic edema, have 100% positive predictive value for diagnosing RCVS or RCVS‐spectrum disorders. In patients without TCH and positive angiography, neuroimaging can discriminate RCVS (no lesion) from PACNS (deep/brainstem infarcts). Ann Neurol 2016;79:882–894

Two patients with familial amyotrophic lateral sclerosis (ALS) and mutations in the gene encoding superoxide dismutase 1 (SOD1) were treated with a single intrathecal infusion of adeno-associated virus encoding a microRNA targeting SOD1. In Patient 1, SOD1 levels in spinal cord tissue as analyzed on autopsy were lower than corresponding levels in untreated patients with SOD1-mediated ALS and in healthy controls. Levels of SOD1 in cerebrospinal fluid were transiently and only slightly lower in Patient 1 but were not affected in Patient 2. In Patient 1, meningoradiculitis developed after the infusion; Patient 2 was pretreated with immunosuppressive drugs and did not have this complication. Patient 1 had transient improvement in the strength of his right leg, a measure that had been relatively stable throughout his disease course, but there was no change in his vital capacity. Patient 2 had stable scores on a composite measure of ALS function and a stable vital capacity during a 12-month period. This study showed that intrathecal microRNA can be used as a potential treatment for SOD1-mediated ALS.

Cerebral amyloid angiopathy (CAA) is characteristically associated with magnetic resonance imaging (MRI) biomarkers of small vessel brain injury, including strictly lobar cerebral microbleeds, cortical superficial siderosis, centrum semiovale perivascular spaces, and white matter hyperintensities. Although these neuroimaging markers reflect distinct pathophysiologic aspects in CAA, no studies to date have combined these structural imaging features to gauge total brain small vessel disease burden in CAA.To investigate whether a composite score can be developed to capture the total brain MRI burden of small vessel disease in CAA and to explore whether this score contributes independent and complementary information about CAA severity, defined as intracerebral hemorrhage during life or bleeding-related neuropathologic changes.This retrospective, cross-sectional study examined a single-center neuropathologic CAA cohort of eligible patients from the Massachusetts General Hospital from January 1, 1997, through December 31, 2012. Data analysis was performed from January 2, 2015, to January 9, 2016. Patients with pathologic evidence of CAA (ie, any presence of CAA from routinely collected brain biopsy specimen, biopsy specimen at hematoma evacuation, or autopsy) and available brain MRI sequences of adequate quality, including T2-weighted, T2*-weighted gradient-recalled echo, and/or susceptibility-weighted imaging and fluid-attenuated inversion recovery sequences, were considered for the study.Brain MRIs were rated for lobar cerebral microbleeds, cortical superficial siderosis, centrum semiovale perivascular spaces, and white matter hyperintensities. All 4 MRI lesions were incorporated into a prespecified ordinal total small vessel disease score, ranging from 0 to 6 points. Associations with severity of CAA-associated vasculopathic changes (fibrinoid necrosis and concentric splitting of the wall), clinical presentation, number of intracerebral hemorrhages, and other imaging markers not included in the score were explored using logistic and ordinal regression.In total, 105 patients with pathologically defined CAA were included: 52 with autopsies, 22 with brain biopsy specimens, and 31 with pathologic samples from hematoma evacuations. The mean (range) age of the patients was 73 (71-74) years, and 55 (52.4%) were women. In multivariable ordinal regression analysis, severity of CAA-associated vasculopathic changes (odds ratio, 2.40; 95% CI, 1.06-5.45; P = .04) and CAA presentation with symptomatic intracerebral hemorrhage (odds ratio, 2.23; 95% CI, 1.07-4.64; P = .03) were independently associated with the total MRI small vessel disease score. The score was associated with small, acute, diffusion-weighted imaging lesions and posterior white matter hyperintensities in adjusted analyses.This study provides evidence of concept validity of a total MRI small vessel disease score in CAA. After further validation, this approach can be potentially used in prospective clinical studies.

Dementia in Alzheimer's disease progresses alongside neurodegeneration1-4, but the specific events that cause neuronal dysfunction and death remain poorly understood. During normal ageing, neurons progressively accumulate somatic mutations5 at rates similar to those of dividing cells6,7 which suggests that genetic factors, environmental exposures or disease states might influence this accumulation5. Here we analysed single-cell whole-genome sequencing data from 319 neurons from the prefrontal cortex and hippocampus of individuals with Alzheimer's disease and neurotypical control individuals. We found that somatic DNA alterations increase in individuals with Alzheimer's disease, with distinct molecular patterns. Normal neurons accumulate mutations primarily in an age-related pattern (signature A), which closely resembles 'clock-like' mutational signatures that have been previously described in healthy and cancerous cells6-10. In neurons affected by Alzheimer's disease, additional DNA alterations are driven by distinct processes (signature C) that highlight C>A and other specific nucleotide changes. These changes potentially implicate nucleotide oxidation4,11, which we show is increased in Alzheimer's-disease-affected neurons in situ. Expressed genes exhibit signature-specific damage, and mutations show a transcriptional strand bias, which suggests that transcription-coupled nucleotide excision repair has a role in the generation of mutations. The alterations in Alzheimer's disease affect coding exons and are predicted to create dysfunctional genetic knockout cells and proteostatic stress. Our results suggest that known pathogenic mechanisms in Alzheimer's disease may lead to genomic damage to neurons that can progressively impair function. The aberrant accumulation of DNA alterations in neurodegeneration provides insight into the cascade of molecular and cellular events that occurs in the development of Alzheimer's disease.

The biophysical properties of neurons are the foundation for computation in the brain. Neuronal size is a key determinant of single neuron input-output features and varies substantially across species1-3. However, it is unknown whether different species adapt neuronal properties to conserve how single neurons process information4-7. Here we characterize layer 5 cortical pyramidal neurons across 10 mammalian species to identify the allometric relationships that govern how neuronal biophysics change with cell size. In 9 of the 10 species, we observe conserved rules that control the conductance of voltage-gated potassium and HCN channels. Species with larger neurons, and therefore a decreased surface-to-volume ratio, exhibit higher membrane ionic conductances. This relationship produces a conserved conductance per unit brain volume. These size-dependent rules result in large but predictable changes in somatic and dendritic integrative properties. Human neurons do not follow these allometric relationships, exhibiting much lower voltage-gated potassium and HCN conductances. Together, our results in layer 5 neurons identify conserved evolutionary principles for neuronal biophysics in mammals as well as notable features of the human cortex.

<h2>Abstract</h2> We evaluated the incidence, distribution, and histopathologic correlates of microvascular brain lesions in patients with severe COVID-19. Sixteen consecutive patients admitted to the intensive care unit with severe COVID-19 undergoing brain MRI for evaluation of coma or neurologic deficits were retrospectively identified. Eleven patients had punctate susceptibility-weighted imaging (SWI) lesions in the subcortical and deep white matter, eight patients had >10 SWI lesions, and four patients had lesions involving the corpus callosum. The distribution of SWI lesions was similar to that seen in patients with hypoxic respiratory failure, sepsis, and disseminated intravascular coagulation. Brain autopsy in one patient revealed that SWI lesions corresponded to widespread microvascular injury, characterized by perivascular and parenchymal petechial hemorrhages and microscopic ischemic lesions. Collectively, these radiologic and histopathologic findings add to growing evidence that patients with severe COVID-19 are at risk for multifocal microvascular hemorrhagic and ischemic lesions in the subcortical and deep white matter.

Mutations in alpha-synuclein (alpha S) and parkin cause heritable forms of Parkinson disease (PD). We hypothesized that neuronal parkin, a known E3 ubiquitin ligase, facilitates the formation of Lewy bodies (LBs), a pathological hallmark of PD. Here, we report that affinity-purified parkin antibodies labeled classical LBs in substantia nigra sections from four related human disorders: sporadic PD, inherited alphaS-linked PD, dementia with LBs (DLB), and LB-positive, parkin-linked PD. Anti-parkin antibodies also detected LBs in entorhinal and cingulate cortices from DLB brain and alphaS inclusions in sympathetic gangliocytes from sporadic PD. Double labeling with confocal microscopy of DLB midbrain sections revealed that approximately 90% of anti-alpha S-reactive LBs were also detected by a parkin antibody to amino acids 342 to 353. Accordingly, parkin proteins, including the 53-kd mature isoform, were present in affinity-isolated LBs from DLB cortex. Fluorescence resonance energy transfer and immunoelectron microscopy showed that alphaS and parkin co-localized within brainstem and cortical LBs. Biochemically, parkin appeared most enriched in cytosolic and postsynaptic fractions of adult rat brain, but also in purified, alpha S-rich presynaptic elements that additionally contained parkin's E2-binding partner, UbcH7. We conclude that parkin and UbcH7 are present with alphaS in subcellular compartments of normal brain and that parkin frequently co-localizes with alpha S aggregates in the characteristic LB inclusions of PD and DLB. These results suggest that functional parkin proteins may be required during LB formation.

We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)–linked mutant PS1 or PS2 significantly attenuated CCE and store depletion–activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid β peptide (Aβ42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Aβ42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.

Amyloid beta-peptide (Abeta) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD). We have previously reported that a well-characterized acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, inhibits Abeta production in cell-based experiments. Here, we assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

To identify gene expression patterns in human dopamine (DA) neurons in the substantia nigra pars compacta (SNc) of male and female control and Parkinson disease (PD) patients, we harvested DA neurons from frozen SNc from 16 subjects (4 male PDs, 4 female PDs, 4 male and 4 female controls) using Laser Capture microdissection and microarrays. We assessed for enrichment of functional categories with a hypergeometric distribution. The data were validated with QPCR. We observed that gender has a pervasive effect on gene expression in DA neurons. Genes upregulated in females relative to males are mainly involved in signal transduction and neuronal maturation, while in males some of the upregulated genes (alpha-synuclein and PINK1) were previously implicated in the pathogenesis of PD. In females with PD we found alterations in genes with protein kinase activity, genes involved in proteolysis and WNT signaling pathway, while in males with PD there were alterations in protein-binding proteins and copper-binding proteins. Our data reveal broad gender-based differences in gene expression in human dopaminergic neurons of SNc that may underlie the predisposition of males to PD. Moreover, we show that gender influences the response to PD, suggesting that the nature of the disease and the response to treatment may be gender-dependent.

Macrophage-1 antigen (Mac-1) (CD11b/CD18), a leukocyte beta2 integrin, facilitates neutrophil adhesion, transendothelial migration, phagocytosis, and respiratory burst, all of which may mediate reperfusion-induced injury to ischemic brain tissue in conditions such as stroke. To determine the role of Mac-1 during ischemia and reperfusion in the brain, we analyzed the effect of transient focal cerebral ischemia in mice genetically engineered with a specific deficiency in Mac-1.Transient focal ischemia/reperfusion was induced by occluding the left middle cerebral artery for 3 hours followed by a 21-hour reperfusion period in Mac-1-deficient (n=12) and wild-type (n=11) mice. Regional cerebral blood flow was determined with a laser-Doppler flowmeter. Brain sections were stained with 2% 2,3,5-triphenyltetrazolium chloride to determine the infarct volume. Neutrophil accumulation was determined by staining the brain sections with dichloroacetate esterase to identify neutrophils.Compared with the wild-type cohort, Mac-1-deficient mice had a 26% reduction in infarction volume (P<0.05). This was associated with a 50%, but statistically insignificant, reduction in the number of extravasated neutrophils in the infarcted areas of the brains in the mutant mice. There were no differences in regional cerebral blood flow between the 2 groups.Mac-1 deficiency reduces neutrophil infiltration and cerebral cell death after transient focal cerebral ischemia. This finding may be related to a reduction in neutrophil extravasation in Mac-1-deficient mice.

Recurrent glioblastomas (rGBM) invariably relapse after initial response to anti-VEGF therapy. There are 2 prevailing hypotheses on how these tumors escape antiangiogenic therapy: switch to VEGF-independent angiogenic pathways and vessel co-option. However, direct evidence in rGBM patients is lacking. Thus, we compared molecular, cellular, and vascular parameters in autopsy tissues from 5 rGBM patients who had been treated with the pan-VEGF receptor tyrosine kinase inhibitor cediranib versus 7 patients who received no therapy or chemoradiation but no antiangiogenic agents. After cediranib treatment, endothelial proliferation and glomeruloid vessels were decreased, and vessel diameters and perimeters were reduced to levels comparable to the unaffected contralateral brain hemisphere. In addition, tumor endothelial cells expressed molecular markers specific to the blood-brain barrier, indicative of a lack of revascularization despite the discontinuation of therapy. Surprisingly, in cediranib-treated GBM, cellular density in the central area of the tumor was lower than in control cases and gradually decreased toward the infiltrating edge, indicative of a change in growth pattern of rGBMs after cediranib treatment, unlike that after chemoradiation. Finally, cediranib-treated GBMs showed high levels of PDGF-C (platelet-derived growth factor C) and c-Met expression and infiltration by myeloid cells, which may potentially contribute to resistance to anti-VEGF therapy. In summary, we show that rGBMs switch their growth pattern after anti-VEGF therapy--characterized by lower tumor cellularity in the central area, decreased pseudopalisading necrosis, and blood vessels with normal molecular expression and morphology--without a second wave of angiogenesis.

We review the clinical, radiologic, and neuropathologic features of the hereditary and sporadic forms of cerebral amyloid angiopathy (CAA) associated with vascular deposition of the beta-amyloid peptide. Amino acid substitutions at 4 sites in the beta-amyloid precursor protein, all situated within the beta-amyloid peptide sequence itself, have been shown to cause heritable forms of CAA. The vascular diseases caused by these mutations are associated primarily with cerebral hemorrhages, white matter lesions, and cognitive impairment, and only variable extents of the plaque and neurofibrillary pathologies characteristic of Alzheimer disease. Sporadic CAA typically presents 20 or more years later than hereditary CAA, but is otherwise characterized by a comparable constellation of recurrent cerebral hemorrhages, white matter lesions, and cognitive impairment. The clinical, radiologic and pathologic similarities between hereditary and sporadic CAA suggest that important lessons for this common age-related process can be learned from the mechanisms by which mutation makes beta-amyloid tropic or toxic to vessels.

Abstract Advanced cerebrovascular β‐amyloid deposition (cerebral amyloid angiopathy, CAA) is associated with cerebral microbleeds, but the precise relationship between CAA burden and microbleeds is undefined. We used T2*‐weighted magnetic resonance imaging (MRI) and noninvasive amyloid imaging with Pittsburgh Compound B (PiB) to analyze the spatial relationship between CAA and microbleeds. On coregistered positron emission tomography (PET) and MRI images, PiB retention was increased at microbleed sites compared to simulated control lesions ( p = 0.002) and declined with increasing distance from the microbleed ( p &lt; 0.0001). These findings indicate that microbleeds occur preferentially in local regions of concentrated amyloid and support therapeutic strategies aimed at reducing vascular amyloid deposition. Ann Neurol 2010

Cerebral volume loss has long been associated with normal aging, but whether this is due to aging itself or to age-related diseases, including incipient Alzheimer disease, is uncertain. To understand the changes that occur in the aging brain, we examined the cerebral cortex of 27 normal individuals ranging in age from 56 to 103 years. None fulfilled the criteria for the neuropathologic diagnosis of Alzheimer disease or other neurodegenerative disease. Seventeen of the elderly participants had cognitive testing an average of 6.7 months prior to death. We used quantitative approaches to analyze cortical thickness, neuronal number, and density. Frontal and temporal neocortical regions had clear evidence of cortical thinning with age, but total neuronal numbers in frontal and temporal neocortical regions remained relatively constant during a 50-year age range. These data suggest that loss of neuronal and dendritic architecture, rather than loss of neurons, underlies neocortical volume loss with increasing age in the absence of Alzheimer disease.

Background and Purpose— Small, asymptomatic microbleeds commonly accompany larger symptomatic macrobleeds. It is unclear whether microbleeds and macrobleeds represent arbitrary categories within a single continuum versus truly distinct events with separate pathophysiologies. Methods— We performed 2 complementary retrospective analyses. In a radiographic analysis, we measured and plotted the volumes of all hemorrhagic lesions detected by gradient-echo MRI among 46 consecutive patients with symptomatic primary lobar intracerebral hemorrhage diagnosed as probable or possible cerebral amyloid angiopathy. In a second neuropathologic analysis, we performed blinded qualitative and quantitative examinations of amyloid-positive vessel segments in 6 autopsied subjects whose MRI scans demonstrated particularly high microbleed counts (&gt;50 microbleeds on MRI, n=3) or low microbleed counts (&lt;3 microbleeds, n=3). Results— Plotted on a logarithmic scale, the volumes of 163 hemorrhagic lesions identified on scans from the 46 subjects fell in a distinctly bimodal distribution with mean volumes for the 2 modes of 0.009 cm 3 and 27.5 cm 3 . The optimal cut point for separating the 2 peaks (determined by receiver operating characteristics) corresponded to a lesion diameter of 0.57 cm. On neuropathologic analysis, the high microbleed-count autopsied subjects showed significantly thicker amyloid-positive vessel walls than the low microbleed-count subjects (proportional wall thickness 0.53±0.01 versus 0.37±0.01; P &lt;0.0001; n=333 vessel segments analyzed). Conclusions— These findings suggest that cerebral amyloid angiopathy-associated microbleeds and macrobleeds comprise distinct entities. Increased vessel wall thickness may predispose to formation of microbleeds relative to macrobleeds.

Hereditary sensory and autonomic neuropathy type 1 (HSAN1) causes sensory loss that predominantly affects the lower limbs, often preceded by hyperpathia and spontaneous shooting or lancinating pain. It is caused by several missense mutations in the genes encoding 2 of the 3 subunits of the enzyme serine palmitoyltransferase (SPT). The mutant forms of the enzyme show a shift from their canonical substrate L-serine to the alternative substrate L-alanine. This shift leads to increased formation of neurotoxic deoxysphingolipids (dSLs). Our initial analysis showed that in HEK cells transfected with SPTLC1 mutants, dSL generation was modulated in vitro in the presence of various amino acids. We therefore examined whether in vivo specific amino acid substrate supplementation influenced dSL levels and disease severity in HSAN1. In mice bearing a transgene expressing the C133W SPTLC1 mutant linked to HSAN1, a 10% L-serine–enriched diet reduced dSL levels. L-serine supplementation also improved measures of motor and sensory performance as well as measures of male fertility. In contrast, a 10% L-alanine–enriched diet increased dSL levels and led to severe peripheral neuropathy. In a pilot study with 14 HSAN1 patients, L-serine supplementation similarly reduced dSL levels. These observations support the hypothesis that an altered substrate selectivity of the mutant SPT is key to the pathophysiology of HSAN1 and raise the prospect of l-serine supplementation as a first treatment option for this disorder.

Although it is clear that astrocytes and microglia cluster around dense-core amyloid plaques in Alzheimer disease (AD), whether they are primarily attracted to amyloid deposits or are just reacting to plaque-associated neuritic damage remains elusive. We postulate that astrocytes and microglia may differentially respond to fibrillar amyloid β. Therefore, we quantified the size distribution of dense-core thioflavin-S (ThioS)-positive plaques in the temporal neocortex of 40 AD patients and the microglial and astrocyte responses in their vicinity (≤50 μm) and performed correlations between both measures. As expected, both astrocytes and microglia were clearly spatially associated with ThioS-positive plaques (p = 0.0001, ≤50 μm vs. >50 μm from their edge), but their relationship to ThioS-positive plaque size differed: larger ThioS-positive plaques were associated with more surrounding activated microglia (p = 0.0026), but this effect was not observed with reactive astrocytes. Microglial response to dense-core plaques seems to be proportional to their size, which we postulate reflects a chemotactic effect of amyloid β. By contrast, plaque-associated astrocytic response does not correlate with plaque size and seems to parallel the behavior of plaque-associated neuritic damage.

The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy is characterized by age-dependent cerebrovascular deposition of amyloid-beta (Abeta) starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months; soluble Abeta levels are elevated long before vascular deposition takes place in this model. It has been suggested that elevated soluble Abeta levels alone are sufficient to impair cerebral blood flow (CBF) regulation thereby contributing to the early progression of Alzheimer's disease. Using laser speckle flowmetry through an intact skull, we studied the impact of elevated soluble Abeta levels and vascular Abeta deposition on a wide range of CBF responses to evaluate vasodilation and vasoconstriction in young or aged Tg2576 mice. Nineteen-month-old Tg2576 with severe vascular Abeta deposits showed an attenuated hyperaemic response during hypercapnia and whisker stimulation compared to wild-type littermates. The anticipated increase in CBF due to isoflurane anaesthesia was also suppressed, as were the typical hypoperfusion responses during cortical spreading depression and alpha-chloralose anaesthesia. The responses of 8-month-old Tg2576 with elevated soluble Abeta levels, but without vascular Abeta deposition, did not differ from age-matched controls. In conclusion, our data suggest that vascular Abeta deposition is associated with impaired vasodilator as well as vasoconstrictor responses to a wide range of stimuli. These responses do not differ from controls when studied non-invasively prior to vascular Abeta deposition, thus challenging the view that elevated soluble Abeta levels are sufficient to cause cerebrovascular dysfunction.

In both trisomy 21 and rare cases of triplication of amyloid precursor protein (APP) Alzheimer’s disease (AD) pathological changes are believed to be secondary to increased expression of APP. We hypothesized that sporadic AD may also be associated with changes in transcription of APP or its metabolic partners. To address this issue, temporal neocortex of 27 AD and 21 non-demented control brains was examined to assess mRNA levels of APP isoforms (total APP, APP containing the Kunitz protease inhibitor domain [APP-KPI] and APP770) and APP metabolic enzymatic partners (the APP cleaving enzymes β-secretase [BACE] and presenilin-1 [PS-1], and putative clearance molecules, low-density lipoprotein receptor protein [LRP] and apolipoprotein E [apoE]). Furthermore, we evaluated how changes in APP at the mRNA level affect the amount of Tris buffer extractable APP protein and Aβ40 and 42 peptides in AD and control brains. As assessed by quantitative PCR, APP-KPI (p = 0.007), APP770 (p = 0.004), PS-1 (p = 0.004), LRP (p = 0.003), apoE (p = 0.0002) and GFAP (p < 0.0001) mRNA levels all increased in AD, and there was a shift from APP695 (a neuronal isoform) towards KPI containing isoforms that are present in glia as well. APP-KPI mRNA levels correlated with soluble APPα-KPI protein (sAPPα-KPI) levels measured by ELISA (τ = 0.33, p = 0.015 by Kendall’s rank correlation); in turn, soluble APPα-KPI protein levels positively correlated with Tris-extractable, soluble Aβ40 (p = 0.046) and 42 levels (p = 0.007). The ratio of soluble APPα-KPI protein levels to total APP protein increased in AD, and also correlated with GFAP protein levels in AD. These results suggest that altered transcription of APP in AD is proportionately associated with Aβ peptide, may occur in the context of gliosis, and may contribute to Aβ deposition in sporadic AD.

The purpose of this study was to characterize the epileptogenicity of tubers and surrounding cortex in patients with tuberous sclerosis complex (TSC). Three pediatric patients with TSC and intractable epilepsy underwent surgical resection of tubers associated with epileptogenic foci. In all patients, presurgical imaging revealed a prominent tuber that correlated on electroencephalography (EEG) with frequent interictal epileptiform discharges and electrographic seizures. Intracranial electrocorticography (ECoG) was performed using subdural grids placed over the tuber and surrounding cortex and depth electrodes positioned directly within the tuber. In all three patients, the depth electrode within the tuber was electrographically silent, whereas the surrounding cortical tissue showed significant epileptiform activity. The tuber and the electrically active adjacent cortex were resected. The patients experienced a drastic reduction in seizure frequency postsurgery. Epileptogenicity of cortical tubers may derive not from the lesion itself, but rather from the perturbation or abnormal development of the surrounding cortex.

Previous clinical studies have documented a close relationship between cerebrovascular disease and risk of Alzheimer's disease. We examined possible mechanistic interactions through use of experimental stroke models in a transgenic mouse model of β-amyloid deposition (APPswe/PS1dE9). Following middle cerebral artery occlusion, we observed a rapid increase in amyloid plaque burden in the region surrounding the infarction. In human tissue samples, however, we were unable to detect a localized increase in amyloid burden adjacent to cerebral infarcts. To resolve this discrepancy, we generated cerebral microstrokes in amyloid precursor protein mouse models with the photosensitive dye Rose bengal, and monitored plaque formation in real time using multiphoton microscopy. We observed a striking increase in the number of new plaques and amyloid angiopathy in the area immediately surrounding the infarcted area; however, the effect was transient, potentially resolving the discord between mouse and human tissue. We did not detect changes in candidate proteins related to β-amyloid generation or degradation such as β-amyloid-converting enzyme, amyloid precursor protein, presenilin 1, neprylisin or insulin-degrading enzyme. Together, these results demonstrate that strokes can trigger accelerated amyloid deposition, most likely through interference with amyloid clearance pathways. Additionally, this study indicates that focal ischaemia provides an experimental paradigm in which to study the mechanisms of plaque seeding and growth.

Modern scientific knowledge of how memory functions are organized in the human brain originated from the case of Henry G. Molaison (H.M.), an epileptic patient whose amnesia ensued unexpectedly following a bilateral surgical ablation of medial temporal lobe structures, including the hippocampus. The neuroanatomical extent of the 1953 operation could not be assessed definitively during H.M.'s life. Here we describe the results of a procedure designed to reconstruct a microscopic anatomical model of the whole brain and conduct detailed 3D measurements in the medial temporal lobe region. This approach, combined with cellular-level imaging of stained histological slices, demonstrates a significant amount of residual hippocampal tissue with distinctive cytoarchitecture. Our study also reveals diffuse pathology in the deep white matter and a small, circumscribed lesion in the left orbitofrontal cortex. The findings constitute new evidence that may help elucidate the consequences of H.M.'s operation in the context of the brain's overall pathology.

Anti-amyloid-beta immunization leads to amyloid clearance in patients with Alzheimer's disease, but the effect of vaccination on amyloid-beta-induced neuronal pathology has not been quantitatively examined. The objectives of this study were to address the effects of anti-amyloid-beta active immunization on neurite trajectories and the pathological hallmarks of Alzheimer's disease in the human hippocampus. Hippocampal sections from five patients with Alzheimer's disease enrolled in the AN1792 Phase 2a trial were compared with those from 13 non-immunized Braak-stage and age-matched patients with Alzheimer's disease, and eight age-matched non-demented controls. Analyses included neurite curvature ratio as a quantitative measure of neuritic abnormalities, amyloid and tau loads, and a quantitative characterization of plaque-associated neuritic dystrophy and astrocytosis. Amyloid load and density of dense-core plaques were decreased in the immunized group compared to non-immunized patients (P < 0.01 and P < 0.001, respectively). The curvature ratio in non-immunized patients with Alzheimer's disease was elevated compared to non-demented controls (P < 0.0001). In immunized patients, however, the curvature ratio was normalized when compared to non-immunized patients (P < 0.0001), and not different from non-demented controls. In the non-immunized patients, neurites close to dense-core plaques (within 50 microm) were more abnormal than those far from plaques (i.e. beyond 50 microm) (P < 0.0001). By contrast, in the immunized group neurites close to and far from the remaining dense-core plaques did not differ, and both were straighter compared to the non-immunized patients (P < 0.0001). Compared to non-immunized patients, dense-core plaques remaining after immunization had similar degree of astrocytosis (P = 0.6060), more embedded dystrophic neurites (P < 0.0001) and were more likely to have mitochondrial accumulation (P < 0.001). In addition, there was a significant decrease in the density of paired helical filament-1-positive neurons in the immunized group as compared to the non-immunized (P < 0.05), but not in the density of Alz50 or thioflavin-S positive tangles, suggesting a modest effect of anti-amyloid-beta immunization on tangle pathology. Clearance of amyloid plaques upon immunization with AN1792 effectively improves a morphological measure of neurite abnormality in the hippocampus. This improvement is not just attributable to the decrease in plaque load, but also occurs within the halo of the remaining dense-core plaques. However, these remaining plaques still retain some of their toxic potential. Anti-amyloid-beta immunization might also ameliorate the hippocampal tau pathology through a decrease in tau phosphorylation. These data agree with preclinical animal studies and further demonstrate that human anti-amyloid-beta immunization does not merely clear amyloid from the Alzheimer's disease brain, but reduces some of the neuronal alterations that characterize Alzheimer's disease.

-Whole-slide imaging technology offers promise for rapid, Internet-based telepathology consultations between institutions. Before implementation, technical issues, pathologist adaptability, and morphologic pitfalls must be well characterized.-To determine whether interpretation of whole-slide images differed from glass-slide interpretation in difficult surgical pathology cases.-Diagnostically challenging pathology slides from a variety of anatomic sites from an outside laboratory were scanned into whole digital format. Digital and glass slides were independently diagnosed by 2 subspecialty pathologists. Reference, digital, and glass-slide interpretations were compared. Operator comments on technical issues were gathered.-Fifty-three case pairs were analyzed. There was agreement among digital, glass, and reference diagnoses in 45 cases (85%) and between digital and glass diagnoses in 48 (91%) cases. There were 5 digital cases (9%) discordant with both reference and glass diagnoses. Further review of each of these cases indicated an incorrect digital whole-slide interpretation. Neoplastic cases showed better correlation (93%) than did cases of nonneoplastic disease (88%). Comments on discordant cases related to digital whole technology focused on issues such as fine resolution and navigating ability at high magnification.-Overall concordance between digital whole-slide and standard glass-slide interpretations was good at 91%. Adjustments in technology, case selection, and technology familiarization should improve performance, making digital whole-slide review feasible for broader telepathology subspecialty consultation applications.

Abstract Objective Mutations in the X‐linked adrenoleukodystrophy (X‐ALD) protein cause accumulation of unbranched saturated very‐long‐chain fatty acids, particularly in brain and adrenal cortex. In humans, the genetic defect causes progressive inflammatory demyelination in the brain, where very‐long‐chain fatty acids accumulate within phospholipid fractions such as lysophosphatidylcholine. Methods To address mechanisms of inflammation, we studied microglial activation in human ALD (10 autopsies) and lysophosphatidylcholine (C24:0) injection into the parietal cortex of mice. Results Unexpectedly, we found a zone lacking microglia within perilesional white matter, immediately beyond the actively demyelinating lesion edge. Surrounding this zone we observed clusters of activated and apoptotic microglia within subcortical white matter. Lysophosphatidylcholine (C24:0) injection in mice led to widespread microglial activation and apoptosis. Interpretation Our data suggest that the distinct mononuclear phagocytic cell response seen in cerebral X‐ALD results, at least in part, from aberrant signaling to cognate receptors on microglia. Our findings support a hypothesis that microglial apoptosis in perilesional white matter represents an early stage in lesion evolution and may be an appropriate target for intervention in X‐ALD patients with evidence of cerebral demyelination. Ann Neurol 2008

1. The pharmacological properties of excitatory amino acid responses on ganglion cells dissociated from the rat retina were examined with the use of the whole‐cell voltage‐clamp technique. 2. L‐Glutamate at a concentration of 50 microM produced inward non‐desensitizing currents at negative holding potentials in nearly every cell tested (83%, n = 18) In physiological solutions, L‐glutamate responses reversed at approximately ‐9 mV, and higher concentrations of this agonist introduced a desensitizing component to the response. 3. At negative holding potentials, kainate (25‐125 microM) produced inward currents in all of the cells tested (n = 37). These currents never desensitized, even at high agonist concentrations, and reversed near ‐6 mV. Currents induced by 50 microM‐kainate were reversibly antagonized by kynurenate (100‐300 microM) but not by 100 microM‐2‐amino‐5‐phosphonovalerate (APV). 4. Quisqualate generated smaller, non‐desensitizing currents in only 50% of the cells tested (n = 38). Quisqualate responses reversed in polarity near ‐4 mV and were maximal at an agonist dose of 25 microM, with higher concentrations introducing a rapidly desensitizing component without a detectable increase in amplitude. Currents produced by quisqualate at a concentration of 50 microM were not antagonized by either 750 microM‐kynurenate or 100 microM‐APV. 5. N‐Methyl‐D‐aspartate (NMDA) produced inward currents at negative holding potentials in 68% of the cells tested (n = 31), but only when magnesium was excluded from the extracellular medium. NMDA currents were non‐desensitizing at agonist concentrations of up to 200 microM, with higher concentrations introducing a rapidly desensitizing component. NMDA (200 microM) responses were blocked by APV (100 microM) and kynurenate (300 microM) and reversed near ‐1 mV. 6. Responses generated by kainate (50‐125 microM) were antagonized by quisqualate (30‐250 microM). This antagonism occurred even in cells having no measurable response to quisqualate alone, suggesting the possibility that quisqualate may be acting both as an agonist, in the 50% of the cells that have the quisqualate‐specific receptor, and as an antagonist, at the kainate‐specific site on all cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Classical immunohistochemical studies in the Alzheimer disease (AD) brain reveal prominent glial reactions, but whether this pathological feature is due primarily to cell proliferation or to a phenotypic change of existing resting cells remains controversial. We performed double-fluorescence immunohistochemical studies of astrocytes and microglia, followed by unbiased stereology-based quantitation in temporal cortex of 40 AD patients and 32 age-matched nondemented subjects. Glial fibrillary acidic protein (GFAP) and major histocompatibility complex II (MHC2) were used as markers of astrocytic and microglial activation, respectively. Aldehyde dehydrogenase 1 L1 and glutamine synthetase were used as constitutive astrocytic markers, and ionized calcium-binding adaptor molecule 1 (IBA1) as a constitutive microglial marker. As expected, AD patients had higher numbers of GFAP(+) astrocytes and MHC2(+) microglia than the nondemented subjects. However, both groups had similar numbers of total astrocytes and microglia and, in the AD group, these total numbers remained essentially constant over the clinical course of the disease. The GFAP immunoreactivity of astrocytes, but not the MHC2 immunoreactivity of microglia, increased in parallel with the duration of the clinical illness in the AD group. Cortical atrophy contributed to the perception of increased glia density. We conclude that a phenotypic change of existing glial cells, rather than a marked proliferation of glial precursors, accounts for the majority of the glial responses observed in the AD brain.

<h2>Summary</h2><h3>Background</h3> Cognitive decline is a debilitating manifestation of disease progression in Parkinson's disease. We aimed to develop a clinical–genetic score to predict global cognitive impairment in patients with the disease. <h3>Methods</h3> In this longitudinal analysis, we built a prediction algorithm for global cognitive impairment (defined as Mini Mental State Examination [MMSE] ≤25) using data from nine cohorts of patients with Parkinson's disease from North America and Europe assessed between 1986 and 2016. Candidate predictors of cognitive decline were selected through a backward eliminated Cox's proportional hazards analysis using the Akaike's information criterion. These were used to compute the multivariable predictor on the basis of data from six cohorts included in a discovery population. Independent replication was attained in patients from a further three independent longitudinal cohorts. The predictive score was rebuilt and retested in 10 000 training and test sets randomly generated from the entire study population. <h3>Findings</h3> 3200 patients with Parkinson's disease who were longitudinally assessed with 27 022 study visits between 1986 and 2016 in nine cohorts from North America and Europe were assessed for eligibility. 235 patients with MMSE ≤25 at baseline and 135 whose first study visit occurred more than 12 years from disease onset were excluded. The discovery population comprised 1350 patients (after further exclusion of 334 with missing covariates) from six longitudinal cohorts with 5165 longitudinal visits over 12·8 years (median 2·8, IQR 1·6–4·6). Age at onset, baseline MMSE, years of education, motor exam score, sex, depression, and β-glucocerebrosidase (<i>GBA</i>) mutation status were included in the prediction model. The replication population comprised 1132 patients (further excluding 14 patients with missing covariates) from three longitudinal cohorts with 19 127 follow-up visits over 8·6 years (median 6·5, IQR 4·1–7·2). The cognitive risk score predicted cognitive impairment within 10 years of disease onset with an area under the curve (AUC) of more than 0·85 in both the discovery (95% CI 0·82–0·90) and replication (95% CI 0·78–0·91) populations. Patients scoring in the highest quartile for cognitive risk score had an increased hazard for global cognitive impairment compared with those in the lowest quartile (hazard ratio 18·4 [95% CI 9·4–36·1]). Dementia or disabling cognitive impairment was predicted with an AUC of 0·88 (95% CI 0·79–0·94) and a negative predictive value of 0·92 (95% 0·88–0·95) at the predefined cutoff of 0·196. Performance was stable in 10 000 randomly resampled subsets. <h3>Interpretation</h3> Our predictive algorithm provides a potential test for future cognitive health or impairment in patients with Parkinson's disease. This model could improve trials of cognitive interventions and inform on prognosis. <h3>Funding</h3> National Institutes of Health, US Department of Defense.

Abstract Context.—Whole-slide imaging technology offers promise for rapid, Internet-based telepathology consultations between institutions. Before implementation, technical issues, pathologist adaptability, and morphologic pitfalls must be well characterized. Objective.—To determine whether interpretation of whole-slide images differed from glass-slide interpretation in difficult surgical pathology cases. Design.—Diagnostically challenging pathology slides from a variety of anatomic sites from an outside laboratory were scanned into whole digital format. Digital and glass slides were independently diagnosed by 2 subspecialty pathologists. Reference, digital, and glass-slide interpretations were compared. Operator comments on technical issues were gathered. Results.—Fifty-three case pairs were analyzed. There was agreement among digital, glass, and reference diagnoses in 45 cases (85%) and between digital and glass diagnoses in 48 (91%) cases. There were 5 digital cases (9%) discordant with both reference and glass diagnoses. Further review of each of these cases indicated an incorrect digital whole-slide interpretation. Neoplastic cases showed better correlation (93%) than did cases of nonneoplastic disease (88%). Comments on discordant cases related to digital whole technology focused on issues such as fine resolution and navigating ability at high magnification. Conclusions.—Overall concordance between digital whole-slide and standard glass-slide interpretations was good at 91%. Adjustments in technology, case selection, and technology familiarization should improve performance, making digital whole-slide review feasible for broader telepathology subspecialty consultation applications.

Individually, heart failure (HF) and Alzheimer's disease (AD) are severe threats to population health, and their potential coexistence is an alarming prospect. In addition to sharing analogous epidemiological and genetic profiles, biochemical characteristics, and common triggers, the authors recently recognized common molecular and pathological features between the 2 conditions. Whereas cognitive impairment has been linked to HF through perfusion defects, angiopathy, and inflammation, whether patients with AD present with myocardial dysfunction, and if the 2 conditions bear a common pathogenesis as neglected siblings are unknown.Here, the authors investigated whether amyloid beta (Aβ) protein aggregates are present in the hearts of patients with a primary diagnosis of AD, affecting myocardial function.The authors examined myocardial function in a retrospective cross-sectional study from a cohort of AD patients and age-matched controls. Imaging and proteomics approaches were used to identify and quantify Aβ deposits in AD heart and brain specimens compared with controls. Cell shortening and calcium transients were measured on isolated adult cardiomyocytes.Echocardiographic measurements of myocardial function suggest that patients with AD present with an anticipated diastolic dysfunction. As in the brain, Aβ40 and Aβ42 are present in the heart, and their expression is increased in AD.Here, the authors provide the first report of the presence of compromised myocardial function and intramyocardial deposits of Aβ in AD patients. The findings depict a novel biological framework in which AD may be viewed either as a systemic disease or as a metastatic disorder leading to heart, and possibly multiorgan failure. AD and HF are both debilitating and life-threatening conditions, affecting enormous patient populations. Our findings underline a previously dismissed problem of a magnitude that will require new diagnostic approaches and treatments for brain and heart disease, and their combination.

Hexanucleotide repeat expansions in chromosome 9 open reading frame 72 (C9orf72) have recently been linked to frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis, and may be the most common genetic cause of both neurodegenerative diseases. Genetic variants at TMEM106B influence risk for the most common neuropathological subtype of FTLD, characterized by inclusions of TAR DNA-binding protein of 43 kDa (FTLD-TDP). Previous reports have shown that TMEM106B is a genetic modifier of FTLD-TDP caused by progranulin (GRN) mutations, with the major (risk) allele of rs1990622 associating with earlier age at onset of disease. Here, we report that rs1990622 genotype affects age at death in a single-site discovery cohort of FTLD patients with C9orf72 expansions (n = 14), with the major allele correlated with later age at death (p = 0.024). We replicate this modifier effect in a 30-site international neuropathological cohort of FTLD-TDP patients with C9orf72 expansions (n = 75), again finding that the major allele associates with later age at death (p = 0.016), as well as later age at onset (p = 0.019). In contrast, TMEM106B genotype does not affect age at onset or death in 241 FTLD-TDP cases negative for GRN mutations or C9orf72 expansions. Thus, TMEM106B is a genetic modifier of FTLD with C9orf72 expansions. Intriguingly, the genotype that confers increased risk for developing FTLD-TDP (major, or T, allele of rs1990622) is associated with later age at onset and death in C9orf72 expansion carriers, providing an example of sign epistasis in human neurodegenerative disease.

The Boston criteria are the basis for a noninvasive diagnosis of cerebral amyloid angiopathy (CAA) in the setting of lobar intracerebral hemorrhage (ICH). We assessed the accuracy of these criteria in individuals with lobar microbleeds (MBs) without ICH.We identified individuals aged >55 years having brain magnetic resonance imaging (MRI) and pathological assessment of CAA in a single academic hospital and a community-based population (Framingham Heart Study [FHS]). We determined the positive predictive value (PPV) of the Boston criteria for CAA in both cohorts, using lobar MBs as the only hemorrhagic lesion to fulfill the criteria.We included 102 individuals: 55 from the hospital-based cohort and 47 from FHS (mean age at MRI 74.7 ± 8.5 and 83.4 ± 10.9 years; CAA prevalence 60% and 46.8%; cases with any lobar MB 49% and 21.3%; and cases with ≥2 strictly lobar MBs 29.1% and 8.5%, respectively). PPV of "probable CAA" (≥2 strictly lobar MBs) was 87.5% (95% confidence interval [CI], 60.4-97.8) and 25% (95% CI, 13.2-78) in hospital and general populations, respectively.Strictly lobar MBs strongly predict CAA in non-ICH individuals when found in a hospital context. However, their diagnostic accuracy in the general population appears limited.

Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aβ aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aβ cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aβ, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein.

The most common monogenic cause of small-vessel disease leading to ischemic stroke and vascular dementia is the neurodegenerative syndrome cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), which is associated with mutations in the Notch 3 receptor. CADASIL pathology is characterized by vascular smooth muscle cell degeneration and accumulation of diagnostic granular osmiophilic material (GOM) in vessels. The functional nature of the Notch 3 mutations causing CADASIL and their mechanistic connection to small-vessel disease and GOM accumulation remain enigmatic. To gain insight into how Notch 3 function is linked to CADASIL pathophysiology, we studied two phenotypically distinct mutations, C455R and R1031C, respectively associated with early and late onset of stroke, by using hemodynamic analyses in transgenic mouse models, receptor activity assays in cell culture, and proteomic examination of postmortem human tissue. We demonstrate that the C455R and R1031C mutations define different hypomorphic activity states of Notch 3, a property linked to ischemic stroke susceptibility in mouse models we generated. Importantly, these mice develop osmiophilic deposits and other age-dependent phenotypes that parallel remarkably the human condition. Proteomic analysis of human brain vessels, carrying the same CADASIL mutations, identified clusterin and collagen 18 α1/endostatin as GOM components. Our findings link loss of Notch signaling with ischemic cerebral small-vessel disease, a prevalent human condition. We determine that CADASIL pathophysiology is associated with hypomorphic Notch 3 function in vascular smooth muscle cells and implicate the accumulation of clusterin and collagen 18 α1/endostatin in brain vessel pathology.

To assess the relative frequency of unique mutations and their associated characteristics in 97 individuals with mutations in progranulin (GRN), an important cause of frontotemporal lobar degeneration (FTLD).A 46-site International Frontotemporal Lobar Degeneration Collaboration was formed to collect cases of FTLD with TAR DNA-binding protein of 43-kDa (TDP-43)-positive inclusions (FTLD-TDP). We identified 97 individuals with FTLD-TDP with pathogenic GRN mutations (GRN+ FTLD-TDP), assessed their genetic and clinical characteristics, and compared them with 453 patients with FTLD-TDP in which GRN mutations were excluded (GRN- FTLD-TDP). No patients were known to be related. Neuropathologic characteristics were confirmed as FTLD-TDP in 79 of the 97 GRN+ FTLD-TDP cases and all of the GRN- FTLD-TDP cases.Age at onset of FTLD was younger in patients with GRN+ FTLD-TDP vs GRN- FTLD-TDP (median, 58.0 vs 61.0 years; P < .001), as was age at death (median, 65.5 vs 69.0 years; P < .001). Concomitant motor neuron disease was much less common in GRN+ FTLD-TDP vs GRN- FTLD-TDP (5.4% vs 26.3%; P < .001). Fifty different GRN mutations were observed, including 2 novel mutations: c.139delG (p.D47TfsX7) and c.378C>A (p.C126X). The 2 most common GRN mutations were c.1477C>T (p.R493X, found in 18 patients, representing 18.6% of GRN cases) and c.26C>A (p.A9D, found in 6 patients, representing 6.2% of cases). Patients with the c.1477C>T mutation shared a haplotype on chromosome 17; clinically, they resembled patients with other GRN mutations. Patients with the c.26C>A mutation appeared to have a younger age at onset of FTLD and at death and more parkinsonian features than those with other GRN mutations.GRN+ FTLD-TDP differs in key features from GRN- FTLD-TDP.

[F-18]-MK-6240, a novel tau positron emission tomography (PET) tracer recently discovered for the in vivo detection of neurofibrillary tangles, has the potential to improve diagnostic accuracy in the detection of Alzheimer disease. We have examined regional and substrate-specific binding patterns as well as possible off-target binding of this tracer on human brain tissue to advance towards its validation. We applied [F-18]-MK-6240 phosphor screen and high resolution autoradiography to postmortem samples from patients with a definite pathological diagnosis of Alzheimer disease, frontotemporal lobar degeneration-tau (Pick's disease, progressive supranuclear palsy and corticobasal degeneration), chronic traumatic encephalopathy, frontotemporal lobar degeneration-Tar DNA-binding protein 43 (TDP-43), dementia with Lewy bodies, cerebral amyloid angiopathy and elderly controls free of pathologic changes of neurodegenerative disease. We also directly compared the binding properties of [F-18]-MK-6240 and [F-18]-AV-1451 in human tissue, and examined potential nonspecific binding of both tau tracers to monoamine oxidases (MAO) by using autoradiography in the presence of selective monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) inhibitors. Our data indicate that MK-6240 strongly binds to neurofibrillary tangles in Alzheimer disease but does not seem to bind to a significant extent to tau aggregates in non-Alzheimer tauopathies, suggesting that it may have a limited utility for the in vivo detection of these pathologies. There is no evidence of binding to lesions containing β-amyloid, α-synuclein or TDP-43. In addition, we identified MK-6240 strong off-target binding to neuromelanin and melanin-containing cells, and some weaker binding to areas of hemorrhage. These binding patterns are nearly identical to those previously reported by our group and others for [F-18]-AV-1451. Of note, [F-18]-MK-6240 and [F-18]-AV-1451 autoradiographic binding signals were only weakly displaced by competing concentrations of selective MAO-B inhibitor deprenyl but not by MAO-A inhibitor clorgyline, suggesting that MAO enzymes do not appear to be a significant binding target of any of these two tracers. Together these novel findings provide relevant insights for the correct interpretation of in vivo [F-18]-MK-6240 PET imaging.

Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer’s disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

Compelling genetic evidence links the amyloid precursor protein (APP) to Alzheimer's disease (AD) and several theories have been advanced to explain the relationship. A leading hypothesis proposes that a small amphipathic fragment of APP, the amyloid β-protein (Aβ), self-associates to form soluble aggregates that impair synaptic and network activity. Here, we used the most disease-relevant form of Aβ, protein isolated from AD brain. Using this material, we show that the synaptotoxic effects of Aβ depend on expression of APP and that the Aβ-mediated impairment of synaptic plasticity is accompanied by presynaptic effects that disrupt the excitatory/inhibitory (E/I) balance. The net increase in the E/I ratio and inhibition of plasticity are associated with Aβ localizing to synapses and binding of soluble Aβ aggregates to synapses requires the expression of APP. Our findings indicate a role for APP in AD pathogenesis beyond the generation of Aβ and suggest modulation of APP expression as a therapy for AD. SIGNIFICANCE STATEMENT Here, we report on the plasticity-disrupting effects of amyloid β-protein (Aβ) isolated from Alzheimer's disease (AD) brain and the requirement of amyloid precursor protein (APP) for these effects. We show that Aβ-containing AD brain extracts block hippocampal LTP, augment glutamate release probability, and disrupt the excitatory/inhibitory balance. These effects are associated with Aβ localizing to synapses and genetic ablation of APP prevents both Aβ binding and Aβ-mediated synaptic dysfunctions. Our results emphasize the importance of APP in AD and should stimulate new studies to elucidate APP-related targets suitable for pharmacological manipulation.

The accumulation of neurofibrillary tangles in Alzheimer’s disease (AD) propagates with characteristic spatiotemporal patterns which follow brain network connections, implying trans-synaptic transmission of tauopathy. Since misfolded tau has been shown to transmit across synapses in AD animal models, we hypothesized that synapses in AD patients may contain misfolded tau. By immunofluorescence imaging of bipartite synapses from AD subjects, we detected tau protein in 38.4% of presynaptic and 50.9% of postsynaptic terminals. The pre/post distribution for hyperphosphorylated tau was 26.9%/30.7%, and for misfolded tau 18.3%/19.3%. In the temporal cortex, microscopic aggregates of tau, containing ultra-stable oligomers, were estimated to accumulate within trillions of synapses, outnumbering macroscopic tau aggregates such as tangles by 10000 fold. Non-demented elderly also showed considerable synaptic tau hyperphosphorylation and some misfolding, implicating the synapse as one of the first subcellular compartments affected by tauopathy. Misfolding of tau protein appeared to occur in situ inside synaptic terminals, without mislocalizing or mistrafficking. Misfolded tau at synapses may represent early signs of neuronal degeneration, mediators of synaptotoxicity, and anatomical substrates for transmitting tauopathy, but its actual role in these processes remain to be elucidated.