Fabio Di Domenico

Alzheimer disease (AD) is the most common form of dementia among the elderly and is characterized by progressive loss of memory and cognition. Epidemiological data show that the incidence of AD increases with age and doubles every 5 years after 65 years of age. From a neuropathological point of view, amyloid-β-peptide (Aβ) leads to senile plaques, which, together with hyperphosphorylated tau-based neurofibrillary tangles and synapse loss, are the principal pathological hallmarks of AD. Aβ is associated with the formation of reactive oxygen (ROS) and nitrogen (RNS) species, and induces calcium-dependent excitotoxicity, impairment of cellular respiration, and alteration of synaptic functions associated with learning and memory. Oxidative stress was found to be associated with type 2 diabetes mellitus (T2DM), which (i) represents another prevalent disease associated with obesity and often aging, and (ii) is considered to be a risk factor for AD development. T2DM is characterized by high blood glucose levels resulting from increased hepatic glucose production, impaired insulin production and peripheral insulin resistance, which close resemble to the brain insulin resistance observed in AD patients. Furthermore, growing evidence suggests that oxidative stress plays a pivotal role in the development of insulin resistance and vice versa. This review article provides molecular aspects and the pharmacological approaches from both preclinical and clinical data interpreted from the point of view of oxidative stress with the aim of highlighting progresses in this field.

The clinical symptoms of Alzheimer disease (AD) include a gradual memory loss and subsequent dementia, and neuropathological deposition of senile plaques and neurofibrillary tangles. At the molecular level, AD subjects present overt amyloid β (Aβ) production and tau hyperphosphorylation. Aβ species have been proposed to overactivate the phosphoinositide3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis, which plays a central role in proteostasis. The current study investigated the status of the PI3K/Akt/mTOR pathway in post-mortem tissue from the inferior parietal lobule (IPL) at three different stages of AD: late AD, amnestic mild cognitive impairment (MCI) and pre-clinical AD (PCAD). Our findings suggest that the alteration of mTOR signaling and autophagy occurs at early stages of AD. We found a significant increase in Aβ (1-42) levels, associated with reduction in autophagy (Beclin-1 and LC-3) observed in PCAD, MCI, and AD subjects. Related to the autophagy impairment, we found a hyperactivation of PI3K/Akt/mTOR pathway in IPL of MCI and AD subjects, but not in PCAD, along with a significant decrease in phosphatase and tensin homolog. An increase in two mTOR downstream targets, p70S6K and 4EBP1, occurred in AD and MCI subjects. Both AD and MCI subjects showed increased, insulin receptor substrate 1, a candidate biomarker of brain insulin resistance, and GSK-3β, a kinase targeting tau phosphorylation. Nevertheless, tau phosphorylation was increased in the clinical groups. The results hint at a link between Aβ and the PI3K/Akt/mTOR axis and provide further insights into the relationship between AD pathology and insulin resistance. In addition, we speculate that the alteration of mTOR signaling in the IPL of AD and MCI subjects, but not in PCAD, is due to the lack of substantial increase in oxidative stress. The figure represents the three different stages of Alzheimer Disease: Preclinical Alzheimer Disease (PCAD), Mild cognitive impairment (MCI) and late stage of Alzheimer Disease. The progression of the disease is associated with a reduction in autophagy (Beclin-1 and LC-3) observed in Inferior parietal lobe of PCAD, MCI, and AD subjects (light red). Related to the autophagy impairment, the graph shows the impairment of PI3K/Akt/mTOR in MCI and AD subjects (dark red).

Compelling evidence indicates that the mammalian target of rapamycin (mTOR) signaling pathway is involved in cellular senescence, organismal aging and age-dependent diseases. mTOR is a conserved serine/threonine kinase that is known to be part of two different protein complexes: mTORC1 and mTORC2, which differ in some components and in upstream and downstream signalling. In multicellular organisms, mTOR regulates cell growth and metabolism in response to nutrients, growth factors and cellular energy conditions. Growing studies highlight that disturbance in mTOR signalling in the brain affects multiple pathways including glucose metabolism, energy production, mitochondrial function, cell growth and autophagy. All these events are key players in age-related cognitive decline such as development of Alzheimer disease (AD). The current review discusses the main regulatory roles of mTOR signalling in the brain, in particular focusing on autophagy, glucose metabolism and mitochondrial functions. Targeting mTOR in the CNS can offer new prospective for drug discovery; however further studies are needed for a comprehensive understanding of mTOR, which lies at the crossroads of multiple signals involved in AD etiology and pathogenesis.

Alzheimer's disease (AD) is the most common form of dementia affecting elderly people. AD is a multifaceted pathology characterized by accumulation of extracellular neuritic plaques, intracellular neurofibrillary tangles (NFTs) and neuronal loss mainly in the cortex and hippocampus. AD etiology appears to be linked to a multitude of mechanisms that have not been yet completely elucidated. For long time, it was considered that insulin signaling has only peripheral actions but now it is widely accepted that insulin has neuromodulatory actions in the brain. Insulin signaling is involved in numerous brain functions including cognition and memory that are impaired in AD. Recent studies suggest that AD may be linked to brain insulin resistance and patients with diabetes have an increased risk of developing AD compared to healthy individuals. Indeed insulin resistance, increased inflammation and impaired metabolism are key pathological features of both AD and diabetes. However, the precise mechanisms involved in the development of AD in patients with diabetes are not yet fully understood. In this review we will discuss the role played by aberrant brain insulin signaling in AD. In detail, we will focus on the role of insulin signaling in the deposition of neuritic plaques and intracellular NFTs. Considering that insulin mitigates beta-amyloid deposition and phosphorylation of tau, pharmacological strategies restoring brain insulin signaling, such as intranasal delivery of insulin, could have significant therapeutic potential in AD treatment.

Oxidative stress is involved in various and numerous pathological states including several age-related neurodegenerative diseases. Peroxidation of the membrane lipid bilayer is one of the major sources of free radical-mediated injury that directly damages neurons causing increased membrane rigidity, decreased activity of membrane-bound enzymes, impairment of membrane receptors and altered membrane permeability and eventual cell death. Moreover, the peroxidation of polyunsaturated fatty acids leads to the formation of aldehydes, which can act as toxic by-products. One of the most abundant and cytotoxic lipid -derived aldehydes is 4-hydroxy 2-nonenal (HNE). HNE toxicity is mainly due to the alterations of cell functions by the formation of covalent adducts of HNE with proteins. A key marker of lipid peroxidation, HNE-protein adducts, were found to be elevated in brain tissues and body fluids of Alzheimer disease, Parkinson disease, Huntington disease and amyotrophic lateral sclerosis subjects and/or models of the respective age-related neurodegenerative diseases. Although only a few proteins were identified as common targets of HNE modification across all these listed disorders, a high overlap of these proteins occurs concerning the alteration of common pathways, such as glucose metabolism or mitochondrial function that are known to contribute to cognitive decline. Within this context, despite the different etiological and pathological mechanisms that lead to the onset of different neurodegenerative diseases, the formation of HNE-protein adducts might represent the shared leit-motif, which aggravates brain damage contributing to disease specific clinical presentation and decline in cognitive performance observed in each case.

Alzheimer disease (AD) is associated with multiple etiologies and pathological mechanisms, among which oxidative stress (OS) appears as a major determinant. Intriguingly, OS arises in various pathways regulating brain functions, and it seems to link different hypotheses and mechanisms of AD neuropathology with high fidelity. The brain is particularly vulnerable to oxidative damage, mainly because of its unique lipid composition, resulting in an amplified cascade of redox reactions that target several cellular components/functions ultimately leading to neurodegeneration. The present review highlights the "OS hypothesis of AD," including amyloid beta-peptide-associated mechanisms, the role of lipid and protein oxidation unraveled by redox proteomics, and the antioxidant strategies that have been investigated to modulate the progression of AD. Collected studies from our groups and others have contributed to unraveling the close relationships between perturbation of redox homeostasis in the brain and AD neuropathology by elucidating redox-regulated events potentially involved in both the pathogenesis and progression of AD. However, the complexity of AD pathological mechanisms requires an in-depth understanding of several major intracellular pathways affecting redox homeostasis and relevant for brain functions. This understanding is crucial to developing pharmacological strategies targeting OS-mediated toxicity that may potentially contribute to slow AD progression as well as improve the quality of life of persons with this severe dementing disorder.

Numerous studies have shown that neuronal lipids are highly susceptible to oxidative stress including in those brain areas directly involved in the neurodegenerative process of Alzheimer's disease (AD). Lipid peroxidation directly damages membranes and also generates a number of secondary biologically active products (toxic aldehydes)that are capable of easily attacking lipids, proteins, and DNA. Accumulating evidence has demonstrated regionally increased brain lipid peroxidation in patients with AD; however, extensive studies on specific targets of lipid peroxidation-induced damage are still missing. The present study represents a further step in understanding the relationship between oxidative modification of protein and neuronal death associated with AD. We used a proteomics approach to determine specific targets of lipid peroxidation in AD brain, both in hippocampus and inferior parietal lobule, by coupling immunochemical detection of 4-hydroxynonenal-bound proteins with 2-D polyacrylamide gel electrophoresis and MS analysis. We identified 4-hydroxynonenal-bound proteins in the hippocampus and inferior parietal lobule brain regions of subjects with AD. The identified proteins play different biological functions including energy metabolism, antioxidant system, and structural proteins, thus impairing multiple molecular pathways. Our results provide further evidence for the role of lipid peroxidation in the pathogenesis of AD.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability characterized by the presence of three copies of chromosome 21 (Chr21). Individuals with DS have sufficient neuropathology for a diagnosis of Alzheimer's disease (AD) after the age of 40 years. The aim of our study is to gain new insights in the molecular mechanisms impaired in DS subjects that eventually lead to the development of dementia. We evaluate the PI3K/Akt/mTOR axis in the frontal cortex from DS cases (under the age of 40 years) and DS with AD neuropathology compared with age-matched controls (Young and Old). The PI3K/Akt/mTOR axis may control several key pathways involved in AD that, if aberrantly regulated, affect amyloid beta (Aβ) deposition and tau phosphorylation. Our results show a hyperactivation of PI3K/Akt/mTOR axis in individuals with DS, with and without AD pathology, in comparison with respective controls. The PI3K/Akt/mTOR deregulation results in decreased autophagy, inhibition of IRS1 and GSK3β activity. Moreover, our data suggest that aberrant activation of the PI3K/Akt/mTOR axis acts in parallel to RCAN1 in phosphorylating tau, in DS and DS/AD. In conclusion, this study provides insights into the neuropathological mechanisms that may be engaged during the development of AD in DS. We suggest that deregulation of this signaling cascade is already evident in young DS cases and persist in the presence of AD pathology. The impairment of the PI3K/Akt/mTOR axis in DS population might represent a key-contributing factor to the neurodegenerative process that culminates in Alzheimer-like dementia.

Alzheimer disease (AD) is the most common form of dementia among the elderly and is characterized by progressive loss of memory and cognition. These clinical features are due in part to the increase of reactive oxygen and nitrogen species that mediate neurotoxic effects. The up-regulation of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system is one of the earlier events in the adaptive response to stress. HO-1/BVR-A reduces the intracellular levels of pro-oxidant heme and generates equimolar amounts of the free radical scavengers biliverdin-IX alpha (BV)/bilirubin-IX alpha (BR) as well as the pleiotropic gaseous neuromodulator carbon monoxide (CO) and ferrous iron. Two main and opposite hypotheses for a role of the HO-1/BVR-A system in AD propose that this system mediates neurotoxic and neuroprotective effects, respectively. This apparent controversy was mainly due to the fact that for over about 20years HO-1 was the only player on which all the analyses were focused, excluding the other important and essential component of the entire system, BVR. Following studies from the Butterfield laboratory that reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative post-translational modifications in the brain of subjects with AD and amnestic mild cognitive impairment (MCI) subjects, a debate was opened on the real pathophysiological and clinical significance of BVR-A. In this paper we provide a review of the main discoveries about the HO/BVR system in AD and MCI, and propose a mechanism that reconciles these two hypotheses noted above of neurotoxic and the neuroprotective aspects of this important stress responsive system.

Cervical cancer lesions are a major threat to the health of women, representing the second most common cancer worldwide. The unanimously recognized etiological factor in the causation of cervical cancer is the infection with human papilloma virus (HPV). HPV infection, although necessary, is not per se sufficient to induce cancer. Other factors have to be involved in the progression of infected cells to the full neoplastic phenotype. Oxidative stress represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. Oxidative stress is known to perturb the cellular redox status thus leading to alteration of gene expression responses through the activation of several redox-sensitive transcription factors. This signaling cascade affects both cell growth and cell death. The ability of naturally occurring antioxidants to modulate cellular signal transduction pathways, through the activation/repression of multiple redox-sensitive transcription factors, has been claimed for their potential therapeutic use as chemopreventive agents. Among these compounds, polyphenols have been found to be promising agents toward cervical cancer. In addition to acting as antioxidants, polyphenols display a wide variety of biological function including induction of apoptosis, growth arrest, inhibition of DNA synthesis and modulation of signal transduction pathways. They can interfere with each stage of carcinogenesis initiation, promotion and progression to prevent cancer development. The present review discusses current knowledge of the major molecular pathways, which are involved in HPV-driven cancerogenesis, and the ability of polyphenols to modulate these pathways. By acting at specific steps of viral transformation cascade, polyphenols have been demonstrated to selectively inhibit tumor cell growth and may be a promising therapeutic tool for treatment of cervical cancer. In addition, recent results obtained in clinical trials using polyphenols are also discussed. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

DS is the most frequent genetic cause of intellectual disability characterized by the anomalous presence of three copies of chromosome 21. One of the peculiar features of DS is the onset of Alzheimer's disease neuropathology after the age of 40years characterized by deposition of senile plaques and neurofibrillary tangles. Growing studies demonstrated that increased oxidative damage, accumulation of unfolded/damaged protein aggregates and dysfunction of intracellular degradative system are key players in neurodegenerative processes. In this study, redox proteomics approach was used to analyze the frontal cortex from DS subjects under the age of 40 compared with age-matched controls, and proteins found to be increasingly carbonylated were identified. Interestingly, our results showed that oxidative damage targets specifically different components of the intracellular quality control system such as GRP78, UCH-L1, V0-ATPase, cathepsin D and GFAP that couples with decreased activity of the proteasome and autophagosome formation observed. We also reported a slight but consistent increase of Aβ 1-42 SDS- and PBS-soluble form and tau phosphorylation in DS versus CTR. We suggest that disturbance in the proteostasis network could contribute to the accumulation of protein aggregates, such as amyloid deposits and NFTs, which occur very early in DS. It is likely that a sub-optimal functioning of degradative systems occur in DS neurons, which in turn provide the basis for further accumulation of toxic protein aggregates. The results of this study suggest that oxidation of protein members of the proteostatis network is an early event in DS and might contribute to neurodegenerative phenomena.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Oxidative and nitrosative stress plays a principal role in the pathogenesis of AD. The induction of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in the brain represents one of the earliest mechanisms activated by cells to counteract the noxious effects of increased reactive oxygen species and reactive nitrogen species. Although initially proposed as a neuroprotective system in AD brain, the HO-1/BVR-A pathophysiological features are under debate. We previously reported alterations in BVR activity along with decreased phosphorylation and increased oxidative/nitrosative posttranslational modifications in the brain of subjects with AD and those with mild cognitive impairment (MCI). Furthermore, other groups proposed the observed increase in HO-1 in AD brain as a possible neurotoxic mechanism. Here we provide new insights about HO-1 in the brain of subjects with AD and MCI, the latter condition being the transitional phase between normal aging and early AD. HO-1 protein levels were significantly increased in the hippocampus of AD subjects, whereas HO-2 protein levels were significantly decreased in both AD and MCI hippocampi. In addition, significant increases in Ser-residue phosphorylation together with increased oxidative posttranslational modifications were found in the hippocampus of AD subjects. Interestingly, despite the lack of oxidative stress-induced AD neuropathology in cerebellum, HO-1 demonstrated increased Ser-residue phosphorylation and oxidative posttranslational modifications in this brain area, suggesting HO-1 as a target of oxidative damage even in the cerebellum. The significance of these findings is profound and opens new avenues into the comprehension of the role of HO-1 in the pathogenesis of AD.

Proteomics techniques are continuously being developed to further understanding of biology and disease. Many of the pathways that are relevant to disease mechanisms rely on the identification of post-translational modifications (PTMs) such as phosphorylation, acetylation, and glycosylation. Much attention has also been focused on oxidative PTMs which include protein carbonyls, protein nitration, and the incorporation of fatty acids and advanced glycation products to amino acid side chains, amongst others. The introduction of these PTMs in the cell can occur due to the attack of reactive oxygen and nitrogen species (ROS and RNS, respectively) on proteins. ROS and RNS can be present as a result of normal metabolic processes as well as external factors such as UV radiation, disease, and environmental toxins. The imbalance of ROS and RNS with antioxidant cellular defenses leads to a state of oxidative stress, which has been implicated in many diseases. Redox proteomics techniques have been used to characterize oxidative PTMs that result as a part of normal cell signaling processes as well as oxidative stress conditions. This review highlights many of the redox proteomics techniques which are currently available for several oxidative PTMs and brings to the reader's attention the application of redox proteomics for understanding disease pathogenesis in neurodegenerative disorders and others such as cancer, kidney, and heart diseases. © 2013 Wiley Periodicals, Inc. Mass Spec Rev 33: 277–301, 2014.

Accumulation of oxidative damage is a common feature of neurodegeneration that, together with mitochondrial dysfunction, point to the fact that reactive oxygen species are major contributors to loss of neuronal homoeostasis and cell death. Among several targets of oxidative stress, free-radical-mediated damage to proteins is particularly important in aging and age-related neurodegenerative diseases. In the majority of cases, oxidative-stress-mediated post-translational modifications cause non-reversible modifications of protein structure that consistently lead to impaired function. Redox proteomics methods are powerful tools to unravel the complexity of neurodegeneration, by identifying brain proteins with oxidative post-translational modifications that are detrimental for protein function. The present review discusses the current literature showing evidence of impaired pathways linked to oxidative stress possibly involved in the neurodegenerative process leading to the development of Alzheimer-like dementia. In particular, we focus attention on dysregulated pathways that underlie neurodegeneration in both aging adults with DS (Down's syndrome) and AD (Alzheimer's disease). Since AD pathology is age-dependent in DS and shows similarities with AD, identification of common oxidized proteins by redox proteomics in both DS and AD can improve our understanding of the overlapping mechanisms that lead from normal aging to development of AD. The most relevant proteomics findings highlight that disturbance of protein homoeostasis and energy production are central mechanisms of neurodegeneration and overlap in aging DS and AD. Protein oxidation affects crucial intracellular functions and may be considered a 'leitmotif' of degenerating neurons. Therapeutic strategies aimed at preventing/reducing multiple components of processes leading to accumulation of oxidative damage will be critical in future studies.

Significance: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder and represents one of the most disabling conditions. AD shares many features in common with systemic insulin resistance diseases, suggesting that it can be considered as a metabolic disease, characterized by reduced insulin-stimulated growth and survival signaling, increased oxidative stress (OS), proinflammatory cytokine activation, mitochondrial dysfunction, impaired energy metabolism, and altered protein homeostasis. Recent Advances: Reduced glucose utilization and energy metabolism in AD have been associated with the buildup of amyloid-β peptide and hyperphosphorylated tau, increased OS, and the accumulation of unfolded/misfolded proteins. Mammalian target of rapamycin (mTOR), which is aberrantly activated in AD since early stages, plays a key role during AD neurodegeneration by, on one side, inhibiting insulin signaling as a negative feedback mechanism and, on the other side, regulating protein homeostasis (synthesis/clearance). Critical Issues: It is likely that the concomitant and mutual alterations of energy metabolism–mTOR signaling–protein homeostasis might represent a self-sustaining triangle of harmful events that trigger the degeneration and death of neurons and the development and progression of AD. Intriguingly, the altered cross-talk between the components of such a triangle of death, beyond altering the redox homeostasis of the neuron, is further exacerbated by increased levels of OS that target and impair key components of the pathways involved. Redox proteomic studies in human samples and animal models of AD-like dementia led to identification of oxidatively modified components of the pathways composing the triangle of death, therefore revealing the crucial role of OS in fueling this aberrant vicious cycle. Future Directions: The identification of compounds able to restore the function of the pathways targeted by oxidative damage might represent a valuable therapeutic approach to slow or delay AD. Antioxid. Redox Signal. 26, 364–387.

Clinical studies suggest a link between peripheral insulin resistance and cognitive dysfunction. Interestingly, post-mortem analyses of Alzheimer disease (AD) subjects demonstrated insulin resistance in the brain proposing a role for cognitive deficits observed in AD. However, the mechanisms responsible for the onset of brain insulin resistance (BIR) need further elucidations. Biliverdin reductase-A (BVR-A) emerged as a unique Ser/Thr/Tyr kinase directly involved in the insulin signaling and represents an up-stream regulator of the insulin signaling cascade. Because we previously demonstrated the oxidative stress (OS)-induced impairment of BVR-A in human AD brain, we hypothesize that BVR-A dysregulation could be associated with the onset of BIR in AD. In the present work, we longitudinally analyze the age-dependent changes of (i) BVR-A protein levels and activation, (ii) total oxidative stress markers levels (PC, HNE, 3-NT) as well as (iii) IR/IRS1 levels and activation in the hippocampus of the triple transgenic model of AD (3xTg-AD) mice. Furthermore, ad hoc experiments have been performed in SH-SY5Y neuroblastoma cells to clarify the molecular mechanism(s) underlying changes observed in mice. Our results show that OS-induced impairment of BVR-A kinase activity is an early event, which starts prior the accumulation of Aβ and tau pathology or the elevation of TNF-α, and that greatly contribute to the onset of BIR along the progression of AD pathology in 3xTg-Ad mice. Based on these evidence we, therefore, propose a new paradigm for which: OS-induced impairment of BVR-A is firstly responsible for a sustained activation of IRS1, which then causes the stimulation of negative feedback mechanisms (i.e. mTOR) aimed to turn-off IRS1 hyper-activity and thus BIR. Similar alterations characterize also the normal aging process in mice, positing BVR-A impairment as a possible bridge in the transition from normal aging to AD.

Down syndrome (DS) is the most common genetic cause of intellectual disability, due to partial or complete triplication of chromosome 21. DS subjects are characterized by a number of abnormalities including premature aging and development of Alzheimer disease (AD) neuropathology after approximately 40 years of age. Several studies show that oxidative stress plays a crucial role in the development of neurodegeneration in the DS population. Increased lipid peroxidation is one of the main events causing redox imbalance within cells through the formation of toxic aldehydes that easily react with DNA, lipids, and proteins. In this study we used a redox proteomics approach to identify specific targets of 4-hydroxynonenal modifications in the frontal cortex from DS cases with and without AD pathology. We suggest that a group of identified proteins followed a specific pattern of oxidation in DS vs young controls, probably indicating characteristic features of the DS phenotype; a second group of identified proteins showed increased oxidation in DS/AD vs DS, thus possibly playing a role in the development of AD. The third group of comparison, DS/AD vs old controls, identified proteins that may be considered specific markers of AD pathology. All the identified proteins are involved in important biological functions including intracellular quality control systems, cytoskeleton network, energy metabolism, and antioxidant response. Our results demonstrate that oxidative damage is an early event in DS, as well as dysfunctions of protein-degradation systems and cellular protective pathways, suggesting that DS subjects are more vulnerable to oxidative damage accumulation that might contribute to AD development. Further, considering that the majority of proteins have been already demonstrated to be oxidized in AD brain, our results strongly support similarities with AD in DS.

Alzheimer’s disease (AD), characterized by progressive loss of memory, language, reasoning and other cognitive functions, including dementia, is characterized pathologically by the presence of senile plaques, neurofibrillary tangles and synapse loss. Increased oxidative/nitrosative stress, decreased antioxidants, mitochondrial damage and other factors play major roles in the development and progression of AD. Strategies to reduce pro-oxidant species to ameliorate AD pathology have been proposed with mixed results. In this review, we focus on the most recent in vitro and in vivo antioxidant approaches for removing oxidant species with relevance to AD, including N-acetyl-l-cysteine, vitamin D, vitamin E, ferulic acid, tricyclodecan-9-yl-xanthogenate, selenium and melatonin as therapeutic stratagems in AD management. In addition, we reviewed the most effective mitochondria targeted antioxidants such as coenzyme Q10 and lipoic acid. We suggest the use of multitargeted approaches by formulas containing one or more antioxidant compounds may be more promising than single-agent approaches.

Free radical-mediated damage to macromolecules and the resulting oxidative modification of different cellular components are a common feature of aging, and this process becomes much more pronounced in age-associated pathologies, including Alzheimer disease (AD). In particular, proteins are particularly sensitive to oxidative stress-induced damage and these irreversible modifications lead to the alteration of protein structure and function. In order to maintain cell homeostasis, these oxidized/damaged proteins have to be removed in order to prevent their toxic accumulation. It is generally accepted that the age-related accumulation of "aberrant" proteins results from both the increased occurrence of damage and the decreased efficiency of degradative systems. One of the most important cellular proteolytic systems responsible for the removal of oxidized proteins in the cytosol and in the nucleus is the proteasomal system. Several studies have demonstrated the impairment of the proteasome in AD thus suggesting a direct link between accumulation of oxidized/misfolded proteins and reduction of this clearance system. In this review we discuss the impairment of the proteasome system as a consequence of oxidative stress and how this contributes to AD neuropathology. Further, we focus the attention on the oxidative modifications of a key component of the ubiquitin-proteasome pathway, UCHL1, which lead to the impairment of its activity.

Down syndrome (DS) individuals, by the age of 40s, are at increased risk to develop Alzheimer-like dementia, with deposition in brain of senile plaques and neurofibrillary tangles. Our laboratory recently demonstrated the disturbance of PI3K/AKT/mTOR axis in DS brain, prior and after the development of Alzheimer Disease (AD). The aberrant modulation of the mTOR signalling in DS and AD age-related cognitive decline affects crucial neuronal pathways, including insulin signaling and autophagy, involved in pathology onset and progression. Within this context, the therapeutic use of mTOR-inhibitors may prevent/attenuate the neurodegenerative phenomena. By our work we aimed to rescue mTOR signalling in DS mice by a novel rapamycin intranasal administration protocol (InRapa) that maximizes brain delivery and reduce systemic side effects. Ts65Dn mice were administered with InRapa for 12 weeks, starting at 6 months of age demonstrating, at the end of the treatment by radial arms maze and novel object recognition testing, rescued cognition. The analysis of mTOR signalling, after InRapa, demonstrated in Ts65Dn mice hippocampus the inhibition of mTOR (reduced to physiological levels), which led, through the rescue of autophagy and insulin signalling, to reduced APP levels, APP processing and APP metabolites production, as well as, to reduced tau hyperphosphorylation. In addition, a reduction of oxidative stress markers was also observed. These findings demonstrate that chronic InRapa administration is able to exert a neuroprotective effect on Ts65Dn hippocampus by reducing AD pathological hallmarks and by restoring protein homeostasis, thus ultimately resulting in improved cognition. Results are discussed in term of a potential novel targeted therapeutic approach to reduce cognitive decline and AD-like neuropathology in DS individuals.

Protein phosphorylation of serine, threonine, and tyrosine residues is one of the most prevalent post-translational modifications fundamental in mediating diverse cellular functions in living cells. Aberrant protein phosphorylation is currently recognized as a critical step in the pathogenesis and progression of Alzheimer disease (AD). Changes in the pattern of protein phosphorylation of different brain regions are suggested to promote AD transition from a presymptomatic to a symptomatic state in response to accumulating amyloid β-peptide (Aβ). Several experimental approaches have been utilized to profile alteration of protein phosphorylation in the brain, including proteomics. Among central pathways regulated by kinases/phosphatases those involved in the activation/inhibition of both pro survival and cell death pathways play a central role in AD pathology. We discuss in detail how aberrant phosphorylation could contribute to dysregulate p53 activity and insulin-mediated signaling. Taken together these results highlight that targeted therapeutic intervention, which can restore phosphorylation homeostasis, either acting on kinases and phosphatases, conceivably may prove to be beneficial to prevent or slow the development and progression of AD.

Several studies suggest that pathological changes in Alzheimer’s disease (AD) brain begin around 10–20 years before the onset of cognitive impairment. Biomarkers that can support early diagnosis and predict development of dementia would, therefore, be crucial for patient care and evaluation of drug efficacy. Although cerebrospinal fluid (CSF) levels of Aβ42, tau, and p-tau are well-established diagnostic biomarkers of AD, there is an urgent need to identify additional molecular alterations of neuronal function that can be evaluated at the systemic level. This study was focused on the analysis of oxidative stress-related modifications of the CSF proteome, from subjects with AD and amnestic mild cognitive impairment (aMCI). A targeted proteomics approach has been employed to discover novel CSF biomarkers that can augment the diagnostic and prognostic accuracy of current leading CSF biomarkers. CSF samples from aMCI, AD and control individuals (CTR) were collected and analyzed using a combined redox proteomics approach to identify the specific oxidatively modified proteins in AD and aMCI compared with controls. The majority of carbonylated proteins identified by redox proteomics are found early in the progression of AD, i.e., oxidatively modified CSF proteins were already present in aMCI compared with controls and remain oxidized in AD, thus suggesting that dysfunction of selected proteins initiate many years before severe dementia is diagnosed. The above findings highlight the presence of early oxidative damage in aMCI before clinical dementia of AD is manifested. The identification of early markers of AD that may be detected peripherally may open new prospective for biomarker studies.

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase involved in the regulation of protein synthesis and degradation, longevity and cytoskeletal formation. The mTOR pathway represents a key growth and survival pathway involved in several diseases such as cancer, obesity, cardiovascular disease and neurodegenerative diseases. Numerous studies linked the alterations of mTOR pathway to age-dependent cognitive decline, pathogenesis of Alzheimer disease (AD) and AD-like dementia in Down syndrome (DS). DS is the most frequent chromosomal abnormality that causes intellectual disability. The neuropathology of AD in DS is complex and involves impaired mitochondrial function, defects in neurogenesis, increased oxidative stress, altered proteostasis and autophagy networks as a result of triplication of chromosome 21(chr 21). The chr21 gene products are considered a principal neuropathogenic moiety in DS. Several genes involved respectively in the formation of senile plaques and neurofibrillary tangles (NFT), two main pathological hallmarks of AD, are mapped on chr21. Further, in subjects with DS the activation of mTOR signaling contributes to Aβ generation and the formation of NFT. This review discusses recent research highlighting the complex role of mTOR associated with the presence of two hallmarks of AD pathology, senile plaques (composed mostly of fibrillar Aß peptides), and NFT (composed mostly of hyperphosphorylated tau protein). Oxidative stress, associated with chr21-related Aβ and mitochondrial alterations, may significantly contribute to this linkage of mTOR to AD-like neuropathology in DS.

Alzheimer's disease (AD) is the most common form of dementia in the elderly followed by vascular dementia. In addition to clinically diagnosed dementia, cognitive dysfunction has been reported in diabetic patients. Recent studies are now beginning to recognize type 2 diabetes mellitus (T2DM), characterized by chronic hyperglycemia and insulin resistance, as a risk factor for AD and other cognitive disorders. While studies on insulin action have remained traditionally in the domain of peripheral tissues, the detrimental effects of insulin resistance in the central nervous system on cognitive dysfunction are increasingly being reported in recent clinical and preclinical studies. Brain functions require continuous supply of glucose and oxygen and a tight regulation of metabolic processes. Loss of this metabolic regulation has been proposed to be a contributor to memory dysfunction associated with neurodegeneration. Within the above scenario, this review will focus on the interplay among oxidative stress (OS), insulin resistance and AMPK dysfunctions in the brain by highlighting how these neurotoxic events contribute to neurodegeneration. We provide an overview on the detrimental effects of OS on proteins regulating insulin signaling and how these alterations impact cell metabolic dysfunctions through AMPK dysregulation. Such processes, we assert, are critically involved in the molecular pathways that underlie AD.

Alzheimer's disease (AD) is the most prevalent form of dementia in the elderly population and has worldwide impact. The etiology of the disease is complex and results from the confluence of multiple mechanisms ultimately leading to neuronal loss and cognitive decline. Among risk factors, aging is the most relevant and accounts for several pathogenic events that contribute to disease-specific toxic mechanisms. Accumulating evidence linked the alterations of the mammalian target of rapamycin (mTOR), a serine/threonine protein kinase playing a key role in the regulation of protein synthesis and degradation, to age-dependent cognitive decline and pathogenesis of AD. To date, growing studies demonstrated that aberrant mTOR signaling in the brain affects several pathways involved in energy metabolism, cell growth, mitochondrial function and proteostasis. Recent advances associated alterations of the mTOR pathway with the increased oxidative stress. Disruption of all these events strongly contribute to age-related cognitive decline including AD. The current review discusses the main regulatory roles of mTOR signaling network in the brain, focusing on its role in autophagy, oxidative stress and energy metabolism. Collectively, experimental data suggest that targeting mTOR in the CNS can be a valuable strategy to prevent/slow the progression of AD.

Down syndrome (DS) is the most frequent genetic cause of intellectual disability and is strongly associated with Alzheimer's disease (AD). Brain insulin resistance greatly contributes to AD development in the general population and previous studies from our group showed an early accumulation of insulin resistance markers in DS brain, already in childhood, and even before AD onset. Here we tested the effects promoted in Ts2Cje mice by the intranasal administration of the KYCCSRK peptide known to foster insulin signaling activation by directly interacting and activating the insulin receptor (IR) and the AKT protein. Therefore, the KYCCSRK peptide might represent a promising molecule to overcome insulin resistance. Our results show that KYCCSRK rescued insulin signaling activation, increased mitochondrial complexes levels (OXPHOS) and reduced oxidative stress levels in the brain of Ts2Cje mice. Moreover, we uncovered novel characteristics of the KYCCSRK peptide, including its efficacy in reducing DYRK1A (triplicated in DS) and BACE1 protein levels, which resulted in reduced AD-like neuropathology in Ts2Cje mice. Finally, the peptide elicited neuroprotective effects by ameliorating synaptic plasticity mechanisms that are altered in DS due to the imbalance between inhibitory vs. excitatory currents. Overall, our results represent a step forward in searching for new molecules useful to reduce intellectual disability and counteract AD development in DS.

Abstract Increasing evidence supports the notion that increased oxidative stress is a fundamental cause in the aging process and in neurodegenerative diseases. As a result, a decline in cognitive function is generally associated with brain aging. Reactive oxygen species (ROS) are highly reactive intermediates, which can modify proteins, nucleic acids, and polyunsaturated fatty acids, leading to neuronal damage. Because proteins are major components of biological systems and play key roles in a variety of cellular functions, oxidative damage to proteins represents a primary event observed in aging and age‐related neurodegenerative disorders. In the present study, with a redox proteomics approach, we identified mitochondrial oxidatively modified proteins as a function of brain aging, specifically in those brain regions, such as cortex and hippocampus, that are commonly affected by the aging process. In all brain regions examined, many of the identified proteins were energy‐related, such as pyruvate kinase, ATP synthase, aldolase, creatine kinase, and α‐enolase. These alterations were associated with significant changes in both cytosolic and mitochondrial redox status in all brain regions analyzed. Our finding is in line with current literature postulating that free radical damage and decreased energy production are characteristic hallmarks of the aging process. In additon, our results further contribute to identifying common pathological pathways involved both in aging and in neurodegenerative disease development. © 2010 Wiley‐Liss, Inc.

Alzheimer's disease (AD) is the most common type of dementia, comprising 60-80% of all reported cases, and currently affects 5.2 million Americans. AD is characterized pathologically by the accumulation of senile plaques (SPs), neurofibrillary tangles (NFTs), and synapse loss. The early stages of memory loss associated with AD have been studied in a condition known as amnestic mild cognitive impairment (MCI), arguably the earliest form of AD. In spite of extensive research across a variety of disciplines, the cause of AD remains elusive. Proteomics techniques have helped to advance knowledge about AD by identifying irregularities in protein expression and post-translational modifications (PTMs) in AD brain. Glycosylation is a less studied PTM with regards to AD and MCI. This PTM is important to study because glycosylation is involved in proper protein folding, protein anchoring to cell membranes, and the delivery of proteins to organelles, and these processes are impaired in AD. Concanavalin-A (Con-A) binds to N-linked glycoproteins, but hydrophobic sites on nonglycoproteins are also known to bind Con-A. To our knowledge, the present study is the first to examine Con-A-associated brain proteins in MCI and AD with focus on the hippocampus and inferior parietal lobule (IPL) brain regions. Proteins found in AD hippocampus with altered levels are glutamate dehydrogenase (GDH), glial fibrillary acidic protein (GFAP), tropomyosin 3 (TPM3), Rab GDP-dissociation inhibitor XAP-4 (XAP4), and heat shock protein 90 (HSP90). Proteins found with altered levels in AD IPL are alpha-enolase, gamma-enolase, and XAP-4. MCI hippocampal proteins with altered levels are dihydropyrimidase-2 (DRP2), glucose-regulated protein 78 (GRP-78), protein phosphatase related protein Sds-22 (Sds22), and GFAP and the only protein found with altered levels in MCI IPL was beta-synuclein. These results are discussed with reference to biochemical and pathological alterations in and progression of AD.

Heat shock proteins (HSPs) are highly regulated proteins that are involved in normal cellular activity and are up-regulated when the cell is exposed to stress such as heat or excess reactive oxygen species (ROS) production. HSPs are molecular chaperones that mediate the proper folding of proteins and promote recovery of the native conformations of proteins lost due to stress. Improperly folded or denatured proteins tend to aggregate and accumulate in cells. A number of neurodegenerative diseases such as Parkinson disease (PD) and Alzheimer disease (AD) have been called “protein misfolding disorders” due their characteristic pathology. Until now the exact mechanism(s) of AD progression and pathogenesis largely remains unknown. Reasoning that stress is present in brain in AD, we tested the suggestion that HSP levels would be increased in amnestic mild cognitive impairment (aMCI), a transition stage between normal aging and AD. Accordingly, in the present study we measured the levels of HSPs in hippocampus, inferior parietal lobule (IPL) and cerebellum of subjects with aMCI. The results show a general induction of HSPs and decreased levels of Thioredoxin 1 in aMCI brain suggesting that alteration in the chaperone protein systems might contribute to the pathogenesis and progression of AD. The results also are consistent with the notion that targeting HSP could be a therapeutic approach to delay the progression of aMCI to AD.

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.

Abstract Purpose : The present study aims to evaluate a set of oxidative stress biomarkers in the amniotic fluid (AF) of women carrying Down syndrome (DS) fetuses that could prove in vivo the early occurrence of oxidative damage in DS. Experimental design : To assess the extent of protein oxidation in DS AF, we measured protein carbonylation and protein‐bound HNE by slot‐blot analysis, total and oxidized GSH levels by enzymatic assay and heat shock proteins (HSPs) thioredoxin (Trx) induction by Western blot. Further, by a redox proteomics approach specific targets of protein carbonylation were identified. Results : We found increased levels of oxidative stress, as indexed by increased protein oxidation, lipid peroxidation, reduction of GSH and Trx levels and induction of the HSP response. By a redox proteomics approach, we identified selective proteins which showed increased oxidation in DS fetuses compared with healthy controls. The identified proteins are involved in iron homeostasis (ceruloplasmin and transferin), lipid metabolism (zinc‐α2‐glycoprotein, retinol‐binding protein 4 and apolipoprotein A1) and inflammation (complement C9, α‐1B‐glycoprotein, collagen α‐1V chain) with critical relevance in the clinical outcome of DS. Conclusions and clinical relevance : Our results indicate that oxidative damage is an early event in the DS pathogenesis and might contribute to the development of deleterious DS phenotypes, including abnormal development and AD‐like neuropathology.

Biliverdin reductase-A (BVR-A) is a pleiotropic enzyme and plays pivotal role in the antioxidant defense against free radicals as well as in cell homeostasis. Together with heme oxygenase, BVR-A forms a powerful system involved in the cell stress response during neurodegenerative disorders including Alzheimer's disease (AD), whereas due to the serine/threonine/tyrosine kinase activity the enzyme regulates glucose metabolism and cell proliferation. In this paper, we report results that demonstrate BVR-A undergoes post-translational oxidative and nitrosative modifications in the hippocampus, but not cerebellum, of subjects with AD and amnestic mild cognitive impairment (MCI). A significant increase of nitrated BVR-A was demonstrated only in AD and MCI hippocampi, whereas no significant modifications were found in cerebellar tissue. In addition, a significant reduction in protein carbonyl-derivatives of BVR-A was found in both AD and MCI hippocampi (15% and 18%, respectively). Biliverdin reductase-bound 4-hydroxynonenals were not modified in hippocampi and cerebella from AD and MCI subjects. These results supported the hypothesis of a prevalence of nitrosative stress-induced modifications on BVR-A structure, and this evidence was confirmed by a significant upregulation of inducible nitric oxide synthase in hippocampal tissue of subjects with AD and MCI that was not present in cerebellum. In conclusion, nitrosative stress-induced modifications on hippocampal BVR-A are an early event in the pathogenesis of AD since they appear also in MCI subjects and could contribute to the antioxidant and metabolic derangement characteristic of these neurodegenerative disorders.

Biliverdin reductase-A is a pleiotropic enzyme involved not only in the reduction of biliverdin-IX-alpha into bilirubin-IX-alpha, but also in the regulation of glucose metabolism and cell growth secondary to its serine/threonine/tyrosine kinase activity. Together with heme oxygenase, whose metabolic role is to degrade heme into biliverdin-IX-alpha, it forms a powerful system involved in the cell stress response during neurodegenerative disorders. In this paper, an up-regulation of the biliverdin reductase-A protein levels was found in the hippocampus of the subjects with Alzheimer disease and arguably its earliest form, mild cognitive impairment. Moreover a significant reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A was found, and this was paralleled by a marked reduction in its reductase activity. Interestingly, the levels of both total and phosphorylated biliverdin reductase-A were unchanged as well as its enzymatic activity in the cerebella. These results demonstrated a dichotomy between biliverdin reductase-A protein levels and activity in the hippocampus of subjects affected by Alzheimer disease and mild cognitive impairment, and this effect likely is attributable to a reduction in the phosphorylation of serine, threonine and tyrosine residues of biliverdin reductase-A. Consequently, not just the increased levels of biliverdin reductase-A, but also its changed activity and phosphorylation state, should be taken into account when considering potential biomarkers for Alzheimer disease and mild cognitive impairment.

Alzheimer disease (AD) is an age-related neurodegenerative disorder characterized by progressive memory loss, inability to perform the activities of daily living and personality changes. Unfortunately, drugs effective for this disease are limited to acetylcholinesterase inhibitors that do not impact disease pathogenesis. Statins, which belong to the class of cholesterol-reducing drugs, were proposed as novel agents useful in AD therapy, but the mechanism underlying their neuroprotective effect is still unknown. In this study, we show that atorvastatin may have antioxidant effects, in aged beagles, that represent a natural higher mammalian model of AD. Atorvastatin (80 mg/day for 14.5 months) significantly reduced lipoperoxidation, protein oxidation and nitration, and increased GSH levels in parietal cortex of aged beagles. This effect was specific for brain because it was not paralleled by a concomitant reduction in all these parameters in serum. In addition, atorvastatin slightly reduced the formation of cholesterol oxidation products in cortex but increased the 7-ketocholesterol/total cholesterol ratio in serum. We also found that increased oxidative damage in the parietal cortex was associated with poorer learning (visual discrimination task). Thus, a novel pharmacological effect of atorvastatin mediated by reducing oxidative damage may be one mechanism underlying benefits of this drug in AD.

Reactive oxygen and reactive nitrogen species oxidize and nitrate DNA, lipid and proteins thus leading to neuronal death. Both endogenous and dietary antioxidants were shown to afford neuroprotection either by scavenging free radicals or inducing antioxidant enzymes. That said, the differential contribution of endogenous versus nutritional antioxidants to prevent neurodegeneration is still debated. In this study the free radical scavenging activity of two endogenous antioxidants, such as bilirubin and its precursor biliverdin, was compared with that of the dietary antioxidant alpha-tocopherol in rat brain microsomes exposed to peroxyl radical or peroxynitrite in vitro. Bilirubin and biliverdin (1–200 μM) inhibited both peroxyl radical- and peroxynitrite-dependent lipid peroxidation with a greater potency and efficacy than alpha-tocopherol. However, both BV and BR displayed greater potency and efficacy in preventing peroxynitrite- than peroxyl radical-induced lipid peroxidation. The greater antioxidant effect of both bilirubin and biliverdin than alpha-tocopherol was also confirmed against peroxyl radical- and peroxynitrite-induced protein oxidation. In conclusion, both bilirubin and biliverdin exhibited a greater antioxidant activity than alpha-tocopherol in preventing oxidative stress damage in rat brain.

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive impairment, inability to perform activities of daily living and mood changes. Statins, long known to be beneficial in conditions where dyslipidemia occurs by lowering serum cholesterol levels, also have been proposed for use in neurodegenerative conditions, including AD. However, it is not clear that the purported effectiveness of statins in neurodegenerative disorders is directly related to cholesterol-lowering effects of these agents; rather, the pleiotropic functions of statins likely play critical roles. The aim of this review is to provide an overview on the new discoveries about the effects of statin therapy on the oxidative and nitrosative stress levels as well as on the modulation of the heme oxygenase/biliverdin reductase (HO/BVR) system in the brain. We propose a novel mechanism of action for atorvastatin which, through the activation of HO/BVR-A system, may contribute to the neuroprotective effects thus suggesting a potential therapeutic role in AD and potentially accounting for the observation of decreased AD incidence with persons on statin.

Genital infection by high risk Human Papillomavirus (HR-HPV), although recognized as the main etio-pathogenetic factor of cervical cancer, is not per se sufficient to induce tumour development. Oxidative stress (OS) represents an interesting and under-explored candidate as a promoting factor in HPV-initiated carcinogenesis. To gain insight into the role of OS in cervical cancer, HPV-16 positive tissues were collected from patients with invasive squamous cervical carcinoma, from patients with High Grade dysplastic HPV lesions and from patients with no clinical evidence of HPV lesions. After virological characterization, modulation of proteins involved in the redox status regulation was investigated. ERp57 and GST were sharply elevated in dysplastic and neoplastic tissues. TrxR2 peaked in dysplastic samples while iNOS was progressively reduced in dysplastic and neoplastic samples. By redox proteomic approach, five proteins were found to have increased levels of carbonyls in dysplastic samples respect to controls namely: cytokeratin 6, actin, cornulin, retinal dehydrogenase and GAPDH. In carcinoma samples the peptidyl-prolyl cis-trans isomerase A, ERp57, serpin B3, Annexin 2 and GAPDH were found less oxidized than in dysplastic tissues. HPV16 neoplastic progression seems associated with increased oxidant environment. In dysplastic tissues the oxidative modification of DNA and proteins involved in cell morphogenesis and terminal differentiation may provide the conditions for the neoplastic progression. Conversely cancer tissues seem to attain an improved control on oxidative damage as shown by the selective reduction of carbonyl adducts on key detoxifying/pro-survival proteins.

Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease of the central nervous system. Several evidences suggest that MS can be considered a multi-factorial disease in which both genetics and environmental factors are involved. Among proposed candidates, growing results support the involvement of oxidative stress (OS) in MS pathology. The aim of this study was to investigate the role of OS in event of exacerbations in MS on serum of relapsing-remitting (RR-MS) patients, either in relapsing or remitting phase, with respect to serum from healthy subjects. We applied proteomics and redox proteomics approaches to identify differently expressed and oxidatively modified proteins in the low-abundant serum protein fraction. Among differently expressed proteins ceruloplasmin, antithrombin III, clusterin, apolipoprotein E, and complement C3, were up-regulated in MS patients compared with healthy controls. Further by redox proteomics, vitamin D-binding protein showed a progressive trend of oxidation from remission to relapse, respect with controls. Similarly, the increase of oxidation of apolipoprotein A-IV confirmed that levels of OS are elevated with the progression of the disease. Our findings support the involvement of OS in MS and suggest that dysfunction of target proteins occurs upon oxidative damage and correlates with the pathology.

Introduction: The mTOR pathway is involved in the regulation of a wide repertoire of cellular functions in the brain and its dysregulation is emerging as a leitmotif in a large number of neurological disorders. In AD, altered mTOR signaling contributes to the inhibition of autophagy deposition of Aβ and tau aggregates and to the alteration of several neuronal metabolic pathways.Areas covered: In this review, we report all the current findings on the use of mTOR inhibitors (rapamycin, rapalogues) in the treatment of AD. These results support the role of mTOR inhibitors as potential therapeutic agents able to reduce AD hallmarks and recover cognitive performances.Expert commentary: Despite mTOR inhibitors appearing to be ideal compounds to counteract AD, further studies are needed in order to gain knowledge on the involvement of aberrant mTOR in AD, and to standardize a valuable therapeutic approach that can be translated to humans.

Bach1, among the genes encoded on chromosome 21, is a transcription repressor, which binds to antioxidant response elements of DNA thus inhibiting the transcription of specific genes involved in the cell stress response including heme oxygenase-1 (HO-1). HO-1 and its partner, biliverdin reductase-A (BVR-A), are upregulated in response to oxidative stress in order to protect cells against further damage. Since oxidative stress is an early event in Down syndrome (DS) and might contribute to the development of multiple deleterious DS phenotypes, including Alzheimer's disease (AD) pathology, we investigated the status of the Bach1/HO-1/BVR-A axis in DS and its possible implications for the development of AD. In the present study, we showed increased total Bach1 protein levels in the brain of all DS cases coupled with reduced induction of brain HO-1. Furthermore, increased oxidative stress could, on one hand, overcome the inhibitory effects of Bach1 and, on the other hand, promote BVR-A impairment. Our data show that the development of AD in DS subjects is characterized by (i) increased Bach1 total and poly-ubiquitination; (ii) increased HO-1 protein levels; and (iii) increased nitration of BVR-A followed by reduced activity. To corroborate our findings, we analyzed Bach1, HO-1, and BVR-A status in the Ts65Dn mouse model at 3 (young) and 15 (old) months of age. The above data support the hypothesis that the dysregulation of HO-1/BVR-A system contributes to the early increase of oxidative stress in DS and provide potential mechanistic paths involved in the neurodegenerative process and AD development.

Context: Advanced Glycation End-Products (AGEs) are signaling proteins associated to several vascular and neurological complications in diabetic and non-diabetic patients.AGEs proved to be a marker of negative outcome in both diabetes management and surgical procedures in these patients.The reported role of AGEs prompted the development of pharmacological inhibitors of their effects, giving rise to a number of both preclinical and clinical studies.Clinical trials with anti-AGEs drugs have been gradually developed and this review aimed to summarize most relevant reports.Evidence Acquisition: Evidence acquisition process was performed using PubMed and ClinicalTrials.govwith manually checked articles.Results: Pharmacological approaches in humans include aminoguanidine, pyridoxamine, benfotiamine, angiotensin converting enzyme inhibitors, angiotensin receptor blockers, statin, ALT-711 (alagebrium) and thiazolidinediones.The most recent promising anti-AGEs agents are statins, alagebrium and thiazolidinediones.The role of AGEs in disease and new compounds interfering with their effects are currently under investigation in preclinical settings and these newer anti-AGEs drugs would undergo clinical evaluation in the next years.Compounds with anti-AGEs activity but still not available for clinical scenarios are ALT-946, OPB-9195, tenilsetam, LR-90, TM2002, sRAGE and PEDF.Conclusions: Despite most studies confirm the efficacy of these pharmacological approaches, other reports produced conflicting evidences; in almost any case, these drugs were well tolerated.At present, AGEs measurement has still not taken a precise role in clinical practice, but its relevance as a marker of disease has been widely shown; therefore, it is important for clinicians to understand the value of new cardiovascular risk factors.Findings from the current and future clinical trials may help in determining the role of AGEs and the benefits of anti-AGEs treatment in cardiovascular disease.

Hyper-active GSK-3β favors Tau phosphorylation during the progression of Alzheimer's disease (AD). Akt is one of the main kinases inhibiting GSK-3β and its activation occurs in response to neurotoxic stimuli including, i.e., oxidative stress. Biliverdin reductase-A (BVR-A) is a scaffold protein favoring the Akt-mediated inhibition of GSK-3β. Reduced BVR-A levels along with increased oxidative stress were observed early in the hippocampus of 3xTg-AD mice (at 6 months), thus suggesting that loss of BVR-A could be a limiting factor in the oxidative stress-induced Akt-mediated inhibition of GSK-3β in AD. We evaluated changes of BVR-A, Akt, GSK-3β, oxidative stress and Tau phosphorylation levels: (a) in brain from young (6-months) and old (12-months) 3xTg-AD mice; and (b) in post-mortem inferior parietal lobule (IPL) samples from amnestic mild cognitive impairment (MCI), from AD and from age-matched controls. Furthermore, similar analyses were performed in vitro in cells lacking BVR-A and treated with H2O2. Reduced BVR-A levels along with: (a) increased oxidative stress; (b) reduced GSK-3β inhibition; and (c) increased Tau Ser404 phosphorylation (target of GSK-3β activity) without changes of Akt activation in young mice, were observed. Similar findings were obtained in MCI, consistent with the notion that this is a molecular mechanism disrupted in humans. Interestingly, cells lacking BVR-A and treated with H2O2 showed reduced GSK-3β inhibition and increased Tau Ser404 phosphorylation, which resulted from a defect of Akt and GSK-3β physical interaction. Reduced levels of Akt/GSK-3β complex were confirmed in both young 3xTg-AD and MCI brain. We demonstrated that loss of BVR-A impairs the neuroprotective Akt-mediated inhibition of GSK-3β in response to oxidative stress, thus contributing to Tau hyper-phosphorylation in early stage AD. Such changes potential provide promising therapeutic targets for this devastating disorder.

Increasing evidences support the notion that the impairment of intracellular degradative machinery is responsible for the accumulation of oxidized/misfolded proteins that ultimately results in the deposition of protein aggregates. These events are key pathological aspects of "protein misfolding diseases", including Alzheimer disease (AD). Interestingly, Down syndrome (DS) neuropathology shares many features with AD, such as the deposition of both amyloid plaques and neurofibrillary tangles. Studies from our group and others demonstrated, in DS brain, the dysfunction of both proteasome and autophagy degradative systems, coupled with increased oxidative damage. Further, we observed the aberrant increase of mTOR signaling and of its down-stream pathways in both DS brain and in Ts65Dn mice. Based on these findings, we support the ability of intranasal rapamycin treatment (InRapa) to restore mTOR pathway but also to restrain oxidative stress resulting in the decreased accumulation of lipoxidized proteins. By proteomics approach, we were able to identify specific proteins that showed decreased levels of HNE-modification after InRapa treatment compared with vehicle group. Among MS-identified proteins, we found that reduced oxidation of arginase-1 (ARG-1) and protein phosphatase 2A (PP2A) might play a key role in reducing brain damage associated with synaptic transmission failure and tau hyperphosphorylation. InRapa treatment, by reducing ARG-1 protein-bound HNE levels, rescues its enzyme activity and conceivably contribute to the recovery of arginase-regulated functions. Further, it was shown that PP2A inhibition induces tau hyperphosphorylation and spatial memory deficits. Our data suggest that InRapa was able to rescue PP2A activity as suggested by reduced p-tau levels. In summary, considering that mTOR pathway is a central hub of multiple intracellular signaling, we propose that InRapa treatment is able to lower the lipoxidation-mediated damage to proteins, thus representing a valuable therapeutic strategy to reduce the early development of AD pathology in DS population.

Dysregulation of insulin signaling pathway with reduced downstream neuronal survival and plasticity mechanisms is a fundamental abnormality observed in Alzheimer's disease (AD) brain. This phenomenon, known as brain insulin resistance, is associated with poor cognitive performance and is driven by the uncoupling of insulin receptor (IR) from its direct substrate (IRS1). Considering that Down syndrome (DS) and AD neuropathology share many common features, we investigated metabolic aspects of neurodegeneration, i.e., brain insulin resistance, in DS and whether it would contribute to early onset AD in DS population. Changes of levels and activation of main brain proteins belonging to the insulin signaling pathway (i.e., IR, IRS1, PTEN, GSK3β, PKCζ, AS160, GLUT4) were evaluated. Furthermore, we analyzed whether changes of these proteins were associated with alterations of: (i) proteins regulating brain energy metabolism; (ii) APP cleavage; and (ii) regulation of synaptic plasticity mechanisms in post-mortem brain samples collected from people with DS before and after the development of AD pathology (DSAD) compared with their age-matched controls. We found that DS cases were characterized by key markers of brain insulin resistance (reduced IR and increased IRS1 inhibition) early in life. Furthermore, downstream from IRS1, an overall uncoupling among the proteins of insulin signaling was observed. Dysregulated brain insulin signaling was associated with reduced hexokinase II (HKII) levels and proteins associated with mitochondrial complexes levels as well as with reduced levels of syntaxin in DS cases. Tellingly, these alterations precede the development of AD neuropathology and clinical presentations in DS. We propose that markers of brain insulin resistance rise earlier with age in DS compared with the general population and may contribute to the cognitive impairment associated with the early development of AD in DS.

A decline in mitochondrial function plays a key role in the aging process and increases the incidence of age-related disorders, including Alzheimer disease (AD). Mitochondria-the power station of the organism-can affect several different cellular activities, including abnormal cellular energy generation, response to toxic insults, regulation of metabolism, and execution of cell death. In AD subjects, mitochondria are characterized by impaired function such as lowered oxidative phosphorylation, decreased adenosine triphosphate production, significant increased reactive oxygen species generation, and compromised antioxidant defense. The current review discusses the most relevant mitochondrial defects that are considered to play a significant role in AD and that may offer promising therapeutic targets for the treatment/prevention of AD. In addition, we discuss mechanisms of action and translational potential of some promising mitochondrial and bioenergetic therapeutics for AD including compounds able to potentiate energy production, antioxidants to scavenge reactive oxygen species and reduce oxidative damage, glucose metabolism, and candidates that target mitophagy. While mitochondrial therapeutic strategies have shown promise at the preclinical stage, there has been little progress in clinical trials. Thus, there is an urgent need to better understand the mechanisms regulating mitochondrial homeostasis in order to identify powerful drug candidates that target 'in and out' the mitochondria to preserve cognitive functions.

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder. The exact mechanism for the AD pathogenesis is not clearly understood. However, a number of hypotheses have been proposed to explain the pathogenesis of AD. One the hypotheses is the oxidative stress hypothesis that is supported by a number of studies which reported an increase in the levels of reactive oxygen/reactive nitrogen species and their products with a concomitant decrease in the levels of antioxidant enzymes in AD brain. In the present study, we measured in AD brain the expression levels of different forms (monomer, dimer and tetramer) of the pro-apoptotic protein, p53, and observed greater levels of p53 monomer and dimer in AD brain compared to control. In addition, we also showed the selective glutathionylation of monomeric and dimeric form of p53 in AD brain. We propose that glutathionylation of p53 may prevent the formation of tetramer, an aggregate form required for effective action of p53, and may be involved in oxidative stress conditions and neurodegeneration observed in this dementing disorder.

Biliverdin reductase-A (BVR-A) is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but through its serine/threonine/tyrosine kinase activity is able to modulate cell signaling networks. BVR-A's involvement in neurodegenerative disorders such as Alzheimer disease (AD) and amnestic mild cognitive impairment was previously described. Statins have been proposed to reduce risk of AD. In this study we evaluated the effect of atorvastatin treatment (80 mg/day for 14.5 months) on BVR-A in the parietal cortex, cerebellum and liver of a well characterized pre-clinical model of AD, the aged beagle. We found that atorvastatin significantly increased BVR-A protein levels, phosphorylation and activity only in parietal cortex. Additionally, we found significant negative correlations between BVR-A and oxidative stress indices, as well as discrimination learning error scores. Furthermore, BVR-A up-regulation and post-translational modifications significantly correlated with β-secretase protein levels in the brain, suggesting a possible role for BVR-A in Aβ formation.

Abstract Background The UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS), which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes. Results The expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized. Conclusions Despite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional) proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Only acetylcholinesterase inhibitors and the NMDA antagonist memantine are approved for AD treatment. Recent preclinical and epidemiological studies proposed statins as novel therapeutics for AD, but the mechanisms of action are still unknown. Here, we demonstrate that atorvastatin (80 mg/d for 14.5 months) treatment resulted in an up-regulation of the inducible isoform of haem oxygenase (HO-1), an enzyme with significant neuroprotective activity. Atorvastatin selectively increased HO-1 in the parietal cortex but not cerebellum. In contrast, HO-2 was increased in cerebellum but not parietal cortex. No changes were observed in HO-1 or HO-2 in the liver. Significant negative correlations between HO-1 and oxidative stress indices and positive correlations with glutathione levels in parietal cortex were found. HO-1 up-regulation significantly correlated with lower discrimination learning error scores in aged beagles. Reference to therapeutic applications of atorvastatin in AD is discussed.

Alzheimer disease (AD) is one of the most disabling disorders of the elderly and the number of people worldwide facing dementia is expected to dramatically increase in the near future. Thus, one of the major concerns of modern society is to identify putative biomarkers that serve as a valuable early diagnostic tool to identify a subset of patients with increased risk to develop AD. An ideal biomarker should be present in blood before dementia is clinically confirmed, have high sensitivity and specificity, and be reproducible. Proteomics platforms offer a powerful strategy to reach these goals and recently have been demonstrated to be promising approaches. However, the high variability of technologies and studied populations has led to contrasting results. To increase specificity, we analyzed both protein expression profiles and oxidative modifications (carbonylation) of plasma proteins in mild cognitive impairment (MCI) and AD subjects compared with age-matched controls. Most of the proteins found to have differential levels in MCI and AD confirmed results already obtained in other cohort studies. Interestingly, we applied for the first time in MCI a redox proteomics approach to specifically identify oxidized proteins. Among them, haptoglobin, one of the most abundantly secreted glycoproteins with chaperone function, was found to be either increasingly downregulated or increasingly oxidized in AD and MCI compared with controls. We also demonstrated that in vitro oxidation of haptoglobin affects the formation of amyloid-β fibrils, thus suggesting that oxidized haptoglobin is not able to act as an extracellular chaperone to prevent or slow formation of amyloid-β aggregates. Another chaperone protein, α2-macroglobulin, was found to be selectively oxidized in AD patients compared with controls. Our findings suggest that alterations in proteins acting as extracellular chaperones may contribute to exacerbating amyloid-β toxicity in the peripheral system and may be considered a putative marker of disease progression.

Alzheimer disease (AD), the most common dementing disorder, is a multifactorial disease with complex etiology. Among different hypotheses proposed for AD one of the most corroborated is the “oxidative stress hypothesis”. Although recent studies extensively demonstrated the specific oxidative modification of selected proteins in the brain of AD patients and how their dysfunction possibly correlates with the pathology, there is still an urgent need to extend these findings to peripheral tissue. So far very few studies showed oxidative damage of proteins in peripheral tissues and current findings need to be replicated. Another limit in AD research is represented by the lack of highly specific diagnostic tools for early diagnosis. For a full screening and early diagnosis, biomarkers easily detectable in biological samples, such as blood, are needed. The search of reliable biomarkers for AD in peripheral blood is a great challenge. A few studies described a set of plasma markers that differentiated AD from controls and were shown to be useful in predicting conversion from mild cognitive impairment, which is considered a prodromal stage, to AD. We review the current state of knowledge on peripheral oxidative biomarkers for AD, including proteomics, which might be useful for early diagnosis and prognosis.

Down syndrome (DS) is one of the most common genetic causes of intellectual disability characterized by multiple pathological phenotypes, among which neurodegeneration is a key feature. The neuropathology of DS is complex and likely results from impaired mitochondrial function, increased oxidative stress, and altered proteostasis. After the age of 40 years, many (most) DS individuals develop a type of dementia that closely resembles that of Alzheimer's disease with deposition of senile plaques and neurofibrillary tangles. A number of studies demonstrated that increased oxidative damage, accumulation of damaged/misfolded protein aggregates, and dysfunction of intracellular degradative systems are critical events in the neurodegenerative processes. This review summarizes the current knowledge that demonstrates a “chronic” condition of oxidative stress in DS pointing to the putative molecular pathways that could contribute to accelerate cognition and memory decline. Proteomics and redox proteomics studies are powerful tools to unravel the complexity of DS phenotypes, by allowing to identifying protein expression changes and oxidative PTMs that are proved to be detrimental for protein function. It is reasonable to suggest that changes in the cellular redox status in DS neurons, early from the fetal period, could provide a fertile environment upon which increased aging favors neurodegeneration. Thus, after a critical age, DS neuropathology can be considered a human model of early Alzheimer's disease and could contribute to understanding the overlapping mechanisms that lead from normal aging to development of dementia.

Several neurodegenerative diseases are characterized by the accumulation of ubiquitin-positive protein aggregates in affected brain regions [1]. These misfolded/aberrant proteins are toxic for neur...

Aims: Among the putative mechanisms proposed to be common factors in Down syndrome (DS) and Alzheimer's disease (AD) neuropathology, deficits in protein quality control (PQC) have emerged as a unifying mechanism of neurodegeneration. Considering that disturbance of protein degradation systems is present in DS and that oxidized/misfolded proteins require polyubiquitinylation for degradation via the ubiquitin proteasome system, this study investigated if dysregulation of protein polyubiquitinylation is associated with AD neurodegeneration in DS. Results: Postmortem brains from DS cases before and after development of AD neuropathology and age-matched controls were analyzed. By selectively isolating polyubiquitinated proteins, we were able to identify specific proteins with an altered pattern of polyubiquitinylation as a function of age. Interestingly, we found that oxidation is coupled with polyubiquitinylation for most proteins mainly involved in PQC and energy metabolism. Innovation: This is the first study showing alteration of the polyubiquitinylation profile as a function of aging in DS brain compared with healthy controls. Understanding the onset of the altered ubiquitome profile in DS brain may contribute to identification of key molecular regulators of age-associated cognitive decline. Conclusions: Disturbance of the polyubiquitinylation machinery may be a key feature of aging and neurodegeneration. In DS, age-associated deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins, which is not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology. Antioxid. Redox Signal. 26, 280–298.

Brain insulin resistance is associated with an increased Aβ production in AD although the molecular mechanisms underlying this link are still largely unknown. Biliverdin reductase-A (BVR-A) is a unique Ser/Thr/Tyr kinase regulating insulin signalling. Studies from our group, demonstrated that BVR-A impairment is among the earliest events favoring brain insulin resistance development. Furthermore, reported a negative association between BVR-A protein levels/activation and BACE1 protein levels in the parietal cortex of aged beagles (an animal model of AD), thus suggesting a possible interaction. Therefore, we aimed to demonstrate that BVR-A impairment is a molecular bridge linking brain insulin resistance with increased Aβ production. Age-associated changes of BVR-A, BACE1, insulin signalling cascade and APP processing were evaluated in the parietal cortex of beagles and experiments to confirm the hypothesized mechanism(s) have been performed in vitro in HEK293APPswe cells. Our results show that BVR-A impairment occurs early with age and is associated with brain insulin resistance. Furthermore, we demonstrate that BVR-A impairment favors CK1-mediated Ser phosphorylation of BACE1 (known to mediate BACE1 recycling to plasma membrane) along with increased Aβ production in the parietal cortex, with age. Overall, our results suggest that the impairment of BVR-A is an early molecular event contributing to both (I) the onset of brain insulin resistance and (II) the increased Aβ production observed in AD. We, therefore, suggest that by targeting BVR-A activity it could be possible to delay the onset of brain insulin resistance along with an improved regulation of the APP processing.

Dysregulation of brain insulin signaling with reduced downstream neuronal survival and plasticity mechanisms are fundamental abnormalities observed in Alzheimer disease (AD). This phenomenon, known as brain insulin resistance, is associated with poor cognitive performance and is driven by the inhibition of IRS1. Since Down syndrome (DS) and AD neuropathology share many common features, we investigated metabolic aspects of neurodegeneration in DS and whether they contribute to early onset AD in DS. We evaluated levels and activation of proteins belonging to the insulin signaling pathway (IR, IRS1, BVR-A, MAPK, PTEN, Akt, GSK3β, PKCζ, AS160, GLUT4) in the frontal cortex of Ts65dn (DS model) (n = 5–6/group) and euploid mice (n = 6/group) at different ages (1, 3, 9 and 18 months). Furthermore, we analyzed whether changes of brain insulin signaling were associated with alterations of: (i) proteins regulating brain energy metabolism (mitochondrial complexes, hexokinase-II, Sirt1); (ii) oxidative stress (OS) markers (iii) APP cleavage; and (iv) proteins mediating synaptic plasticity mechanisms (PSD95, syntaxin-1 and BDNF). Ts65dn mice showed an overall impairment of the above-mentioned pathways, mainly characterized by defects of proteins activation state. Such alterations start early in life (at 1 month, during brain maturation). In particular, accumulation of inhibited IRS1, together with the uncoupling among the proteins downstream from IRS1 (brain insulin resistance), characterize Ts65dn mice. Furthermore, reduced levels of mitochondrial complexes and Sirt1, as well as increased indices of OS also were observed. These alterations precede the accumulation of APP-C99 in Ts65dn mice. Tellingly, oxidative stress levels were negatively associated with IR, IRS1 and AS160 activation as well as mitochondrial complexes levels in Ts65dn mice, suggesting a role for oxidative stress in the observed alterations. We propose that a close link exists among brain insulin resistance, mitochondrial defects and OS that contributes to brain dysfunctions observed in DS, likely favoring the development of AD in DS.

A major challenge in neurobiology is the identification of the mechanisms by which protein misfolding leads to cellular toxicity. Many neurodegenerative disorders, in which aberrant protein conformers aggregate into pathological inclusions, present the chronic activation of the PERK branch of the unfolded protein response. The adaptive effects of the PERK pathway include reduction of translation by transient inhibition of eIF2α and antioxidant protein production via induction of Nrf2 transcription factor. In contrast, PERK prolonged activation leads to sustained reduction in protein synthesis and induction of cell death pathways. To further investigate the role of the PERK pathway in neurodegenerative disorders, we focused on Down syndrome (DS), in which aging confers a high risk of Alzheimer disease (AD). By investigating human DS frontal cortices, we found early and sustained PERK activation associated with the induction of eIF2α and ATF4 downstream signals. We also observed that the Nrf2 response is uncoupled from PERK and its antioxidant effects are repressed in a mechanism implicating the transcription repressor Bach1. The pharmacological inhibition of PERK in DS mice reduced eIF2α-related translational repression and promoted Nrf2 nuclear translocation, favoring the rescue of Nrf2/Bach1 imbalance. The further analysis of peripheral cells from living DS individuals provided strong support of the pathological link between PERK and trisomy 21. Our results suggest that failure to regulate the PERK pathway is a peculiar characteristic of DS pathology and it may represent an essential step to promote cellular dysfunction, which actively contributes in the brain to the early development of AD.

Several studies showed increased oxidative and nitrosative stress in plasma from patients with Alzheimer's disease (AD), however, little and controversial knowledge has emerged about the antioxidant functionality of the heme oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system in blood. The current study reports increased levels of both HO-1 and BVR-A in plasma from probable AD patients, as a result of the increased oxidative environment. However, the increase of oxidative stress in plasma result also in the increase of BVR-A 3-nitrotyrosine levels and the decrease of BVR-A phosphotyrosine levels and reductase activity, suggesting that nitrosative stress play the prominent oxidative role in plasma during AD. Our data on HO-1/BVR-A status in plasma closely correlate with recent reports in hippocampus of subjects with AD and arguably its early form, mild cognitive impairment. Moreover, we show that alterations on HO-1/BVR-A system are tightly connected with cognitive decline indexed by Mini-Mental Status Exam scores. We hypothesize that the HO-1/BVR-A system status in plasma might reflect the ongoing situation in the brain, offering an important biochemical tool for the potential prediction of AD at the earliest stages of the disease.

Aims: The human LRRK2 gene has been identified as the most common causative gene of autosomal-dominantly inherited and idiopathic Parkinson disease (PD). The G2019S substitution is the most common mutation in LRRK2. The R1441C mutation also occurs in cases of familial PD, but is not as prevalent. Some cases of LRRK2-based PD exhibit Tau pathology, which suggests that alterations on LRRK2 activity affect the pathophysiology of Tau. To investigate how LRRK2 might affect Tau and the pathophysiology of PD, we generated lines of C. elegans expressing human LRRK2 [wild-type (WT) or mutated (G2019S or R1441C)] with and without V337M Tau. Expression and redox proteomics were used to identify the effects of LRRK2 (WT and mutant) on protein expression and oxidative modifications. Results: Co-expression of WT LRRK2 and Tau led to increased expression of numerous proteins, including several 60S ribosomal proteins, mitochondrial proteins, and the V-type proton ATPase, which is associated with autophagy. C. elegans expressing mutant LRRK2 showed similar changes, but also showed increased protein oxidation and lipid peroxidation, the latter indexed as increased protein-bound 4-hydroxy-2-nonenal (HNE). Innovation: Our study brings new knowledge about the possible alterations induced by LRRK2 (WT and mutated) and Tau interactions, suggesting the involvement of G2019S and R1441C in Tau-dependent neurodegenerative processes. Conclusion: These results suggest that changes in LRRK2 expression or activity lead to corresponding changes in mitochondrial function, autophagy, and protein translation. These findings are discussed with reference to the pathophysiology of PD. Antioxid. Redox Signal. 17, 1490–1506.

Free radical-mediated damage to proteins is particularly important in aging and age-related neurodegenerative diseases, because in the majority of cases it is a non-reversible phenomenon that requires clearance systems for removal. Major consequences of protein oxidation are loss of protein function and the formation of large protein aggregates, which are often toxic to cells if allowed to accumulate. Deposition of aggregated, misfolded, and oxidized proteins may also result from the impairment of protein quality control (PQC) system, including protein unfolded response, proteasome, and autophagy. Perturbations of such components of the proteostasis network that provides a critical protective role against stress conditions are emerging as relevant factor in triggering neuronal death. In this outlook paper, we discuss the role of protein oxidation as a major contributing factor for the impairment of the PQC regulating protein folding, surveillance, and degradation. Recent studies from our group and from others aim to better understand the link between Down syndrome and Alzheimer’s disease neuropathology. We propose oxidative stress and alteration of proteostasis network as a possible unifying mechanism triggering neurodegeneration.

Down syndrome (DS) is the most common genetic cause of intellectual disability, resulting from trisomy of chromosome 21. The main feature of DS neuropathology includes early onset of Alzheimer’s disease (AD), with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activ ated in the presence of AD neuropathology in DS, thus we measured proteins associated with upstream and downstream pathways of p53 in the frontal cortex from DS cases with and without AD pathology and from Ts65Dn mice, at different ages. We observed increased acetylation and phosphorylation of p53, coupled to reduced MDM2/p53 complex level and lower levels of SIRT1. Activation of p53 was associated with a number of targets (BAX, PARP1, caspase-3, p21, heat shock proteins, and PGC1α) that were modulated in both DS and DS/AD compared with age-matched controls. In particular, the most relevant changes (increased p-p53 and acetyl-p53 and reduced formation of MDM2/p53 complex) were found to be modified only in the presence of AD pathology in DS. In addition, a similar pattern of alterations in the p53 pathway was found in Ts65Dn mice. These results suggest that p53 may integrate different signals, which can result in a pro-apoptotic-phenotype contributing to AD neuropathology in people with DS.

Neurodegenerative diseases are characterized by increased levels of oxidative stress and an altered mammalian target of rapamycin (mTOR)/autophagy axis; however, the mutual relationship between these two events is controversial. Previous studies in Down's syndrome (DS) and Alzheimer's disease (AD) suggested that the accumulation of protein oxidative damage results from the increased free radical production, mainly related to metabolic alterations, mitochondrial degeneration and amyloid-β deposition, and aberrant activity of protein degradative systems.This study analyzed mTOR signaling in Ts65Dn mice, a model of DS, at 6 and 12 months of age compared with euploid mice showing the early aberrant hyperphosphorylation of mTOR coupled with the reduction of autophagosome formation. Moreover, the evaluation of protein oxidation shows an increase in protein nitration and protein-bound 4-hydroxynonenal in 12-month-old Ts65Dn mice suggesting the potential involvement of altered autophagy in the buildup of protein oxidative damage. In addition, data obtained on cell culture support the protective role of autophagy in reducing protein oxidation.Overall, this study provides further evidence for the role of mTOR hyperactivation and reduced autophagy in the accumulation of protein oxidative damage during DS and AD pathologies.

PET scan analysis demonstrated the early reduction of cerebral glucose metabolism in Alzheimer disease (AD) patients that can make neurons vulnerable to damage via the alteration of the hexosamine biosynthetic pathway (HBP). Defective HBP leads to flawed protein O-GlcNAcylation coupled, by a mutual inverse relationship, with increased protein phosphorylation on Ser/Thr residues. Altered O-GlcNAcylation of Tau and APP have been reported in AD and is closely related with pathology onset and progression. In addition, type 2 diabetes patients show an altered O-GlcNAcylation/phosphorylation that might represent a link between metabolic defects and AD progression. Our study aimed to decipher the specific protein targets of altered O-GlcNAcylation in brain of 12-month-old 3×Tg-AD mice compared with age-matched non-Tg mice. Hence, we analysed the global O-GlcNAc levels, the levels and activity of OGT and OGA, the enzymes controlling its cycling and protein specific O-GlcNAc levels using a bi-dimensional electrophoresis (2DE) approach. Our data demonstrate the alteration of OGT and OGA activation coupled with the decrease of total O-GlcNAcylation levels. Data from proteomics analysis led to the identification of several proteins with reduced O-GlcNAcylation levels, which belong to key pathways involved in the progression of AD such as neuronal structure, protein degradation and glucose metabolism. In parallel, we analysed the O-GlcNAcylation/phosphorylation ratio of IRS1 and AKT, whose alterations may contribute to insulin resistance and reduced glucose uptake. Our findings may contribute to better understand the role of altered protein O-GlcNAcylation profile in AD, by possibly identifying novel mechanisms of disease progression related to glucose hypometabolism.

Biliverdin reductase-A (BVR-A) is a serine/threonine/tyrosine kinase involved in the regulation of insulin signaling. In vitro studies have demonstrated that BVR-A is a substrate of the insulin receptor and regulates IRS1 by avoiding its aberrant activation, and in animal model of obesity the loss of hepatic BVR-A has been associated with glucose/insulin alterations and fatty liver disease. However, no studies exist in humans. Here, we evaluated BVR-A expression levels and activation in peripheral blood mononuclear cells (PBMC) from obese subjects and matched lean controls and we investigated the related molecular alterations of the insulin along with clinical correlates. We showed that BVR-A levels are significantly reduced in obese subjects and associated with a hyper-activation of the IR/IRS1/Akt/GSK-3β/AS160/GLUT4 pathway. Low BVR-A levels also associate with the presence of obesity, metabolic syndrome, NASH and visceral adipose tissue inflammation. These data suggest that the reduction of BVR-A may be responsible for early alterations of the insulin signaling pathway in obesity and in this context may represent a novel molecular target to be investigated for the comprehension of the process of insulin resistance development in obesity.

Down syndrome (DS) is the most common genomic disorder characterized by the increased incidence of developing early Alzheimer’s disease (AD). In DS, the triplication of genes on chromosome 21 is intimately associated with the increase of AD pathological hallmarks and with the development of brain redox imbalance and aberrant proteostasis. Increasing evidence has recently shown that oxidative stress (OS), associated with mitochondrial dysfunction and with the failure of antioxidant responses (e.g., SOD1 and Nrf2), is an early signature of DS, promoting protein oxidation and the formation of toxic protein aggregates. In turn, systems involved in the surveillance of protein synthesis/folding/degradation mechanisms, such as the integrated stress response (ISR), the unfolded stress response (UPR), and autophagy, are impaired in DS, thus exacerbating brain damage. A number of pre-clinical and clinical studies have been applied to the context of DS with the aim of rescuing redox balance and proteostasis by boosting the antioxidant response and/or inducing the mechanisms of protein re-folding and clearance, and at final of reducing cognitive decline. So far, such therapeutic approaches demonstrated their efficacy in reverting several aspects of DS phenotype in murine models, however, additional studies aimed to translate these approaches in clinical practice are still needed.

Abstract Introduction Intellectual disability, accelerated aging, and early‐onset Alzheimer‐like neurodegeneration are key brain pathological features of Down syndrome (DS). Although growing research aims at the identification of molecular pathways underlying the aging trajectory of DS population, data on infants and adolescents with DS are missing. Methods Neuronal‐derived extracellular vesicles (nEVs) were isolated form healthy donors (HDs, n = 17) and DS children (n = 18) from 2 to 17 years of age and nEV content was interrogated for markers of insulin/mTOR pathways. Results nEVs isolated from DS children were characterized by a significant increase in pIRS1 Ser636 , a marker of insulin resistance, and the hyperactivation of the Akt/mTOR/p70S6K axis downstream from IRS1, likely driven by the higher inhibition of Phosphatase and tensin homolog (PTEN). High levels of pGSK3β Ser9 were also found. Conclusions The alteration of the insulin‐signaling/mTOR pathways represents an early event in DS brain and likely contributes to the cerebral dysfunction and intellectual disability observed in this unique population.

Alzheimer disease (AD) is the most common type of dementia and is characterized pathologically by the presence of neurofibrillary tangles (NFTs), senile plaques (SPs), and loss of synapses. The main component of SP is amyloid-beta peptide (Aβ), a 39 to 43 amino acid peptide, generated by the proteolytic cleavage of amyloid precursor protein (APP) by the action of beta- and gamma-secretases. The presenilins (PS) are components of the γ-secretase, which contains the protease active center. Mutations in PS enhance the production of the Aβ42 peptide. To date, more than 160 mutations in PS1 have been identified. Many PS mutations increase the production of the β-secretase-mediated C-terminal (CT) 99 amino acid-long fragment (CT99), which is subsequently cleaved by γ-secretase to yield Aβ peptides. Aβ has been proposed to induce oxidative stress and neurotoxicity. Previous studies from our laboratory and others showed an age-dependent increase in oxidative stress markers, loss of lipid asymmetry, and Aβ production and amyloid deposition in the brain of APP/PS1 mice. In the present study, we used APP (NLh)/APP(NLh) × PS-1(P246L)/PS-1(P246L) human double mutant knock-in APP/PS-1 mice to identify specific targets of brain protein carbonylation in an age-dependent manner. We found a number of proteins that are oxidatively modified in APP/PS1 mice compared to age-matched controls. The relevance of the identified proteins to the progression and pathogenesis of AD is discussed.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory, reasoning and other cognitive functions. Pathologically, patients with AD are characterized by deposition of senile plaques (SPs), formed by β-amyloid (Aβ), and neurofibrillary tangles (NTFs) that consist of aggregated hyperphosphorylated tau protein. The accumulation of insoluble protein aggregates in AD brain can be associated with an impairment of degradative systems. This current study investigated if the disturbance of protein polyubiquitination is associated with AD neurodegeneration. By using a novel proteomic approach, we found that 13 brain proteins are increasingly polyubiquitinated in AD human brain compared to age-matched controls. Moreover, the majority of the identified proteins were previously found to be oxidized in our prior proteomics, and these proteins are mainly involved in protein quality control and glucose metabolism. This is the first study showing alteration of the poly-ubiquitin profile in AD brain compared with healthy controls. Understanding the onset of the altered ubiquitin profile in AD brain may contribute to identification of key molecular regulators of cognitive decline. In AD, deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins that are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology and cognitive decline.

Lysosomal acid lipase (LAL) plays a key role in intracellular lipid metabolism. Reduced LAL activity promotes increased multi-organ lysosomal cholesterol ester storage, as observed in two recessive autosomal genetic diseases, Wolman disease and Cholesterol ester storage disease. Severe liver steatosis and accelerated liver fibrosis are common features in patients with genetic LAL deficiency. By contrast, few reliable data are available on the modulation of LAL activity in vivo and on the epigenetic and metabolic factors capable of regulating its activity in subjects without homozygous mutations of the Lipase A gene. In the last few years, a less severe and non-genetic reduction of LAL activity was reported in children and adults with non-alcoholic fatty liver disease (NAFLD), suggesting a possible role of LAL reduction in the pathogenesis and progression of the disease. Patients with NAFLD show a significant, progressive reduction of LAL activity from simple steatosis to non-alcoholic steatohepatitis and cryptogenic cirrhosis. Among cirrhosis of different etiologies, those with cryptogenic cirrhosis show the most significant reductions of LAL activity. These findings suggest that the modulation of LAL activity may become a possible new therapeutic target for patients with more advanced forms of NAFLD. Moreover, the measurement of LAL activity may represent a possible new marker of disease severity in this clinical setting.

The disturbance of protein O-GlcNAcylation is emerging as a possible link between altered brain metabolism and the progression of neurodegeneration. As observed in brains with Alzheimer’s disease (AD), flaws of the cerebral glucose uptake translate into reduced protein O-GlcNAcylation, which promote the formation of pathological hallmarks. A high-fat diet (HFD) is known to foster metabolic dysregulation and insulin resistance in the brain and such effects have been associated with the reduction of cognitive performances. Remarkably, a significant role in HFD-related cognitive decline might be played by aberrant protein O-GlcNAcylation by triggering the development of AD signature and mitochondrial impairment. Our data support the impairment of total protein O-GlcNAcylation profile both in the brain of mice subjected to a 6-week high-fat-diet (HFD) and in our in vitro transposition on SH-SY5Y cells. The reduction of protein O-GlcNAcylation was associated with the development of insulin resistance, induced by overfeeding (i.e., defective insulin signaling and reduced mitochondrial activity), which promoted the dysregulation of the hexosamine biosynthetic pathway (HBP) flux, through the AMPK-driven reduction of GFAT1 activation. Further, we observed that a HFD induced the selective impairment of O-GlcNAcylated-tau and of O-GlcNAcylated-Complex I subunit NDUFB8, thus resulting in tau toxicity and reduced respiratory chain functionality respectively, highlighting the involvement of this posttranslational modification in the neurodegenerative process.

The cells possess several mechanisms to counteract the over-production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), including enzymes such as superoxide dismutase, catalase and glutathione peroxidase. Moreover, an important sensor involved in the anti-oxidant response is KEAP1-NRF2-ARE signaling complex. Under oxidative stress (OS), the transcription factor NRF2 can dissociate from the KEAP1-complex in the cytosol and translocate into the nucleus to promote the transcriptional activation of anti-oxidant genes, such as heme oxygenase 1 and NADPH quinone oxidoreductase. Within this context, the activation of NRF2 response is further regulated by BACH1, a transcription repressor, that compete with the KEAP1-NRF2-ARE complex. In this work, we focused on the role of BACH1/NRF2 ratio in the regulation of the anti-oxidant response, proposing their antithetical relation as a valuable target for a therapeutic strategy to test drugs able to exert neuroprotective effects, notably in aging and neurodegenerative diseases. Among these, Down syndrome (DS) is a complex genetic disorder characterized by BACH1 gene triplication that likely results in the impairment of NRF2 causing increased OS. Our results revealed that BACH1 overexpression alters the BACH1/NRF2 ratio in the nucleus and disturbs the induction of antioxidant response genes ultimately resulting in the accumulation of oxidative damage both in Ts2Cje mice (a mouse model of DS) and human DS lymphoblastoid cell lines (LCLs). Based on this evidence, we tested Caffeic Acid Phenethyl Ester (CAPE) and the synthetic analogue VP961, which have been proven to modulate NRF2 activity. We showed that CAPE and VP961 administration to DS LCLs was able to promote NRF2 nuclear translocation, which resulted in the amelioration of antioxidant response. Overall, our study supports the hypothesis that BACH1 triplication in DS subjects is implicated in the alteration of redox homeostasis and therapeutic strategies to overcome this effect are under investigation in our laboratory.

UV solar radiation is the major environmental risk factor for malignant melanoma. A great effort is currently posed on the search of new compounds able to prevent or reduce UV-mediated cell damage. Ferulic acid is a natural compound recently included in the formulation of solar protecting dermatological products. The purpose of the present work was to assess whether its ethyl ester derivative, FAEE, could protect skin melanocytes from UV-induced oxidative stress and cell damage. Experiments on human melanocytes irradiated with UVB showed that FAEE treatment reduced the generation of ROS, with a net decrease of protein oxidation. FAEE treatment was accompanied by an induction of HSP70 and heme oxygenase, by a marked suppression of PARP activation and a significant suppression of apoptosis. Moreover FAEE prevented iNOS induction, thus suppressing the secondary generation of NO-derived oxidizing agents. FAEE may represent a potentially effective pharmacological approach to reduce UV radiation-induced skin damage.

Lectin affinity chromatography is a powerful separation technique that fractionates proteins by selectively binding to specific carbohydrate moieties characteristic of protein glycosylation type. Wheat germ agglutinin (WGA) selectively binds terminal N-acetylglucosamine (O-GlcNAc) and sialic acid moieties characteristic of O-linked glycosylation. The current study utilizes WGA affinity chromatography to fractionate proteins from hippocampus and inferior parietal lobule (IPL) from subjects with Alzheimer's disease (AD) and arguably its earliest form, mild cognitive impairment (MCI). Proteins identified by proteomics that were fractionated from MCI and AD hippocampus by WGA affinity chromatography with altered levels compared with age-matched controls included GP96, γ-enolase, glutamate dehydrogenase, glucosidase IIα, 14-3-3ϵ, 14-3-3γ, 14-3-3ζ, tropomyosin-2, calmodulin 2, gelsolin, β-synuclein, α1-antichymotrypsin, and dimethylguanosine tRNA methyltransferase. Proteins identified by proteomics that were fractionated from MCI and AD IPL by WGA affinity chromatography showing altered levels compared with age-matched controls included protein disulfide isomerase, calreticulin, and GP96. The proteins described in this study are involved in diverse processes, including glucose metabolism, endoplasmic reticulum (ER) functions, chaperoning, cytoskeletal assembly, and proteolysis, all of which are affected in AD. This study, the first to use proteomics to identify WGA-fractionated proteins isolated from brains from subjects with MCI and AD, provides additional information about the active proteome of the brain throughout AD progression.

Signal transduction and activator of transcription-3 (STAT3) plays an important role in neuronal survival, regeneration and repair after brain injury. We previously demonstrated that STAT3 is activated in brain after cerebral ischemia specifically in neurons. The effect was sex-specific and modulated by sex steroids, with higher activation in females than males. In the current study, we used a proteomics approach to identify downstream proteins affected by ischemia in male and female wild-type (WT) and neuron-specific STAT3 knockout (KO) mice. We established four comparison groups based on the transgenic condition and the hemisphere analyzed, respectively. Moreover, the sexual variable was taken into account and male and female animals were analyzed independently. Results support a role for STAT3 in metabolic, synaptic, structural and transcriptional responses to cerebral ischemia, indeed the adaptive response to ischemia/reperfusion injury is delayed in neuronal-specific STAT3 KO mice. The differences observed between males and females emphasize the importance of sex-specific neuronal survival and repair mechanisms, especially those involving antioxidant and energy-related activities, often caused by sex hormones.

The basal oxidative and nitrosative stress levels measured in cytosol, mitochondria, and nuclei as well as in the whole homogenate obtained from the brain of wild type (wt) and p53 knockout [p53((-/-))] mice were evaluated. We hypothesized that the loss of p53 could trigger the activation of several protective mechanisms such as those involving thioredoxin-1 (Thio-1), the heme-oxygenase-1/biliverdin reductase-A (HO-1/BVR-A) system, manganese superoxide dismutase (MnSOD), the IkB kinase type β (IKKβ)/nuclear factor kappa-B (NF-kB), and the nuclear factor-erythroid 2 (NF-E2) related factor 2 (Nrf-2).A decrease of protein carbonyls, protein-bound 4-hydroxy-2-nonenal (HNE), and 3-nitrotyrosine (3-NT) was observed in the brain from p53((-/-)) mice compared with wt. Furthermore, we observed a significant increase of the expression levels of Thio-1, BVR-A, MnSOD, IKKβ, and NF-kB. Conversely a significant decrease of Nrf-2 protein levels was observed in the nuclear fraction isolated from p53((-/-)) mice. No changes were found for HO-1.This is the first study of basal oxidative/nitrosative stress in in vivo conditions of brain obtained from p53((-/-)) mice. New insights into the role of p53 in oxidative stress have been gained.We demonstrated, for the first time, that the lack of p53 reduces basal oxidative stress levels in mice brain. Due to the pivotal role that p53 plays during cellular stress response our results provide new insights into novel therapeutic strategies to modulate protein oxidation and lipid peroxidation having p53 as a target. The implications of this work are profound, particularly for neurodegenerative disorders.

Tauopathies, the most common of which is Alzheimer's disease (AD), constitute the most crippling neurodegenerative threat to our aging population. Tauopathic patients have significant cognitive decline accompanied by irreversible and severe brain atrophy, and it is thought that neuronal dysfunction begins years before diagnosis. Our current understanding of tauopathies has yielded promising therapeutic interventions but have all failed in clinical trials. This is partly due to the inability to identify and intervene in an effective therapeutic window early in the disease process. A major challenge that contributes to the definition of an early therapeutic window is limited technologies. To address these challenges, we modified and adapted a manganese-enhanced magnetic resonance imaging (MEMRI) approach to provide sensitive and quantitative power to detect changes in broad neuronal function in aging mice. Considering that tau tangle burden correlates well with cognitive impairment in Alzheimer's patients, we performed our MEMRI approach in a time course of aging mice and an accelerated mouse model of tauopathy. We measured significant changes in broad neuronal function as a consequence of age, and in transgenic mice, before the deposition of bona fide tangles. This MEMRI approach represents the first diagnostic measure of neuronal dysfunction in mice. Successful translation of this technology in the clinic could serve as a sensitive diagnostic tool for the definition of effective therapeutic windows.

Metformin is the first-line therapy for diabetes, even in children, and a promising attractive candidate for drug repurposing. Mitochondria are emerging as crucial targets of metformin action both in the periphery and in the brain. The present study evaluated whether treatment with metformin may rescue brain mitochondrial alterations and contrast the increased oxidative stress in a validated mouse model of Rett syndrome (RTT), a rare neurologic disorder of monogenic origin characterized by severe behavioral and physiological symptoms. No cure for RTT is available. In fully symptomatic RTT mice (12 months old MeCP2-308 heterozygous female mice), systemic treatment with metformin (100 mg/kg ip for 10 days) normalized the reduced mitochondrial ATP production and ATP levels in the whole-brain, reduced brain oxidative damage, and rescued the increased production of reactive oxidizing species in blood. A 10-day long treatment with metformin also boosted pathways related to mitochondrial biogenesis and antioxidant defense in the brain of metformin-treated RTT mice. This treatment regimen did not improve general health status and motor dysfunction in RTT mice at an advanced stage of the disease. Present results provide evidence that systemic treatment with metformin may represent a novel, repurposable therapeutic strategy for RTT.

Compelling evidence supports the role of oxidative stress in Alzheimer’s disease (AD) pathophysiology. Interestingly, Herpes simplex virus-1 (HSV-1), a neurotropic virus that establishes a lifelong latent infection in the trigeminal ganglion followed by periodic reactivations, has been reportedly linked both to AD and to oxidative stress conditions. Herein, we analyzed, through biochemical and redox proteomic approaches, the mouse model of recurrent HSV-1 infection we previously set up, to investigate whether multiple virus reactivations induced oxidative stress in the mouse brain and affected protein function and related intracellular pathways. Following multiple HSV-1 reactivations, we found in mouse brains increased levels of oxidative stress hallmarks, including 4-hydroxynonenal (HNE), and 13 HNE-modified proteins whose levels were found significantly altered in the cortex of HSV-1-infected mice compared to controls. We focused on two proteins previously linked to AD pathogenesis, i.e., glucose-regulated protein 78 (GRP78) and collapsin response-mediated protein 2 (CRMP2), which are involved in the unfolded protein response (UPR) and in microtubule stabilization, respectively. We found that recurrent HSV-1 infection disables GRP78 function and activates the UPR, whereas it prevents CRMP2 function in mouse brains. Overall, these data suggest that repeated HSV-1 reactivation into the brain may contribute to neurodegeneration also through oxidative damage.

Biliverdin reductase-A (BVR-A) impairment is associated with increased accumulation of oxidatively-damaged proteins along with the impairment of autophagy in the brain during neurodegenerative disorders. Reduced autophagy inhibits the clearance of misfolded proteins, which then form neurotoxic aggregates promoting neuronal death. The aim of our study was to clarify the role for BVR-A in the regulation of the mTOR/autophagy axis by evaluating age-associated changes (2, 6 and 11 months) in cerebral cortex samples collected from BVR-A knock-out (BVR-A-/-) and wild-type (WT) mice. Our results show that BVR-A deficiency leads to the accumulation of oxidatively-damaged proteins along with mTOR hyper-activation in the cortex. This process starts in juvenile mice and persists with aging. mTOR hyper-activation is associated with the impairment of autophagy as highlighted by reduced levels of Beclin-1, LC3, LC3II/I ratio, Atg5-Atg12 complex and Atg7 in the cortex of BVR-A-/- mice. Furthermore, we have identified the dysregulation of AMP-activated protein kinase (AMPK) as a critical event driving mTOR hyper-activation in the absence of BVR-A. Overall, our results suggest that BVR-A is a new player in the regulation of autophagy, which may be targeted to arrive at novel therapeutics for diseases involving impaired autophagy.

Protein homeostasis or "proteostasis" represent the process that regulates the balance of the intracellular functional and "healthy" proteins. Proteostasis is fundamental to preserve physiological metabolic processes in the cell and it allow to respond to any given stimulus as the expression of components of the proteostasis network is customized according to the proteomic demands of different cellular environments. In conditions that promote unfolding/misfolding of proteins chaperones act as signaling molecules inducing extreme measures to either fix the problem or destroy unfolded proteins. When the chaperone machinery fails under pathological insults unfolded proteins induce the endoplasmic reticulum (ER) stress activating the unfolded protein response (UPR) machinery. The activation of the UPR restores ER proteostasis primarily through the transcriptional remodeling of ER protein folding, trafficking, and degradation pathways, such as the ubiquitin proteasome system (UPS). If these mechanisms do not manage to clear the aberrant proteins, proteasome overload and become defective, and misfolded proteins may form aggregates thus extending the UPR mechanism. These aggregates are then attempted to be cleared by macroautophagy. Impaired proteostasis promote the accumulation of misfolded proteins that exacerbate the damage to chaperones, surveillance systems and/or degradative activities. Remarkably, the removal of toxic misfolded proteins is critical for all cells, but it is especially significant in neurons since these cannot be readily replaced. In neurons, the maintenance of efficient proteostasis is essential to healthy aging since the dysregulation of the proteostasis network can lead to neurodegenerative disease. Each of these brain pathologies is characterized by the repeated misfolding of one of more peculiar proteins, which evade both the protein folding machinery and cellular degradation mechanisms and begins to form aggregates that nucleate out into large fibrillar aggregates. In this chapter we describe the mechanisms, associated with faulty proteostasis, that promote the formation of protein aggregates, amyloid fibrils, intracellular, and extracellular inclusions in the most common nondegenerative disorders also referred to as protein misfolding disorders.

Metformin is the most common anti-diabetic drug and a promising therapy for disorders beyond diabetes, including Rett syndrome (RTT), a rare neurologic disease characterized by severe intellectual disability. A 10-day-long treatment rescued aberrant mitochondrial activity and restrained oxidative stress in a female RTT mouse model. However, this treatment regimen did not improve the phenotype of RTT mice. In the present study, we demonstrate that a 4-month-long treatment with metformin (150 mg/Kg/day, delivered in drinking bottles) provides a selective normalization of cognitive flexibility defects in RTT female mice at an advanced stage of disease, but it does not affect their impaired general health status and abnormal motor skills. The 4-month-long treatment also rescues the reduced activity of mitochondrial respiratory chain complex activities, the defective brain ATP production and levels as well as the increased production of reactive oxidizing species in the whole blood of RTT mice. A significant boost of PGC-1α-dependent pathways related to mitochondrial biogenesis and antioxidant defense occurs in the brain of RTT mice that received the metformin treatment. Further studies will have to verify whether these effects may underlie its long-lasting beneficial effects on brain energy metabolism.

Alzheimer's disease (AD) is a progressive form of dementia characterized by increased production of amyloid-β plaques and hyperphosphorylated tau protein, mitochondrial dysfunction, elevated oxidative stress, reduced protein clearance, among other. Several studies showed systemic modifications of immune and inflammatory systems due, in part, to decreased levels of CD3+ lymphocytes in peripheral blood in AD. Considering that oxidative stress, both in the brain and in the periphery, can influence the activation and differentiation of T-cells, we investigated the 3-nitrotyrosine (3-NT) proteome of blood T-cells derived from AD patients compared to non-demented (ND) subjects by using a proteomic approach. 3-NT is a formal protein oxidation and index of nitrosative stress. We identified ten proteins showing increasing levels of 3-NT in CD3+ T-cells from AD patients compared with ND subjects. These proteins are involved in energy metabolism, cytoskeletal structure, intracellular signaling, protein folding and turnover, and antioxidant response and provide new insights into the molecular mechanism that impact reduced T-cell differentiation in AD. Our results highlight the role of peripheral oxidative stress in T-cells related to immune-senescence during AD pathology focusing on the specific targets of protein nitration that conceivably can be suitable to further therapies. Further, our data demonstrate common targets of protein nitration between the brain and the periphery, supporting their significance as disease biomarkers.

Down syndrome (DS) is the most common chromosomal disorder and the leading genetic cause of intellectual disability in humans, which results from the triplication of chromosome 21. DS individuals have an increased risk of developing Alzheimer's disease (AD)-like pathology and dementia by the age of 40 due to the triplication of several genes involved in the formation of amyloid plaques and tau tangles. Further, DS and AD are characterized by the aberrant accumulation of unfolded/misfolded proteins resulting from over-burdened protein quality control systems. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Long-term activation of the UPR mediates neuronal dysfunction in AD. We hypothesized that the UPR is impacted in a mouse model of DS. To test this, we performed gene and protein expression analysis of ER stress markers in the Ts65Dn mouse model of DS at 3, 9, and 18 months. We identified activation of the PERK pathway in Ts65Dn DS mice at 3 months of age compared to euploid controls. We also determined that the early and overt UPR activation decreased with age, the UPR signal was significantly reduced by 18 months. Our data suggest that UPR activation in DS mouse models occurs early before consistent brain neurodegeneration and might be an essential contributor to dys-proteostasis.

Protein O-GlcNAcylation is a nutrient-related post-translational modification that, since its discovery some 30 years ago, has been associated with the development of neurodegenerative diseases. As reported in Alzheimer's disease (AD), flaws in the cerebral glucose uptake translate into reduced hexosamine biosynthetic pathway flux and subsequently lead to aberrant protein O-GlcNAcylation. Notably, the reduction of O-GlcNAcylated proteins involves also tau and APP, thus promoting their aberrant phosphorylation in AD brain and the onset of AD pathological markers. Down syndrome (DS) individuals are characterized by the early development of AD by the age of 60 and, although the two conditions present the same pathological hallmarks and share the alteration of many molecular mechanisms driving brain degeneration, no evidence has been sought on the implication of O-GlcNAcylation in DS pathology. Our study aimed to unravel for the first time the role of protein O-GlcNacylation in DS brain alterations positing the attention of potential trisomy-related mechanisms triggering the aberrant regulation of OGT/OGA cycle. We demonstrate the disruption of O-GlcNAcylation homeostasis, as an effect of altered OGT and OGA regulatory mechanism, and confirm the relevance of O-GlcNAcylation in the appearance of AD hallmarks in the brain of a murine model of DS. Furthermore, we provide evidence for the neuroprotective effects of brain-targeted OGA inhibition. Indeed, the rescue of OGA activity was able to restore protein O-GlcNAcylation, and reduce AD-related hallmarks and decreased protein nitration, possibly as effect of induced autophagy.

Doxorubicin (DOX) is an anticancer drug used for the treatment of solid tumors. The ability of DOX to treat cancer is not specific to cancer cells; some of the cells that are normal may also become targets of DOX, thereby altering the normal cellular functions and eventual cell loss. DOX effects have been studied in detail in heart because of its ability to cause cardiomyopathy. The exact mechanism of DOX-induced cardiomyopathy is not completely understood. One of organs that can be affected by DOX is thymus. DOX treatment leads to degeneration of thymus; however, since thymus undergoes age-dependent degeneration, researchers have understudied the effect of DOX on thymus. In the present investigation, we studied the effects of DOX on thymus, an organ that is important for the T-cell maturation. DOX treatment led to loss of cortical cells, decrease lymphopoiesis and increased the number of Hassells corpuscles, a marker of thymus aging. Proteomics analysis led to identification of a number of thymic proteins whose expression are altered by in vivo DOX treatment. Taken together, these results are consistent with the notion that DOX-treatment leads to thymic senescence.

Diabetes mellitus (DM) remains the main predictor of restenosis rates and cardiovascular events following successful percutaneous coronary intervention (PCI) despite the use of drug-eluting stents (DES). HbA1c <6.0% is considered an index of optimized metabolic control in patients with DM, but several studies are downsizing its role in the clinical management of these patients. Increasing evidence points at the role of advanced glycation end products (AGEs) in restenosis pathogenesis independently on Hb1AC levels. Thus, we investigated the predictive value of preprocedural AGE levels for in-stent restenosis in a population of euglycaemic diabetic patients undergoing PCI with DES implantation. One hundred twenty-five consecutive patients with DM in optimized glycemic control admitted for stable angina pectoris and treated with elective DES implantation at a tertiary hospital were prospectively included. The primary end point of the ARMYDA-AGEs study was to compare rates of angiographic ISR at 6 months after the intervention according to pre-PCI levels of AGEs. Secondary end points were the correlations of AGE levels with occurrence of periprocedural myocardial damage, major adverse cardiac events, and in-stent late loss at 6-month control coronary angiography. AGE levels >17 μM was found to be an independent predictor of ISR at 6 months and stent lumen loss. AGEs failed to predict occurrence of secondary endpoints. In conclusion, elevated AGE levels predict occurrence of in-stent restenosis after DES implantation in patients with DM on optimized glycemic control and might represent a dosable marker of adverse outcome after PCI. Diabetes mellitus (DM) remains the main predictor of restenosis rates and cardiovascular events following successful percutaneous coronary intervention (PCI) despite the use of drug-eluting stents (DES). HbA1c <6.0% is considered an index of optimized metabolic control in patients with DM, but several studies are downsizing its role in the clinical management of these patients. Increasing evidence points at the role of advanced glycation end products (AGEs) in restenosis pathogenesis independently on Hb1AC levels. Thus, we investigated the predictive value of preprocedural AGE levels for in-stent restenosis in a population of euglycaemic diabetic patients undergoing PCI with DES implantation. One hundred twenty-five consecutive patients with DM in optimized glycemic control admitted for stable angina pectoris and treated with elective DES implantation at a tertiary hospital were prospectively included. The primary end point of the ARMYDA-AGEs study was to compare rates of angiographic ISR at 6 months after the intervention according to pre-PCI levels of AGEs. Secondary end points were the correlations of AGE levels with occurrence of periprocedural myocardial damage, major adverse cardiac events, and in-stent late loss at 6-month control coronary angiography. AGE levels >17 μM was found to be an independent predictor of ISR at 6 months and stent lumen loss. AGEs failed to predict occurrence of secondary endpoints. In conclusion, elevated AGE levels predict occurrence of in-stent restenosis after DES implantation in patients with DM on optimized glycemic control and might represent a dosable marker of adverse outcome after PCI.

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)-scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O2- to H2O2 and molecular oxygen (O2). Among the three forms of SOD identified, manganese-containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2(-/+) ) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole-brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53(-/-) × SOD2(-/+) and p53(-/-) × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole-brain homogenate. The analysis of well-known antioxidant systems such as thioredoxin-1 and Nrf2/HO-1/BVR-A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels.

Abstract Increasing evidence supports the role of oxidative stress in cancer development. Ultraviolet (UV) irradiation is one of the major sources of oxidative stress through the generation of reactive oxygen species (ROS). Besides the physiological function of ROS in cellular homeostasis, accumulating reports suggest that ROS are involved in all stages of multistep cancer development. In order to investigate the involvement of oxidative damage into the mechanisms of tumour progression, we used a parallel proteomic approach to analyse the protein expression profile and to identify oxidatively modified proteins in human papillomavirus (HPV)‐transformed keratinocytes (HK‐168 cells) upon ultraviolet B (UVB) exposure. The HK‐168 cells were obtained from normal human epidermal keratinocytes transfected with the whole genome of the high‐risk HPV type 16, unanimously recognized as an etiological agent of cervical carcinoma. Because of its year‐long latency, this tumour offers a convenient model to study the role of environmental concurring agents in the multistep malignant progression. By the protein expression profile, we identified 21 proteins that showed different expression levels in HK‐168 cells treated with UVB in comparison with untreated cells. Focusing on the oxidative modifications occurring at the protein level, we identified five proteins that showed elevated protein carbonyls levels: α‐enolase, heat shock protein 75, annexin 2, elongation factor Tu and elongation factor γ. Our results indicate that UVB‐induced oxidative stress perturbs the normal redox balance and shifts HPV‐transformed keratinocytes into a state in which the carbonylation of specific proteins is systematically induced. We suggest that UVB‐induced modulation of protein expression combined with oxidative modification lead to protein dysfunction that might contribute to the malignant progression of transformed cells.

Although the role of STAT3 in cell physiology and tissue development has been largely investigated, its involvement in the development and maintenance of nervous tissue and in the mechanisms of neuroprotection is not yet known. The potentially wide range of STAT3 activities raises the question of tissue- and gender-specificity as putative mechanisms of regulation. To explore the function of STAT3 in the brain and the hypothesis of a gender-linked modulation of STAT3, we analyzed a neuron-specific STAT3 knockout mouse model investigating the influence of STAT3 activity in brain protein expression pattern in both males and females in the absence of neurological insult. We performed a proteomic study aimed to reveal the molecular pathways directly or indirectly controlled by STAT3 underscoring its role in brain development and maintenance. We identified several proteins, belonging to different neuronal pathways such as energy metabolism or synaptic transmission, controlled by STAT3 that confirm its crucial role in brain development and maintenance. Moreover, we investigated the different processes that could contribute to the sexual dimorphic behavior observed in the incidence of neurological and mental disease. Interestingly both STAT3 KO and gender factors influence the expression of several mitochondrial proteins conferring to mitochondrial activity high importance in the regulation of brain physiology and conceivable relevance as therapeutic target.

ABSTRACT Down syndrome (DS) is the most common condition with intellectual disability and is caused by trisomy of Homo sapiens chromosome 21 (HSA21). The increased dosage of genes on HSA21 is the cause for the initial neurodevelopmental disorder and for further development of cognitive decline, however the molecular mechanisms promoting brain pathology along ageing are still missing. One of the major challenges in the study of DS is the lack of reliable murine model able to accurately replicate genotypic and phenotypic aspects observed in humans along ageing. Preclinical studies in DS were pioneered using the Ts65Dn murine model, which despite its genetic limitations, has been extremely helpful in characterising the progression of brain degeneration. The novel Ts66Yah model represents an evolution of the Ts65Dn, with phenotypes only induced by trisomic HSA21 homologous genes, closer to human DS condition. In this study, we confirmed the behavioural features of Ts66Yah mice with improvement in the detection of spatial memory defects and also a new anxiety-related phenotype. The molecular characterisation of Ts66Yah demonstrated the aberrant regulation of redox balance, proteostasis, stress response, metabolic pathways, programmed cell death and synaptic plasticity. Intriguingly, the genotype-related alterations of those pathways occur early promoting the alteration of brain development and the onset of a condition of premature aging. Overall, data collected in Ts66Yah provide novel and consolidated insights, devoid of genome bias, concerning trisomy-driven processes that contribute to brain pathology in conjunction with aging. This, in turn, aids in bridging the existing gap in comprehending the intricate nature of DS phenotypes.

Redox reactions play a critical role for intracellular processes, including pathways involved in metabolism and signaling. Reactive oxygen species (ROS) act either as second messengers or generators of protein modifications, fundamental mechanisms for signal transduction. Disturbance of redox homeostasis is associated with many disorders. Among these, Alzheimer's disease is a neurodegenerative pathology that presents hallmarks of oxidative damage such as increased ROS production, decreased activity of antioxidant enzymes, oxidative modifications of macromolecules, and changes in mitochondrial homeostasis. Interestingly, alteration of redox homeostasis is closely associated with defects of energy metabolism, involving both carbohydrates and lipids, the major energy fuels for the cell. As the brain relies exclusively on glucose metabolism, defects of glucose utilization represent a harmful event for the brain. During aging, a progressive perturbation of energy metabolism occurs resulting in brain hypometabolism. This condition contributes to increase neuronal cell vulnerability ultimately resulting in cognitive impairment. The current review discusses the crosstalk between alteration of redox homeostasis and brain energy defects that seems to act in concert in promoting Alzheimer's neurodegeneration.

Down syndrome (DS) is the most common condition with intellectual disability and is caused by trisomy of Homo sapiens chromosome 21 (HSA21). The increased dosage of genes on HSA21 is associated with early neurodevelopmental changes and subsequently at adult age with the development of Alzheimer-like cognitive decline. However, the molecular mechanisms promoting brain pathology along aging are still missing. The novel Ts66Yah model represents an evolution of the Ts65Dn, used in characterizing the progression of brain degeneration, and it manifest phenotypes closer to human DS condition. In this study we performed a longitudinal analysis (3–9 months) of adult Ts66Yah mice. Our data support the behavioural alterations occurring in Ts66Yah mice at older age with improvement in the detection of spatial memory defects and also a new anxiety-related phenotype. The evaluation of hippocampal molecular pathways in Ts66Yah mice, as effect of age, demonstrate the aberrant regulation of redox balance, proteostasis, stress response, metabolic pathways, programmed cell death and synaptic plasticity. Intriguingly, the genotype-driven changes observed in those pathways occur early promoting altered brain development and the onset of a condition of premature aging. In turn, aging may account for the subsequent hippocampal deterioration that fall in characteristic neuropathological features. Besides, the analysis of sex influence in the alteration of hippocampal mechanisms demonstrate only a mild effect. Overall, data collected in Ts66Yah provide novel and consolidated insights, concerning trisomy-driven processes that contribute to brain pathology in conjunction with aging. This, in turn, aids in bridging the existing gap in comprehending the intricate nature of DS phenotypes.

Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer's disease (AD), although the molecular changes preceding overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dysregulates Akt-GSK3β complex in response to insulin, hindering the accumulation of pGSK3βS9 into the mitochondria. This event impairs oxidative phosphorylation and fosters the activation of the mitochondrial Unfolded Protein Response (UPRmt). Remarkably, we unveil that BVR-A is required to shuttle pGSK3βS9 into the mitochondria. Our data sheds light on the intricate interplay between insulin signaling and mitochondrial metabolism in the brain unraveling potential targets for mitigating the development of brain insulin resistance and neurodegeneration.

Molecular mechanisms underlying abdominal aneurysm (AAA) formation and rupture are not well understood. Early detection and repair of AAA may reduce the high mortality rates associated with rupture. Serum proteomics allows the detection of alterations in the expression of proteins, guiding further studies on these target molecules as potential markers. Analysis of proteomic profile of asymptomatic patients with AAA allows the identification of reliable predictors or markers of disease presence or progression. A proteomics approach based on two-dimensional electrophoresis and mass spectrometry was used to compare serum proteomic profiles of patients with AAA who are candidates for surgical repair compared with healthy controls. We analyzed in parallel the proteomic profile of subjects with cardiac heart failure to discriminate these two pathologies, which show similar pattern of systemic inflammation process. We identified in AAA subjects four serum proteins that show altered expression profile and that could be specifically linked to AAA pathology. We discuss the role of our identified proteins with their possible implications in disease outcome. This approach could provide an initial screening tool that may drive the basis for further research in the field of cardiovascular diseases. These results need to be validated in larger studies to find potential markers of AAA presence or progression to use in clinical settings. A proteomics approach was used to compare serum proteomic profiles of patients with abdominal aortic aneurysm who are candidates for surgical repair compared with healthy controls. Four serum proteins showed altered expression profile that could be correlated with the pathology. This approach could provide an initial screening tool that may drive the basis for further research in the field of cardiovascular diseases.

Alzheimer disease (AD) is the most common form of dementia among the elderly and is characterized by progressive loss of memory and cognition. Amyloid-ß-peptide (Aß) forms senile plaques, which, together with hyperphosphorylated tau-based neurofibrillary tangles, are the hallmarks of AD neuropathology. Evidence support the involvement of immune system in AD progression and current concepts regarding its pathogenesis include the participation of inflammatory and autoimmune components in the neurodegenerative process. Pathologically, immune system components have been detected in the brain, cerebrospinal fluid (CSF) and in serum of AD subjects and their trend of variation correlates with disease progression. However, patients with AD present significantly lower levels of antibody immunoreactivity against Aß in serum and CSF than healthy controls suggesting that a depletion of such patrolling system is involved in the deposition of toxic aggregates in AD. Within this frame, incomplete and often controversial results are reported about CNS immune/ autoimmune responses during AD, and a better comprehension of such processes is needed. Our research will aim to shed light on the nature and potential role of autoantibodies in CSF and serum from AD and amnestic mild cognitive impairment (aMCI) patients compared to healthy subjects by using an immunoproteomics approach. Our method allows recognition of natural occurring antibodies by the identification of brain antigen targeted by human IgGs. Overall our data reveal that the alterations of autoantibodies profile both in CSF and serum follow disease staging and progression. However, we demonstrate a fair overlap between CSF and serum suggesting the existence of different immunogenic events. Interestingly, CSF autoantibodies recognized, among others, key players of energy metabolic pathway, including glycolysis and TCA cycle, found oxidatively modified in AD brain studies. These data suggest a potential casual sequence between oxidative damage at brain level, autoantibodies presence in CSF and reduced energy metabolism of AD patients. Keywords: Autoantibodies, autoimmunity, Alzheimer disease, cerebrospinal fluid, immunoproteomics, mild cognitive impairment.

Abstract Background Diabetic patients exhibit an increased risk of saphenous graft occlusion after coronary bypass. Advanced glycation end products (AGEs) are ubiquitous signalling proteins that are associated with vascular and neurological complication of diabetes. The aim of this study is to verify whether AGE levels may promote endothelial cell alterations responsible for vein graft failure. Methods Segments of saphenous vein were obtained from both normal people and diabetic patients (HbA 1c &lt; 6.0%) at the time of coronary surgery. Cultured endothelial cells were incubated in the absence/presence of AGEs (2 and 20 µM), and mRNA and protein for both receptor of AGEs (RAGE) and peroxisome proliferator‐activated receptors‐γ (PPAR‐γ) were analysed by real‐time polymerised chain reaction (PCR) and Western blot analysis. In the same fashion, the cell release of reactive oxygen species (ROS) was estimated in the absence/presence of AGEs by spectrofluorimetric analysis. Finally, neutrophil‐endothelial adhesion was evaluated in saphenous vein segments with and without the addition of AGEs. Results AGEs activated in a dose‐dependent manner the expression of RAGE and inhibited PPAR‐γ expression in endothelial cells as testified by both reverse transcription‐PCR (RT‐PCR) and Western blot analysis. Stimulation of cultured endothelial cells with AGEs significantly enhanced intracellular ROS formation in a dose‐dependent manner. Finally, neutrophil‐endothelial adhesion was significantly increased after incubation of control veins with AGEs. Conclusions These findings indicate that even in diabetic patients with HbA 1c &lt; 6.0%, elevated serum levels of AGE determine a sort of a pro‐thrombotic state, providing a common mechanism that could explain the increased rate of vein graft occlusion in this population. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.