Chiara Lanzillotta

Oxidative stress, protein modification and Alzheimer disease

Alzheimer disease (AD) is a progressive neurodegenerative disease that affects the elderly population with complex etiology. Many hypotheses have been proposed to explain different causes of AD, but the exact mechanisms remain unclear. In this review, we focus attention on the oxidative-stress hypothesis of neurodegeneration and we discuss redox proteomics approaches to analyze post-mortem human brain from AD brain. Collectively, these studies have provided valuable insights into the molecular mechanisms involved both in the pathogenesis and progression of AD, demonstrating the impairment of numerous cellular processes such as energy production, cellular structure, signal transduction, synaptic function, mitochondrial function, cell cycle progression, and degradative systems. Each of these cellular functions normally contributes to maintain healthy neuronal homeostasis, so the deregulation of one or more of these functions could contribute to the pathology and clinical presentation of AD. In particular, we discuss the evidence demonstrating the oxidation/dysfunction of a number of enzymes specifically involved in energy metabolism that support the view that reduced glucose metabolism and loss of ATP are crucial events triggering neurodegeneration and progression of AD.

Targeting mTOR to reduce Alzheimer-related cognitive decline: from current hits to future therapies

ABSTRACT 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.

Increased mammalian target of rapamycin signaling contributes to the accumulation of protein oxidative damage in a mouse model of down's syndrome

Background: 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 Downs syndrome (DS) and Alzheimers 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. Summary: 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. Key Messages: 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.

Activation of p53 in Down Syndrome and in the Ts65Dn Mouse Brain is Associated with a Pro-Apoptotic Phenotype

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 Alzheimers disease (AD), with deposition of senile plaques and tangles. We hypothesized that apoptosis may be activated 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.

Identification of changes in neuronal function as a consequence of aging and tauopathic neurodegeneration using a novel and sensitive magnetic resonance imaging approach

Tauopathies, the most common of which is Alzheimers 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 Alzheimers 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.

Protein nitration profile of CD3+ lymphocytes from Alzheimer disease patients: Novel hints on immunosenescence and biomarker detection

&NA; Alzheimers disease (AD) is a progressive form of dementia characterized by increased production of amyloid‐&bgr; 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. Graphical abstract Figure. No caption available. HighlightsAD is characterized by modifications of the immune systems associated with reduced T‐cells activity.We identified increased protein nitration levels in T‐cells from AD patients compared to healthy subjects.The protein showing increased nitration are involved in pathways crucial to maintain neuronal homeostasis.Our data provide new insights into the molecular mechanisms that lead to reduced T‐cell differentiation in AD.The finding of common nitration targets between the brain and the periphery support their use in disease prediction.

Therapeutic potential of rescuing protein O-GlcNAcylation in tau-related pathologies

O-GlcNAcylation is the non-canonical glycosylation of nucleocytoplasmic proteins with a single O-linked N-acetylglucosamine (O-GlcNAc) moiety. The dynamic cycling of O-GlcNAc on proteins is regulated by the concerted actions of two enzymes: the O-GlcNAc transferase (OGT) and a neutral β-hexosaminidase known as O-GlcNAcase (OGA). The O-GlcNAcylation of proteins occurs on serine and threonine residues, which can be commonly occupied by phosphate groups. Therefore, O-GlcNAcylation and phosphorylation are mutually related and by this cells can modulate a variety of signaling pathways and transcription factor in response to nutrients or stress [1]. O-GlcNAcylation is the product of nutrient flux through the hexosamine biosynthetic pathway (HBP), which integrates glucose, amino acid, fatty acid, and nucleotide metabolism to generate the donor substrate for O-GlcNAcylation, uridine diphosphate GlcNAc (UDP-GlcNAc). Approximately 2–5% of all glucose entering the cell is channeled into the HBP to generate UDPGlcNAc. Glutamine–fructose6-phosphate amide transferase (GFAT), the rate-limiting enzyme of the HBP that catalyzes the formation of glucosamine 6-phosphate, is shown to be subject to feedback inhibition by UDPGlcNAc. Because O-GlcNAcylation depends on the availability of UDP-GlcNAc, and in turn intracellular UDP-GlcNAc level determines OGT activity, O-GlcNAcylation is considered a valuable intracellular sensor of glucose metabolism that can be directly regulated in a glucose-responsive manner.

Proteomic identification of altered protein O-GlcNAcylation in a triple transgenic mouse model of Alzheimer's disease

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.

POST-INJURY PERK INHIBITION IN MOUSE MODEL OF TAUOPATHY

Background:Pathological aggregations of tau and amyloid beta proteins are the hallmarks of Alzheimer’s disease. Previous work from our group has shown Abl-selective tyrosine kinase inhibitors stimulate beclin-mediated autophagy and promote clearance of neurodegenerative proteins. Specifically, the drugs nilotinib and bosutinib decrease levels of pathological proteins and reverse motor and cognitive decline in mouse models of neurodegenerative disease. Further, we have shown pazopanib, an FDA-approved inhibitor of the tyrosine kinases VEGFR, PDGFRa, PDGFRb, and c-KIT, penetrates the blood-brain barrier and decreases p-tau levels in TauP301L mice. These experiments aim to confirm and expand upon the initial findings as well as determine the effects of pazopanib on amyloid beta in 3x-APP mice. Methods:Male and female TauP301L, 3x-APP, and non-transgenic littermates approximately 12-17 months old were treated with 5mg/kg pazopanib (roughly half the clinically-used dose) or vehicle (DMSO) intraperitoneally (IP) for 3-4 weeks. Phosphorylated tau levels were measured by Western blot and enzyme-linked immunosorbent assay in brain homogenates, and immunohistochemistry in 20mm brain sections, fixed in 4% paraformaldehyde. Serum was collected to assess kidney and liver function. Ab levels and inflammation were measured using MILLIPLEX ELISA. Autophagy markers were measured via Western blot. Results: Pazopanib treatment does not alter weight, liver (ALT), or kidney injury markers in TauP301L and 3x-APP mouse cohorts. Pazopanib significantly reduces levels of p-tau (T181, T231) in TauP301L mice. Further, brain levels of beclin-1 and p-mTOR/mTOR were unchanged. In 3x-APP mice, treatment does not alter Ab40 or Ab42levels. However, pazopanib significantly reverses levels of IP-10, MIP-1a, MIP-1b, and RANTES toward control levels. Conclusions: Pazopanib (5mg/kg) appears to be a safe, well-tolerated drug that significantly reduces p-tau levels in TauP301L mice in a manner likely independent of beclin or mTOR-mediated autophagy and reverses inflammation in 3x-APP mice. Future studies aim to determine which specific tyrosine kinase target(s) is complicit in p-tau clearance.