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Postsynaptic Proteome of Non-Demented Individuals with Alzheimer’S Disease Neuropathology

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Postsynaptic Proteome of Non-Demented Individuals with Alzheimer’S Disease Neuropathology Journal of Alzheimer’s Disease 65 (2018) 659–682 659 DOI 10.3233/JAD-180179 IOS Press Postsynaptic Proteome of Non-Demented Individuals with Alzheimer’s Disease Neuropathology Olga Zolochevskaa,2, Nicole Bjorklunda,1,2, Randall Woltjerb, John E. Wiktorowiczc and Giulio Taglialatelaa,∗ aDepartment of Neurology, University of Texas Medical Branch, Galveston, TX, USA bDepartment of Pathology, Oregon Health and Science University, Portland, OR, USA cDepartment of Biochemistry and Molecular Biology, Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, TX, USA Handling Associate editor: Daniela Puzzo Accepted 5 July 2018 Abstract. Some individuals, here referred to as Non-Demented with Alzheimer’s Neuropathology (NDAN), retain their cognitive function despite the presence of amyloid plaques and tau tangles typical of symptomatic Alzheimer’s disease (AD). In NDAN, unlike AD, toxic amyloid-␤ oligomers do not localize to the postsynaptic densities (PSDs). Synaptic resistance to amyloid-␤ in NDAN may thus enable these individuals to remain cognitively intact despite the AD-like pathology. The mechanism(s) responsible for this resistance remains unresolved and understanding such protective biological processes could reveal novel targets for the development of effective treatments for AD. The present study uses a proteomic approach to compare the hippocampal postsynaptic densities of NDAN, AD, and healthy age-matched persons to identify protein signatures characteristic for these groups. Subcellular fractionation followed by 2D gel electrophoresis and mass spectrometry were used to analyze the PSDs. We describe fifteen proteins which comprise the unique proteomic signature of NDAN PSDs, thus setting them apart from control subjects and AD patients. Keywords: Alzheimer’s disease, non-demented with AD-like pathology, postsynaptic density proteome, synapse INTRODUCTION history/genetics (apolipoprotein ε, APOE; amyloid- ␤ precursor protein, A␤PP; presenilin, PSEN1 and Alzheimer’s disease (AD) is the most common PSEN2), brain injury, cardiovascular health, physical form of dementia, affecting more than 5 million activity, and social interactions among others [2–4]. Americans [1]. Old age is the greatest risk factor for While available therapies have limited efficacy and AD, which thus affects over one third of people older only temporarily mitigate cognitive decline, currently than 85 years of age. Other risk factors include family there are no disease-modifying treatments for AD. Typical AD pathology is characterized by two 1Present address: Department of Medicine, Albert Einstein main neurodegenerative processes: amyloidogenesis College of Medicine, Bronx, NY, USA and neurofibrillary degeneration [5]. Amyloidogen- 2These authors contributed equally to this work. ␤ ∗ esis leads to deposition of extracellular amyloid- Correspondence to: Giulio Taglialatela, Department of Neu- ␤ rology, University of Texas Medical Branch, 301 University Blvd., (A ) peptide, while neurofibrillary tangles (NFTs) Galveston, TX 77555-1045, USA. Tel.: +1 409 772 1679; E-mail: contain hyperphosphorylated tau protein. However, [email protected]. there is no correlation between presence of mature ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial License (CC BY-NC 4.0). 660 O. Zolochevska et al. / Proteome of AD Resistant Synapses A␤ deposits and NFTs with the cell loss or cog- at the PSDs of NDAN subjects that specifically mark nitive decline [6–10]. On the other hand, presence the ability of their PSDs in the hippocampus to reject of soluble oligomeric species of A␤ and tau in binding of toxic A␤ oligomers. To test our hypothesis, the brain correlates with cognitive dysfunction [7, in the present work we performed proteomic analy- 11–13]. Early oligomers, the most toxic forms of sis of the PSDs isolated from healthy control, AD, misfolded A␤ and tau forming during the aggre- and NDAN individuals. The protein levels in AD and gation process, can accumulate at the synapse and NDAN were compared to control, in addition to direct synergistically impair dendritic spines in the hip- NDAN versus AD comparison. As a result, we identi- pocampus [7, 14–19]. Microscopic changes in the fied a unique PSD protein signature of NDAN which brain begin long before memory loss [20]. Synapse consists of fifteen proteins, setting them apart from loss is believed to occur at early stages of the dis- control and AD. ease [20, 21], while cell death occurs at later stages and contributes to ultimate cognitive decline [20]. METHODS AD has a long asymptomatic stage and with the development of improved neuroimaging techniques Case subjects it has become evident that some individuals with AD-like neuropathology do not exhibit any cognitive Frozen mid-hippocampus tissue was obtained from decline during their lifetime ([8, 20, 22]; reviewed by the Oregon Brain Bank at Oregon Health and Sci- [23]). Such individuals can be described as “asymp- ence University (OHSU) in Portland, OR. Donor tomatic at risk for AD dementia”, or “not normal, subjects were enrolled and clinically evaluated in not MCI (mild cognitive impairment)” [22]. We refer studies at the NIH-sponsored Layton Aging and to this cohort of individuals as Non-Demented with AD Center (ADC) at OHSU. Subjects were par- Alzheimer’s Neuropathology (NDAN) [24]. Several ticipants in brain aging studies at the ADC and research groups, including ours, are trying to under- received annual neurological and neuropsycholog- stand the mechanisms involved in preservation of ical evaluations, with a Clinical Dementia Rating cognition in NDAN, with the goal of identifying treat- (CDR) assigned by an experienced clinician. Con- ment targets to develop novel therapeutic concepts trols and NDAN had normal cognitive and functional based on intrinsic resistance to AD [6, 8, 22, 24–30]. examinations with CDR <1. The AD subjects were Synaptic dysfunction in AD is observed as a result diagnosed by a clinical team consensus confer- of A␤ oligomers association with the postsynap- ence, met the National Institute for Neurological and tic density (PSD) [16]. At the PSD, A␤ oligomers Communicative Disorders and Stroke-Alzheimer’s oppose expression of long-term potentiation (LTP), Disease and Related Disorder Association diagnos- modify protein content, and induce dendritic spine tic criteria for clinical AD, had a CDR of greater shrinkage and eventually loss [16, 31, 32]. Since the than 1.0 and neuropathologic confirmation at autopsy size of the PSD is proportional to the strength of (after informed consent). Tissue use conformed the synapse, A␤-driven synapse damage can result to institutional review board-approved protocols. in the loss of cognitive function. In AD, plasticity Neuropathologic assessment conformed to National and cognition are affected through the perturbation Institute on Aging-Reagan consensus criteria. All of Ca2+ /calmodulin-dependent protein kinase II-␣ brain tissue was examined by a neuropathologist (CaMKII) autophosphorylation [32]. Association of for neurodegenerative pathology including NFTs and A␤ oligomers with the PSD implicates dephospho- neuritic plaques. Using standardized CERAD criteria rylation (deactivation) of CREB (cAMP response [34], cases were assigned an amyloid score based on element-binding protein factor), which in turn affects the deposition of amyloid plaques in the brain (0 = no transcription of genes regulating long-term changes plaques, 1 = sparse plaques, 2 = moderate plaques and in synaptic strength [33]. 3 = dense plaques) and a Braak stage (0–6; with 6 We have previously reported that NDAN synapses being the most severe) indicative of the level and reject A␤ oligomers, which could explain why location of hyperphosphorylated tau tangles [35]. NDAN subjects remain cognitively intact. Our lab- In addition to the pathological information detailed oratory has demonstrated for the first time that the above, demographical data were received along with PSD of NDAN subjects is free of A␤ oligomers [24]. the frozen tissue. These included age, sex, and MMSE Based on this observation, we hypothesized that there score [34] for each case. Several AD patients pre- might be unique changes in protein expression levels sented with relatively high MMSE scores, which is O. Zolochevska et al. / Proteome of AD Resistant Synapses 661 attributable to multiple factors: 1) the last MMSE was Synaptic junctions were obtained by incubating the collected several months prior to death, 2) the diag- synaptosomes in pH = 6 buffer (as described above) nosis is mainly based on consensus CDR assessments and then centrifuging at 40,000 × g for 30 min at and several tests beyond MMSE, 3) even milder 4◦C. The supernatant (containing synaptic vesicles) impairments sometimes led to the diagnosis of AD and the pellet were collected separately. The pellet with the understanding that this was earlier-stage dis- was solubilized and incubated in pH = 8 buffer (as ease. When these scores are looked at as a whole, it described above) and then centrifuged at 40,000 × g can be appreciated that these patients do tend to be for 30 min at 4◦C to generate the PSD pellet. This in earlier stages of dementia, both in terms of their pellet was solubilized in 1% SDS. clinical ratings and in terms of their pathologies, with intermediate (3–4) versus high (5–6) Braak scores. Proteomics
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