Alterations In Synaptic Proteins Research Articles

A Strategy for Allowing Earlier Diagnosis and Rigorous Evaluation of BACE1 Inhibitors in Preclinical Alzheimer’s Disease

Given continued failure of BACE1 inhibitor programs at symptomatic and prodromal stages of Alzheimer’s disease (AD), clinical trials need to target the earlier preclinical stage. However, trial design is complex in this population with negative diagnosis of classical hippocampal amnesia on standard memory tests. Besides recent advances in brain imaging, electroencephalogram, and fluid-based biomarkers, new cognitive markers should be established for earlier diagnosis that can optimize recruitment to BACE1 inhibitor trials in presymptomatic AD. Notably, accelerated long-term forgetting (ALF) is emerging as a sensitive cognitive measure that can discriminate between asymptomatic individuals with high risks for developing AD and healthy controls. ALF is a form of declarative memory impairment characterized by increased forgetting rates over longer delays (days to months) despite normal storage within the standard delays of testing (20–60 min). Therefore, ALF may represent a harbinger of preclinical dementia and the impairment of systems memory consolidation, during which memory traces temporarily stored in the hippocampus become gradually integrated into cortical networks. This review provides an overview of the utility of ALF in a rational design of next-generation BACE1 inhibitor trials in preclinical AD. I explore potential mechanisms underlying ALF and relevant early-stage biomarkers useful for BACE1 inhibitor evaluation, including synaptic protein alterations, astrocytic dysregulation and neuron hyperactivity in the hippocampal-cortical network. Furthermore, given the physiological role of the isoform BACE2 as an AD-suppressor gene, I also discuss the possible association between the poor selectivity of BACE1 inhibitors and their side effects (e.g., cognitive worsening) in prior clinical trials.

Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Circadian rhythm disruptions associated with opioid use disorder in synaptic proteomes of human dorsolateral prefrontal cortex and nucleus accumbens

Opioid craving and relapse vulnerability is associated with severe and persistent sleep and circadian rhythm disruptions. Understanding the neurobiological underpinnings of circadian rhythms and opioid use disorder (OUD) may prove valuable for developing new treatments for opioid addiction. Previous work indicated molecular rhythm disruptions in the human brain associated with OUD, highlighting synaptic alterations in the dorsolateral prefrontal cortex (DLPFC) and nucleus accumbens (NAc)—key brain regions involved in cognition and reward, and heavily implicated in the pathophysiology of OUD. To provide further insights into the synaptic alterations in OUD, we used mass-spectrometry based proteomics to deeply profile protein expression alterations in bulk tissue and synaptosome preparations from DLPFC and NAc of unaffected and OUD subjects. We identified 55 differentially expressed (DE) proteins in DLPFC homogenates, and 44 DE proteins in NAc homogenates, between unaffected and OUD subjects. In synaptosomes, we identified 161 and 56 DE proteins in DLPFC and NAc, respectively, of OUD subjects. By comparing homogenate and synaptosome protein expression, we identified proteins enriched specifically in synapses that were significantly altered in both DLPFC and NAc of OUD subjects. Across brain regions, synaptic protein alterations in OUD subjects were primarily identified in glutamate, GABA, and circadian rhythm signaling. Using time-of-death (TOD) analyses, where the TOD of each subject is used as a time-point across a 24-h cycle, we were able to map circadian-related changes associated with OUD in synaptic proteomes associated with vesicle-mediated transport and membrane trafficking in the NAc and platelet-derived growth factor receptor beta signaling in DLPFC. Collectively, our findings lend further support for molecular rhythm disruptions in synaptic signaling in the human brain as a key factor in opioid addiction.

Spreading of Tau Protein Does Not Depend on Aggregation Propensity

The stereotypical progression of Tau pathology during Alzheimer disease has been attributed to trans-neuronal spreading of misfolded Tau proteins, followed by prion-like templated aggregation of Tau. The nature of Tau and the cellular mechanisms of Tau spreading are still under debate. We hypothesized that Tau’s propensity for aggregation would correlate with its ability to spread across synapses and propagate pathology. To study the progressive propagation of Tau proteins in brain regions relevant for Alzheimer disease, we used mice expressing near-physiological levels of full-length human Tau protein carrying pro-aggregant (TauΔK280, TauΔK) or anti-aggregant (TauΔK280-PP, TauΔK−PP) mutations in the entorhinal cortex (EC). To enhance Tau expression in the EC, we performed EC injections of adeno-associated virus (AAV) particles encoding TauΔK or TauΔK−PP. The brains of injected and non-injected EC/TauΔK and EC/TauΔK−PP mice were studied by immunohistological and biochemical techniques to detect Tau propagation to dentate gyrus (DG) neurons and Tau-induced pathological changes. Pro- and anti-aggregant mice had comparable low transgene expression (~0.2 times endogenous mouse Tau). They accumulated human Tau at similar rates and only in expressing EC neurons, including their axonal projections of the perforant path and presynaptic terminals in the molecular layer of the DG. Pro-aggregant EC/TauΔK mice showed misfolded Tau and synaptic protein alterations in EC neurons, not observed in anti-aggregant EC/TauΔK−PP mice. Additional AAV-mediated expression of TauΔK or TauΔK−PP in EC/TauΔK or EC/TauΔK−PP mice, respectively, increased the human Tau expression to ~0.65 times endogenous mouse Tau, with comparable spreading of TauΔK and TauΔK−PP throughout the EC. There was a low level of transcellular propagation of Tau protein, without pathological phosphorylation or misfolding, as judged by diagnostic antibodies. Additionally, TauΔK but not TauΔK−PP expression induced hippocampal astrogliosis. Low levels of pro- or anti-aggregant full-length Tau show equivalent distributions in EC neurons, independent of their aggregation propensity. Increasing the expression via AAV induce local Tau misfolding in the EC neurons, synaptotoxicity, and astrogliosis and lead to a low level of detectable trans-neuronal spreading of Tau. This depends on its concentration in the EC, but, contrary to expectations, does not depend on Tau’s aggregation propensity/misfolding and does not lead to templated misfolding in recipient neurons.

Impact of Adolescent Nicotine Exposure in Pre- and Post-natal Oxycodone Exposed Offspring.

Perinatal exposure to prescription opioids pose a critical public health risk. Notably, research has found significant neurodevelopmental and behavioral deficits between in utero (IUO) and postnatal (PNO) oxycodone-exposed offspring but there is a notable gap in knowledge regarding the interaction of these groups to other drug exposure, particularly nicotine exposure. Nicotine's widespread use represents a ubiquitous clinical interaction that current research does not address. Children often experiment with drugs and risky behavior; therefore, adolescence is a key timepoint to characterize. This study employed an integrated systems approach to investigate escalating nicotine exposure in adolescence and subsequent nicotine withdrawal in the IUO- and PNO-offspring. Western blot analysis found synaptic protein alterations, especially upregulation of synaptophysin in IUO-withdrawal animals. RT-qPCR further validated immune dysfunction in the central nervous system (CNS). Peripheral nicotine metabolism was consistent with increased catabolism of nicotine concerning IUO animals. Lastly, behavioral assays found subtle deficits to withdrawal in nociception and anxiety-like behavior. This study showed, for the first time, the vulnerabilities of PNO- and IUO-exposed groups concerning nicotine use during early adolescence and withdrawal. Graphical Abstract.

Stereotactic Delivery of Helper-dependent Adenoviral Viral Vectors at Distinct Developmental Time Points to Perform Age-dependent Molecular Manipulations of the Mouse Calyx of Held.

Synapses are specialized structures that enable neuronal communication, which is essential for brain function and development. Alterations in synaptic proteins have been linked to various neurological and neuropsychiatric disorders. Therefore, manipulating synaptic proteins in vivo can provide insight into the molecular mechanisms underlying these disorders and aid in developing new therapeutic strategies. Previous methods such as constitutive knock-out animals are limited by developmental compensation and off-target effects. The current approach outlines procedures for age-dependent molecular manipulations in mice using helper-dependent adenovirus viral vectors (HdAd) at distinct developmental time points. Using stereotactic injection of HdAds in both newborn and juvenile mice, we demonstrate the versatility of this method to express Cre recombinase in globular bushy cells of juvenile Rac1fl/fl mice to ablate presynaptic Rac1 and study its role in synaptic transmission. Separately, we overexpress CaV2 α1 subunits at two distinct developmental time points to elucidate the mechanisms that determine presynaptic CaV2 channel abundance and preference. This method presents a reliable, cost-effective, and minimally invasive approach for controlling gene expression in specific regions of the mouse brain and will be a powerful tool to decipher brain function in health and disease. Key features Virus-mediated genetic perturbation in neonatal and young adult mice. Stereotaxic injection allows targeting of brain structures at different developmental stages to study the impact of genetic perturbation throughout the development.

Translational potential of synaptic alterations in Alzheimer's disease patients and amyloid precursor protein knock-in mice.

Synaptic dysfunction is an early event in Alzheimer's disease. Post-mortem studies suggest that alterations in synaptic proteins are associated with cognitive decline in Alzheimer's disease. We measured the concentration of three synaptic proteins, zinc transporter protein 3, dynamin1 and AMPA glutamate receptor 3 in cerebrospinal fluid of subjects with mild cognitive impairment (n = 18) and Alzheimer's disease (n = 18) and compared the levels to cognitively and neurologically healthy controls (n = 18) by using ELISA assay. In addition, we aimed to assess the translational potential of these synaptic proteins in two established amyloid precursor protein knock-in Alzheimer's disease mouse models by assessing the cerebrospinal fluid, hippocampal and cortical synaptic protein concentrations. Using ELISA, we measured in parallel these three proteins in cerebrospinal fluid and/or brain of 12- and 24-month-old AppNL-F and AppNL-G-F knock-in mice and AppWt control mice. The regional distribution and expression of these proteins were explored upon aging of the App knock-in models by quantitative immunofluorescence microscopy. Notably, we found a significant increase in concentrations of zinc transporter protein 3 and AMPA glutamate receptor 3 in cerebrospinal fluid of both patient groups compared with cognitively healthy controls. Dynamin1 concentration was significantly higher in Alzheimer's disease patients. Remarkably, patients with mild cognitive impairment who converted to Alzheimer's disease (n = 7) within 2 years exhibited elevated baseline cerebrospinal fluid zinc transporter protein 3 concentrations compared with mild cognitive impairment patients who did not convert (n = 11). Interestingly, similar to the alterations in Alzheimer's disease subjects, cerebrospinal fluid AMPA glutamate receptor 3 concentration was significantly higher in AppNL-G-F knock-in mice when compared with wild-type controls. Furthermore, we have detected age and brain regional specific changes of the three synaptic proteins in the hippocampus and prefrontal cortex of both AppNL-F and AppNL-G-F knock-in mice. Notably, all the three cerebrospinal fluid synaptic protein concentrations correlated negatively with concentrations in hippocampal lysates. The elevated zinc transporter protein 3 concentrations in the cerebrospinal fluid of converter versus non-converter mild cognitive impairment patients suggests a prospective role of zinc transporter 3 in differentiating dementia patients of the biological continuum of Alzheimer's disease. The increased cerebrospinal fluid concentrations of synaptic proteins in both patient groups, potentially reflecting synaptic alterations in the brain, were similarly observed in the amyloid precursor protein knock-in mouse models highlighting the translational potential of these proteins as markers for synaptic alterations. These synaptic markers could potentially help reduce the current disparities between human and animal model-based studies aiding the translation of preclinical discoveries of pathophysiological changes into clinical research.

Cerebrospinal Fluid Biomarkers of Synaptic Dysfunction are Altered in Parkinson's Disease and Related Disorders.

Synaptic dysfunction and degeneration are central contributors to the pathogenesis and progression of parkinsonian disorders. Therefore, identification and validation of biomarkers reflecting pathological synaptic alterations are greatly needed and could be used in prognostic assessment and to monitor treatment effects. To explore candidate biomarkers of synaptic dysfunction in Parkinson's disease (PD) and related disorders. Mass spectrometry was used to quantify 15 synaptic proteins in two clinical cerebrospinal fluid (CSF) cohorts, including PD (n1 =51, n2 =101), corticobasal degeneration (CBD) (n1 =11, n2 =3), progressive supranuclear palsy (PSP) (n1 =22, n2 =21), multiple system atrophy (MSA) (n1 =31, n2 =26), and healthy control (HC) (n1 =48, n2 =30) participants, as well as Alzheimer's disease (AD) (n2 =23) patients in the second cohort. Across both cohorts, lower levels of the neuronal pentraxins (NPTX; 1, 2, and receptor) were found in PD, MSA, and PSP, compared with HC. In MSA and PSP, lower neurogranin, AP2B1, and complexin-2 levels compared with HC were observed. In AD, levels of 14-3-3 zeta/delta, beta- and gamma-synuclein were higher compared with the parkinsonian disorders. Lower pentraxin levels in PD correlated with Mini-Mental State Exam scores and specific cognitive deficits (NPTX2; rho=0.25-0.32, P < 0.05) and reduced dopaminergic pre-synaptic integrity as measured by DaTSCAN (NPTX2; rho=0.29, P=0.023). Additionally, lower levels were associated with the progression of postural imbalance and gait difficulty symptoms (All NPTX; β-estimate=-0.025 to -0.038, P < 0.05) and cognitive decline (NPTX2; β-estimate=0.32, P=0.021). These novel findings show different alterations of synaptic proteins in parkinsonian disorders compared with AD and HC. The neuronal pentraxins may serve as prognostic CSF biomarkers for both cognitive and motor symptom progression in PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Cerebrospinal fluid biomarker panel of synaptic dysfunction in Alzheimer's disease and other neurodegenerative disorders.

Synaptic degeneration is a key part of the pathophysiology of neurodegenerative diseases, and biomarkers reflecting the pathological alterations are greatly needed. Seventeen synaptic proteins were quantified in a pathology-confirmed cerebrospinal fluid cohort of patients with Alzheimer's disease (AD; n=63), frontotemporal lobar degeneration (FTLD; n=53), and Lewy body spectrum of disorders (LBD; n=21), as well as healthy controls (HC; n=48). Comparisons revealed four distinct patterns: markers decreased across all neurodegenerative conditions compared to HC (the neuronal pentraxins), markers increased across all neurodegenerative conditions (14-3-3 zeta/delta), markers selectively increased in AD compared to other neurodegenerative conditions (neurogranin and beta-synuclein), and markers selectively decreased in LBD and FTLD compared to HC and AD (AP2B1 and syntaxin-1B). Several of the synaptic proteins may serve as biomarkers for synaptic dysfunction in AD, LBD, and FTLD. Additionally, differential patterns of synaptic protein alterations seem to be present across neurodegenerative diseases. A panel of synaptic proteins were quantified in the cerebrospinal fluid using mass spectrometry. We compared Alzheimer's disease, frontotemporal degeneration, and Lewy body spectrum of disorders. Pathology was confirmed by autopsy or familial mutations. We discovered synaptic biomarkers for synaptic degeneration and cognitive decline. We found differential patterns of synaptic proteins across neurodegenerative diseases.

Alterations in brain synaptic proteins and mRNAs in mood disorders: a systematic review and meta-analysis of postmortem brain studies.

The pathophysiological mechanisms underlying bipolar (BD) and major depressive disorders (MDD) are multifactorial but likely involve synaptic dysfunction and dysregulation. There are multiple synaptic proteins but three synaptic proteins, namely SNAP-25, PSD-95, and synaptophysin, have been widely studied for their role in synaptic function in human brain postmortem studies in BD and MDD. These studies have yielded contradictory results, possibly due to the small sample size and sourcing material from different cortical regions of the brain. We performed a systematic review and meta-analysis to understand the role of these three synaptic proteins and other synaptic proteins, messenger RNA (mRNA) and their regional localizations in BD and MDD. A systematic literature search was conducted and the review is reported in accordance with the MOOSE Guidelines. Meta-analysis was performed to compare synaptic marker levels between BD/MDD groups and controls separately. 1811 papers were identified in the literature search and screened against the preset inclusion and exclusion criteria. A total of 72 studies were screened in the full text, of which 47 were identified as eligible to be included in the systematic review. 24 of these 47 papers were included in the meta-analysis. The meta-analysis indicated that SNAP-25 protein levels were significantly lower in BD. On average, PSD-95 mRNA levels were lower in BD, and protein levels of SNAP-25, PSD-95, and syntaxin were lower in MDD. Localization analysis showed decreased levels of PSD-95 protein in the frontal cortex. We found specific alterations in synaptic proteins and RNAs in both BD and MDD. The review was prospectively registered online in PROSPERO international prospective register of systematic reviews, registration no. CRD42020196932.

Further Characterization of Intrastriatal Lipopolysaccharide Model of Parkinson's Disease in C57BL/6 Mice.

Parkinson’s disease (PD) is the most common movement disorder, characterized by progressive degeneration of the nigrostriatal pathway, which consists of dopaminergic cell bodies in substantia nigra and their neuronal projections to the striatum. Moreover, PD is associated with an array of non-motor symptoms such as olfactory dysfunction, gastrointestinal dysfunction, impaired regulation of the sleep-wake cycle, anxiety, depression, and cognitive impairment. Inflammation and concomitant oxidative stress are crucial in the pathogenesis of PD. Thus, this study aimed to model PD via intrastriatal injection of the inflammagen lipopolysaccharide (LPS)to investigate if the lesion causes olfactory and motor impairments, inflammation, oxidative stress, and alteration in synaptic proteins in the olfactory bulb, striatum, and colon. Ten µg of LPS was injected unilaterally into the striatum of 27 male C57BL/6 mice, and behavioural assessment was conducted at 4 and 8 weeks post-treatment, followed by tissue collection. Intrastriatal LPS induced motor impairment in C57BL/6 mice at 8 weeks post-treatment evidenced by reduced latency time in the rotarod test. LPS also induced inflammation in the striatum characterized by increased expression of microglial marker Iba-1 and astrocytic marker GFAP, with degeneration of dopaminergic neuronal fibres (reduced tyrosine hydroxylase immunoreactivity), and reduction of synaptic proteins and DJ-1 protein. Additionally, intrastriatal LPS induced inflammation, oxidative stress and alterations in synaptic proteins within the olfactory bulb, although this did not induce a significant impairment in olfactory function. Intrastriatal LPS induced mild inflammatory changes in the distal colon, accompanied by increased protein expression of 3-nitrotyrosine-modified proteins. This model recapitulated the major features of PD such as motor impairment and degeneration of dopaminergic neuronal fibres in the striatum, as well as some pathological changes in the olfactory bulb and colon; thus, this model could be suitable for understanding clinical PD and testing neuroprotective strategies.

Changes in synaptic proteins of the complex PSD-95/NMDA receptor/nNOS and mitochondrial dysfunction after levocabastine treatment

Nitric oxide generation is related to the activity of certain proteins located at synaptic sites. Previous findings show that NOS activity, nNOS protein expression, respiratory parameters and mitochondrial complex activities are altered in rat cerebral cortex by administration of levocabastine, an antagonist of histamine H1 and neurotensin NTS2 receptors. ATP provision by mitochondria may play an important role in the functional interaction between synaptic proteins NMDA receptor and PSD-95 with NO synthesis. In this context, our purpose was to evaluate the effect of levocabastine administration on protein expression of PSD-95, GluN2B and iNOS, as well as on mitochondrial ATP production. Male Wistar rats received a single (i.p.) dose of levocabastine (50 μg/kg) or saline solution (controls) and were decapitated 18 h later. Mitochondrial and synaptosomal membrane fractions were isolated from cerebral cortex by differential and sucrose gradient centrifugation. Expression of synaptic proteins was evaluated by Western blot assays in synaptosomal membrane fractions. Oxygen consumption, mitochondrial membrane potential and ATP production rate were determined in fresh crude mitochondrial fractions. After levocabastine treatment, protein expression of PSD-95, GluN2B and β-actin decreased 97, 45 and 55%, respectively, whereas that of iNOS enhanced 3.5-fold versus controls. In crude mitochondrial fractions levocabastine administration reduced roughly 15% respiratory control rate as assayed with malate-glutamate or succinate as substrates, decreased mitochondrial membrane potential (21%), and ATP production rates (57%). Results suggested that levocabastine administration induces alterations in synaptic proteins of the protein complex PSD-95/NMDA receptor/nNOS and in neuron cytoskeleton. Mitochondrial bioenergetics impairment may play a role in the functional link between synaptic proteins and NO synthesis.

Molecular Imaging in Alzheimer's Disease: Linking pathologic, neurotransmitter, synaptic and clinical findings relevant to geriatric psychiatry

The pathogenesis of Alzheimer's disease (AD) remains elusive and rests predominantly on the observed accumulations of amyloid beta and phosphorylated tau protein aggregates that are thought to play key roles in initiating or propagating the disease. However, other processes including changes in synaptic proteins, neurotransmitter loss, inflammation and cerebrovascular disease have been suggested as important etiologies as well. Furthermore, the manifestations of AD are heterogeneous. A majority of individuals experience neuropsychiatric symptoms during the disease process, but the onset and composition of these symptoms vary widely. Until recently, much of what is known about AD pathology has been learned from post mortem studies and animal models. The advancements of positron emission tomography (PET) imaging allow in vivo investigations into molecular changes associated with AD. Molecular imaging using PET allows us to study multiple biomarkers both early in the disease process and longitudinally. PET imaging can be used in concert with other imaging modalities, such as MRI, and with assessments of cognition and neuropsychiatric symptoms to investigate the molecular underpinnings of AD. It is of great importance for clinicians to learn about these innovative and translational tools which will ultimately lead to the identification of disease biomarkers and therapeutic targets. The goal of this session is to illustrate the importance of in vivo molecular imaging in the study of AD with a specific emphasis on PET. Dr. Adam Mecca will present results of cross-sectional studies that investigate synaptic protein alterations in AD, and the association between synaptic density and tau accumulation. These investigations utilize specific PET tracers to quantify synaptic density and tau. Dr. Gwenn Smith will describe longitudinal changes in serotonin (5-HT), tau and amyloid in relationship to cognitive decline and neuropsychiatric symptoms in individuals with mild cognitive impairment and normal cognition. Dr. Jennifer Gatchel will present work describing associations between subjective cognitive and depressive symptoms and in vivo cerebral tau and amyloid measurements. Dr. Nancy Donovan (Chair) will describe a longitudinal study examining the combined effects of widowhood status and PET-derived amyloid measures on cognitive outcomes in cognitively normal older adults. Finally, Dr. Gatchel (Discussant) will lead the speakers in a panel discussion, along with Q&A involving session attendees.

Hippocampal synaptoproteomic changes of susceptibility and resilience of male rats to chronic social isolation

The susceptibility of an individual to chronic social isolation (CSIS) stress may cause major depression (MD) whereby some individuals are resistant to the stress. Recent studies relate MD with altered expression of synaptic proteins in specific brain regions. To explore the neurobiological underpinnings and identify candidate biomarkers of susceptibility or resilience to CSIS, a comparative proteomic approach was used to map hippocampal synaptic protein alterations of rats exposed to 6 weeks of CSIS, an animal model of depression. This model generates two stress-response phenotypes: CSIS-sensitive (depressive-like behaviour) and CSIS-resilience assessed by means of sucrose preference and forced swim tests. Our aim was to characterize the synaptoproteome changes representative of potential long-term changes in protein expression underlying susceptibility or resilience to stress. Proteomic data showed increased expression of glycolytic enzymes, the energy-related mitochondrial proteins, actin cytoskeleton, signalling transduction and synaptic transmission proteins in CSIS-sensitive rats. Protein levels of glutamate-related enzymes such as glutamate dehydrogenase and glutamine synthetase were also increased. CSIS-resilient rats showed similar proteome changes, however with a weaker increase compared to CSIS-sensitive rats. The main difference was observed in the level of protein expression of vesicle-mediated transport proteins. Nonetheless, only few proteins were uniquely up-regulated in the CSIS-resilient rats, whereby Cytochrome b-c1 complex subunit 2, mitochondrial (Uqcrc2) and Voltage-dependent anion-selective channel protein 1 (Vdac1) were uniquely down-regulated. Identified altered activated pathways and potential protein biomarkers may help us better understand the molecular mechanisms underlying synaptic neurotransmission in MD or resilience, crucial for development of new therapeutics.

MOLECULAR IMAGING IN ALZHEIMER'S DISEASE: LINKING PATHOLOGIC, NEUROTRANSMITTER, SYNAPTIC AND CLINICAL FINDINGS RELEVANT TO GERIATRIC PSYCHIATRY

The pathogenesis of Alzheimer's disease (AD) remains elusive and rests predominantly on the observed accumulations of amyloid beta and phosphorylated tau protein aggregates that are thought to play key roles in initiating or propagating the disease. However, other processes including changes in synaptic proteins, neurotransmitter loss, inflammation and cerebrovascular disease have been suggested as important etiologies as well. Furthermore, the manifestations of AD are heterogeneous. A majority of individuals experience neuropsychiatric symptoms during the disease process, but the onset and composition of these symptoms vary widely. Until recently, much of what is known about AD pathology has been learned from post mortem studies and animal models. The advancements of positron emission tomography (PET) imaging allow in vivo investigations into molecular changes associated with AD. Molecular imaging using PET allows us to study multiple biomarkers both early in the disease process and longitudinally. PET imaging can be used in concert with other imaging modalities, such as MRI, and with assessments of cognition and neuropsychiatric symptoms to investigate the molecular underpinnings of AD. It is of great importance for clinicians to learn about these innovative and translational tools which will ultimately lead to the identification of disease biomarkers and therapeutic targets. The goal of this session is to illustrate the importance of in vivo molecular imaging in the study of AD with a specific emphasis on PET. Dr. Adam Mecca will present results of cross-sectional studies that investigate synaptic protein alterations in AD, and the association between synaptic density and tau accumulation. These investigations utilize specific PET tracers to quantify synaptic density and tau. Dr. Gwenn Smith will describe longitudinal changes in serotonin (5-HT), tau and amyloid in relationship to cognitive decline and neuropsychiatric symptoms in individuals with mild cognitive impairment and normal cognition. Dr. Jennifer Gatchel will present work describing associations between subjective cognitive and depressive symptoms and in vivo cerebral tau and amyloid measurements. Dr. Nancy Donovan (Chair) will describe a longitudinal study examining the combined effects of widowhood status and PET-derived amyloid measures on cognitive outcomes in cognitively normal older adults. Finally, Dr. Gatchel (Discussant) will lead the speakers in a panel discussion, along with Q&A involving session attendees.

ADVANCES IN THE MOLECULAR IMAGING OF ALZHEIMER'S DISEASE: NEUROBIOLOGICAL MECHANISMS OF DISEASE AND NEUROPSYCHIATRIC SYMPTOMS: Session 209

The pathogenesis of Alzheimer's disease (AD) remains elusive. The observed accumulations of beta amyloid and phosphorylated tau protein aggregates are thought to play key roles in initiating or propagating the disease. However, other processes including changes in synaptic proteins, neurotransmitter loss, inflammation and cerebrovascular disease have been suggested as important etiologies as well. Importantly, the recent, unprecedented advancements of positron emission tomography (PET) radiotracer chemistry allow investigations into molecular changes associated with AD. Until recently, much of what is known about AD pathology has been learned from post mortem studies and animal models. The advancements of PET imaging allows quantitative in vivo measurements of specific proteins within many brain regions. Because the manifestations of AD are heterogeneous. The use of PET imaging can be used to improve the understanding of diverse symptoms. For example, a majority of individuals experience neuropsychiatric symptoms during the disease process, but the onset and composition of these symptoms vary widely. Molecular imaging using PET allows us to study neurobiological processes both early in the disease process and longitudinally. PET imaging can be used in concert with other imaging modalities such as MRI, as well as assessments of cognition and neuropsychiatric symptoms to investigate the molecular underpinnings of AD. It is of great importance for clinicians to learn about these innovative and translational tools which will ultimately lead to the identification of disease biomarkers and therapeutic targets. The goal of this session is to illustrate the importance of in vivo molecular imaging in the study of AD with a specific emphasis on PET. Dr. Adam Mecca (Chair) will present results of cross-sectional studies that investigate synaptic protein alterations in individuals with AD. These investigations utilize specific PET tracers to quantify synaptic density and metabotrophic glutamate receptor subtype 5 (mGluR5). Dr. Nancy Donovan will describe research that examines the relationships between cortical and subcortical brain amyloid quantified using PET, anxiety, and AD risk in cognitively normal older adults. Dr. Jennifer Gatchel will present work on the association between depressive symptoms and in vivo cerebral tau and amyloid measurements. Dr. Gwenn Smith will describe the role of serotonin (5-HT), tau and amyloid b in cognitive decline in individuals with mild cognitive impairment and normal cognition. Finally, Dr. Donovan (Discussant) will lead the speakers in a panel discussion, along with Q&A involving session attendees.

Curcumin can influence synaptic dysfunction in APPswe/PS1dE9 mice

ObjectiveSynaptic loss in the hippocampus in Alzheimer's disease (AD) has been shown to be closely associated with the cognitive impairment. Synaptic dysfunction is a pathological feature that occurs prior to synaptic loss and mainly depends on structural changes and alterations of synaptic proteins. Evidence has suggested that curcumin, obtained from the traditional Chinese medicine—Turmeric (Curcuma longa L.), can ameliorate cognitive impairment, but few studies have focused on the mechanism by which curcumin affects synapses at early stages of AD. Therefore, we performed a study to investigate whether curcumin exerted its effect on synapses at the early stage in AD. MethodsWe used 3-month-old APPswe/PS1dE9 mice and wild type (WT) littermates of the APPswe/PS1dE9 mice from the same colony as the normal controls. Seventy-five APPswe/PS1dE9 mice were allocated to the Model group, Rosiglitazone group, and Curcumin groups randomly. The Wild and Model groups were orally administered an equal amount of 0.5% carboxymethyl cellulose (CMC). We observed the ultrastructure of synapses in the CA1 area of hippocampus and analyzed the expression levels of PSD95 and Shank1, two important synapse-associated proteins, in APPswe/PS1dE9 mice by immunohistochemical staining and western blot after gavage for three months. ResultsOur findings showed that curcumin treatment not only improved the quantity and ultrastructure of synapses but also increased the expression of PSD95 and Shank1. ConclusionThe results indicate that curcumin improves synaptic dysfunction and the potential mechanism may involve improving the structure of synapses and regulating the synapse related proteins PSD95 and Shank1.

Cognitive impairments associated with alterations in synaptic proteins induced by the genetic loss of adenosine A2A receptors in mice

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A2A adenosine receptor subtype (A2AR) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A2AR in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A2AR constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A2AR knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders.

Sex differences in the effects of pre- and postnatal caffeine exposure on behavior and synaptic proteins in pubescent rats

Few studies have addressed the effects of caffeine in the puberty and/or adolescence in a sex dependent manner. Considering that caffeine intake has increased in this population, we investigated the behavioral and synaptic proteins changes in pubescent male and female rats after maternal consumption of caffeine. Adult female Wistar rats started to receive water or caffeine (0.1 and 0.3g/L in drinking water; low and moderate dose, respectively) during the active cycle at weekdays, two weeks before mating. The treatment lasted up to weaning and the offspring received caffeine until the onset of puberty (30–34days old). Behavioral tasks were performed to evaluate locomotor activity (open field task), anxious-like behavior (elevated plus maze task) and recognition memory (object recognition task) and synaptic proteins levels (proBDNF, BDNF, GFAP and SNAP-25) were verified in the hippocampus and cerebral cortex. While hyperlocomotion was observed in both sexes after caffeine treatment, anxiety-related behavior was attenuated by caffeine (0.3g/L) only in females. While moderate caffeine worsened recognition memory in females, an improvement in the long-term memory was observed in male rats for both doses. Coincident with memory improvement in males, caffeine increased pro- and BDNF in the hippocampus and cortex. Females presented increased proBDNF levels in both brain regions, with no effects of caffeine. While GFAP was not altered, moderate caffeine intake increased SNAP-25 in the cortex of female rats. Our findings revealed that caffeine promoted cognitive benefits in males associated with increased BDNF levels, while females showed less anxiety. Our findings revealed that caffeine promotes distinct behavioral outcomes and alterations in synaptic proteins during brain development in a sex dependent manner.

Electroconvulsive Seizures in Rats and Fractionation of Their Hippocampi to Examine Seizure-induced Changes in Postsynaptic Density Proteins

Electroconvulsive seizure (ECS) is an experimental animal model of electroconvulsive therapy, the most effective treatment for severe depression. ECS induces generalized tonic-clonic seizures with low mortality and neuronal death and is a widely-used model to screen anti-epileptic drugs. Here, we describe an ECS induction method in which a brief 55-mA current is delivered for 0.5 s to male rats 200 - 250 g in weight via ear-clip electrodes. Such bilateral stimulation produced stage 4 - 5 clonic seizures that lasted about 10 s. After the cessation of acute or chronic ECS, most rats recovered to be behaviorally indistinguishable from sham "no seizure" rats. Because ECS globally elevates brain activity, it has also been used to examine activity-dependent alterations of synaptic proteins and their effects on synaptic strength using multiple methods. In particular, subcellular fractionation of the postsynaptic density (PSD) in combination with Western blotting allows for the quantitative determination of the abundance of synaptic proteins at this specialized synaptic structure. In contrast to a previous fractionation method that requires large amount of rodent brains, we describe here a small-scale fractionation method to isolate the PSD from the hippocampi of a single rat, without sucrose gradient centrifugation. Using this method, we show that the isolated PSD fraction contains postsynaptic membrane proteins, including PSD95, GluN2B, and GluA2. Presynaptic marker synaptophysin and soluble cytoplasmic protein α-tubulin were excluded from the PSD fraction, demonstrating successful PSD isolation. Furthermore, chronic ECS decreased GluN2B expression in the PSD, indicating that our small-scale PSD fractionation method can be applied to detect the changes in hippocampal PSD proteins from a single rat after genetic, pharmacological, or mechanical treatments.