Agenor Limon - US grants

PROJECT SUMMARY Initial stages of Alzheimer?s disease (AD) appear to be correlated with elevated electrical activity and synaptic abnormalities in brain regions first affected by pathology. This pathologically shifting towards excitation suggests that there are alterations in the synaptic excitation and inhibition balance (E/I ratio) within these areas (e.g., entorhinal cortex), which in turn may accelerate activity-dependent AD pathology. However, there are no quantitative, regional measurements of the E/I ratio in the human brain, and alterations in this measure in AD are unknown. In preliminary work leading to this proposal, we have found evidence of inhibitory signaling disturbances at early stages of AD. Levels of Gephyrin expression, an inhibitory postsynaptic synaptic density (iPSD) protein, are reduced in entorhinal cortex neuronal cell bodies of postmortem brain from donors diagnosed with mild cognitive impairment (MCI), a prodromal stage of AD. In addition, by microtransplanting receptors from temporal cortices of human AD donors into Xenopus oocytes, we discovered electrophysiological abnormalities of GABA receptors (GABAARs) suggesting that inhibitory tone is reduced in AD. Importantly though, it is not known whether these collective alterations also occur at the level of synapses in AD or if they are emergent in MCI. Given these preliminary findings, we hypothesize that 1) MCI is characterized by abnormally large E/I ratios in brain regions particularly affected early on in AD and 2) E/I ratio imbalance is driven by impairment in the clustering of synaptic excitatory or inhibitory receptors, or by alteration of the electrophysiological properties of major synaptic glutamate and GABA receptors (GluRs and GABAARs). This general hypothesis will be evaluated in two Specific Aims. Aim 1 will test whether there are specific pro- excitatory alterations in the ratio of excitatory to inhibitory postsynaptic density (ePSD/iPSD) proteins in MCI and AD versus controls. Studies will use Fluorescence Deconvolution Tomography (FDT), developed by part of our research group, whereby immunolabeling of PSD markers are measured within the size constraints of synapses from 3D reconstructions; FDT analysis will determine if pro-excitatory E/I ratios based on counts, volume, and intensity of synaptic markers characterize and differentiate MCI from control and AD cases. Complementing the anatomical work, Aim 2 will test whether electrophysiological alterations of synaptic receptors contribute to larger E/I ratio in MCI and AD using the Microtransplantation of Synaptic Membranes (MSM), a novel technique that allows for electrophysiological studies of GluRs and GABAARs from postmortem human brain tissue. Understanding the degree to which abnormal synaptic E/I ratios are present in MCI and/or AD, and which PSD receptors or proteins are affected, would greatly facilitate targeted pharmacological interventions aimed at restoring E/I balance and may provide substantial benefit to patients showing early signs of cognitive decline by delaying or stopping the progression to AD which currently affects ~5.3 million Americans.

Project Summary Alterations of synaptic function in individuals with schizophrenia have been found in transcriptomics, proteomics, and genome wide association studies. Impaired synaptic glutamatergic (excitatory) and GABAergic (inhibitory) neurotransmission in affected brain regions (e.g. dorsolateral prefrontal cortex; DLPFC) is thought to be involved in the core symptoms of schizophrenia. However, there are no quantitative measurements of synaptic function in the human DLPFC, therefore concrete and specific functional alterations of ion fluxes of glutamate and GABA receptors are lacking. We have begun to address this problem by directly measuring AMPA- and GABA receptor-mediated synaptic currents in postmortem brains from subjects with schizophrenia and contrasting to controls. We demonstrate in our preliminary work that the function of synaptic receptors is maintained in postmortem brains and is significantly decreased in schizophrenia compared to controls. Our overarching hypothesis is that reductions in both inhibitory and excitatory currents underlie synaptic deficits in schizophrenia. We will test rigor and reproducibility of this hypothesis in two larger independent case-control cohorts. These deficits in synaptic currents can be statistically modeled with proteomic and transcriptomic data which will be useful in downstream studies that pharmacologically challenge activation of these currents. In our model, we suggest that an unequal loss of GABAergic and glutamatergic transmission potentially biases circuits towards producing increased inhibition by dual complementary mechanisms. Our novel molecular evidence will be tested in three complementary aims. Aim 1 will test whether there are electrophysiological alterations of the excitatory (E) to inhibitory (I) balance (E/I ratio) in the DLPFC of subjects with schizophrenia compared to controls, by using microtransplantation of synaptic membranes, a novel method that allows for electrophysiological studies of synaptic receptors from postmortem human brain. Aim 2 will test the hypothesis that integration of proteomic, transcriptomic, and electrophysiological data in the same subjects predicts synaptic dysfunction and E/I ratio alterations at the molecular level in SZ. We will use mRNA-Seq in conjunction with label free liquid chromatography-MS (LC-MS/MS) to characterize major synaptic elements with modulatory capacity on GABA and glutamate receptors. Aim 3 will test rigor and reproducibility of electrophysiological data across different brain banks, by using an independent cohort from the NIHM Human Brain Collection Core. Understanding the relationships between parallel transcriptomic and proteomic data sets with the actual dysfunction of synaptic receptors would greatly facilitate targeted pharmacological interventions that help persons suffering with schizophrenia. This approach could benefit other neuropsychiatric disorders involving mood, behavior, and cognition by targeting potential alterations in synaptic function.

PROJECT SUMMARY/ABSTRACT There is ample scientific evidence that toxic oligomers of A? and tau, considered the major neuropathological factors in Alzheimer?s disease (AD) lead to synaptic failure and dementia, but is also well recognized that some individuals can accumulate significant AD neuropathology without clinical manifestations. The established existence of these non-demented with high pathology (NDAN) individuals, suggests that if binding of A? oligomers to specific AMPA and NMDA receptors trigger Ca2+ disruption and synaptic failure in AD, then modifications in the pharmacology or abundance of these receptors may underlie synaptic protection in NDAN individuals. However, little is known about the pharmacological sensitivity of human native glutamate receptors to A? and tau oligomers in AD, or their role in resilience, as information primarily has come from studies in animal models of AD or protein expression systems. Here we will fill in this critical gap of our current knowledge by identifying the synaptic proteins involved in glutamatergic signaling and Ca2+ dysregulation in AD, evaluating whether modifications of these proteins correlate with metrics of synaptic preservation in NDAN individuals, and determining whether A? and tau oligomers differentially activate human native synaptic receptors in NDAN individuals compared to those in AD and controls. The present proposal will test the hypothesis that differences in the regional abundance of Ca2+-permeable glutamate receptors and/or their pharmacodynamics profile in response to oligomeric species distinguishes NDAN from AD and control subjects. We will test our hypothesis in a rigorously characterized cohort of postmortem NDAN, AD, and age-matched normal cognitive control brains, using two unique and innovative approaches: Microtransplantation of Synaptic Membranes (MSM) to evaluate receptor electrical activity of native receptors complexes isolated from autopsy human brain, and Electrophysiologically-anchored Dataset Analysis (EDA) to identify proteins that correlate with receptors? activity. The rationale for this project is that determining the pharmacological sensitivity of the natural targets of toxic oligomers is likely to offer a strong framework whereby novel strategies to AD therapy can be developed. Two complementary aims are proposed. Aim 1 will identify the impact of gene expression modifications on the abundance and function of Ca2+ permeable human native glutamate receptors in NDAN compared to AD and control cases, and aim 2 will evaluate the efficacy of A? and tau oligomers to activate human native synaptic glutamate receptors from NDAN, AD and control cases. At the end of the proposed research we expect to have defined the mechanisms whereby oligomers trigger synaptic Ca2+ dysregulation in AD but not in NDAN subjects. These results will lay the foundations for the future development of innovative target-directed pharmacologic interventions to promote synaptic resilience in AD as a clinically valuable, effective therapeutic approach.

PROJECT SUMMARY/ABSTRACT Finding a resolving cure for Alzheimer's Disease (AD) is an urgent critical need and there is ample consensus that successful treatments should target early disease events. Among these, synaptic dysfunction induced by soluble oligomers of tau (TauO) is recognized as one of the earliest key events in AD and related disorders, where disease-specific TauO conformers (strains) may drive the diverse clinical presentations. Thus, blocking synaptic demise induced by TauO conformers is a highly desirable therapeutic strategy, its potential effectiveness supported by the existence of resilient individuals who remain Non-Demented despite the CNS presence of late-stage Braak AD Neuropathology (here termed NDAN) that present with functional synapses devoid of TauO. Notably, late Braak stage pathology paralleled by intact synapses in NDAN suggests the novel concept that trans-synaptic spreading of tau and tau-driven synaptic toxicity are two distinct phenomena that coexist in AD and other symptomatic tauopathies but not in NDAN, where the toxic impact of TauO on synapses (but not spreading) is curbed. While such evidence indicates that rejecting synaptic tau toxic impact as a protective mechanism can be effectively enabled in the human brain despite the spread of AD neuropathology, a therapeutic strategy to achieve such a game-changing goal remains missing. Filling this critical knowledge gap by laying the molecular foundations for the development of such therapeutic strategy is the overall objective of the present project. Our central hypothesis is that dysfunctional engagement of synapses by TauO is determined by the type of tau strain and its binding to LRP1 (a synaptic receptor essential for tau uptake and spreading), which differentially affects LRP1-containing protein complexes in vulnerable vs. resilient synapses. We will test our central hypothesis by evaluating binding dynamic and functional impact of disease-specific brain-derived TauO conformers on human synapses as a function of LRP1-mediated toxicity (Aim 1) and by evaluating the functional response of human synapses from different tauopathies (AD, PSP) and resilience status (NDAN, PART) to TauO as a function of the synaptic LRP1 (Aim 2). At the completion of the proposed studies, we expect to have documented a previously unappreciated mechanism of synaptic interaction of TauO conformers as well as a phenomenon of synaptic resistance modulated by LRP1. This discovery will have a significant impact in the development of an innovative treatment concept for AD centered on sustaining synaptic resistance to toxic oligomers, a strategy expected effective as demonstrated by NDAN subjects. A uniquely qualified investigative team has been assembled to successfully accomplish this project, bringing together expertise in AD molecular neurobiology (Taglialatela), human synaptic physiology (Limon) and function (Krishnan) and biochemistry of tau oligomers (Kayed).