Angus C. Nairn - US grants

Cell growth and differentiation are regulated by a large array of overlapping signalling pathways which coordinate both mRNA transcription and protein translation. While many individual details have been elucidated, it is clear that more information is necessary to fully understand the normal and aberrant regulation of these complex pathways. Many growth factors cause rapid and transient increases in intracellular calcium levels, however, the physiological consequences of this are not known. Calcium is widely recognized as an important messenger in eukaryotic systems. In many instances, its actions are mediated by binding to calmodulin, and strong evidence indicates, in turn, that the effects of calmodulin are often achieved through the regulation of protein phosphorylation. A potential target for this ubiquitous signalling pathway is elongation factor 2(EF-2), a key component in protein synthesis and hence cell growth. EF-2 is subject to two different types of post-translation modification. The protein is ADP-ribosylated by diphtheria toxin resulting in inhibition of protein synthesis and cell death. EF-2 is also phosphorylated by a specific calcium/calmodulin- dependent protein kinase, CaM kinase III, resulting in its complete inactivation. The exact physiological role of EF-2 phosphorylation is unknown; however, recent studies suggest a potential role in regulation of cell growth. EF-2 is rapidly phosphorylated in cells in response to a variety of growth factors and mitogens and this is associated with transient inhibition of protein synthesis. CaM kinase III levels are very high in rapidly growing cells but are down-regulated in non-dividing cells, and results using site-directed mutagenesis suggest that phosphorylation of EF-2 is necessary for cell viability. Finally, this signalling system is conserved in species as diverse as man and yeast. The overall aim of the proposed studies is to characterize the biochemical and physiological consequences of EF-2 phosphorylation. Specifically, the hypothesis will be tested in both mammalian and yeast cells, that EF-2 phosphorylation and inhibition of protein synthesis influences translation of specific proteins by affecting mRNA selection and/or mRNA stability. Results from these studies will lead to a greater understanding of the regulation by calcium of growth and differentiation in normal and cancerous cells.

Many vital functions of nerve cells arc regulated by reversible protein phosphorylation, including the control of neurotransmitter release, signalling by neurotransmitter receptors and ion channels, and various aspects of gene expression, neuronal differentiation and development. A systematic investigation of the distribution of protein kinases and their substrates in the rat basal ganglia has identified a family of substrates for cAMP-dependent protein kinase (PKA). One of these substrates, termed DARPP-32, was found to be highly enriched in medium-sized spiny neurons that possess D I dopamine receptors, and is likely to be involved in the physiological effects of dopamine mediated through dopamine-sensitive adenylyl cyclase. Biochemical studies have suggested that DARPP-32 functions to inhibit PP1, a multi-functional serine/threonine protein phosphatase. The high levels of DARPP-32 in dopaminoceptive neurons in the basal ganglia and its ability to potently inhibit protein phosphatase-1 (PP1) has suggested that DARPP-32 is central to the interactions of dopamine and other neurotransmitters acting on D1 dopaminoceptive neurons. In particular, medium-spiny neurons receive an extensive excitatory input from glutamate. containing cortical neurons, and dopamine, by stimulating D1 receptors, inhibits the responsiveness of striatal neurons to glutamate. Thus, DARPP-32 and PP1 are likely to be involved in the interaction of glutamate and dopamine in the striatum. Recent studies have identified that Na+K+ATPase, as well as Na+ and Ca2+ channels, are modulated via phosphorylation by variouS protein kinases and are likely to be targets for dephosphorylation by PP1. A complete biochemical analysis of these various downstream targets for PP1 and the regulation of their dephosphorylation by DARPP-32 will, therefore, be necessary to fully understand the actions of dopamine and other neurotransmitters that influence this signal transduction pathway. In addition, the elucidation of the structural basis for the ability of phospho-DARPP-32 to inhibit PP1 will further the understanding of dopamine signalling and will hopefully allow the design of specific agonists and antagonists that influence this signalling pathway. Such reagents may lead to the development of new therapeutic approaches to the treatment of such diseases as Parkinson's disease and schizophrenia, that result from disruption of normal dopaminergic neurotransmission.

A systematic investigation of protein phosphorylation in the rat central nervous system has revealed that a family of substrates for cAMP-dependent protein kinase, termed DARPPs (for dopamine and cAMP-regulated phosphoproteins), are highly enriched in athe basal ganglia. In this brain region, dopamine, through binding to D1 receptors, increases cAMP levels leading to activation of PKA and phosphorylation of this family of substrates. Studies over the last few years now indicate that members of the DARPP family mediate many of the actions of dopamine in this brain region. There is also considerable evidence to indicate that the acute and chronic actions of psychomotor stimulants (e.g. cocaine and amphetamine) are mediated through augmentation of neurotransmission in mesolimbic and nigrostriatal dopamine systems. The major aim of the present proposal will be to study the role of two of the DARPP family, namely DARPP-21 and DARPP- 16, in the actions of these psychomotor stimulants. Specific Aims will be: I. to use gene-targeting techniques to "knock-out" the genes for DARPP-21 and DARPP-16 in mice. This will provide information in an intact animal model concerning the physiological role these proteins play in dopaminergic neurotransmission and ina the actions of psychomotor stimulants; II. to identify the function of DARPP-21 and DARPP-16 through the use of the yeast two-hybrid method and other affinity methods to identify the targets for each of these two proteins. These studies will include characterization of each DARPP-21 and DARPP-16 binding protein using biochemical, immunological and structural methods, including cloning of cDNAs for each protein, and examination of how phosphorylation of each DARPP influences its interaction with identified binding proteins. The role played by intracellular targets for DARPP-21 and DARPP-16 in the actions of cocaine and amphetamines will also be studied; III. to purify and characterize other novel DARPPs that are localized in the neostriatum and nucleus accumbens. The characterization of basal ganglia phosphoproteins, within specific neurons which are affected by psychostimulants, will provide a rational new approach to developing drugs that specifically affect these phosphoproteins or their targets. Thus, these studies may have potential implication int he development of new therapeutic approaches to the treatment of such drugs of abuse.

The several hundred members of the Eukaryotic protein kinase superfamily characterized to date share a similar catallytic domain structure, consisting of 12 conserved subdomains. Recently, a new class of protein kinases, represented by eukaryotic elongation factor-2 kinase, has been discovered with completely different structure. We are attempting to identify the ATP binding site of this new class of protein kinase and also gain an understanding of its regulation via phosphorylation.

calmodulin dependent protein kinase; phosphorylation; biomedical resource; macromolecule; clinical research; mass spectrometry;

calcium channel; phosphorylation; biomedical resource; macromolecule; clinical research; mass spectrometry;

cyclin dependent kinase; phosphorylation; biomedical resource; macromolecule; clinical research; mass spectrometry;

laboratory mouse

Considerable evidence indicates that the acute and chronic actions of psychomotor stimulants (e.g. cocaine and amphetamine), as well as of other drugs of abuse, involve modulation of neurotransmission in mesolimbic and nigrostriatal dopamine systems. Our previous studies have revealed that a family of substrates for cAMP-dependent protein kinase, including DARPP-32, RCS (Regulator of Calmodulin Signaling, previously termed ARPP-21), and ARPP-16, are highly enriched in the basal ganglia, including the neostriatum and nucleus accumbens. Previous studies have indicated that a critical target for DARPP-32 is the serine/threonine protein phosphatase, PPL In recent studies we have found that RCS interacts with the Ca2+-binding protein calmodulin and in turn regulates the serine/threonine protein phosphatase, calcineurin (or PP2B). In other studies, we have found that ARPP-16 is likely to regulate the stability of GAP-43 mRNA. In Project III, we propose to study the role(s) of RCS and ARPP-16 in mediating or modulating the actions of psychostimulants. In addition, we propose to study the roles of the three isoforms of PP1 (PPla, (3 and y) in the actions of psychostimulants. The Specific Aims are: Aim I: Characterization of RCS - We will analyze in RCS knockout mice, several aspects of animal behavior and physiology that are modulated by psychostimulants. These studies will include acute and chronic motor-stimulant properties and drug reinforcing properties of cocaine and amphetamine in RCS mutant mice. We will also characterize molecular targets for RCS that may be involved in mediating the actions of psychostimulants. Studies will include analysis of RCS and regulation of calcineurin, and of RCS and regulation of other calmodulin-binding proteins. Aim II: Characterization of ARPP-16 - We will analyze in ARPP-16 knockout mice, several aspects of animal behavior and physiology that are modulated by psychostimulants. These studies will include acute and chronic motorstimulant properties and drug reinforcing properties of cocaine and amphetamine in RCS mutant mice. We will also characterize the regulation of ARPP-16 by phosphorylation at novel sites and study the phosphorylation of these sites in response to psychostimulant treatment. Aim HI: Characterization of PP1 isoforms - We will investigate the role of PPla, (3 and y isoforms in the actions of psychostimulants. These will include acute and chronic motor-stimulant properties and drug reinforcing properties of cocaine and amphetamine in RCS mutant mice. Results from our studies will complement other Projects of this Program Project grant. Together, these studies will hopefully lead to elucidation of the biochemical pathways through which drugs of abuse act in the brain, and to an increased likelihood that therapeutic agents will be developed that will prevent or reverse molecular adaptations within these pathways.

Project 2 - Aging is associated with a decline in cognitive performance. PFC shows particularly large functional deterioration with age. The molecular mechanisms responsible for this decline are poorly understood. Our preliminary data suggests that aging is associated with increased cAMP signaling in the PFC. Antagonizing cAMP signaling in the PFC improves working memory performance and increasing cAMP signaling in the PFC causes further declines in working memory. We have also shown that HCN1 and 2 channels are expressed on dendritic spines where cAMP can affect channel opening and reduce the magnitude of synaptic inputs. Aged PFC shows reduced expression of RNA for PDE4A which would be expected to cause increased cAMP levels. Western blots demonstrate increased HCN1 expression in aged PFC. We hypothesize that cAMP levels increase with aging and that the deleterious effects of cAMP are mediated by HCN ion channels. To test this hypothesis we will measure cAMP levels in the prefrontal cortex of young, aging, and aged rats. We will then overexpress PDE4A using viral methods and measure working memory performance. We predict that increasing PDE4A expression to that seen in young animals will improve working memory performance in aged rats. We will then examine the role of HCN channels by overexpressing or knocking down HCN 1/2 expression and measure working memory. We predict that reducing HCN expression will reverse the effects of aging on PFC function and that overexpression will cause further deterioration. We will use cDNA microarrays and protein profiling approaches to examine RNA and protein expression changes associated with aging in the PFC to generate additional hypotheses. Genes identified in this manner will be targeted for overexpression and knockdown studies. We believe that this work will identify components of the cAMP signaling pathway that underlie decreased working memory performance associated with aging. Lay language: Certain cognitive capacities become less efficient with aging. One such capacity known as working memory plays an important role in carrying out tasks associated with daily living and is mediated by the prefrontal cortex. We have determined that age-related changes in cyclic AMP signaling in the prefrontal cortex may cause reduced working memory performance. This proposal seeks to uncover the molecular mechanisms responsible for these changes.

Project 3 will address the major theme of the Conte center Identification of cell type-specific actions of antipsychotic drugs by taking primarily biochemical approaches to characterize key signaling proteins believed to be involved in the effects of antipsychotic drugs on specific aspects of neuronal function within corticostriatal circuits. In Aim 1, Project 3 will follow up from recent studies carried out in collaboration with Project 1 that have found that the protein DARPP-32, which is highly enriched in all striatal medium spiny neurons (MSNs), regulates chromatin function through a novel mechanism involving phosphorylation/dephosphorylation and nuclear translocation. Other studies indicate that DARPP-32 is required for the effects of haloperidol on regulation of MSN gene expression. Project 3 will elucidate the role of DARPP-32's nuclear function in the actions of antipsychotic drugs. These studies will be carried out in collaboration with Dr. Nestler, and will be integrated with Project 4's studies of chromatin remodeling. In Aim 2, building on results obtained in collaboration with Dr. Surmeier (Project 5) that show specific effects of the typical antipsychotic haloperidol on the dendritic morphology of striatonigral and striatopallidal (MSNs), Project 3 will identify the biochemical basis for the observed effects of haloperidol. In collaboration with Projects 2 and 5, these studies will be expanded to analysis of the biochemical effects of atypical antipsychotic drugs on the structure of MSNs, as well as of both typical and atypical classes of drug on corticostriatal projection neurons. In these studies, Project 3 will make use of translational profiling data from Projects 1 and 2, as well as gene expression data from Project 4, to identify candidate genes involved in control of MSN structure. In Aim 3, Project 3 will work together with Project 2 in the development of new methods to exploit the power of transgenic mouse models to allow for systematic biochemical and proteomic analysis of specific neuronal cell populations within corticostriatal circuits. Project 3 will also contribute biochemical expertise to ongoing studies by the other projects, as well as contribute to future studies that are carried out collectively by the Conte Center as a result of new discoveries made.

Considerable evidence indicates that the acute and chronic actions of psychomotor stimulants (e.g. cocaine and amphetamine), as well as of other drugs of abuse, involve modulation of neurotransmission in mesolimbic and nigrostriatal dopamine systems. Our previous studies have revealed that a family of substrates for cAMP-dependent protein kinase, including DARPP-32, RCS (Regulator of Calmodulin Signaling, previously termed ARPP-21), and ARPP-16, are highly enriched in medium spiny neurons of the basal ganglia, including the neostriatum and nucleus accumbens. Our ongoing research has also identified a new member of this family of striatal phosphoproteins as RaplGAP, a protein involved in the control of the small GTPase Rap. Other ongoing work has identified critical roles for novel isoforms of the serine/threonine protein phosphatase PP2A in control of the nucleo-cytoplasmic trafficking of DARPP-32, a process that is critical for the ability of DARPP-32 to mediate the actions of pyschostimulants. Moreover, we have found that ARPP-16 interacts with and may act to inhibit PP2A. Since the serine/threonine protein phosphatase, PPI, is a direct target for DARPP-32, and RCS controls PP2B activity indirectly, this work indicates that dopamine action in striatal neurons is likely to be largely mediated via the control of protein phosphatases. To address questions raised by these ongoing studies we propose two broad Specific Aims in Project 3 of the Program Project Grant. In Aim I we will study the role of Rap GTPase, and its modulators RapGAP and EPAC in the actions of psychostimulants. In Aim II we will study the role of novel isoforms of PP2A in the actions of psychostimulants. Aim II will also include analysis of novel functions of PPI isoforms, the targets for DARPP-32 in striatal neurons. Results from our studies will complement the other two Projects of this Program Project grant. In addition, we will also carry out a number of collaborative studies with Projects 1 and 2, including studies of WAVEI phosphorylation with Project 1 and phosphoproteomic studies of mGluRS-dependent signaling in striatal neurons.

SUMMARY FOR BIOMARKERS/PATHOLOGY CORE The Biomarkers/Pathology Core will provide post-mortem diagnoses and biomarker analyses for patients and control subjects enrolled in the Clinical Core and for other well documented AD cases and controls. The approaches will be state-of-the-art and consistent with 21st century brain banking procedures, and will extend neuropathology core procedures to include targeted mass spectrometric assays to identify and quantitate AD- related biomarkers, as well as to establish induced pluripotent stem cells (iPSCs) from AD patients, innovative approaches which are essential to meet the increasingly sophisticated needs of the AD research community. Thus, the Biomarkers/Pathology Core will provide staff, technical resources, laboratory facilities and expertise for the three main Aims. Aim 1 will establish a Yale Neuropathology Core that will conduct postmortem examinations on Yale-ADRC patients and for other well documented AD cases and controls; and maintain a bank of unfixed frozen and fixed tissue from the ADRC control and patients with AD or other dementing neurodegenerative conditions. Aim 2 will establish and apply quantitative targeted mass spectrometric assays for the analysis of human AD brain samples and biofluids. This will include analysis of unfractionated and fractionated brain tissue, and CSF using MRM and SWATH mass spectrometry methods. Aim 3 will establish procedures for generation of Induced Pluripotent Stem Cells (iPSCs) from human AD patients. The Biomarkers/Pathology Core will closely interact with the other Cores of the Yale ADRC and provide tissue, methodologies and advice to Projects and Pilot Projects users in the ADRC, Yale researchers with an interest in AD, neurodegenerative disease and aging, and with other ADRCs. 1

? DESCRIPTION (provided by applicant): We propose to maintain and continuously improve the Yale/NIDA Neuroproteomics Center that brings exceptionally strong Yale programs in proteomics and signal transduction in the brain together with neuroscientists from nine other institutions across the U.S. to identify adaptive changes in protein signaling that occur in response to substances of abuse. Twenty-three faculties with established records of highly innovative research into the molecular actions of psychoactive addictive drugs, as well as of other basic aspects of neurobiology, will work together in a unique synergy with the Keck Foundation Biotechnology Laboratory to create the Yale/NIDA Neuroproteomics Center. The main goal of the Center, whose theme is Proteomics of Altered Signaling in Addiction, is to use cutting edge proteomic technologies to analyze neuronal signal transduction mechanisms and the adaptive changes in these processes that occur in response to drugs of abuse. With Co-Directors Drs. Angus Nairn (Psychiatry) and Kenneth Williams (Mol. Biophys. & Biochem.) in the Administration Core, the Center includes Discovery Proteomics (DPC) and Targeted Proteomics (TPC) Cores. Biophysical technologies from the DPC will extend protein profiling analyses into the functional domain while lipid analyses from the DPC will positively leverage proteome level analyses to provide an increasingly biological systems level approach. A Bioinformatics and Biostatistics Core, which includes high performance computing and the Yale Protein Expression Database, provides essential support that will positively leverage the value of each of the proteomic technology cores. A Pilot Research Project Core is a cornerstone in our efforts to encourage strong mentoring relationships that will help attract and train future outstanding scientists. Behavioral adaptations that accompany drug addiction are believed to result from both short and long-term adaptive changes in brain reward centers. Thus, exposure to drugs of abuse regulates intracellular signaling processes that alter gene expression, protein translation, and protein post-translational modifications. Repeated exposure to drugs of abuse leads to stable alterations in these signaling systems that are critical for the changes in brain chemistry and structure of the addicted brain. The Center's research goals include analysis of the actions of cannabis, cocaine, nicotine, and opioids on these intracellular signaling pathways in brain reward areas and development of methods that enable proteomic analysis of single types of neurons that define the circuits that underlie the actions and addictive properties of drugs of abuse. Targeted and data- independent MS analyses of signaling proteins implicated in the actions of drugs of abuse will be used to analyze the impact of substance abuse on the neuroproteome with motif-based, Top-Down MS/MS, and other approaches being used to study protein post-translational modifications. A major initiative led by the Bioinformatics and Biostatistics Core will be to develop novel methods for deep integration of genomic, transcriptomic, and proteomic data with brain region and cell type-specificity.

? DESCRIPTION (provided by applicant): The behavioral adaptations that accompany drug addiction likely result from short and long term regulatory changes in gene expression, protein translation, and protein modification. These three mechanisms underlie stable alterations in brain chemistry and structure of specific neuronal sub-types in specific circuits in the addicted brain. While the effects of drugs of abuse on transcription have been studied in depth little is known about how they regulate protein expression via translational control. Psychostimulant effects are acutely sensitive to protein synthesis inhibitors, yet systemic injection of psychostimulants inhibits protein synthesis in the dorsal and ventral striatum, indicating that regulation of translation of specific mRNAs is complex. Indeed, our preliminary studies suggest a novel role for regulation of protein elongation in striatal neurons in the acute actions of cocaine. While current methods, termed TRAP or Ribo-tag, allow quantification of ribosome bound RNA transcripts in specific neuronal cell types, they do not allow assessment of the dynamics or control of protein translation, i.e. the translatome, at the level of either translaton initiation or elongation. We will extend TRAP using highly complementary biochemical methods for the profiling of ribosome-bound RNA, ribosome footprints, and nascent polypeptides from specific neuronal cell types. These data will be interrogated using a deeply integrated computational analysis infrastructure to enable quantitative assessment of the cell type- specific translatome at isoform-level, rather than gene-level, resolution. In the proposed studies, we will use existing TRAP mice to comprehensively profile the translatome of either D1- or D2-receptor containing medium spiny neurons of the striatum in cohorts of acute cocaine-treated and control animals. In Aim 1a: Profiling total-RNA and ribosome footprint RNA from specific neural cell types, we will modify the existing TRAP protocol to enable parallel profiling of the ribosome-associated transcriptome (by RNA-seq) and ribosome occupancy (by ribosome foot printing). In Aim 1b: Profiling nascent chain proteins from specific neural cell types, we will analyze nascent chain proteins by puromycin incorporation into purified ribosomes, which we coin protein-TRAP (pTRAP), followed by tandem mass-spectrometry. In Aim 2: Integrative statistical analysis, we will integrate the three-level measurements of the translatome provided by Aim 1 using our recently developed expectation-maximization algorithm that probabilistically assigns footprints and/or peptides to specific isoforms based on transcript abundances obtained from RNA-seq. The resulting isoform-level quantitation will enable analysis of translational control at unprecedented resolution and accuracy, in addition to precise characterization of novel mechanisms of translational control such as upstream open reading frames and stalled ribosomes. Through the analysis of cohorts of control and cocaine-treated animals, we will classify sets of isoforms that respond in a variety of ways to different mechanisms of translational control in specific striatal neurons, setting the stage for more in-depth studies in other models of cocaine addiction.

There are currently no practical biomarkers for Alzheimer?s Disease (AD) that can routinely stage, assess prognosis, and monitor treatment response. AD is a complex disease of overlapping pathophysiologies that lead to eventual synapse loss and progressive dementia. The core AD biomarkers, amyloid-b and tau (total and phospho-) measured in cerebrospinal fluid (CSF), provide a strong indication of the presence or absence of substantial AD pathology, but as yet poorly reflect disease stage or progression. In order to diagnose the disease earlier, learn about the time-course of concurrent pathophysiologies, and maximize the potential for staging, tracking and treatment, it is critical that more useful AD biomarkers are characterized. A collaboration is proposed between the Yale and Massachusetts ADRCs that will leverage the individual strengths of these two centers to discover pathway driven novel biomarkers of AD. Using the large numbers of clinically characterized biofluid samples available from the Massachusetts ADRC, the Yale ADRC will employ their expertise in quantitative targeted LC- MS/MS technology to assess over one hundred analytes in CSF, with an extension of the most informative to blood. LC-MS/MS is inherently specific, and enables simultaneous testing of many markers from a small biofluid volume. Use of Data-Independent-Acquisition (DIA) LC- MS/MS techniques allows for primary data to be reanalyzed for retrospective analysis of new biomarkers, providing a reproducible yet flexible discovery pipeline. In Aim 1, a panel of over 100 proteins in CSF from pathways involved in AD pathophysiology will be subjected to comprehensive technical qualification. Using samples from the placebo arm of a clinical trial, peptides will be tested for linearity, inter-assay, intra-assay and short term biotemporal stability. All high performing peptides will then be quantified in a high contrast sample of 60 Cognitively unimpaired-adults (CU-A) and 60 dementia due to AD cases. They will be assessed for their ability to distinguish AD from CU-A, and for their correlation with markers of synaptic function. In Aim 2, technically qualified peptides will be quantified in 3 tailored sample collections to assess their performance in AD staging, prognosis and differential diagnosis. In Aim 3, the most informative analytes from Aims 1 & 2 will be quantified in blood, either by LC-MS/MS, or by sensitive or ultra-sensitive ELISA approaches. Peptide abundance will be compared across biofluids, and the diagnostic utility of these markers in blood assessed.

The Biomarker Core will play a key role in the proposed Yale ADRC by managing the Biospecimen Repository consisting of both standard and novel biospecimens, and through the development and application of cutting-edge proteomic and epigenetic assays and bioinformatics approaches to integrate high-dimensional multi-omics data. Methods and biospecimens under the jurisdiction of the Yale ADRC Biomarker Core will facilitate the development and validation of promising biomarkers of Alzheimer?s disease (AD) susceptibility, improve AD risk prediction, and identify promising directions for development of targeted interventions. The Biomarker Core will work closely with the Data Management and Statistics Core (DMSC) in the management of the Biospecimens Repository. We will also facilitate the integration of multi-omics data with data generated by the Clinical and Imaging Cores to assess and validate both standard and novel biomarkers aimed at capturing AD heterogeneity, susceptibility, and progression. We will work with the DMSC to utilize statistical modeling of cognitive trajectories and decline, and/or considerations of mortality selection or other biases when evaluating the reliability, validity, and generalizability of AD-related biomarkers. Finally, we will work with the Education Component and Outreach Core to facilitate training and outreach in AD Biomarker acquisition and use. Specific Aims include: Aim 1: Manage Yale ADRC Biospecimen Repository. Working closely with the Clinical, Neuropathology, and DMS Cores, and NCRAD, we will manage the Yale ADRC biorepository and provide biospecimens to ADRC and non-ADRC investigators. Aim 2: Assess, track and validate biofluid and tissue biomarkers of AD. We will use standardized ELISA-based assays for Abeta1-40, Abeta1-42, t-tau and p- tau in CSF in existing and future biofluid samples from Yale ADRC Biorepository, as well as utilize state-of-the- art targeted mass spectrometric assays to analyze selected biomarkers in biofluid and brain tissue from the Yale ADRC Biorepository and other ADRC-affiliated research projects. Aim 3: Assess, track and validate systems-level biomarkers based on high-dimensional omics data. We will assess DNA methylation (DNAm) in biofluids from the Yale ADRC Biorepository and other ADRC-affiliated research projects, and use it to calculate both validated and novel biomarker measures of epigenetic age and AD heterogeneity. Working with the DMSC, we will use systems biology approaches to integrate the DNAm and proteomic data with data available via the Clinical and Imaging Cores. Aim 4: Facilitate training and Outreach in AD Biomarker acquisition and use.

The Biostatistics and Bioinformatics Core (BBC) supports statistical, bioinformatic and computational needs of the Discovery and Targeted Proteomics Cores, as well as Center Investigators, their postdoctoral associates and students, and Pilot Grant awardees. The BBC has four inter-related Specific Aims: 1) Biostatistics; 2) Bioinformatics; 3) High Performance Computing; 4) Training and Education. In Aim 1, we will provide statistical guidance on experimental design and data analysis, including sample quality assessment, and exploratory analysis for a wide range of types of proteomics data sets; continue to develop and add more features/functions to ProteomicsBrowser, a proteomics data analysis and visualization tool developed by the BBC to assist Center users in better interpreting complex proteomics data; develop and implement novel statistical methods to impute missing information in proteomics data; and develop and implement an online tool for proteomics data preprocessing, including data normalization, batch effect correction, and missing data imputation. In Aim 2, we will provide advanced bioinformatics software and approaches to assist Center investigators and Pilot Grant awardees in fully interpreting their comparative protein and protein post-translational modification profiling data; we will leverage information from single-cell RNA-seq by incorporating stochastic expression at the cell-type-level into an analytic framework to deconvolve tissue-level transcriptomics (RNA-seq) into fractions of constituent cell types for individual samples, and identify genes and cell types showing significant discrepancies between RNA and protein levels; and develop a unified computational framework for the detection of allele-specific peptides and allele-specific events from existing whole-genome sequencing, whole-transcriptome sequencing, and proteomics data generated from post-mortem human brain samples. In Aim 3, we will provide continued support for large-scale peptide sequence alignment and support novel pipelines to integrate genomic, transcriptomic, and proteomic datasets; work closely with the bioinformatics and biostatistics teams to help benchmark, scale, optimize, and speed up computing tasks involving large-scale data analyses and database queries; and explore alternatives to traditional high performance computing environments such as container systems and private cloud computing. In Aim 4, we will provide training and education in biostatistics, bioinformatics, database and high performance computing through interaction and collaboration with the Center investigators, including working closely with the Yale Medical Library Bioinformatics Support Program and other Yale organizations.

Epilepsy is one of the most common neurological disorders globally, estimated to affect 3.4 million people in the United States and 50 million people globally. Alarmingly, even in high-income countries, epilepsy in one- third of patients resists conventional drug intervention, highlighting the need to further understand the pathogenesis and molecular changes associated with this disease. Developmental and epileptic encephalopathy (DEE) is a relatively new concept in epilepsy research that describes genetic disorders in which a variation in a gene, such as a mutation, causes increased epileptiform activity and slowing of mental development. Recently, several developmental neurological conditions, such as Rett syndrome, cerebral palsy, and mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations have been associated with mutations in the microtubule-associated serine/threonine (MAST) protein kinase family. Specifically, our colleagues have discovered seven individuals with DEE with de novo missense mutations in the MAST3 gene: G510S, G515S, L516P, and V551L. Our group recently showed in brain that MAST3 phosphorylates ARPP-16, a small heat- and acid-stable protein enriched in striatum and cortex, at Ser46, converting it into a potent inhibitor of the serine/threonine phosphatase PP2A. Preliminary results suggest the DEE mutations in MAST3 cause a gain-of-function increase in MAST3 activity, promoting ARPP-16 phosphorylation at Ser46 which would lead to subsequent inhibition of specific forms of PP2A. Interestingly, other recent studies have identified loss of function mutations in the catalytic C, and substrate targeting B subunits, of PP2A in individuals with intellectual disability and developmental delay, the majority of which also have epilepsy. We hypothesize that increased MAST3 activity, and selective perturbation of PP2A signaling, may in part play a causative role in the development of childhood neurological conditions. The proposed research contains two specific aims to test the hypothesis that the DEE MAST3 mutations increase kinase activity and may change its interactome, causing detrimental changes in neuronal development. Aim 1 will determine which proteins and substrates interact with MAST3, how this interactome changes when the DEE MAST3 mutations are introduced, and how this affects kinase activity. This aim will be achieved using in vitro biochemical techniques, such as kinase assays with recombinant proteins, and pulldown assays in primary neuron cultures to identify interacting proteins and substrates. Aim 2 will investigate the role of the DEE MAST3 mutations in neuronal development and function using primary neuron cultures to visualize dendritic arborization and spines, morphological changes, and electrophysiological properties. Results gained from this study will further elucidate the potential for MAST3 to be a prominent force in the development of several neurological conditions early in life. Understanding this mechanism should lead to new diagnostic tools and interventions aimed at improving the quality of life of individuals affected by these disorders.