Jason P. Weick - US grants

DESCRIPTION (provided by applicant): We are primarily interested in determining the mechanisms by which L-type calcium channels, and not other calcium entry routes, influence neuronal plasticity via changes in gene expression and protein synthesis. Specifically, we believe different alpha1 subunits of L-type calcium channels are uniquely responsible for activation of the transcription factors CREB and NFATc4, both of which regulate cellular excitability. The experiments we will use to address this question are organized into three specific aims: 1) Determine the molecular components of (alpha1C that are necessary and sufficient for activation of CREB. 2) Establish whether it is the (alpha1C and/or alpha1D comprised L-type calcium channel that regulates NFATc4 activation. 3) Elucidate the mechanism by which L-type calcium channels regulate NFAT-dependent transcription. Our overall goal is to comprehend why the expression of many genes influencing cell excitability are tightly regulated by calcium entry specifically through L-type calcium channels. Thus, we will gain insight into the mechanisms surrounding a variety of cellular events, including those linked to development, learning and memory, hyperalgesia, drug addiction and aging.

DESCRIPTION (provided by applicant): The success of neuronal cell replacement therapy depends on the ability of transplanted cells to synaptically integrate with host tissue. Complete integration requires that neurons can both send and receive synaptic information, as well as to modify their synaptic strength in response to changes in the cellular behavior of synaptically connected neurons. Due to limited cell tracking and stimulation techniques, previous reports have shown only that transplanted neurons can receive information from host neurons via synaptic stimulation. Thus, no direct evidence exists for their ability to send information to host cells or undergo synaptic plasticity. To test these hypotheses, we propose to use the light-activated Channelrhodopsin-2 (ChR2) ion channel linked to the mCherry fluorophore in human embryonic stem cell (hESC)-derived neurons. Following transplantation of hESC-derived forebrain-patterned neurons to the mouse hippocampus, we will use light stimulation to selectively activate human neurons while recording mouse cells. We hypothesize that light stimulation (and subsequent action potential generation) will give rise to robust synaptic activation of host neurons. Application of various light stimulus protocols will then test whether human neurons can trigger short-term and long-term forms of synaptic plasticity in host cells. In parallel, we will perform similar experiments on mixed cultures of ChR2-expressing and non-expressing hESC-derived neurons. Here, application of light stimulation protocols will test whether hESC-derived neurons can undergo post-synaptic changes required for enduring changes in synaptic efficacy. Together, these data seek to provide evidence of the ability of human neurons to act as a fully functional unit within a neural network in host tissues. PUBLIC HEALTH RELEVANCE: Successful neuronal cell replacement is thought to rely on the integration of transplanted cells with host tissue via the formation of synaptic connections. Until recently, technical limitations have prevented the determination of whether stem cell-derived neurons can send information to host cells or undergo changes in synaptic efficacy that are necessary for true circuit integration. The proposed research will use the newly characterized light activated Channelrhodopsin-2 in human embryonic cell-derived neurons to regulate the excitability of transplanted neurons to test these hypotheses.

SUMMARY Stroke represents a major health issue not only as a cause of death but because most people survive their first stroke, leaving victims permanently disabled. With an aging baby boom generation in the United States, incidence of stroke and costs to society propose to reach epidemic proportions within the next two decades. While prevention efforts have significantly reduced lethality, few effective treatment options exist to provide patients with a means of recovery, and none exist to address the root cause of the problem, loss of brain tissue. Recently however, successful outcomes from cell replacement therapies in pre-clinical studies have led to a number of clinical trials which have demonstrated safety and efficacy for a variety of cell types. Interestingly, many of the mechanisms attributed to transplanted mediated recovery converge on the activity- regulated release of paracrine factors from transplant to host. In the current study, we propose first to validate that transplanted human pluripotent stem cell-derived neurons (hPSNs) improve behavioral recovery in a mouse model of focal ischemia that is amenable to mechanistic and manipulation studies (Specific Aim 1). We will then use this model to test whether optogenetic stimulation of hPSNs can augment behavioral recovery from stroke (Specific Aim 2). Lastly, multiple lines of evidence suggest that functional integration of transplanted cells with host tissue is critical for long-term benefits of cell replacement. In support of this, we have recently demonstrated in uninjured animals that human embryonic stem cell-derived neurons (hPSNs) can functionally integrate with host circuitry after transplantation, and can cause changes to overall excitability in vitro. In the Specific Aim 3 we will test whether and how transplanted hPSNs are functionally integrating with host circuits in vivo to understand how we might augment future intervention strategies. The impact of answering these questions proposes to improve efficacy of cell-based therapeutic interventions.

PROJECT SUMMARY/ABSTRACT The objective of this project is to create an optical strategy with high spatio-temporal specificity to regulate the expression of genes that may provide benefit to neurons prone to degeneration. While we will focus on the clearance of pathological forms of microtubule associated protein tau (MAPT), our study will lay the foundation for a variety of future examinations of regulated biological pathways related to disease. Previously reported in expression systems, our study will seek to be the first to use plant phytochromes that are bi-directionally regulated by red light (ON) and far red light (OFF), to induce transcription of a master autophagy regulating protein Transcription Factor EB (TFEB) in neurons. We will then test the capacity of this system to: (i) reduce the presence of pathological MAPT in vitro using cell lines expressing mutant MAPT and human pluripotent stem cells from patients with Down syndrome (DS) and sporadic Alzheimer's disease (AD), (ii) validate that TFEB is causative for, and the autophagy-inducing effects are positively correlated with, reductions in MAPT, and (iii) test whether application of light-induced TFEB expression in the hippocampus of a mouse model of AD can reduce cell death and cause improvements in learning and memory. The main hypothesis is that repeated, transient increases in autophagy can be used to inhibit MAPT-mediated degeneration in brains that cause various forms of tauopathy. Previous studies have shown that sustained increases in TFEB-mediated autophagy can clear MAPT aggregates and improve cell survival. However, chronic induction of autophagy can itself be deleterious and may exacerbate other neurological insults highly prevalent in aging individuals, the target population for tauopathy treatments. Thus, we believe the proposed strategy will provide therapeutic benefits (increased autophagy and reduced MAPT pathology) while reducing the likelihood of side effects such as autophagy-mediated cell death in response to additional cell stresses (e.g. ischemia/vascular dementia). Furthermore, once developed, this system may be applied to many neurological disorders for which regulated gene expression may be desirable, including Parkinson?s disease, Epilepsy, Amyotrophic Lateral Sclerosis, and multiple forms of poly-glutamine disorders (e.g. Huntington?s disease) to name a few.

PROJECT SUMMARY Schizophrenia (SCZ) and Bipolar disorder (BD) are heterogeneous psychiatric disorders with severe socioeconomic impacts and unknown pathogenesis. Circular RNAs (circRNAs) are a novel category of non- coding RNAs that are derived from the back-splicing and covalent joining of exons and introns of protein- coding genes, yet lack the capacity to become translated into protein. Recent studies have suggested that circRNAs are relatively enriched in the brain, are preferentially generated from brain plasticity-associated genes, and are abundant in dendrites and synapses. However, very little is known about the function of circRNAs in the human brain and their potential involvement in neuropsychiatric disease. Here we carried out systematic profiling of circRNA expression in a large cohort of human postmortem brains from subjects with SCZ and BD and uncovered a subset of differentially expressed circRNAs produced from genes with known links to synaptic plasticity and neuronal excitability. We propose to study the function of the evolutionary conserved, neuronal-enriched circRNA, circHomer1, which is reduced in both the prefrontal cortex (PFC) of both BD and SCZ postmortem brains and in patient-derived neuronal cultures. We hypothesize that circHomer1 inhibits glutamatergic synaptic transmission and neuronal excitiability via inhibiting the expression and synaptic localization of plasticity-related Homer protein homolog 1 long isoform B (HOMER1B) mRNA, thereby disrupting PFC functions. We intend to knockdown circHomer1 expression in both induced pluripotent stem cell (iPSC)-derived neuronal cultures and mouse PFC and examine its role in neuronal fuction and psychiatric disease-related behavior. We will test our hypothesis via three specific aims: 1) Test the hypothesis that circHomer1 and HOMER1B mRNA levels are differentially altered in a cell-specific manner in human PFC and iPSC-derived neuronal cultures from patients with psychiatric disease. 2) Test the hypothesis that circHomer1 regulates synaptic efficacy and neuronal excitability through inhibition of HOMER1B localization. 3) Test the hypothesis that circHomer1 deficits in the PFC influence neuronal firing, cognitive flexibility, and sensorimotor gating.

Unique patterns of synaptic connectivity between neurons, and the differential strength of those synapses, are fundamental to the information processing capability of the brain. Synaptic strength is determine by the number, composition, and post-translational modifications of post-synaptic AMPA receptors (AMPARs). These features of AMPARs are regulated by a host of second messenger pathways, scaffolding proteins, and trafficking proteins in post-synaptic densities (PSDs). For synapses that display primarily postsynaptic plasticity, the proteins responsible for regulating AMPAR surface expression are thought to be shared across glutamatergic PSDs. Thus, it remains unknown whether fundamental differences in synaptic strength between synapses exist due to unique protein signatures of individual PSDs. Neuron-specific genes (NSG1-3) encode single transmembrane proteins involved in the secretory trafficking of multiple scaffold and signaling proteins, including postsynaptic AMPARs. Studies in cultured cells as well as acute hippocampal slice preparations have established that disrupting NSG1-3 function independently causes severe alterations in basal synaptic activity and plasticity. Interestingly, our published and preliminary evidence show that NSG1 and NSG2 chronically reside within a subset of synapses in excitatory hippocampal neurons. In addition, our data show that knockout (KO) of these proteins differentially affects network function and induces behavioral deficits. This study will be significant because it will identify whether multiple members of the NSG family are restricted to a unique subpopulation of excitatory synapses, and confer unique functional properties via the promotion of AMPAR surface expression. We will use a combination of validated and novel techniques to address these important questions in three specific aims: Specific Aim 1. Determine whether NSG proteins define unique population(s) of excitatory synapses. We will use in vitro time lapse, and ex vivo imaging to determine whether NSG1 and NSG2 are specifically targeted to a subset of hippocampal synapses or trafficked between them. Specific Aim 2. Determine whether individual NSG proteins differentially affect synaptic function. Using physiological recordings and glutamate uncaging we will determine whether NSG1/2 are differentially involved in promoting surface AMPAR expression during basal or activity-dependent conditions. Specific Aim 3: Determine whether KO of NSG proteins leads to specific, dissociable behavioral deficits. Using established and novel behavioral tests in single and double KO mice we will determine whether NSG1/2 proteins play unique or overlapping roles in shaping motor, affective, and cognitive function in live animals.