Georgia Panagiotakos - US grants

DESCRIPTION (provided by applicant): Mutations in L-type voltage gated calcium channels (LTCs) are associated with autism and other neurodevelopmental disorders. The goal of this project is to investigate how LTCs regulate neural progenitor cell (NPC) proliferation and differentiation. Neural development involves a series of coordinated events that balance maintenance and proliferation of NPCs with the differentiation of daughter cells to generate the neuronal populations that comprise the mature cerebral cortex. LTCs such as CaV1.2 convert electrical signals into calcium signals that control biochemical pathways and activate programs of gene expression in the developing nervous system. A gain of function mutation in CaV1.2 leads to Timothy Syndrome (TS), a multi- systemic disorder characterized by autism and mental retardation. Our laboratory has generated a transgenic mouse model for TS and I have found that there is a significant increase in proliferation of NPCs in the ventricular zone (VZ) of these animals. Using an in vitro system to study neuronal differentiation, I have also found that over-expression of wildtype CaV1.2 or CaV1.2 containing the TS mutation (TS-CaV1.2) results in a dramatic impairment of neuronal differentiation. Furthermore, blocking endogenous LTCs also reduces NPC differentiation in vitro. The goal of this project is to extend these initial findings by investigating both the regulation of CaV1.2 during development and the mechanisms by which these channels control NPC differentiation in vitro and in vivo. These studies will expand our understanding of how activity regulates brain development and will provide new insights into the underlying pathophysiology of autism and mental retardation. PUBLIC HEALTH RELEVANCE: Autism spectrum disorders affect more than a million children in the US. The research described here focuses on an inherited form of autism called Timothy Syndrome that is caused by a mutation in the voltage-gated calcium channel CaV1.2. This work will explore the mechanisms by which this mutation influences neurogenesis in the developing brain and will provide insights into cellular and molecular mechanisms that may cause autism in Timothy Syndrome, as well as other autism spectrum disorders.

PROJECT SUMMARY/ABSTRACT Mutations in the calcium channel Cav1.2 and downstream calcium signaling proteins in the calcineurin (CaN)/ NFAT pathway, in particular the kinase Dyrk1a, have been reproducibly associated with neuropsychiatric disorders, including autism spectrum disorders (ASD). These genetic findings implicate calcium signaling dysfunction in psychiatric disease and underscore a critical gap in our knowledge of how calcium signals are initiated and transduced in the developing brain. Our long-term goal is to understand how intracellular calcium elevations in neural progenitor cells (NPCs) direct their differentiation into neurons and glia, with an eye towards uncovering how mutations in calcium signaling proteins alter their developmental functions to promote disease. In this proposal, we focus on two distinct aspects of calcium signaling: detectors and sensors that initiate calcium responses to extrinsic cues or depletion of intracellular calcium stores, and molecular pathways that act as downstream transducers of calcium signals. We have found that utilization of two disease-relevant Cav1.2 exons is dynamically regulated in the embryonic cortex, and that an ASD-associated mutation in Cav1.2 prevents this developmental splicing switch in channel transcripts, which in turn alters the differentiation of specific cortical neuron subtypes. Similarly, we have also found that splicing of STIM2, a calcium sensor involved in store operated calcium entry (SOCE) in response to ER calcium depletion, is developmentally regulated to generate two isoforms with opposing effects on SOCE. Altering the relative levels of these isoforms in NPCs using in utero electroporation bidirectionally modulates cell cycle exit in vivo. Finally, in mice bearing a forebrain-specific deletion of Dyrk1a, a kinase that antagonizes CaN/NFAT signaling, we have observed broad misregulation of NPC function and differentiation. Building on these published and preliminary studies, the central objective of this proposal is to interrogate specific mechanisms by which intracellular calcium signals link extracellular cues with intrinsic differentiation programs and to elucidate how alternative splicing refines these signals. The proposed studies will test the hypotheses that calcium entry, regulated by precisely-timed exon utilization, orchestrates differentiation programs in the developing cortex (Aim1), and that downstream cell type-specific calcium signaling via the CaN/NFAT pathway is a key mechanism involved in the regulation of NPC function and differentiation (Aim2). This research will broadly impact the field of developmental neuroscience by elucidating the developmental regulation of calcium signaling in differentiating cells, building a foundation for future studies aimed at understanding how extracellular cues and intracellular calcium dynamics converge to regulate brain development. Our results will also have significant translational potential by providing new insights into mechanisms underlying the pathophysiology of psychiatric disorders.

PROJECT SUMMARY/ABSTRACT Mutations in DYRK1A, which encodes a ubiquitously expressed kinase that antagonizes the calcium- dependent calcineurin (CaN)/NFAT signaling pathway, have been reproducibly linked to neurodevelopmental disease. DYRK1A loss of function has been associated with syndromic intellectual disability and autism spectrum disorders (ASD), and increased DYRK1A activity is thought to underlie aspects of Down Syndrome pathophysiology. These genetic clues underscore DYRK1A dosage-dependent regulation of nervous system development; however, the precise mechanisms by which DYRK1A executes its roles in the developing brain remain poorly understood. Our long-term goal is to understand how DYRK1A acts in specific cell types of the embryonic cerebral cortex to influence the commitment of neural stem and progenitor cells to specific neural fates. In the proposed studies, we focus on NFAT-dependent transcriptional mechanisms as primary effectors of DYRK1A activity in neural stem cells and their progeny. We have found that deleting Dyrk1a specifically in the developing cortex differentially impacts calcium signaling in neural stem/progenitor cells and neurons of both the mouse and human. Loss of one or both copies of Dyrk1a results in dose-dependent cortical thinning, depletion of radial glia stem cells, reduced astrocyte abundance, neuronal cell death, and shifts in excitatory neuron differentiation. Our previous studies uncovered similar changes in the generation of excitatory neuron subtypes resulting from the mutation in the Cav1.2 calcium channel that gives rise to the syndromic ASD Timothy Syndrome. Imbalances in these same excitatory neuron types have also been linked to neuropsychiatric syndromes and channelopathies, hinting that calcium-regulated molecular mechanisms may represent a core substrate underlying cellular phenotypes in ASD and other neurodevelopmental disorders. In line with this idea, we have found that the effects of Dyrk1a deletion during cortical development are propagated by CaN/NFAT signaling, and we have used CUT&RUN sequencing to begin to identify targets of NFAT transcription factors in the developing brain. Building on these strong published and preliminary findings, the central objective of this proposal is to define cell type-specific mechanisms by which DYRK1A regulates the development of the cortex. The proposed research tests the ideas that NFAT transcriptional targets underlie deficits in stem cell maintenance and differentiation resulting from cortex-specific Dyrk1a inactivation (AIM 1), that DYRK1A and calcium signaling through CaN/NFAT play key roles in cortical astrogliogenesis (AIM 2), and that cell type-specific NFAT targets contribute to DYRK1A signaling specificity (AIM 3). These studies will build a foundation for future research expanding our knowledge of how calcium signaling regulates brain development and how ubiquitously expressed disease-relevant genes exert specific functions in different cell types. Our results will also provide therapeutic entry points for convergent intracellular mechanisms driving neurodevelopmental disorders.