Shelley Halpain - US grants

This proposal addresses the molecular control of neuronal cytoskeletal proteins by extracellular signals. Understanding how the neuronal cytoskeleton is regulated is important because several disease processes, including Alzheimer's disease and schizophrenia are thought to affect the cytoskeleton and thereby alter neuronal function. During outgrowth, development, and synaptogenesis, the morphology of neurons is controlled by local environmental cues like growth factors and neurotransmitters. The microstructure of adult neurons is also thought to be modified in response to synaptic or neurohumoral activity. We propose to examine the cellular basis for morphological plasticity in neurons by focusing on the regulation of the phosphorylation state and function of the microtubule- associated protein MAP2. MAP2 is thought to play an important role in the development and maintenance of dendritic morphology via its interaction with microtubules and other cytoskeletal elements. Multiple protein kinases and protein phosphatases are known to modify MAP2 phosphorylation and MAP2 function in vitro, but little is known about the properties of MAP2 phosphorylation in vivo. Our previous studies revealed that one signal which alters MAP2 phosphorylation in intact neurons is activation of excitatory amino acid receptors. MAP2 becomes rapidly and selectively dephosphorylated as a result of N-methyl-D- aspartate receptor activation. This effect appears to involve a novel signal transduction pathway for NMDA receptors. In the proposed studies, we will define the signal transduction pathways which modulate the phosphorylation state of MAP2 in hippocampal slices and cultured hippocampal neurons. Hippocampal cells metabolically labeled with 32P- orthophosphate will be used to determine the mechanism, receptor specificity and temporal properties of excitatory amino acid-induced dephosphorylation of MAP2. The stimulation of MAP2 phosphorylation by neurotransmitter and growth factor-dependent protein kinase activity will also be examined. Two forms of MAP2 will be studied: conventional MAP2 found in adult neurons, and an alternatively spliced variant unique to immature neurons called MAP2c. This variant lacks a large middle portion of adult MAP2, and thus contains a relatively small number of phosphorylation sites. The immature form of MAP2 will help us to define which phosphorylation sites are under the control of NMDA receptors. Once we identify the kinase and phosphatase pathways for MAP2 and MAP2c regulation, we will determine the precise location of phosphorylation sites on the smaller protein MAP2c. Such information will be important in eventually determining the role of phosphorylation of MAP2 and MAP2c in regulating neuronal morphology. These results will contribute to an understanding of how extracellular signals regulate MAP2 function in vivo. More generally, these experiments will allow formulation of a molecular model for transmembrane regulation of the neuronal cytoskeleton.

DESCRIPTION (provided by applicant): Abnormal development of neuronal circuitry is thought to underlie the characteristic behavioral manifestations of autism spectrum disorder (ASD) and related disorders like Rett syndrome. The aberrant size and function of specific brain regions that has been documented in ASD is likely to result from aberrant development of neurons and synapses, features that can be quantified using in vitro approaches. This application proposes to develop an inexpensive, cell-based assay of neuronal morphogenesis and synaptogenesis that will facilitate both basic research and pre-clinical drug development for ASD. The morphology-based assay will be amenable to automated, high-content screening of neurons derived from either animal models or from induced pluripotent stem cells (iPS cells) from human patients with ASD and related disorders. Neurite outgrowth, dendritic branching, spine shape, and synaptic markers will be quantified. Proof of concept experiments will be conducted to evaluate differences in neuronal development in iPS cells (supplied by collaborator) from Rett syndrome patients compared to unaffected individuals. PUBLIC HEALTH RELEVANCE: This pilot project will develop quantitative, microscopy-based assays of neuronal development that can be used to evaluate neurons derived from lines of induced pluripotent stem cells of patients with autism and related disorders. The assay will facilitate a) the assessment of heterogeneity in neuronal development among patient populations and across individuals with autistic syndromes;b) investigation of molecular mechanisms underlying abnormal neuronal development;and c) screening of pharmaceutical compounds that may improve normal neuronal development in autism.

Project Summary Proper neural networks rely on the neurons' ability to generate neurites (axons and dendrites) and form synaptic connections with appropriate partners. Impairments in neuronal morphology and synapse composition in specific subtypes of neurons have been described in various mental health conditions, ranging from schizophrenia and bipolar disorder to autism spectrum disorder (ASD). Such altered neuronal morphology and synapse assembly presumably underlie disrupted neural circuit function, and therefore disrupted behavioral patterns. The objective of Project 4 is to create a suite of cellular imaging-based assays in hIPSC-derived cultures of differentiated neurons. These assays are based on quantitative fluorescence microscopy, and will focus on specific aspects of neuritogenesis and synaptogenesis that are relevant to ASD. The assays will be compatible with high-content screening technology, and will be applied to engineered hIPSC ASD models.