2011 — 2012 |
Garrett, Andrew [⬀] |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Intracellular Signaling by Dscam During Retinal Development
DESCRIPTION (provided by applicant): In order for the nervous system to develop and function normally, many processes must occur. Neurons must be spaced appropriately, they must send out axons and dendrites that extend through the tissue to develop arbors and find targets, and they must form synapses to communicate with partners. Defects at any of these points can lead to dysfunction and neurodevelopmental disorders. The Down syndrome cell adhesion molecule (Dscam) gene is on the region of chromosome 21 that is associated with trisomies in Down syndrome. In the mouse retina, Dscam and the very similar Dscam Like (DscamL1) are involved in adhesive masking, a cellular process important for self-avoidance, allowing cell spacing and dendrite arborization. The Dscams are also involved in some aspects of synapse development. There are a few proteins known to interact with the Dscams: Pak1 (p21-activated kinase) can be activated by Dscam, and the MAGI (membrane- associated guanylate kinase with inverted domain structure) family of scaffolding molecules interacts with the c-terminus of the Dscams. The aim of the experiments described in this proposal is to elucidate the signaling mechanisms downstream of the Dscams during adhesive masking and synapse development. The overall hypothesis is that Dscams regulate adhesive masking early in development through activation of Pak1, and are important for synapse maturation later in development through interactions with the MAGI proteins. To test this hypothesis, retina ganglion cells will be cultured in a system that allows the assessment of adhesive masking and the manipulation of gene expression. Experiments will also be performed in the mouse by making new mouse lines in which Dscam and DscamL1 have targeted mutations that do not allow the proteins to interact with the MAGIs. It is expected that the results will show that the Dscams carry out their different functions through distinct signaling mechanisms. These findings will have implications for the mechanisms of the Dscams'possible role in the pathology of Down syndrome and other neurodevelopmental disorders including congenital retinopathies. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to study the molecular mechanisms by which the mouse ortholog of Down syndrome cell adhesion molecule (Dscam) and the similar Dscam Like (DscamL1) function in the cellular recognition events that direct retinal development. In humans, Dscam is in region of Chromosome 21 associated with Down syndrome trisomies, and understanding the basic molecular mechanisms of the Dscams'function will help to define its possible role in the phenotypes associated with Down syndrome and other neurodevelopmental disorders. Studying these mechanisms in the retina may provide insight into human congenital retinopathies as well.
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0.915 |
2021 |
Garrett, Andrew [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Cell Adhesion Molecule Function in Retinal Development
ABSTRACT Neural circuit formation requires a series of highly diverse and specific cell-cell recognition steps, many mediated by cell adhesion molecules (CAMs). Indeed, mutations that disrupt CAMs or their regulation are associated with circuit level neurodevelopmental disorders from dyslexia to schizophrenia. Our model is the mouse retina, an extension of the central nervous system where ~100 types of neurons organize into dedicated circuits that encode the features of the visual world. We focus here on the gamma-protocadherins (?-Pcdhs), 22 CAMs expressed from a single gene cluster that generate many thousands of distinct homophilic recognition complexes. The ?-Pcdhs are critical regulators of neuronal self-avoidance in starburst amacrine cells (SACs), and cell survival and in many other types of neurons in the retina. The mechanisms through which the ?-Pcdhs serve these functions are unknown, as is the importance of ?-Pcdh isoform diversity. We used a CRISPR/Cas9 approach to generate an unbiased allelic series of mouse mutants with between 1 and 21 intact ?-Pcdh isoforms. From these, we learned that one isoform, ?C4, is essential for neuronal survival, suggesting that this isoform functions differently from the other 21. We propose to define the mechanisms of self-avoidance and neuronal survival, and to use our allelic series to determine the level of isoform diversity required for normal neural circuit formation. Our central hypotheses are that: 1) a high level of ?-Pcdh isoform diversity enables neurons to distinguish between ?self? and ?non-self? to mediate self-avoidance while permitting interaction with neighboring neurons through mechanisms common to all isoforms; and 2) neuronal survival, in contrast, requires interactions specific to the ?C4 isoform. In Specific Aim 1, we will use a strategic subset of our reduced-diversity mutants to determine the extent of isoform diversity required for self/non-self discrimination in SACs, neurons essential for the motion detection circuit in the retina. We will analyze this circuit at two levels: A) morphology of contacts between SACs, and B) the electrophysiological function of direction-selective retinal ganglion cells, the downstream neurons in the circuit. In Specific Aim 2, we will define the molecular mechanisms of self-avoidance using in vivo gene delivery to manipulate candidate pathways and map essential domains. In Specific Aim 3 we will uncover the mechanisms through which ?C4 promotes neuronal survival. We will use retinal electroporation to map critical protein domains, complemented by a discovery-based proteomics approach to find isoform-specific protein interactions for ?C4. These studies will allow us to better understand how the ?-Pcdhs contribute to cell-cell recognition and neural circuit formation in the retina and provide insight into processes disrupted by neurodevelopmental disorders.
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0.915 |