Date of Degree

9-2015

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Itzhak Mano

Subject Categories

Genetics

Keywords

Cell death; Cytohesin/GRP-1; Excitotoxicity; Glutamate; Insulin

Abstract

Excitotoxicity is a form of neurodegeneration that serves as the main underlying cause of brain damage in stroke/brain ischemia, and a contributing factor in a range of neurological diseases such as Epilepsy, ALS, Alzheimer, and Huntington's disease. In excitotoxicity, over-activation of glutamate receptors causes necrotic neuronal cell death. In spite of intense study of excitotoxicity, the molecular mechanisms that lead from glutamate receptor activation to necrotic death remain a mystery. Aging neurons are known to be more vulnerable to excitotoxicity and less likely to recover, but the underlying reasons for the increased cellular vulnerability are unknown. To gain insight into the core process of excitotoxicity and factors that affect neuronal vulnerability, we turn to a model system that offers simplified but conserved signaling cascades and strong research tools. We therefore study excitotoxicity in the nematode C. elegans, in which a strong track record of studies of cell death mechanisms and signaling cascades suggests that this genetically accessible model system can help illuminate critical and conserved cellular processes. Our model combines a deletion of glutamate transporter 3 (glt-3) in a sensitized background (nuIs5), resulting in glutamate-triggered necrotic cell death. Previously, other investigators showed the role of the evolutionary conserved Insulin/IGF Signaling pathway (IIS) in longevity and cell stress resistance. Recently, we were able to show that the IIS cascade also controls vulnerability of postsynaptic neurons to excitotoxicity, by regulating the nuclear/cytoplasmic localization of the neuroprotective transcription factor FOXO3/DAF-16. Active IIS signaling causes FOXO/DAF-16 to be sequestered in the cytoplasm, while IIS inhibition allows FOXO/DAF-16 to enter the nucleus and activate protective genes. To gain further insight into the ability of this signaling cascade to regulate neurodegeneration, we study factors that are upstream and downstream of the IIS cascade and may be involved in excitotoxicity. As a preliminary step, I confirmed the involvement of IIS pathway in excitotoxicity. I worked towards preparing an excitotoxicity strain that is permissible for RNAi effect in neurons, to facilitate the ability to screen for the effect of FOXO/DAF-16 candidate target genes on neuroprotection. I then examined potential upstream modulators of IIS, and focused on the relationship between the Cytohesin/GRP-1 protein complex (previously shown to control insulin-regulated metabolism in mammals) and IIS in excitotoxicity. I observed that mutations that are expected to decrease the complex's activity and its potentiation of IIS signaling reduced susceptibility to excitotoxicity, while an over-activation of this complex increased neuronal vulnerability. These results support that the Cytohesin/GRP-1 complex regulates the ability of the IIS cascade to modulate excitotoxicity. As a preliminary step to examine a possible direct communication between glutamate receptors and Cytohesin/GRP-1 IIS complexes, I examined their cellular localization. I observed a rough co-localization of the Cytohesin/GRP-1 complex components PPK-1 and GRP-1 and the glutamate receptor subunit GLR-1. Put together, these observations suggest that the Cytohesin/GRP-1 complex modulates IIS cascade's ability to regulate the susceptibility to excitotoxic neurodegeneration, and that glutamate might directly regulate this signaling cascade. We therefore provide novel insights into conserved signaling cascades that control neuronal sensitivity in nematode excitotoxicity. We hope that these new insights could inspire new research directions in the search for therapeutic interventions in stroke and brain ischemia.

Included in

Genetics Commons

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