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The Orphan G Protein-Coupled Receptor, Gpr161, Regulates Adult Neurogenesis Under Normal Conditions And Post-Traumatic Brain Injury

Myka Francesca R. Ababon
M.S., University of the Philippines - 2010

Thesis Advisor: James H. Millonig, Ph.D.
Graduate Program in Cell & Developmental Biology

CABM Room 010

Thursday, January 12, 2017
10:00 a.m.


Adult neurogenesis occurs in specialized stem cell regions within the adult central nervous system. Brain damage from injury, stroke and neurodegenerative diseases is irreversible, with no known treatment. In addition to their roles under normal conditions, these adult neural stem cells proliferate in response to neurodegeneration caused by brain injury or disease. Therefore, understanding the molecular mechanisms regulating adult neurogenesis will help identify targets for therapeutics to aid repair. We demonstrated that Gpr161, an orphan G protein coupled receptor (GPCR) that regulates the retinoic acid signaling pathway during neurulation, is expressed in these adult CNS stem cell populations. Because embryonic neurodevelopment and adult neurogenesis are known to be regulated by similar signaling pathways, we tested the hypothesis that Gpr161 regulates adult neurogenesis under normal conditions and post-traumatic brain injury (TBI). We first investigated the effect of the hypomorphic Gpr161vl allele on adult neurogenesis in vitro and and in vivo. Next, we looked at the effect of Gpr161 knockdown and overexpression in SGZ neural stem cells in vitro and in vivo under normal conditions and after traumatic brain injury. Lastly, we explored candidate signaling pathways downstream of Gpr161 in regulating adult neurogenesis.
First, using the hypomorphic Gpr161vl allele, we demonstrated in vitro that stem cell number and self-renewal is decreased in Gpr161vl/vl compared to wild-type. The mutation also significantly reduces stem cell proliferation and increases apoptosis. To exclude the developmental consequences of the Gpr161vl allele, we performed in vitro lentiviral knockdown in SGZ-derived neurosphere cultures. Similar to Gpr161vl, Gpr161 knockdown also decreased self-renewal ability and increased apoptosis; however, it did not affect proliferation. On the other hand, lentiviral Gpr161 overexpression resulted in a significant increase self-renewal ability and proliferation, coupled with a decrease in apoptosis. Next, we conducted Gpr161 knockdown and overexpression in the adult SGZ in vivo. Under normal conditions, lentiviral knockdown of Gpr161 in the hippocampus of adult mice did not affect proliferation or differentiation but increased apoptotic cells, consistent with in vitro data. Gpr161 overexpression increased both proliferation (EdU+ cells) and differentiation (Dcx+) cells. Investigating the effects of altered Gpr161 levels post-injury, Gpr161 knockdown decreased the proliferative response and increased apoptosis. In contrast, overexpression of Gpr161 was able to enhance the stem cell proliferative response to injury. To investigate whether this increased response translates to improved cognitive recovery, we conducted the Morris water maze test. Due to inherent strain limitations, we were unable to obtain relevant data from the water maze test.
Lastly, to investigate the signaling pathway downstream of Gpr161, we hypothesized that Gpr161 regulates adult neurogenesis through crosstalk between cAMP and RA signaling: Gpr161 activates cAMP and subsequent protein kinase A (PKA) phosphorylation of RA receptor alpha (RARa), increasing RARa nuclear translocation and transactivation activity, and increasing downstream RA signaling. Through immunocytochemistry for phosphorylated RARa (phosphoRARa) in our neurosphere cultures, we observed a decrease in nuclear staining of phosphoRARa in the knockdown and an increase in the overexpression. Our results demonstrate that Gpr161 is a novel regulator of adult neurogenesis, and possibly through downstream cAMP and RA signaling, is both necessary and sufficient to regulate neurogenesis of adult neural stem cells in response to injury. Currently, 50-60% of available drugs target GPCRs, making them the most important family of pharmaceutical targets. Based on our study, Gpr161 may be a potential pharmaceutical target to treat brain damage.

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