Date of Award

2014-01-01

Degree Name

Master of Science

Department

Biological Sciences

Advisor(s)

Manuel Miranda- Arango

Abstract

Glycine is an important inhibitory neurotransmitter that is localized in the caudal areas of the nervous system and plays an important role in mediating many important functions related to breathing, spinal reflexes, nociception etc. Termination of neurotransmission is achieved by the re-uptake of glycine back into the pre- synaptic neuron and neighboring glial cells by the two glycine transporters namely Glycine transporter 1 (GlyT1) and Glycine transporter 2 (GlyT2). These transporters play an essential role in regulating glycinergic inhibition in the central nervous system by controlling the duration and intensity of neurotransmission and therefore the efficacy of synaptic inhibition as well.

The GlyTs share 50 % amino acid sequence identity and similar topology; however, unique to GlyT2 is the presence of a 200-amino acid long N-terminal tail which is not present in any other related transporter such as the monoamine or GABA transporters, and its functional aspects have not been explored to date. The importance of this N- terminal domain can be gauged from the fact that many individuals diagnosed with sporadic Hyperekplexia, a neurological disorder characterized by excessive startle response, carry point, nonsense and frameshift mutations along the GlyT2 sequence, including the N-terminal domain (Davies, Chung et al. 2010). The aim of this research is to understand the role of this N-terminal domain of GlyT2 in regulating the function and anchorage of the transporter and to study its expression in the glycine- rich areas of the nervous system like the brainstem, cerebellum and the spinal cord. This is accomplished by performing Yeast Two Hybrid assay in order to identify proteins that could interact with the N- terminal domain of GlyT2. Out of the 74 clones identified in the screen, the first 26 proteins have been identified and are mainly cytoskeletal proteins as well as some involved in signaling cascades, thus hinting at the important role of the N- terminal domain

vii

of GlyT2 in anchoring the protein at the plasma membrane and perhaps, influencing its functioning at the glycinergic synapse.

Although it is well known that GlyT2 is abundant in the spinal cord and caudal regions of the CNS, the location of glycinergic nuclei, axonal projections and connections have not been explored in detail. The efforts to study these neuronal circuits have been limited, due in part, to the essential function of GlyT2 for survival and the lack of reagents such as highly specific antibodies. To map the neuronal pathways containing GlyT2, a transgenic mouse was developed and kindly provided by Dr. H. U. Zeilhofer, University of Erlangen-Nurnberg, Germany (Zeilhofer et al. (2005) J. Comp. Neurol. 482, 123-41) which expresses the green fluorescent protein (GFP) under the control of the GlyT2 promoter. Coronal and sagittal sections of the transgenic mice brains reveal that the majority of GFP signal is localized to the medulla, pons and midbrain. Several nuclei containing GFP are restricted to the Colliculi and several areas of the Pons in the brain stem. Consistent with these findings, we have identified several GFP-, GlyT2- positive neurons in the Central nucleus if the Inferior Colliculus (CIC) and several areas of the brainstem such as the ventral cochlear nuclei (VCA), and several nuclei which make up the Superior Olivary Complex (SOC). The existence of GlyT2 in the areas involved in audition could provide important insights into the importance of glycinergic inhibition in mediating audition and could possible lead to further research in understanding the important sense of audition and treat maladies associated with it.

The work described in this Thesis has provided insights into the importance of GlyT2 in mediating glycinergic inhibition and could serve as a foundation to identify glycinergic circuits in the CNS, which can be manipulated by optogenetic techniques to gain a better understanding of glycinergic neurotransmission.

Language

en

Provenance

Received from ProQuest

File Size

67 pages

File Format

application/pdf

Rights Holder

Shweta Lavania

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Included in

Biology Commons

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