Characterization of Nestin Proteins in the Goldfish: Implications for Regeneration of Adult Dopaminergic Neurons

Title: Characterization of Nestin Proteins in the Goldfish: Implications for Regeneration of Adult Dopaminergic Neurons
Authors: Venables, Maddie Jolyane
Date: 2016
Abstract: Nestin is a type VI intermediate filament protein that marks proliferative cells in the central and peripheral nervous system of vertebrates during development and adulthood. Nestin is not only expressed in progenitor cells of neuronal tissues but is also present in muscle, heart, lung, pancreas and skin follicle tissues. The goal of this thesis is to investigate and characterize the nestin protein in goldfish and relate nestin expression to neuroregeneration and brain plasticity events in the adult goldfish forebrain. Currently little is known about nestin function and regulation in vertebrates, especially in fish. In this study we used Rapid amplification of cDNA ends PCR (RACE-PCR) to isolate goldfish nestin mRNA. We uncovered several different mRNA transcripts. PCR analysis and sequencing further identified three different nestin transcripts of 4003, 2446, and 2126 nucleotides with a predicted protein length of 860, 274, and 344 amino acids respectively. We next applied a multiple-antigenic peptide (MAP) strategy to generate a polyclonal goldfish-specific nestin antibody against a 23 amino acid sequence located at the N-terminal end of goldfish nestin. Western blotting revealed the existence of three different nestin protein isoforms (nestin A, B and C); the first report of nestin isoforms in teleost species. Nestin expression and distribution in the goldfish brain is complex and revealed both individual and tissue-dependent variations. The most remarkable finding following principal component analysis of the western blot data was the uniqueness of the pituitary, hypothalamus and telencephalon. These tissues are proliferative in nature containing progenitor and proliferative cellular pools that are involved in important biological axes such as the motor and reproductive axis. Interestingly, all three tissues were able to change their proliferative cellular profile of nestin protein expression to alleviate the detrimental effects of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) upon administration. The toxin MPTP destroys dopamine neurons in the fish brain leading to motor deficits and reproductive difficulties. The incorporation of 5-bromo-2’-deoxyuriding (BrdU) into newly synthesized DNA revealed an upregulation of BrdU immunolabeling following MPTP administration in the area telencephali pars dorsalis (Vd) and along the ventricular surface area of the telencephalon suggesting the generation of new neurons in the adult central nervous system. This thesis reports novel nestin isoforms and illustrates regenerative events occurring in the goldfish telencephalon following a neurotoxic insult. This work provides a framework for future investigations of the differential roles and regulation of the nestins to better understand seasonal neuronal plasticity, neuronal regeneration and neuronal circuitry in teleost.
CollectionThèses, 2011 - // Theses, 2011 -