UVM Theses and Dissertations
Format:
Print
Author:
Otto, Simone
Dept./Program:
Neuroscience Graduate Program
Year:
2013
Degree:
Ph. D.
Abstract:
The development of the nervous system is a carefully orchestrated dance of highly conserved programs interacting with, and being altered by, the unique environment in which they find themselves. One developmentally relevant interaction found in the avian ciliary ganglion, involving Prostate Stem Cell Antigen (PSCA) and [alpha]7 containing nicotinic acetylcholine receptors (nAChRs), is both unique and interesting [alpha]7 nAChRs have a high permeability to Ca², fundamental to their function; but potentially neurotoxic. One mechanism of controlling this is inhibition of [alpha]7 nAChR opening. Prototoxins, endogenous 3-fingered proteins that resemble of [alpha]-bungarotoxin[alpha]btx), are thought to inhibit nAChRs. PSCA is a prototoxin upregulated during development int~eavian ciliary ganglion that decreases responsiveness of [alpha]7 nAChRs. The goal of this research was to determine what conditions upregulate PSCA and under what conditions PSCA colocalizes with [alpha]7 nAChRs.
In vivo, PSCA transcript levels in the developing ciliary ganglion increase many fold from E.8-14. When neurons are isolated at E8 or E14 and placed in cell culture, PSCA levels neither decrease nor increase. In addition, multiple conditions were tried and failed to cause upregulation of psca. These include cocultures with iris or choroid, inducing depolarization by increasing [K+] to 25 mM, nor the addition of brain-derived neurotrophic factor, activin, basic fibroblast growth factor, Fe-ovotransferrin or neuregulin. However, coculture with Schwann cells increased PSCA to levels comparable to those seen in vivo.
To determine whether PSCA co-localized with [alpha]7 nAChRs, we expressed a V5epitope tagged PSCA using a retroviral vector, then monitored cell surface V5-PSCA together with [alpha]7 nAChRs using deconvolution restoration microscopy. A Pearson's coefficient of colocalization was used to quantify the level of colocalization of cell surface V5-PSCA to [alpha]7 nAChRs. Results indicate PSCA colocalizes with [alpha]7 nAChRs in ciliary neurons with a significantly higher mean Pearson's coefficient than in choroid neurons. To determine if the level of colocalization in the ciliary neurons was coincidental to the limitations of pseudospine structure, the F-actin substructure of the pseudospine was disrupted using latrunculin-A. That colocalization was not altered supports an interpretation of PSCA interacting with [alpha]7 nAChRs.
To determine if PSCA colocalization with [alpha]7 AChRs was responsive to receptor activity, embryos were exposed to constant light during development as a mechanism to drive increased [alpha]7 nAChR activity through its role in the circuitry of the pupillary light reflex. Embryos exposed to constant light exhibited increased ciliary ganglion volume and a rescue of neurons from naturally occurring cell death. In addition, exposure to constant light significantly increased PSCA levels as well as colocalization with [alpha]7 nAChRs in both ciliary and choroid neurons. By contrast, blocking nicotinic receptor activity resulted in significantly lower colocalization in light-incubated embryos. In addition, neurons cultured for 24 hours had lower colocalization of PSCA and [alpha]7 nAChRs than acutely cultured neurons. These results support a role for PSCA dynamically interacting with [alpha]7 nAChRs in accordance with the activity level of the receptor.
Collectively these studies support the concept that PSCA inhibits [alpha]7 nAChR activity previously established in the Nishi Laboratory and point to Schwann cells as a potential factor in its upregulation. In addition they underscore the importance of environmental factors in understanding normal development with particular emphasis on how these factors might alter the interactions of prototoxins with their target molecules.
In vivo, PSCA transcript levels in the developing ciliary ganglion increase many fold from E.8-14. When neurons are isolated at E8 or E14 and placed in cell culture, PSCA levels neither decrease nor increase. In addition, multiple conditions were tried and failed to cause upregulation of psca. These include cocultures with iris or choroid, inducing depolarization by increasing [K+] to 25 mM, nor the addition of brain-derived neurotrophic factor, activin, basic fibroblast growth factor, Fe-ovotransferrin or neuregulin. However, coculture with Schwann cells increased PSCA to levels comparable to those seen in vivo.
To determine whether PSCA co-localized with [alpha]7 nAChRs, we expressed a V5epitope tagged PSCA using a retroviral vector, then monitored cell surface V5-PSCA together with [alpha]7 nAChRs using deconvolution restoration microscopy. A Pearson's coefficient of colocalization was used to quantify the level of colocalization of cell surface V5-PSCA to [alpha]7 nAChRs. Results indicate PSCA colocalizes with [alpha]7 nAChRs in ciliary neurons with a significantly higher mean Pearson's coefficient than in choroid neurons. To determine if the level of colocalization in the ciliary neurons was coincidental to the limitations of pseudospine structure, the F-actin substructure of the pseudospine was disrupted using latrunculin-A. That colocalization was not altered supports an interpretation of PSCA interacting with [alpha]7 nAChRs.
To determine if PSCA colocalization with [alpha]7 AChRs was responsive to receptor activity, embryos were exposed to constant light during development as a mechanism to drive increased [alpha]7 nAChR activity through its role in the circuitry of the pupillary light reflex. Embryos exposed to constant light exhibited increased ciliary ganglion volume and a rescue of neurons from naturally occurring cell death. In addition, exposure to constant light significantly increased PSCA levels as well as colocalization with [alpha]7 nAChRs in both ciliary and choroid neurons. By contrast, blocking nicotinic receptor activity resulted in significantly lower colocalization in light-incubated embryos. In addition, neurons cultured for 24 hours had lower colocalization of PSCA and [alpha]7 nAChRs than acutely cultured neurons. These results support a role for PSCA dynamically interacting with [alpha]7 nAChRs in accordance with the activity level of the receptor.
Collectively these studies support the concept that PSCA inhibits [alpha]7 nAChR activity previously established in the Nishi Laboratory and point to Schwann cells as a potential factor in its upregulation. In addition they underscore the importance of environmental factors in understanding normal development with particular emphasis on how these factors might alter the interactions of prototoxins with their target molecules.