Regulated survival, proliferation and differentiation of cells in the nervous
system is crucial for development. Much of regulation is controlled by hormones.
There is abundant evidence that a member of the glucagon superfamily, pituitary
adenylate cyclase-activating polypeptide (PACAP), is important in this process. PACAP
functions have been described in the peripheral and central nervous systems of many
species. Although the primary function of PACAP is not known, its high conservation
and presence in all species examined to date suggest it is vital to normal development.
My thesis objective was to determine the response of early CNS neuroblasts to
PACAP, in conjunction with another glucagon superfamily member, growth hormone releasing hormone (GHRH). GHRH is best known for causing release of growth hormone from the pituitary, but it also has functions in nervous system development.
Because PACAP and GHRH are encoded on the same gene in non-mammalian vertebrates, it is possible that they have similar or coordinated functions. PACAP affects development by altering levels of proliferation and differentiation and decreasing
apoptosis. For these reasons, I focused my research in these areas.
Using neuroblast-enriched cultures from embryonic day 3.5 chick, my first goal was to show that PACAP and GHRH affected these cells. Radioimmunoassays for cAMP revealed that all but one form of PACAP, and only one form of GHRH, caused an
increase in cAMP relative to controls. As to the former, comparison of differing PACAP structures suggested that conservation at the amino terminus was important in binding the hormone to the receptor. The fact that PACAP, but not GHRH, increased
cAMP, indicated that evolution of PACAP and GHRH had altered their functions. Chick neuroblasts were also shown to produce PACAP and its primary receptor, suggesting an autocrine/paracrine role for PACAP.
My next goal was to examine the nature of the downstream effects of increased cAMP. To study cell cycle, I developed a protocol using proliferating cell nuclear antigen (PCNA) and propidium iodide (PI), in fixed cell populations. PCNA is present
in low amounts in non-cycling cells, but rises sharply in actively proliferating cells. The PI helped delineate cell cycle compartments, because in permeabilized cells it binds to and quantifies DNA. Changes in G0, G1, S and G2/M were recorded using flow cytometry. Because the cells were producing PACAP and most were cycling, rather than add more PACAP I chose to block the PACAP receptor. This caused cell cycle exit. I also blocked the cell cycle at two points, and showed that exogenous PACAP could release some cells from the block, and return them to cycling. PACAP affected apoptosis also, but because the protocol was not designed to measure this, I adopted another protocol using flow cytometry. With live cells, and fluorescein diacetate, which is retained and fluoresces in healthy cells, and PI, which enters only cells with damaged membranes, I used the characteristic of apoptotic cells to die with membranes intact to confirm increased apoptosis when the PACAP receptor was blocked.
This left the question of whether PACAP affected differentiation. The cell cycle protocol had shown some cells were still quiescent, not dying, at 24 h, so I hypothesized that they might be differentiating. I used proteomics to test this. With isotope-coded affinity tagged (ICAT) analysis, I measured changes in protein content in cells that had been treated with the receptor blocker, compared to control. This confirmed previous work and my hypothesis that some cells were differentiating. Because this technique is not commonly used in molecular biology, I also evaluated the effectiveness of the technique. My work showed that endogenous PACAP keeps chick neuroblasts alive and cycling, but will allow some to differentiate rather than die, when the hormone is withdrawn. Obviously, PACAP plays a crucial role in early chick brain development. / Graduate
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/7910 |
| Date | 12 April 2017 |
| Creators | Erhardt, Nola Marlene |
| Contributors | Sherwood, Nancy |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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