The study presented here used "omic" technology to look at the mechanism behind the selective delayed death of hippocampus CA1 neurons after transient global brain ischemia. The main findings are summarized: 1. The main form of ELAV protein family member detected in CA1/CA3 in Hu protein immunoprecipitation and polysomes was HuB (Rel-N1). HuB is present in control CA3, 8 hr reperfused CA3, and 8 hr reperfused CA1, but absent from control CA1. AUF-1, hnRNP K, hnRNP M were also absent from control CA1 following Hu protein immunoprecipitation and Western blot, suggesting that HuB bound AUF-1, hnRNP K, hnRNP M in all experimental groups except control CA1. 2. mRNA populations were different between sucrose pad preparation and sucrose gradient preparations of polysomes, although both were enriched with ARE-mRNA. This suggests different RNA binding complexes were isolated by the two methods. 3. Polysomes fractionation on sucrose pad and Hu protein immunoprecipitations using post-mitochondrial supernatants from homogenized brain regions were shown by 316 liquid chromatography mass spectroscopy to be over 75% contaminated by neuron debris, cytoskeleton and internal membrane structures, in spite of showing no contamination by Western blots of organelle markers. This suggests proteomics should become the accepted standard for validating purity of reactions derived from homogenized tissues. To summarize the results, I have worked up a consistent method of isolating polysomes from whole animal model, which has less contamination than the sucrose density gradient method. Both results from Hu IP and polysomes experiments show that control CA1 is in a different state compared with control CA3. My results suggest that the selective vulnerability of CA1 after ischemia reperfusion injury may be due, in part, to the fact that CA1 is "weaker" from the beginning. This finding is significant as it shifts the focus of research from studying the difference of ischemia reperfusion injury to the different initial states of CA1 and CA3 neurons. This study has also reformed our general idea as revealed by the high resolution of proteomics, which is superior to Western blotting for detecting contamination of samples. It is shown here that contaminationmakes up a large proportion of subcellular fractionations. This result suggests proteomics should be the new standard for quantifying contaminants, particularly in fractions obtained from whole tissues in animal experimental models.
|Advisor:||DeGracia, Donald J.|
|Commitee:||Chen, Xuequn, Evans, David R., Jena, Bhanu P.|
|School:||Wayne State University|
|School Location:||United States -- Michigan|
|Source:||DAI-B 76/02(E), Dissertation Abstracts International|
|Keywords:||ARE-mRNAs, Hu immunoprecipitation, HuB, Microarray, Polysomes, Proteomics|
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