Pathophysiology

Adult neurogenesis is affected in neurodegenerative diseases and also represents an important therapeutic target. The goal of this dissertation research was to test the hypothesis that regeneration of neurons and glia in the adult brain can be manipulated by neurotrophic drugs in the context of two mouse models of neurodegenerative disease : Parkinson's disease and Huntington's disease.... These findings have implications for the pathophysiology of Huntington's disease and neurodegeneration in general. Specific alterations to the SVZ neurogenic niche parallel some of the pre-motor symptoms of Parkinson's disease and Huntington's disease. This dissertation research contributes to the growing body of literature concerning the pharmacological modulation of SVZ-derived neurogenesis designed to attenuate the progressive loss of neurons in neurodegenerative diseases and perhaps delay the onset of symptoms.

p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway.

Huntington's Disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the Huntingtin gene IT15. In this study we demonstrated that Bcl-2 interacting mediator of cell death Extra Long (BimEL) protein expression was significantly increased in cells expressing mutant Huntingtin (mHtt). Moreover, striatal BimEL expression remained high in an R6/2 HD mouse model throughout the disease progression. Utilizing novel BimEL phospho-mutants we demonstrated the phosphorylation of Ser65 to be important for the stabilization of BimEL. We provided evidence that impaired proteasome function, increased JNK activity and reduced striatal BDNF lead to changes in the phosphorylation of BimEL, thereby promoting its stabilization specifically within the striatum of R6/2 mice. Furthermore, knocking down BimEL expression prevented mHtt-induced cell death in a HD cell culture. Taken together, these findings suggest that BimEL may contribute to the selective neurodegeneration and pathogenesis of HD.

In sea turtles, the study of the etiology and development of fibropapillomatosis is not fully understood. Sea turtle fibropapillomatosis is a disease characterized by the proliferation of skin fibropapillomas and occasional internal fibromas. In this study, sea turtle fibropapilloma tumor and healthy tissue samples were used to look at VEGF, BCL-2 and Bax expression. Cancer tumors have a well established pattern of protein expression that involves overexpression of vascular endothelial growth factor (VEGF), responsible for the growth of new blood vessels, and a high BCL-2 to Bax ratio that leads to uncontrolled cell proliferation. Real time PCR was used to analyze VEGF expression, and Western blot techniques were used to measure BCL-2 and Bax expression. The results indicated that expression of VEGF was not significantly higher in tumor vs. skin tissue. For the differential expression of BCL-2 and Bax, the results were not in agreement with the established levels found in cancer studies, showing no significant change in BCL-2 expression and significantly higher levels of Bax in tumor vs. healthy tissue.

Transgenic mice were generated to express a restrictive cardiomyopathy (RCM) human cardiac troponin I (cTnI) R192H mutation in the heart. My study's objective was to assess cardiac function during the development of diastolic dysfunction and to gain insight into the pathophysiological impact of the RCM cTnI mutation. Cardiac function was monitored in cTnI193His mice and wild-type littermates for a period of 12 months. It progressed gradually from abnormal relaxation to diastolic dysfunction characterized with micro- echocardiography by a reversed E/A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. The negative impact of cTnI193His on cardiac function was further demonstrated in isolated mouse working heart preparations. Dobutamine stimulation increased heart rate in cTnI193His mice but did not improve CO. The cTnI193His mice had a phenotype similar to that in human RCM patients carrying the cTnI mutation characterized morphologically by enlarged atria and restricted ventricle and functionally by diastolic dysfunction.

Two major troponin I (TnI) genes, fetal TnI (ssTnI) and adult TnI (cTnI), are expressed in the mammalian heart under the control of a developmentally regulated program. In this study, the up-stream domain (~1,800 bp) of mouse fetal TnI gene has been cloned and characterized. There is a high homology of this region among mouse, rat and human. Transfection assays indicated that conserved GA-rich sequences, CREB and a CCAAT box within the first 300 bp upstream of the transcription start site were critical for the gene expression. Electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays revealed binding proteins to CREB site in nuclear extracts from myocardial cells. Thyroid hormone (T3) caused a significant inhibitory effect on ssTnI expression in myocardial cells. Cardiac troponin I (cTnI) mutations have been linked to the development of restrictive cardiomyopathy (RCM) in human patients. We modeled one mutation in human cTnI Cv terminus, arginine1 92 histidine (R192H) by cardiac specific expression of the mutated protein (cTnI193His in mouse sequence) in transgenic mice. The main functional alteration detected in cTnI193His mice by ultrasound cardiac imaging examinations was impaired cardiac relaxation manifested by a decreased left ventricular end diastolic dimension (LVEDD) and an increased end diastolic dimension in both atria. Echocardiography revealed a series of changes on the transgenic mice including a reversed E-to-A ratio, increased deceleration time, and prolonged isovolumetric relaxation time. At the age of 12 months, cardiac output in cTnI193His mice was significantly declined, and some transgenic mice showed congestive heart failure. The negative impact of cTnI193His on ventricular contraction and relaxation was further demonstrated in isolated mouse working heart preparations.

Caloric restriction (CR), the reduction of nutrient intake short of malnutrition, extends the lifespan of various organisms and can improve measures of human health. Whether mechanisms of lifespan extension are conserved between humans and model organisms is unknown. In mammals, implementing CR is easily achieved by providing a restricted group with a fraction of the food consumed by an "ad libitum" fed group, which has unlimited food access. Due to the difficulty in directly controlling Drosophila food intake, caloric restriction, performed similarly to the mammalian paradigm, has never been tested in flies. Here, we demonstrate a system that allows measurement of food intake throughout life. This system will be used to measure fly lifespan under caloric restriction analogous to current mammalian studies. Our work will help tease apart the differences between the various caloric and dietary restriction paradigms in Drosophila, strengthening our understanding of how fly models relate to mammalian systems.

Aging is a process characterized by accumulated oxidative damage to DNA, proteins, and lipids,which leads to the gradual degeneration of cellular activity. Mitochondria play a central role in aging because they produce both cellular energy and oxidative stress. As resultof accumulated oxidative damage, mitochondria function decays, which leads to a cellular energy deficit and compromises cellular function. Iron is an essential nutrient reequired by mitodhondria to function optimally. It has been proved that iron supplementation increases the lifespan of several yeast strains, including superoxide dismutase mutants. We are interested in finding where the iron is going and what it might be doing that is beneficial to the cell. We have used Saccharomyces cerevisiae as our molecular model of aging. Our results indicate that the extra iron is being transported into the mitochoindria.