­^ ¤å ºK ­n We investigated the role of heat shock proteins (HSPs), particularly HSP60, HSP70 or HSP90 in E. coli lipopolysaccharide (LPS)-induced naïve pheochrommocytoma cell (PC12) death. PC12 cells seeded at a density of 1x105 cells per poly-L-lysine-coated 3.5 cm diameter polystyrene dish were incubated with LPS (1 mg/ml; serotype O55:B5) for 3, 6, 12, or 24 hr. Cell viability was measured by trypan blue test, and expression of HSP60, HSP70, and HSP90 were detected by Western blot analysis. We found that the viability of PC12 cell decreased significantly after treatment with LPS for 12 hr, and viability was only 30% at 24 hr post-treatment. Western blot analysis revealed that LPS-induced PC12 cell death was associated with an increase in HSP70 or HSP60. HSP70 was markedly up-regulation at 12 hr; and both HSP70 and HSP60 increased significantly by over 1000% and 200%, respectively, 24 hr after administration of LPS. There was no significant change in HSP90 level 3, 6, 12, or 24 hr after LPS treatment. To further investigate the role of HSP70, 60, or HSP90 in LPS-induced PC12 cell death, we treated PC12 cells with hsps antisense oligonucleotide (AODN) for 24 hr. The effects of LPS on cell viability and HSP60, HSP70, or HSP90 expression were again tested. We found that suppression of HSP70 or HSP60 expression accelerated the process of LPS-induced cell death. A reduction in HSP90 level, however, had little effect. The study revealed that HSP70 and HSP60 played an anti-death role during LPS-induced PC12 cell death, and HSP90 did not appear to be involved.

Damage and/or loss of functional neurons can lead to detrimental cognitive and paralyzing effects in humans. Prime examples of such negative situations are well documented in patients with Parkinson's and Alzheimer's disease. In recent years, the utilization of neural stem cells and their derivation into neurons have been the focus of many research endeavors. The main reason for this is because neural stem cells are multi-potent and can differentiate into neurons, astrocytes, and oligodendrocytes. The research that will be detailed in this thesis involves the potential use of inorganic nanostructured materials to efficiently deliver bioactive molecules (i.e., retinoic acid, kinase inhibitors) to cells that can modulate the differentiation potential of certain cells into neurons. Specifically, PC12 (derived from rat pheochromocytoma) cells, as a neural model, was treated with select nanostructured materials with and without neuron inducers (molecules and ions) and the results were analyzed via biochemical assays and live-cell fluorescence microscopy. This thesis will include an in depth look into the cytocompatibility of the tested nanostructured materials that include silica nanoparticles, titanate nanotube microspheres, and carbon microparticles. / Bioengineering / Accompanied by two .avi files.

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Indiana University-Purdue University Indianapolis (IUPUI) / Acute and chronic activation of both ionotropic and metabotropic glutamate (glut) receptors is implicated in many neurodegenerative disorders including AD, dementia, epilepsy, stroke and neurotrauma. TRH and glut receptors (ionotropic & metabotropic) receptors are differentially coexpressed in granule and pyramidal neurons of the hippocampus. The author shows TRH to be protective when added to cultured pituitary adenoma (GH-3) cells and neuron-like pheochromocytoma (PC12) cells either prior to, during, or after glut-induced toxicity (Endo. Soc. Abs. 01), and also shows that the possible neuroprotective mechanism may involve heterologous downregulation of the metabotropic glut receptors, using superfused hippocampal slices and noting a reduction of Gαq/11 (SFN Abs. 02). He has also demonstrated that TRH protected against glut toxicity in fetal cortical cultures (Endo. Soc. Abs. 04). To extend these studies he used 14-day cultured rat fetal hippocampal neurons (Day E17) to determine if TRH is protective against toxicity induced by specific ionotropic and metabotropic glut agonists. Neuronal viability and integrity were assessed by trypan blue exclusion and LDH release after 18 hrs following 30 min exposure to glut agonists. Ten µM dihydroxyphenylglycine (DHPG, a Group 1 receptor agonist) + 30 µM N-methyl-D-aspartate (NMDA)-induced toxicity (42% vs contr. P<0.05); whereas, concurrent and continued treatment with 10 uM but not 1uM 3Me-HTRH resulted in less neuronal death and damage (86% vs contr P<0.05; 53% vs contr. P>0.05) respectively. DHPG treatment alone (10 µM) for 30 min. was non-toxic by both criteria (90% vs contr. P<0.05). The data suggest that TRH may be a selective modulator of glut-induced toxicity.

L'exocytose régulée, est un processus qui permet la communication entre les cellules à travers la sécrétion des hormones et des neurotransmetteurs. Dans les neurones et les cellules neuroendocrines, l'exocytose est strictement contrôlée par des signaux extracellulaires tels que le potentiel trans-membranaire et la fixation des ligands sur des récepteurs. Des progrès substantiels ont été effectués afin de comprendre le mécanisme moléculaire de l'exocytose. Les composants majeurs de la machinerie de sécrétion ont été dévoilés. Maintenant, la question qui émerge concerne le rôle de la plateforme de protéines qui semble avoir une action coordonnée entre chaque protéine. Dans le cas de la famille des annexines, qui est bien connue pour son action dans l'exocytose, leurs modes d'interactions séquentielles ou concertées avec d'autres protéines ainsi que leurs effets régulateurs sur l'exocytose ne sont pas encore bien établis. Des résultats précédents indiquent que l'Annexine A6 (AnxA6) affecte l'homéostasie du calcium et la sécrétion de la dopamine à partir des cellules PC12, utilisées comme un modèle cellulaire de neurosécrétion (Podszywalow Bartnicka et al., 2010). Afin de déterminer l'effet inhibiteur de l'AnxA6 sur l'exocytose de la dopamine, nous cherchons des partenaires moléculaires de l'AnxA6 dans les cellules PC12. Nous faisons l'hypothèse que l'AnxA6 interagit avec la PLD1, une enzyme active dans l'étape de la fusion des vésicules avec la membrane plasmique. En utilisant la microscopie confocale et la microscopie à onde évanescente, nous avons trouvé que l'isoforme 1 de l'AnxA6 et la PLD1 sont tous les deux recrutés sur la surface des vésicules au cours de la stimulation des cellules PC12. AnxA6 inhibait l'activité de la PLD comme indiqué par notre méthode d'analyse enzymatique au moyen de la spectroscopie infrarouge. En conclusion, nous proposons que l'AnxA6 n'est pas seulement impliquée dans la réorganisation des membranes par ses capacités à se lier avec des phospholipides négativement chargés et avec le cholestérol, mais elle influence également l'activité de la PLD1, changeant la composition lipidique des membranes / The regulated exocytosis is a key process allowing cell-cell communication through the release of hormone and neurotransmitters. In neurons and neuroendocrine cells, it is strictly controlled by extracellular signal such as transmembrane potential and ligand bindings to receptors. Substantial progress has been made to understand the molecular mechanism of exocytosis. Major components of secretory machinery have been brought to light. Now the emergent question concerns the role of scaffolding proteins that are thought to coordinate the action of each other. In the case of annexin family well known to be involved in exocytosis, their modes of –sequential or concerted- interactions with other proteins, and their regulatory effects on exocytosis are not very well established. Previous findings indicated that Annexin A6 (AnxA6) affected calcium homeostasis and dopamine secretion from PC12 cells, used as cellular model of neurosecretion (Podszywalow-Bartnicka et al., 2010). To determine the inhibitory effect of AnxA6 on exocytosis of dopamine, we were looking for molecular partners of AnxA6 in PC12 cells. We hypothesized that AnxA6 interacts with phospholipase D1 (PLD1), an enzyme involved in the fusion step. By using confocal microscopy and total internal reflection fluorescence microscopy, we found that isoform 1 of AnxA6 and Phospholipase D1 are both recruited on the surface of vesicles upon stimulation of PC12 cells. AnxA6 inhibited phospholipase D activity as revealed by our enzymatic assay based on infrared spectroscopy. To conclude, we propose that AnxA6 is not only implicated in membrane organization by its capacity to bind to negative charged phospholipids and to cholesterol, but AnxA6 is also affecting PLD1 activity, changing membrane lipids composition