Charles E. Schmidt College of Medicine

Experimentally naive rats show variance in their locomotor reactivity to novelty, some displaying higher (HR) while others displaying lower (LR) reactivity, associated with vulnerability to stress. LRHR phenotype is proposed as an antecedent to the development of stress hyper responsiveness. Results presented here show emergence of antidepressive-like behavior following peripubertal-juvenile exposure to chronic variable physical (CVP) and chronic variable social stress (CVS) in HR rats, and depressive-like behavior following CVP in the LRs. The antidepressive-like behavior in HR rats was accompanied by increased levels of acetylated Histone3 (acH3) and acetylated Histone4 (acH4) at the hippocampal brain-derived neurotrophic factor (BDNF) P2 and P4 promoters respectively. This effect may mediate increased mossy fibre (MF) terminal field size, particularly the suprapyramidal mossy fibre projection volume (SP-MF), in the HR animals following both stress regimens. These findings show that chronic variable stress during adolescence induces individual differences in molecular, neuromorphological and behavioral parameters between LRs and HRs, which provides further evidence that individual differences in stress responsiveness is an important factor in resistance or vulnerability to stress-induced depression and/or anxiety.

Sjèogren's Syndrome (SS) is a chronic, inflammatory autoimmune disease affecting mostly the exocrine cells of lacrimal and salivary glands, leading to diminished secretory function and resulting in keratoconjunctivitis sicca (dry eye disease) and/or stomatitis sicca (dry mouth disease). Despite several decades of studies focusing on autoimmune diseases and dry eye diseases, the exact etiology and mechanisms of SS remain unknown. Besides the fact that SS is often unreported, unrecognized and untreated, today's therapies rely exclusively on treating the symptoms after disease progression; there exists neither prevention therapy nor cure for SS. In addition, SS has been diagnosed predominantly in post-menopausal women with the female to male ratio reaching 9:1, suggesting a role of ovarian sex hormones in the pathogenesis of SS. However, not all postmenopausal women develop SS, indicating the contribution of other factors such as a genetic background to the onset of SS. In the present study, ovariectomized (OVX) NOD.B10.H2b mice provide a model of menopause with a genetic predisposition to SS, as compared to non-predisposed C57BL/10 mice. Both strands of mice were either sham operated, OVX, OVX and treated with 17(Sb (Bestradiol (E2), or OVX and treated with dihydrotestosterone (DHT). Lacrimal glands were collected 3, 7, 21, and 30 days after surgery and processed for RNA analysis by rt-qPCR and protein assays by ELISA to evaluate cytokine expression and concentrations of IL- 1\U+fffd\, TNF-a, IFN-(Sd(B, IL-10, and IL-4 on a timeline. Overall, our results showed a significant increase in IL-1\U+fffd\ TNF-a, IL-10, and IL-4 expression and levels in the lacrimal glands of OVX NOD.B10.H2b mice as compared to sham operated animals, and treatment with E2 or DHT at time of OVX prevented the increase in cytokine expression and levels.

Restrictive cardiomyopathy (RCM) is a cardiac muscle disorder characterized by increased ventricular stiffness and diastolic dysfunction. Patients with RCM often present severe cardiac problems which usually lead to heart failure and sudden death. No effective treatment is available for RCM which makes the finding of novel efficient therapies an urgent necessity. Great progress in molecular biology techniques and advances in transgenic animal development provide great opportunities for the study of RCM and other cardiovascular diseases encountered in clinical patients.... Our laboratory is among the first to generate transgenic mouse models of RCM based on cardiac troponin I (cTnI) missense mutations. In this study, transgenic mice that suffer from RCM have been generated to understand the factors behind the diastolic dysfunction associated with that myocardial disease.... The information obtained from this study allows a better understanding of the role of troponin in RCM and the factors behind the physiopathology of the disease. It will also offer a therapeutic strategy taking into account the physiological characteristic of RCM.

The function and role of PAK6, serine/threonone kinase, in cancer progressionhas not yet been clearly identified. Several studies reveal that PAK6 may participate in key changes contributing to cancer progression such as cell survival, cell motility, and invasiveness. Basedon the membrane localization of PAK6 in prostate and breast cancer cells,we speculated that PAK6 plays a rolein cancer progression cells by localizing on the membrane and modifying proteins linked to motility and proliferation. We isolated the raft domain of breast cancer cells expressing either wild type (WT), constitutively active (SN), or kinase dead PAK6 (KM) and found that PAK6 is a membrane associated kinase which translocates from the plasma membrane to the cytosol when activated. The downstream effects of PAK6 are unknown ; however, results from cell proliferation assays suggest a growth regulatory mechanism.

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.

Sjèogren's syndrome (S) is a chronic autoimmune disease characterized by ocular and oral dryness and primarily affects post menopausal women. In the present study we investigated the time course of lymphocytic infiltration, apoptosis, caspase-3 activity and different cytokines levels in the lacrimal glands of both genetically predisposed and control mice to elucidate immunopathological mechanism leading to dry eye. The results of our experiments showed that ovariectomy accelerated pathological findings of SS by increasing lympocytic infiltration, cytokine production, lacrimal gland cell death and cleaved caspase-3 activity, and these effects were more pronounced and persistent in the genetically predisposed mouse model of SS. In addition, we observed that lymphocytic infiltration occurred earlier compared to apoptosis which may perpetuate immune mediated destruction of lacrimal epithelial cells. Furthermore, treatment with physioloigical doses of 17-B Estradiol (E2) or DIhydrotestosterone (DHT) prevented all these pathological events observed after ovariectomy.

Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD.

The turtle is a unique model of anoxic survival. The turtle's brain can tolerate total oxygen deprivation for hours to days as well as prevent high levels of mitochondrial-derived free radicals upon re-oxygenation. Because of its ability to prevent elevated free radical generation, the turtle has also become recognized as a model of exceptional longevity. We are employing the turtle model for an investigation into the regulation of a key antioxidant enzyme system - methionine sulfoxide reductases (Msrs), primarily MsrA and MsrB. The Msr system is capable of reversing oxidation of methionines in proteins and Msr subtypes have been implicated in protecting tissues against oxidative stress, as well as, enhancing the longevity of organisms from yeast to mammals. Preliminary data, unpublished results, indicate that MsrA protein and transcripts are elevated by anoxia. A recent study on Caenorhabditis elegans demonstrated that FOXO is involved in activation of the MsrA promoter. Using the turtle MsrA promoter sequence we worked to determine which regions in the promoter are necessary for activation by anoxia. The results of the present study were 1) to prepare a TAT-FOXO3a fusion protein which could penetrate animal cells and 2) to construct a FOXO3a expression vector for transcription studies on MsrA expression.

We employed three genotypes of GAD 65, wildtype (GAD 65 +/+), heterozygous (GAD 65 +/-) and knockout (GAD 65 -/-) to investigate the role of GAD 65 in survival of pancreatic islets. We analyzed the mRNA expression of pro-survival proteins including Bcl2 and Bax in pancreas of wildtype, heterozygous and knockout using Reverse Transcriptase Polymerase Chain Reaction (RTPCR). The level of expression of Bcl2 mRNA was down regulated in knockout mice pancreas and Bax to Bcl2 ratio was found higher in knockout mice pancreas suggesting higher cell death rate. However, further studies are required to recognize and understand the specific connections between apoptotic pathways and GAD 65 in pancreatic islets.

Molecular chaperones guide peptide fold conformation throughout the lifetime of the peptide. One network of chaperone proteins involved in this activity, Heat shock protein 70s (Hsp70s), are well characterized at restoring peptide fold, utilizing J-domain containing protein chaperone cofactors to activate Hsp70 activity. DnaJ (Hsp40) homolog, subfamily C, member 25 (DNAJC25) is a class III transmembrane J-domain containing protein that to date is underrepresented in the literature. Recently, Hejtmancik et al. 2012. (unpublished data) have revealed that missense mutation to DNACJ25 at Pro90Leu (P90L) is strongly correlated with inherited Closed-Angle Glaucoma. Inherited mutations are well characterized for Open-Angle Glaucoma, however, prior to this finding, were unknown for Closed-Angle Glaucoma. In this report, analysis of the in vitro chaperone activity of DNAJC25 w+ and P90L is assessed utilizing an Hsp70 mediated Glucose-6-Phosphate Dehydrogenase refolding system, SWISS-MODEL predictions are performed for the J-domain structure of DNAJC25 w+ and P90L with consequent analysis of DNAJC25 Pro90 conservation relative to other type I, II, and III J-domain containing proteins. DNAJC25 P90L demonstrated decreased chaperone activity in vitro compared to w+ DNAJC25.