Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 199-250.

A proteína miostatina, é um regulador negativo do crescimento muscular e a modulação de sua expressão pode consistir em tratamento para distrofias musculares. Nós estudamos expressão endógena da miostatina no músculos gastrocnêmio e diafragma de 4 modelos murinos de degeneração muscular: os camundongos Dmdmdx, SJL/J, Largemyd e Lama2dy-2J/J. Observamos que a miostatina é menos expressa no músculo gastrocnêmio do que diafragma normal, refletindo um músculo mais sujeito a lesão. Nas quatro linhagens distróficas a miostatina é menos expressa do que em camundongos normais, tanto no músculo gastrocnêmio como diafragma, sem diferença entre os dois. A analise comparativa da degeneração e regeneração muscular mostrou maior correlação da inibição da miostatina com o padrão de degeneração. Nossos resultados sugerem que o processo de degeneração, quando iniciado, e independentemente de seu grau, causa molecular primária, ou músculo afetado, parece atuar de forma similar na inibição da expressão da miostatina, possivelmente como estimulo a regeneração do dano. / Myostatin is a negative regulator of muscle growth, and its inhibition has been considered a therapeutic strategy for muscular dystrophies. We evaluated the endogenous expression of myostatin in the gastrocnemius and diaphragm muscles from 4 mouse dystrophic models including Dmdmdx, SJL/J>, Largemyd and Lama2dy2J/J. In normal mice, we observed that myostatin is less expressed in the gastrocnemius than in the diaphragm, reflecting a muscle most prone to lesions. In the 4 dystrophic models, myostatin expression was reduced, in both gastrocnemius and diaphragm muscles. The comparative analysis of the histopathology of the muscles with the expression of myostatin showed a stronger correlation with the pattern of degeneration then regeneration. Our results suggest that, when started, the process of degeneration of the muscle, independently of the primary molecular defect, or degree, seems to act in a similar pathway leading to the inhibition of the expression of myostatin in the affected muscles, possibly as a stimulus to regeneration of damage.

Infiltration of myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis in many types of cancer. The polypeptide chemokine PK2 (Bv8, PROK2) has been shown to regulate myeloid cell mobilization from the bone marrow, leading to activation of the angiogenic process, as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 were shown to display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. In this study we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the context of glioblastoma and pancreatic cancer xenograft tumor models. For the highly vascularized glioblastoma, PKRA7 was associated with decreased blood vessel density and increased necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 appears to be mediated by the blockage of myeloid cell migration/infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of certain pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both types of tumor. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by two distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy. / Dissertation

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. In this study, corticosterone (CORT) was orally administrated to rats for 21 days to mimic stress status. In situ hybridization measurements showed that CORT ingestion significantly increased mRNA levels of norepinephrine transporter (NET) and dopamine β-hydroxylase (DBH) in the LC region. Immunofluorescence staining and western blotting revealed that CORT treatment also increased protein levels of NET and DBH in the LC, as well as NET protein levels in the hippocampus, the frontal cortex and the amygdala. However, CORT-induced increase in DBH protein levels only appeared in the hippocampus and the amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by simultaneous treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone (s.c. for 21 days). Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. In addition, behavioral tasks showed that CORT ingestion facilitated escape in avoidance trials using an elevated T-maze, but interestingly, there was no significant effect on the escape trial. Corticosteroid receptor antagonists failed to counteract this response in CORT-treated rats. In the open-field task, CORT treatment resulted in less activity in a defined central zone compared to controls and corticosteroid receptor antagonist treatment alleviated this increase. In conclusion, this study demonstrates that chronic exposure to CORT results in a phenotype that mimics stress-induced alteration of noradrenergic phenotypes, but the effects on behavior are task dependent. As the sucrose consumption test strongly suggests CORT ingestion-induced depression-like behavior, further elucidation of underlying mechanisms may improve our understanding of the correlation between stress and the development of depression.

Un nouveau facteur de transcription, appartenant à la famille des protéines à domaine bHLH, a récemment été isolé dans notre laboratoire. Initialement appelé « clone bc8 » puis HRT1, ce facteur présentait des similitudes avec les protéines Hairy and Enhancer of split qui interviennent notamment dans le phénomène d’inhibition latérale lors de la formation du tissu neural. Des études d’hybridation in situ réalisées chez l'embryon de xénope ont suggéré un rôle important de XHRT1, la protéine HRT1 de xénope, dans le développement neural. Nous avons recherché les partenaires protéiques de XHRT1 par la technique du double-hybride afin de mieux comprendre son mécanisme d’action moléculaire dans la neurogenèse.<p>Tout d’abord nous avons construit les outils appropriés pour l’élaboration du travail, à savoir, les clones de levures exprimant les appâts spécifiques des domaines de la protéine étudiée et la création d’une banque d’ADNc du xénope au stade de la neurulation. Ensuite, trois criblages ont été réalisés. Dans le premier cas, nous avons recherché les partenaires des domaines bHLH et Orange (bHLH-O). Le domaine bHLH est en effet responsable de la dimérisation de ce type de protéine. Le domaine Orange qui suit le domaine bHLH, pourrait participer dans le choix du partenaire d’hétérodimérisation. Nous avons isolé deux facteurs de type bHLH-Orange apparentés à HRT1, XHairy1 et XHairy2b et confirmé leur interaction avec XHRT1. Les domaines impliqués dans ces interactions sont les bHLH-O pour les trois facteurs. Ce même criblage nous a permis d’isoler un nouvel ADNc qui code une protéine sans domaine apparent connu actuellement. Nous avons montré que cette protéine reconnaissait spécifiquement le domaine Orange de HRT1 mais pas celui des autres facteurs de type bHLH-O. Elle a été baptisée BOIP pour Bc8 Orange Interacting Protein. Le rôle physiologique de cette interaction n’a pu être démontré. Nous avons établi que la protéine BOIP pouvait aussi s’homodimériser. Nous avons aussi déterminé son profil d’expression chez le xénope et la souris. Son transcrit est hautement présent dans les testicules adultes. La protéine pourrait donc jouer un rôle important dans la spermatogenèse. Les deux autres criblages, utilisant les domaines situés dans la partie C-terminale de XHRT1, ont apporté des nouveaux partenaires potentiels, mais ces interactions n’ont pu être confirmées dans un système indépendant. <p>Enfin, en étudiant plus en détail les interactions entre XHRT1 et XHairy1 ou XHairy2b, nous avons mis à jour une possible fonction de spécificité dans le choix du partenaire dans la région C-terminale de HRT1. La formation de ces dimères pourrait jouer un rôle dans la formation du tube neural mais également dans d’autres différenciations tissulaires.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Neurogenin 3 and its downstream target NeuroD are basic helix-loop-helix transcription factors which promote endocrine differentiation in the gastrointestinal tract. However, mice lacking Ngn3 still produce several hormones in the stomach. Lineage tracing mouse models demonstrated that a majority of hormone cells in the corpus region of the stomach did not express Ngn3 or NeuroD during differentiation. Serotonin and histamine cells were entirely NeuroD-independently derived, and serotonin cells were additionally entirely Ngn3-independently derived. In this study, we isolated serotonin and histamine cells from the gastric corpus of transgenic mice expressing the fluorescent marker CFP. Serotonin cells expressed multiple mast cell markers by RT-PCR, and were found to be nearly absent in a mast cell-deficient mouse model. Labeled bone marrow transplant mice showed all serotonin cells derived from bone marrow. Histamine-expressing ECL cells, while lacking NeuroD, did not appear to express granulocyte or mast cell markers by analytical flow cytometry and RT-PCR, and resemble other enteroendocrine cell populations. Mouse gastric corpus serotonin cells, but not antral serotonin cells, are bone marrow-derived mast cells.

Indiana University-Purdue University Indianapolis (IUPUI) / Posttraumatic epilepsy, the development of temporal lobe epilepsy (TLE) following traumatic brain injury, accounts for 20% of symptomatic epilepsy. Reorganization of mossy fibers within the hippocampus is a common pathological finding of TLE. Normal mossy fibers project into the CA3 region of the hippocampus where they form synapses with pyramidal cells. During TLE, mossy fibers are observed to innervate the inner molecular layer where they synapse onto the dendrites of other dentate granule cells, leading to the formation of recurrent excitatory circuits. To date, the molecular mechanisms contributing to mossy fiber sprouting are relatively unknown. Recent focus has centered on the involvement of tropomycin-related kinase receptor B (TrkB), which culminates in glycogen synthase kinase 3β (GSK3β) inactivation. As the neurite outgrowth promoting collapsin response mediator protein 2 (CRMP2) is rendered inactive by GSK3β phosphorylation, events leading to inactivation of GSK3β should therefore increase CRMP2 activity. To determine the involvement of CRMP2 in mossy fiber sprouting, I developed a novel tool ((S)-LCM) for selectively targeting the ability of CRMP2 to enhance tubulin polymerization. Using (S)-LCM, it was demonstrated that increased neurite outgrowth following GSK3β inactivation is CRMP2 dependent. Importantly, TBI led to a decrease in GSK3β-phosphorylated CRMP2 within 24 hours which was secondary to the inactivation of GSK3β. The loss of GSK3β-phosphorylated CRMP2 was maintained even at 4 weeks post-injury, despite the transience of GSK3β-inactivation. Based on previous work, it was hypothesized that activity-dependent mechanisms may be responsible for the sustained loss of CRMP2 phosphorylation. Activity-dependent regulation of GSK3β-phosphorylated CRMP2 levels was observed that was attributed to a loss of priming by cyclin dependent kinase 5 (CDK5), which is required for subsequent phosphorylation by GSK3β. It was confirmed that the loss of GSK3β-phosphorylated CRMP2 at 4 weeks post-injury was likely due to decreased phosphorylation by CDK5. As TBI resulted in a sustained increase in CRMP2 activity, I attempted to prevent mossy fiber sprouting by targeting CRMP2 in vivo following TBI. While (S)-LCM treatment dramatically reduced mossy fiber sprouting following TBI, it did not differ significantly from vehicle-treated animals. Therefore, the necessity of CRMP2 in mossy fiber sprouting following TBI remains unknown.

No / The ability of neurons to modulate synaptic strength underpins synaptic plasticity, learning and memory, and adaptation to sensory experience. Despite the importance of synaptic adaptation in directing, reinforcing, and revising the behavioral response to environmental influences, the cellular and molecular mechanisms underlying synaptic adaptation are far from clear. Brain-derived neurotrophic factor (BDNF) is a prime initiator of structural and functional synaptic adaptation. However, the signaling cascade activated by BDNF to initiate these adaptive changes has not been elucidated. We have previously shown that BDNF activates mitogen- and stress-activated kinase 1 (MSK1), which regulates gene transcription via the phosphorylation of both CREB and histone H3. Using mice with a kinase-dead knock-in mutation of MSK1, we now show that MSK1 is necessary for the upregulation of synaptic strength in response to environmental enrichment in vivo. Furthermore, neurons from MSK1 kinase-dead mice failed to show scaling of synaptic transmission in response to activity deprivation in vitro, a deficit that could be rescued by reintroduction of wild-type MSK1. We also show that MSK1 forms part of a BDNF- and MAPK-dependent signaling cascade required for homeostatic synaptic scaling, which likely resides in the ability of MSK1 to regulate cell surface GluA1 expression via the induction of Arc/Arg3.1. These results demonstrate that MSK1 is an integral part of a signaling pathway that underlies the adaptive response to synaptic and environmental experience. MSK1 may thus act as a key homeostat in the activity- and experience-dependent regulation of synaptic strength.

No / AIMS/HYPOTHESIS: We previously demonstrated that animals fed a high-fat (HF) diet for 10 weeks developed insulin resistance and behavioural inflexibility. We hypothesised that intervention with metformin would diminish the HF-feeding-evoked cognitive deficit by improving insulin sensitivity. METHODS: Rats were trained in an operant-based matching and non-matching to position task (MTP/NMTP). Animals received an HF (45% of kJ as lard; n = 24), standard chow (SC; n = 16), HF + metformin (144 mg/kg in diet; n = 20) or SC + metformin (144 mg/kg in diet; n = 16) diet for 10 weeks before retesting. Body weight and plasma glucose, insulin and leptin were measured. Protein lysates from various brain areas were analysed for alterations in intracellular signalling or production of synaptic proteins. RESULTS: HF-fed animals developed insulin resistance and an impairment in switching task contingency from matching to non-matching paradigm. Metformin attenuated the insulin resistance and weight gain associated with HF feeding, but had no effect on performance in either MTP or NMTP tasks. No major alteration in proteins associated with insulin signalling or synaptic function was detected in response to HF diet in the hypothalamus, hippocampus, striatum or cortex. CONCLUSIONS/INTERPRETATION: Metformin prevented the metabolic but not cognitive alterations associated with HF feeding. The HF diet protocol did not change basal insulin signalling in the brain, suggesting that the brain did not develop insulin resistance. These findings indicate that HF diet has deleterious effects on neuronal function over and above those related to insulin resistance and suggest that weight loss may not be sufficient to reverse some damaging effects of poor diet.