Skeletal muscle protein synthesis is regulated by the mammalian target of rapamycin (mTOR) signaling pathway. The first objective was to optimize the methodological procedures for assessing mTOR signaling in horses. The response of mTOR signaling (P-Akt Ser473, P-S6K1 Thr389, P-rpS6 Ser235/26 & 240/244, and P-4EBP1 Thr37/46 by Western blotting techniques) to meal consumption was determined at three gluteal muscle biopsy depths (6, 8, and 10 cm), and the repeatability of the contralateral side at 8 cm during 5 days of repeated biopsies. There was no effect (P > 0.05) of sampling side or biopsy depth on mTOR signaling in mature horses. During repeated biopsies there was an increase (P < 0.05) in downstream (P-S6K1 Thr389, P-rpS6 Ser235/236 & 240/244 and P-4EBP1 Thr389) mTOR signaling in response to feeding. The second objective was to characterize alterations in mTOR signaling throughout the equid lifespan. Adolescent horses (yearlings and two year olds) studied in the postprandial had a lowered (P < 0.05) activation of downstream mTOR signaling with aging. There was a lower (P < 0.05) abundance of P-S6K1 Thr389 in aged horses (23.5 years old) than in mature horses (11 years old) during the post-absorptive state. The final objective was to assess mTOR signaling during acute and chronic inflammation. Acute inflammation occurred during 5 days of repeated biopsies, and chronic inflammation is characteristic of the aged. During acute inflammation, characterized by increased muscle mRNA expression of inflammatory cytokines, there was an increase (P < 0.05) in downstream mTOR signaling. Chronic inflammation resulted in a decrease (P < 0.05) in the abundance of P-S6K1 Thr389. Phenylbutazone was administered to reduce (P < 0.05) acute and chronic inflammation in muscle. Phenylbutazone administration during acute inflammation reduced (P < 0.05) the activation of downstream mTOR signaling and trended to increase (P = 0.09) P-S6K1 Thr389 abundance during chronic inflammation. Whole-body protein synthesis determined using isotope infusion techniques increased (P < 0.05) when chronic inflammation was reduced due to phenylbutazone administration. This research provides new standards for muscle biopsy collection when examining mTOR signaling, and insight into management and feeding practices for adolescent and aging horses.

Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

Lithium is given to millions of bipolar disorder or post-traumatic disorder patients. The recent studies also support a role for lithium in treating neurodegenerative disease such as Parkinson's disease and stroke. Lithium treatment leads to lithium nephropathy, which includes lithium-induced nephrogenic diabetic insipidus (NDI), lithium-induced renal cell proliferation leading to the formation of microcysts in the kidney, and lithium-induced renal fibrosis. However, there is still a gap in understanding the mechanisms and signaling pathways involved in regulating lithium-induced nephropathy. mTOR pathway activation and primary cilia are known to be associated with the abnormal renal cell proliferation and the formation of renal cysts in polycystic kidney disease, a renal disease model similar to our lithium model. The activation of hedgehog pathway is associated with the renal fibrosis observed in the unilateral ureteral obstruction and unilateral ischemia reperfusion injury models of chronic renal injury. Thus, I hypothesize that mTOR signaling pathway, primary cilia and hedgehog pathway may all contribute to lithium-induced nephropathy. To address the hypothesis that the mTOR signaling pathway may be responsible for lithium-induced renal collecting duct proliferation, mTOR pathway activation was assessed in lithium-treated mice and lithium-treated mouse inner medullary collecting duct (mIMCD3) cells. Lithium activated mTOR signaling pathway in renal collecting duct cells both in vivo and in vitro. Rapamycin, an inhibitor of mTOR, blocked lithium-induced renal cell proliferation in renal cortex and medulla in vivo and in renal collecting duct cells in vitro, supporting the hypothesis. However, rapamycin did not improve lithium-induced reduction of urine osmolality, suggesting mTOR signaling pathway may not contribute to lithium-induced NDI. To address the hypothesis that primary cilia may be necessary for lithium-induced mTOR activation and renal cell proliferation, primary cilia deficient cells were used to assess mTOR pathway activation and cell proliferation in response to lithium treatment. The absence of primary cilia abolished lithium-induced activation of mTOR pathway and cell proliferation, which supports the hypothesis. To address the hypothesis that lithium elongates primary cilia length, which is mediated by mTOR signaling pathway, primary cilia length alternation was assessed in the kidney and in mIMCD3 cells in response to lithium treatment. Lithium increased primary cilia length in renal collecting duct cells of cortex, outer medulla, and inner medulla kidney regions in vivo and in mIMCD3 cells in vitro. Rapamycin reversed lithium-induced elongation of primary cilia in renal cortical and outer medullary collecting duct cells in vivo, and blocked the increase of primary cilia length in mIMCD3 cells in vitro, which support the hypothesis. To address the hypothesis that lithium activates the hedgehog pathway in a Smoothened (smo, a key regulator of the hedgehog pathway)-dependent manner in renal collecting duct cells, mIMCD3 cells were treated with lithium or lithium/Smo inhibitor or lithium/Smo activator. Hedgehog signaling pathway is activated by lithium in mIMCD3 cells, which is partially Smo-dependent. However, the role of hedgehog signaling pathway in regulating lithium-induced fibrosis was not assessed in the study. Future studies are required to determine the role of the hedgehog pathway in the lithium model.

Os transtornos do espectro autista (TEA) são distúrbios neuropsiquiátricos bastante comuns, graves, e que propiciam grande impacto social e financeiro. A identificação de vias moleculares e processos celulares alterados que são compartilhados pelos pacientes, mesmo que estes apresentem causas etiológicas distintas, pode contribuir de forma significativa para o entendimento de sua patofisiologia desses transtornos. Ainda, a identificação destas vias pode propiciar o desenvolvimento de abordagens terapêuticas mais eficientes, uma vez que o uso de medicamentos nos TEA ainda é inadequado, envolvendo baixa melhora funcional e diversos efeitos colaterais, como o ganho excessivo de peso e anormalidades metabólicas associadas. Neste trabalho, selecionamos como uma primeira abordagem o estudo da via de sinalização PI3K-mTOR em pacientes com TEA não-sindrômico, via esta envolvida com diversos aspectos do desenvolvimento e funcionamento neuronal, assim como com a patofisiologia de síndromes monogênicas que apresentam alta prevalência de TEA em seu quadro clínico. Foram utilizadas como modelo experimental in vitro células-tronco mesenquimais provenientes de polpa de dente decíduo (SHEDs) de pacientes e indivíduos controles. Os resultados aqui obtidos sugerem a importância da desregulação da via PI3K/mTOR na patofisiologia de uma parcela importante dos casos de TEA não-sindrômico. Ainda, observamos que as células dos pacientes com alterações nessa via de sinalização apresentam maior capacidade proliferativa, e que a modulação deste fenótipo alterado por meio do uso concomitante de inibidores de PI3K e mTOR nas células de um destes pacientes sugere esta via como um alvo promissor para o desenvolvimento de novas abordagens terapêuticas para estes pacientes. Em seguida, na tentativa de desvendar os mecanismos subjacentes aos efeitos metabólicos adversos associados com o uso de antipsicóticos prescritos para o tratamento de pacientes com TEA, investigamos os efeitos destes psicofármacos sobre a biologia do tecido adiposo humano. Foram utilizadas como modelos in vitro células-tronco (ADSCs) e adipócitos maduros derivados de tecido adiposo humano de indivíduos controles. Os resultados obtidos sugerem que a ação direta dos antipsicóticos com alta propensão ao ganho de peso (como a olanzapina e a clozapina) sobre a proliferação, diferenciação, e o metabolismo do tecido adiposo humano parece não constituir um mecanismo importante associado ao ganho de peso apresentado pelos pacientes, e que a ação desses medicamentos sobre os sistemas centrais que regulam o peso e o metabolismo deve ser o mecanismo principal levando aos efeitos metabólicos adversos. Juntos, os resultados gerados neste trabalho podem, de certa forma, contribuir para da farmacoterapia dos TEA / Autism spectrum disorders (ASD) are common neuropsypchiatric disorders, which has serious social and economic impacts. Identification of common molecular and cellular processes altered in patients, despite the underlying genetic heterogeneity, can contribute significantly to our understanding of the disease pathophysiology and can help to develop more effective treatments, since available pharmacotherapy for ASD is inefficient and frequently associated with adverse side effects, such as weight gain and metabolic disturbances. Here, we used patient-derived Stem cells from Human Exfoliated Deciduous teeth (SHEDs) as an intro model system to investigate whether non-syndromic ASD patients show altered regulation of PI3K/mTOR signaling pathway, which is involved in multiple aspects of neuronal development and physiology, and in the pathogenesis of monogenic syndromes that share features with ASD. Our results suggest that dysregulation of PI3K/mTOR-linked networks play an important role in the pathogenesis of a subgroup of non-syndromic ASD. In addition, we found enhanced proliferative capacity in cells with altered PI3K/mTOR activity, which was rescued in one of these patients through combined pharmacological inhibition of both PI3K and mTOR kinase activity, suggesting that PI3K-mTOR signaling is a promising target for the development of new therapeutic approaches for these individuals. Next, in an attempt to better understand the mechanisms underlying the metabolic side effects of the antipsychotics prescribed for ASD treatment, we investigated the effects of some of these drugs on the biology of human adipose tissue using as in vitro model systems human adipose-derived stem cells (ADSCs) and mature adipocytes. Our results suggest that a direct and potent effect of antipsychotics with high weight gain liability (such as clozapine and olanzapine) on cell proliferation, differentiation, and metabolism of human adipose tissue is not an important mechanism by which these drugs induce metabolic disturbances. Consequently, our results suggest that these side effects may mainly reflect the action of these drugs on central pathways involved in weight control and metabolism. Together, our results can, to some extent, contribute to improving pharmacotherapy of ASD

The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations.

Insulin plays a vital role in whole-body metabolism and provides a major anabolic stimulus for cellular signaling pathways, including those involved in the metabolism of glucose and protein. Consequently, insulin dysregulation (ID) is known to alter molecular signal transduction in insulin-sensitive tissues such as skeletal muscle, thereby disrupting glucose metabolism and compromising protein synthetic capacity. Our first objective was to induce ID in healthy horses by administering dexamethasone (DEX), a potent glucocorticoid, for 21 days. We evaluated the effects on insulin-stimulated muscle protein signaling components involved in the mammalian target of rapamycin (mTOR) pathway. DEX-induced ID reduced insulin-stimulated activation of downstream (rpS6, 4EBP-1) mTOR signaling and increased atrogin-1 abundance, a marker for protein breakdown (P < 0.05). Additionally, 21 days of DEX elevated plasma amino acids levels in insulin-stimulated conditions, indicative of reduced uptake or increase release into circulation (P < 0.05). The second objective was to evaluate the short-term effects of DEX treatment in healthy horses. Plasma insulin, glucose and amino acid dynamics and activation of mTOR signaling pathways following an oral sugar test (OST) or intake of a high protein meal were evaluated before and after 7 days of DEX treatment, and after 7 days of no treatment. Seven days of DEX treatment increased basal levels of glucose, insulin and several amino acids (P < 0.05). Additionally horses treated with DEX had an exacerbated insulin response to the OST and consumption of the high protein meal in comparison to control horses (P < 0.05). The majority of blood metabolites returned to basal levels after 7 days of recovery from DEX treatment, indicating these effects were transient. Short-term DEX treatment decreased overall activation of mTOR and FoxO3 but increased total FoxO3 and IRS-1 abundance (P < 0.05). Postprandial activation of rpS6 was greater in horses treated with DEX for 7 days but was lower in those horses after 7 days of recovery from treatment (P < 0.05). Postprandial activation of ULK and AMPK tended to be greater in DEX treated horses (P < 0.1). Akt phosphorylation and mysotatin abundance were lower after the OST in DEX treated horses (P < 0.05). The final objective was to evaluate whether similar changes in postprandial metabolic responses would be seen in horses with naturally occurring ID. Plasma insulin, glucose and amino acid responses following ingestion of a high protein meal were determined in mature horses with equine metabolic syndrome (EMS). Horses with EMS had higher basal plasma insulin concentrations but lower levels of aspartate, glutamate, asparagine and plasma urea nitrogen in comparison to healthy controls (P < 0.05). Consumption of a high protein meal resulted in a 9-fold greater insulin response and higher postprandial levels of various amino acids (P < 0.05). Together this research indicates that ID affects whole body protein metabolism by altering cellular signaling pathways in healthy and diseased horses.

Os transtornos do espectro autista (TEA) são distúrbios neuropsiquiátricos bastante comuns, graves, e que propiciam grande impacto social e financeiro. A identificação de vias moleculares e processos celulares alterados que são compartilhados pelos pacientes, mesmo que estes apresentem causas etiológicas distintas, pode contribuir de forma significativa para o entendimento de sua patofisiologia desses transtornos. Ainda, a identificação destas vias pode propiciar o desenvolvimento de abordagens terapêuticas mais eficientes, uma vez que o uso de medicamentos nos TEA ainda é inadequado, envolvendo baixa melhora funcional e diversos efeitos colaterais, como o ganho excessivo de peso e anormalidades metabólicas associadas. Neste trabalho, selecionamos como uma primeira abordagem o estudo da via de sinalização PI3K-mTOR em pacientes com TEA não-sindrômico, via esta envolvida com diversos aspectos do desenvolvimento e funcionamento neuronal, assim como com a patofisiologia de síndromes monogênicas que apresentam alta prevalência de TEA em seu quadro clínico. Foram utilizadas como modelo experimental in vitro células-tronco mesenquimais provenientes de polpa de dente decíduo (SHEDs) de pacientes e indivíduos controles. Os resultados aqui obtidos sugerem a importância da desregulação da via PI3K/mTOR na patofisiologia de uma parcela importante dos casos de TEA não-sindrômico. Ainda, observamos que as células dos pacientes com alterações nessa via de sinalização apresentam maior capacidade proliferativa, e que a modulação deste fenótipo alterado por meio do uso concomitante de inibidores de PI3K e mTOR nas células de um destes pacientes sugere esta via como um alvo promissor para o desenvolvimento de novas abordagens terapêuticas para estes pacientes. Em seguida, na tentativa de desvendar os mecanismos subjacentes aos efeitos metabólicos adversos associados com o uso de antipsicóticos prescritos para o tratamento de pacientes com TEA, investigamos os efeitos destes psicofármacos sobre a biologia do tecido adiposo humano. Foram utilizadas como modelos in vitro células-tronco (ADSCs) e adipócitos maduros derivados de tecido adiposo humano de indivíduos controles. Os resultados obtidos sugerem que a ação direta dos antipsicóticos com alta propensão ao ganho de peso (como a olanzapina e a clozapina) sobre a proliferação, diferenciação, e o metabolismo do tecido adiposo humano parece não constituir um mecanismo importante associado ao ganho de peso apresentado pelos pacientes, e que a ação desses medicamentos sobre os sistemas centrais que regulam o peso e o metabolismo deve ser o mecanismo principal levando aos efeitos metabólicos adversos. Juntos, os resultados gerados neste trabalho podem, de certa forma, contribuir para da farmacoterapia dos TEA / Autism spectrum disorders (ASD) are common neuropsypchiatric disorders, which has serious social and economic impacts. Identification of common molecular and cellular processes altered in patients, despite the underlying genetic heterogeneity, can contribute significantly to our understanding of the disease pathophysiology and can help to develop more effective treatments, since available pharmacotherapy for ASD is inefficient and frequently associated with adverse side effects, such as weight gain and metabolic disturbances. Here, we used patient-derived Stem cells from Human Exfoliated Deciduous teeth (SHEDs) as an intro model system to investigate whether non-syndromic ASD patients show altered regulation of PI3K/mTOR signaling pathway, which is involved in multiple aspects of neuronal development and physiology, and in the pathogenesis of monogenic syndromes that share features with ASD. Our results suggest that dysregulation of PI3K/mTOR-linked networks play an important role in the pathogenesis of a subgroup of non-syndromic ASD. In addition, we found enhanced proliferative capacity in cells with altered PI3K/mTOR activity, which was rescued in one of these patients through combined pharmacological inhibition of both PI3K and mTOR kinase activity, suggesting that PI3K-mTOR signaling is a promising target for the development of new therapeutic approaches for these individuals. Next, in an attempt to better understand the mechanisms underlying the metabolic side effects of the antipsychotics prescribed for ASD treatment, we investigated the effects of some of these drugs on the biology of human adipose tissue using as in vitro model systems human adipose-derived stem cells (ADSCs) and mature adipocytes. Our results suggest that a direct and potent effect of antipsychotics with high weight gain liability (such as clozapine and olanzapine) on cell proliferation, differentiation, and metabolism of human adipose tissue is not an important mechanism by which these drugs induce metabolic disturbances. Consequently, our results suggest that these side effects may mainly reflect the action of these drugs on central pathways involved in weight control and metabolism. Together, our results can, to some extent, contribute to improving pharmacotherapy of ASD

In order to function, proteins must be folded into their native shape. While this can sometimes occur spontaneously, the process can be hindered by thermodynamic barriers, trapped intermediates, and aggregation prone hydrophobic interactions. Molecular chaperones are proteins that help client proteins or substrates overcome these barriers so that they can be folded properly. One such chaperone is the chaperonin CCT, a large MDa protein made up of 16 paralogous subunits that form a double ring structure. CCT encapsulates its substrates in a central cavity, where they are sequestered and folded, using ATP binding and hydrolysis to drive conformational changes in the CCT-substrate complex. CCT mediates the folding of many substrates involved in a variety of cellular process, including the cytoskeletal proteins actin and tubulin, and the G protein subunit Gabg, which signals downstream of GPCRs in a variety of cellular processes. We showed that CCT is responsible for folding the b-propeller containing proteins, mLST8 and Raptor, which are subunits of the mTOR complexes. The mTOR complexes (mTORC1 and mTORC2) are master regulators of cell growth and survival by controlling processes such as protein synthesis, energy metabolism, cell survival pathways and autophagy. CCT folds mLST8 and Raptor and help them assemble into the mTOR complexes. As a result, CCT is required for functional mTOR signaling. Furthermore, we solved a 4.0 Ǻ resolution structure of mLST8 bound to CCT. Surprisingly, mLST8 is found in the center of the folding cavity, in between the rings, despite previous evidence suggesting that substrates bind only in the apical domains. Given its role in folding and assembling the mTOR complexes, G proteins, and many other proteins involved in cell survival pathways, CCT has been implicated in cancer. CCT upregulation often correlates with a worse prognosis, likely because uncontrolled growth requires increased chaperone capacity. The peptide CT20P has been shown to have cytotoxic effects in cancer cells, likely through its binding to CCT. We characterized CT20P, showing that it binds to CCT and inhibits its substrate-folding functions in cells. We specifically showed that a GFP-CT20P fusion protein inhibited the assembly of two important signaling complexes Gbg and mTORC1. These results show that CT20P is a useful inhibitor for the study of CCT function.

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