A distrofia muscular progressiva no cão Golden Retriever (GRMD) é uma miopatia genética homóloga à Distrofia Muscular de Duchenne (DMD) que acomete humanos. A utilização do modelo canino é considerada ideal uma vez que a progressão da doença, as manifestações clínicas e a massa corporal se assemelham às apresentadas por meninos doentes. Sabe-se que a degradação de proteínas musculares e atrofia muscular estão intimamente relacionadas com a atuação do sistema catalítico ubiquitina-proteassoma. Estudos utilizando camundongos com distrofia muscular e músculos de humanos DMD (in vitro) tratados com inibidores de proteassoma (MG-132 e bortezomibe) apresentaram restauração da distrofina e das proteínas associadas à distrofina e melhora do fenótipo histopatológico. Nesta pesquisa, foram avaliados cinco cães GRMD, dois tratados com Bortezomibe e três controles. Coletou-se biopsia muscular antes de iniciar o tratamento e ao término do mesmo, para os testes de histologia, ultraestrutura e imunohistoquímica. Foram realizados semanalmente testes bioquímicos e em cada ciclo de aplicação do bortezomibe foi mensurada a taxa de inibição do proteassoma. Na primeira biopsia todos os cães apresentaram morfologia histopatológica das fibras musculares semelhantes. Ao final do tratamento, os cães tratados apresentaram menor deposição de tecido conjuntivo e infiltração de células inflamatórias que os cães não tratados julgados por meio da histologia, morfometria do colágeno e ultraestrutura. Na ultraestrutura observamos infiltração de macrófagos nas fibras, material degenerado, fibroblastos ativados e deposição e tecido conjuntivo em endomísio e perimísio das fibras. Pela análise de imunohistoquímica, não constatamos presença de distrofina na membrana sarcoplasmática de ambos os grupos avaliados. Entretanto, os cães tratados apresentaram maior expressão das proteínas α e β distroglicano, o que sugere uma melhora no fenótipo histopatológico da doença. Nos cães não tratados observou-se maior expressão de phospho- NFκB e TGF-β1 sugerindo maior ativação de fatores pró-apoptóticos e moléculas inflamatórias, e maior deposição de tecido conjuntivo, respectivamente. Os testes de inibição do proteassoma indicaram maior inibição nas células sanguíneas uma hora após a aplicação do bortezomibe e estes de comportaram de forma dose-dependente. Concluímos que os inibidores de proteassoma podem melhorar a morfologia das fibras musculares dos cães GRMD tratados, diminuindo a deposição de colágeno e infiltração de células inflamatórias, bem como restaurar parte das proteínas associadas à distrofina na membrana sarcoplasmática das fibras musculares. / The Golden Retriever Muscular Dystrophy (GRMD) is a genetic myopathy homologue to Duchenne Muscular Dystrophy (DMD) in human. Use of canine model is the better because the disease development, clinical signs and body mass are closed to the sick children. It has been known muscle protein proteolysis and muscle atrophy are related with ubiquitin-proteasome pathway. Researches using mdx mice and human muscle (in vitro) treated with proteasome inhibitor (MG-132 and bortezomib) showing rescue of the dystrophin and associated proteins and improvement of histopatologycal phenotype. In this research, were analyzed five GRMD dogs, two dogs were treated with Bortezomib and three dogs were control. Muscle biopsies were collected before the treatment and after the treatment to histology, ultrastructure and immunohistochemical muscle analysis. Biochemistry analyses were made once a week and measurement of proteasome inhibition was analysed in each cycle of Bortezomib administration. In the first byopsy, all the dogs showing closed histophatological morphology of muscle fibers. At the end of treatment, the treated dogs had lower connective tissue deposition and inflammatory cells infiltration than untreated dogs by histology, collagen morphometry and ultrastructural analysis. We noted by ultrastructural analysis macrophages inside the fibers, degenerated products, activated fibroblasts and connective tissue deposition in edomisium and perimisium of the fibers. The dystrophin immunohistochemistry was not presence in sarcoplasmatic membrane in both groups. However, the treated dogs showing bigger expression of α and β-dystroglycan, this fact means improved of disease histopathology phenotype. The untreated dogs had bigger expression of phospho-NFκB and TGF-β1, suggesting bigger activation of pro-apoptotic factors, inflammatory molecules and bigger connective tissue deposition respectively. The proteasome inhibition tests indicated bigger inhibition in the blood cells one hour post doses of bortezomib and was dose-dependent pathway. In conclusion, the proteasome inhibitors may improve the appearance of GRMD muscle fibers, lowered the connective tissue deposition and infiltration of inflammatory cells, likewise to rescue the dystrophin- associated proteins in the muscle fiber membrane.

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

No description available.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 147-167).

A distrofia muscular progressiva no cão Golden Retriever (GRMD) é uma miopatia genética homóloga à Distrofia Muscular de Duchenne (DMD) que acomete humanos. A utilização do modelo canino é considerada ideal uma vez que a progressão da doença, as manifestações clínicas e a massa corporal se assemelham às apresentadas por meninos doentes. Sabe-se que a degradação de proteínas musculares e atrofia muscular estão intimamente relacionadas com a atuação do sistema catalítico ubiquitina-proteassoma. Estudos utilizando camundongos com distrofia muscular e músculos de humanos DMD (in vitro) tratados com inibidores de proteassoma (MG-132 e bortezomibe) apresentaram restauração da distrofina e das proteínas associadas à distrofina e melhora do fenótipo histopatológico. Nesta pesquisa, foram avaliados cinco cães GRMD, dois tratados com Bortezomibe e três controles. Coletou-se biopsia muscular antes de iniciar o tratamento e ao término do mesmo, para os testes de histologia, ultraestrutura e imunohistoquímica. Foram realizados semanalmente testes bioquímicos e em cada ciclo de aplicação do bortezomibe foi mensurada a taxa de inibição do proteassoma. Na primeira biopsia todos os cães apresentaram morfologia histopatológica das fibras musculares semelhantes. Ao final do tratamento, os cães tratados apresentaram menor deposição de tecido conjuntivo e infiltração de células inflamatórias que os cães não tratados julgados por meio da histologia, morfometria do colágeno e ultraestrutura. Na ultraestrutura observamos infiltração de macrófagos nas fibras, material degenerado, fibroblastos ativados e deposição e tecido conjuntivo em endomísio e perimísio das fibras. Pela análise de imunohistoquímica, não constatamos presença de distrofina na membrana sarcoplasmática de ambos os grupos avaliados. Entretanto, os cães tratados apresentaram maior expressão das proteínas α e β distroglicano, o que sugere uma melhora no fenótipo histopatológico da doença. Nos cães não tratados observou-se maior expressão de phospho- NFκB e TGF-β1 sugerindo maior ativação de fatores pró-apoptóticos e moléculas inflamatórias, e maior deposição de tecido conjuntivo, respectivamente. Os testes de inibição do proteassoma indicaram maior inibição nas células sanguíneas uma hora após a aplicação do bortezomibe e estes de comportaram de forma dose-dependente. Concluímos que os inibidores de proteassoma podem melhorar a morfologia das fibras musculares dos cães GRMD tratados, diminuindo a deposição de colágeno e infiltração de células inflamatórias, bem como restaurar parte das proteínas associadas à distrofina na membrana sarcoplasmática das fibras musculares. / The Golden Retriever Muscular Dystrophy (GRMD) is a genetic myopathy homologue to Duchenne Muscular Dystrophy (DMD) in human. Use of canine model is the better because the disease development, clinical signs and body mass are closed to the sick children. It has been known muscle protein proteolysis and muscle atrophy are related with ubiquitin-proteasome pathway. Researches using mdx mice and human muscle (in vitro) treated with proteasome inhibitor (MG-132 and bortezomib) showing rescue of the dystrophin and associated proteins and improvement of histopatologycal phenotype. In this research, were analyzed five GRMD dogs, two dogs were treated with Bortezomib and three dogs were control. Muscle biopsies were collected before the treatment and after the treatment to histology, ultrastructure and immunohistochemical muscle analysis. Biochemistry analyses were made once a week and measurement of proteasome inhibition was analysed in each cycle of Bortezomib administration. In the first byopsy, all the dogs showing closed histophatological morphology of muscle fibers. At the end of treatment, the treated dogs had lower connective tissue deposition and inflammatory cells infiltration than untreated dogs by histology, collagen morphometry and ultrastructural analysis. We noted by ultrastructural analysis macrophages inside the fibers, degenerated products, activated fibroblasts and connective tissue deposition in edomisium and perimisium of the fibers. The dystrophin immunohistochemistry was not presence in sarcoplasmatic membrane in both groups. However, the treated dogs showing bigger expression of α and β-dystroglycan, this fact means improved of disease histopathology phenotype. The untreated dogs had bigger expression of phospho-NFκB and TGF-β1, suggesting bigger activation of pro-apoptotic factors, inflammatory molecules and bigger connective tissue deposition respectively. The proteasome inhibition tests indicated bigger inhibition in the blood cells one hour post doses of bortezomib and was dose-dependent pathway. In conclusion, the proteasome inhibitors may improve the appearance of GRMD muscle fibers, lowered the connective tissue deposition and infiltration of inflammatory cells, likewise to rescue the dystrophin- associated proteins in the muscle fiber membrane.

O camundongo mdx é um modelo animal muito utilizado para estudar a distrofia muscular degenerativa de Duchenne (DMD) e apesar de o modelo apresentar uma degeneração mais branda, inúmeras pesquisas com este animal são realizadas devido à alta reprodutibilidade de fenótipos, obtenção comercial de várias linhagens e baixo custo de manutenção. A degeneração muscular progressiva ocorre devido a uma mutação genética que culmina na ausência ou diminuição da produção da proteína distrofina. Esta alteração gera uma cascata de eventos que pioram a degeneração muscular. Atualmente, a terapia preconizada é o uso de corticóide, que visa modular a inflamação muscular e possui uma série de efeitos colaterais, por isso uma série de estudos propondo outras formas de tratamento vem sendo desenvolvidos. Um tratamento promissor parece ser a terapia celular com células-tronco mesenquimais, a qual pode interferir no processo degenerativo por imunomodulação celular. Como a acupuntura pode controlar o processo inflamatório de doenças degenerativas, tal qual na doença de Parkinson, acredita-se que o uso consorciado das duas terapias possa diminuir o efeito degenerativo da DMD. Nesta pesquisa foram avaliados 22 camundongos mdx, machos, 4 a 6 semanas de idade. As células-tronco utilizadas para a aplicação foram de polpa dentária humana carregando a proteína verde fluorescente (EGFP) e a quantidade aplicada em cada acuponto foi de 1x104 células. Os acupontos selecionados foram B47 (Hunmen), B49 (Yishe) e B52 (Zhishi). Os animais foram distribuídos aleatoriamente em quatro tratamentos (A) células-tronco em acupontos falsos, (B) solução fisiológica em acupontos verdadeiros, (C) células-tronco em acupontos verdadeiros e (D) controle, sem nenhum tratamento. No total três injeções foram realizados em um período de 56 dias de estudo. As avaliações do tratamento foram feitas através das análises da força de tração dos músculos, da creatinofosfoquinase sérica (CPK), histológica através da estruturação das miofibrilas e morfometria do colágeno e imuno-histoquímica pela expressão da proteína distrofina. Ao décimo dia após a terceira aplicação de células-tronco observa-se uma diferença somente no grupo controle, a qual manifestou um aumento da CPK em relação aos outros tratamentos e sem diferença estatística para força de tração pelos membros torácicos. Na histologia dos músculos tibial cranial e diafragma foram observou-se infiltrados inflamatórios, regeneração tecidual e diminuição do colágeno nos grupos tratados p<0,05. Na imuno-histoquímica verificou-se uma melhora da quantidade de distrofina nos animais tratados principalmente no tratamento células-tronco em acupontos verdadeiros p< 0,05 e, pela análise do rastreamento das células-tronco com EGFP não se pode determinar o seu caminho uma vez que não observamos imunorreatividade nas análises utilizando o anti-GFP. Conclui-se que os tratamentos com terapia celular nos acupontos verdadeiros, acupontos falsos e solução fisiológica nos acupontos podem melhorar a doença degenerativa muscular no mdx pelo aumento da expressão da proteína distrofina. / Muscular dystrophy in mdx mouse is an animal model used to study Duchenne muscular dystrophy (DMD) in humans. Although this model presents a milder degeneration, there are several studies with this animal due to the high reproducibility of phenotypes, obtaining various lines available and low maintenance cost. It is known that progressive muscle degeneration occurs due to a genetic mutation that culminates in the absence or decreased production of dystrophin protein. This change generates a cascade of events which aggravate the muscle degeneration. Currently, the recommended therapy is the use of corticosteroids to modulate muscle inflammation; however, there are a number of collateral effects, so a series of studies suggesting other forms of treatment have been developed. A promising treatment seems to be a therapy with mesenchymal stem cells, which can interfere with the degenerative process by cellular immunomodulation. As acupuncture can control the inflammatory process of degenerative diseases such as Parkinson\'s disease, the use of both syndication therapies may reduce the DMD degenerative effect. In this study 22 mdx mice, males, 4-6 weeks of age were evaluated. Stem cells were used for the application of human dental pulp carrying the green fluorescent protein (EGFP) and the quantity applied to each acupoint was 1x104 cells. The acupoints selected were B47 (Hunmen), B49 (Yishe) and B52 (Zhishi). The animals were randomly assigned to four treatments groups: (A) stem cells in false acupoints, (B) saline in true acupoints, (C) stem cells in true acupoints and (D) control without any treatment. In total, three injections were performed over a period of 56 days of study. Evaluations of treatment were made by analyzing the tensile strength of muscles, measuring the serum creatine phosphokinase (CPK), structuring of myofibrils by histology and morphometry of collagen immunohistochemistry and by the amount of dystrophin protein. At day ten after the third application of stem cells there is a difference only in the control group, which manifests an increase in CPK compared to other treatments and no statistical difference in tensile strength. In the histology of cranial tibial muscles and diaphragm, inflammatory infiltrates were observed, as well as tissue regeneration and decreased collagen in the treated groups (p <0.05). On immunohistochemistry there was an improvement in the amount of dystrophin in animals treated mainly in acupoints true (p <0.05) and through analysis and tracking of stem cells with EGFP it was not possible to determine its path once immunoreactivity was not observed in analyzes using anti-GFP. We conclude that treatment with stem cell therapy on true and false acupoints and with saline solution on acupoints can improve muscular degenerative disease in mdx by increased expression of dystrophin protein.

A Distrofia Muscular de Duchenne (DMD) é uma doença genética neuromuscular hereditária, ligada ao cromossomo X, sendo encontrada em humanos do sexo masculino. Esta doença muscular é descrita em outras espécies. O modelo de estudo pré-clínico GRMD (Golden Retrievier Muscular Dystrophy) apresenta sintomas clínicos fenotipicamente característicos da DMD em humanos e, por esta razão, tem sido amplamente utilizado como modelo de estudos pré-clínicos. O objetivo da presente pesquisa foi avaliar o tecido muscular, no modelo canino distrófico, ao longo da gestação. Quatro fêmeas, portadoras do gene distrófico, foram inseminadas com sêmen fresco de cães distróficos. No 25&ordm; dia, pós-inseminação, as fêmeas foram submetidas a exames de ultrassonografia para confirmar a gestação. As fêmeas gestantes passaram por uma ovariosalpingohisterectomia (OSH) para a retirada dos embriões e fetos nos seguintes períodos gestacionais: 28&ordm; , 33&ordm; , 38&ordm; e 42&ordm; dias. Em seguida fragmentos de tecido muscular foram analisados macroscopicamente e microscopicamente. Para verificar expressões proteicas, amostras de tecido foram submetidas a técnicas imunológicas, e PCR para distrofina, miostatina, e utrofina. Aos, 33&ordm; e 38&ordm; dias de gestação, no grupo distrófico, foram observadas características teciduais que corroboram com desenvolvimento tardio do tecido muscular. Os resultados para detecção proteica sugerem que, a distrofina, miostatina e utrofina foram expressas igualmente nos grupos controle e distrófico, durante todos os períodos do desenvolvimento gestacional analisado. Por fim, os dados sugerem que animais distróficos apresentam músculo sadio durante a fase gestacional, o que pode ser benéfico para testes farmacológicos em idade precoce. / Duchenne Muscular Dystrophy (DMD) is a hereditary neuromuscular genetic disease linked to the X chromosome, being found in male humans. This muscle disease is described in other species. The pre-clinical GRMD (Golden Retrievier Muscular Dystrophy) study model presents phenotypically characteristic clinical symptoms of DMD in humans and,for this reason, has been widely used as a model for preclinical studies. The aim of the present study was to evaluate the muscular tissue, in the dystrophic canine model, throughout the gestation. Four females, carriers of the dystrophic gene, were inseminated with fresh semen from dystrophic dogs. On the 25th day, post-insemination, the females were submitted to ultrasonography to confirm the pregnancy. The pregnant females underwent an ovariosalpingohisterectomy (OSH) for the removal of the embryos and fetuses in the following gestational periods: 28&ordm;, 33&ordm;, 38&ordm; and 42&ordm; days. Then fragments of muscle tissue were analyzed macroscopically and microscopically. To verify protein expression, tissue samples were submitted to immunological techniques, and PCR for dystrophin, myostatin, and utrophin. At the 33 and 38th days of gestation, tissue characteristics were observed in the dystrophic group, which corroborate the late development of muscle tissue. The results for protein detection suggest that dystrophin, myostatin and utrophin were also expressed in the control and affected groups, during all periods of the gestational development analyzed. Lastly, the data suggest that dystrophic animals present healthy muscle during the gestational phase, which may be beneficial for pharmacological tests at an early age.

Myoclonus Dystonia (MD) is an autosomal dominant movement disorder characterized by bilateral myoclonic jerks paired with dystonia 1. Mutations have been mapped to the ε-sarcoglycan (SGCE) gene in about 40% of patients 2,92. The purpose of this project was to examine the properties of SGCE in the central nervous system (CNS) and use this knowledge to elucidate the pathology of MD. Although Sgce is a member of the sarcoglycan complex (SGC) in other tissues, little is known about its interactions in the CNS. The vast majority of mutations in SGCE alter the translational reading frame. Proteins arising from these rare mutations are less stable than the wild type (WT) and undergo preferential degradation via the ubiquitin proteasome system 3. As this locus is maternally imprinted, patients with MD are effectively null for sgce expression 73,91. Therefore, Sgce knock out (KO) models should approximate MD conditions both in vivo and in vitro. As there are no current treatments for MD, in sight into the pathology of the disease will aid in eventual treatments and help bring patients some relief by finally understanding their disease. Since a large percentage of MD patients are without the sgce protein, identifying what this protein’s function is and how its absence effects normal processing in the brain should help to identify the underlying cellular pathology which produces the MD phenotype. This research was performed under the hypothesis that, in neuronal cells, sgce interacts with a group of proteins that together play a role in stabilization and localization of ion channels and signaling proteins at the cell membrane. The aims were to: (1) Build a MD mouse model with either a conditional knock-out (cKO) or a conditional gene repair (cGR) mutation; (2) Use neuroblastoma cells to identify the other proteins which interact with sgce in neurons, and; (3) Determine if there is a disruption of the localization of the sgce-complex members due to the loss of sgce. Recombineering was used to complete the constructs for two transgenic mouse models: One model for the KO of exon 4 of sgce and one for the cGR in intron 1. Primary neurosphere lines from two previously generated chimeras were developed, as well as from a WT mouse. These neurosphere cell lines allowed comparisons of RT-PCR results from a heterogeneous neurological cell population to neuroblastoma cell lines. mRNA is present in neuronal cells for many of the DGC associated proteins. It was confirmed that the KD of sgce results in a reduction of nNOS protein and in increased proliferation of NIE cells. By using a nitrite/nitrate assay as well as studies with L-NAME, it was confirmed that this increased proliferation was in fact due to a lack of nNOS function. These proliferation changes did not occur in N2A cells, which do not express high levels of nNOS during proliferation, further confirming nNOS’s role in the proliferation changes. Using qRT-PCR, KD of sgce was shown to result in significant changes in the transcript levels for many DGC associated proteins. This suggests that a DGC-like complex is forming in neuronal cells. Also, as a result of difficulties with the research, it became clear that over-expression of sgce causes cell death. This observation was quantified using cell counts and TUNEL staining, both showing significant results. Additionally, several new constructs were created which will hopefully be of use for future students wanting to study sgce’s functions. New shRNA targeting sgce and sgcb have been made and both constructs result in reducing the expression of sgce. Seven different flag-tagged sgces have been created and some of these have been transferred into a tet-inducible system, which should circumvent the problem of over-expression. Finally GFP-tagged constructs for sgce and sgcb have been made and pooled clones have been developed. These tools will hopefully enable future students to continue to tease apart sgce’s function(s).

Claudin-5 is a transmembrane cell junction protein that is a component of tight junctions in endothelial cell layers. We have previously shown that claudin-5 also localizes to lateral membranes of murine cardiomyocytes at their junction with the extracellular matrix. Claudin-5 levels are specifically reduced in myocytes from a mouse model of muscular dystrophy with cardiomyopathy. To establish whether claudin-5 is similarly specifically reduced in human cardiomyopathy, we compared the levels of claudin-5 with other cell junction proteins in 62 cardiomyopathic end-stage explant samples. We show that claudin-5 levels are reduced in at least 60% of patient samples compared with non-failing controls. Importantly, claudin-5 reductions can be independent of connexin-43, a gap junction protein previously reported to be reduced in failing heart samples. Other cell junction proteins including α-catenin, β-catenin, γ-catenin, desmoplakin, and N-cadherin are reduced in only a small number of failing samples and only in combination with reduced claudin-5 or connexin-43 levels. We also show that reduced claudin-5 levels can be present independently from dystrophin alterations, which are known to be capable of causing and resulting from cardiomyopathy. These data are the first to show alterations of a tight junction protein in human cardiomyopathy samples and suggest that claudin-5 may participate in novel mechanisms in the pathway to end-stage heart failure.