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1.
J Back Musculoskelet Rehabil ; 32(6): 885-895, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30958331

RESUMO

BACKGROUND: Healthy individuals who develop low back pain (LBP) during standing (standing intolerant) respond favorably to stabilization-based exercise interventions. People with clinical LBP meeting clinical prediction rules for stabilization-based exercise share characteristics with standing intolerant individuals. OBJECTIVE: To investigate the impact of stabilization-based exercise on standing tolerance, muscle activation and clinical measures in individuals with LBP meeting clinical prediction rules for stabilization-based exercise. METHODS: Participants with and without LBP completed testing pre- and post-6 weeks of progressive home exercise intervention. Testing included clinical examination and electromyography during sagittal and frontal plane movements. LBP was also assessed by visual analogue scale (VAS) during standing. Outcomes included clinical findings, muscle sequencing, and VAS in standing. RESULTS: The LBP group had non-significant decreases in Oswestry Disability Index (-2.1%, p= 0.22), baseline VAS (-7.1 mm, p= 0.11), lumbopelvic reversal (p= 0.06) and positive active hip abduction test (p= 0.06). Significant improvements were seen in standing VAS (-5.6 mm, p< 0.001). The LBP group had beneficial changes in activation strategies in standing flexion (p< 0.05) following intervention, with no changes during frontal plane movement strategies. CONCLUSIONS: Individuals with LBP meeting clinical prediction rules for stabilization-based exercise demonstrated increased standing tolerance and sagittal plane muscle sequencing following a 6-week intervention.


Assuntos
Eletromiografia , Terapia por Exercício , Dor Lombar/reabilitação , Músculo Esquelético/fisiologia , Posição Ortostática , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Dor Lombar/fisiopatologia , Masculino , Pessoa de Meia-Idade , Escala Visual Analógica , Adulto Jovem
2.
Toxicol Appl Pharmacol ; 298: 56-65, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-26905748

RESUMO

Fumonisin B1 (FB1) is a mycotoxin produced by a common fungal contaminant of corn. Administration of FB1 to pregnant LM/Bc mice induces exencephaly in embryos, and ingestion of FB1-contaminated food during early pregnancy is associated with increased risk for neural tube defects (NTDs) in humans. FB1 inhibits ceramide synthase enzymes in sphingolipid biosynthesis, causing sphinganine (Sa) and bioactive sphinganine-1-phosphate (Sa1P) accumulation in blood, cells, and tissues. Sphingosine kinases (Sphk) phosphorylate Sa to form Sa1P. Upon activation, Sphk1 associates primarily with the plasma membrane, while Sphk2 is found predominantly in the nucleus. In cells over-expressing Sphk2, accumulation of Sa1P in the nuclear compartment inhibits histone deacetylase (HDAC) activity, causing increased acetylation of histone lysine residues. In this study, FB1 treatment in LM/Bc mouse embryonic fibroblasts (MEFs) resulted in significant accumulation of Sa1P in nuclear extracts relative to cytoplasmic extracts. Elevated nuclear Sa1P corresponded to decreased histone deacetylase (HDAC) activity and increased histone acetylation at H2BK12, H3K9, H3K18, and H3K23. Treatment of LM/Bc MEFs with a selective Sphk1 inhibitor, PF-543, or with ABC294640, a selective Sphk2 inhibitor, significantly reduced nuclear Sa1P accumulation after FB1, although Sa1P levels remained significantly increased relative to basal levels. Concurrent treatment with both PF-543 and ABC294640 prevented nuclear accumulation of Sa1P in response to FB1. Other HDAC inhibitors are known to cause NTDs, so these results suggest that FB1-induced disruption of sphingolipid metabolism leading to nuclear Sa1P accumulation, HDAC inhibition, and histone hyperacetylation is a potential mechanism for FB1-induced NTDs.


Assuntos
Núcleo Celular/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Fibroblastos/efeitos dos fármacos , Fumonisinas/toxicidade , Histona Desacetilases/metabolismo , Defeitos do Tubo Neural/metabolismo , Esfingosina/análogos & derivados , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Embrião de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/patologia , Camundongos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/patologia , Cultura Primária de Células , Espectrometria de Massas por Ionização por Electrospray , Esfingosina/metabolismo , Espectrometria de Massas em Tandem
3.
Toxicol Sci ; 150(1): 161-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26719367

RESUMO

FTY720 (fingolimod) is a U.S. Food and Drug Administration-approved drug to treat relapsing remitting multiple sclerosis. FTY720 treatment in pregnant inbred LM/Bc mice results in approximately 60% of embryos having a neural tube defect (NTD). Sphingosine kinases (Sphk1, Sphk2) phosphorylate FTY720 in vivo to form the bioactive metabolite FTY720-1-phosphate (FTY720-P). Cytoplasmic FTY720-P is an agonist for 4 of the 5 sphingosine-1-phosphate (S1P) receptors (S1P1, 3-5) and can also act as a functional antagonist of S1P1, whereas FTY720-P generated in the nucleus inhibits histone deacetylases (HDACs), leading to increased histone acetylation. This study demonstrates that treatment of LM/Bc mouse embryonic fibroblasts (MEFs) with FTY720 results in a significant accumulation of FTY720-P in both the cytoplasmic and nuclear compartments. Elevated nuclear FTY720-P is associated with decreased HDAC activity and increased histone acetylation at H3K18 and H3K23 in LM/Bc MEFs. Treatment of LM/Bc MEFs with FTY720 and a selective Sphk2 inhibitor, ABC294640, significantly reduces the amount of FTY720-P that accumulates in the nucleus. The data provide insight into the relative amounts of FTY720-P generated in the nuclear versus cytoplasmic subcellular compartments after FTY720 treatment and the specific Sphk isoforms involved. The results of this study suggest that FTY720-induced NTDs may involve multiple mechanisms, including: (1) sustained and/or altered S1P receptor activation and signaling by FTY720-P produced in the cytoplasm and (2) HDAC inhibition and histone hyperacetylation by FTY720-P generated in the nucleus that could lead to epigenetic changes in gene regulation.


Assuntos
Núcleo Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Defeitos do Tubo Neural/induzido quimicamente , Organofosfatos/toxicidade , Esfingosina/análogos & derivados , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Fibroblastos/metabolismo , Histona Desacetilases/metabolismo , Histonas/efeitos dos fármacos , Camundongos Endogâmicos , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/metabolismo , Organofosfatos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Esfingosina/metabolismo , Esfingosina/toxicidade
4.
Neurogenetics ; 16(4): 277-85, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26051944

RESUMO

The progressive and permanent loss of cerebellar Purkinje cells (PC) is a hallmark of many inherited ataxias. Mutations in several genes involved in the regulation of Ca(2+) release from intracellular stores by the second messenger IP3 have been associated with PC dysfunction or death. While much is known about the defects in production and response to IP3, less is known about the defects in breakdown of the IP3 second messenger. A mutation in Inpp4a of the pathway is associated with a severe, early-onset PC degeneration in the mouse model weeble. The step preceding the removal of the 4-phosphate is the removal of the 5-phosphate by Inpp5a. Gene expression analysis was performed on an Inpp5a (Gt(OST50073)Lex) mouse generated by gene trap insertion using quantitative real-time PCR (qRT-PCR), immunohistochemistry, and Western blot. Phenotypic analyses were performed using rotarod, ß-galactosidase staining, and phosphatase activity assay. Statistical significance was calculated. The deletion of Inpp5a causes an early-onset yet slowly progressive PC degeneration and ataxia. Homozygous mutants (90%) exhibit perinatal lethality; surviving homozygotes show locomotor instability at P16. A consistent pattern of PC loss in the cerebellum is initially detectable by weaning and widespread by P60. Phosphatase activity toward phosphoinositol substrates is reduced in the mutant relative to littermates. The ataxic phenotype and characteristics neurodegeneration of the Inpp5a (Gt(OST50073)Lex) mouse indicate a crucial role for Inpp5a in PC survival. The identification of the molecular basis of the selective PC survival will be important in defining a neuroprotective gene applicable to establishing a disease mechanism.


Assuntos
Ataxia Cerebelar/genética , Deleção de Genes , Monoéster Fosfórico Hidrolases/genética , Animais , Ataxia Cerebelar/embriologia , Ataxia Cerebelar/enzimologia , Ataxia Cerebelar/patologia , Modelos Animais de Doenças , Inositol Polifosfato 5-Fosfatases , Camundongos , Atividade Motora/genética , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Células de Purkinje/patologia , Teste de Desempenho do Rota-Rod
5.
Birth Defects Res A Clin Mol Teratol ; 94(10): 790-803, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22991331

RESUMO

BACKGROUND: Fumonisin B(1) (FB(1)) is a mycotoxin produced by a common fungal contaminant of corn. Ingestion of FB(1)-contaminated food is associated with increased risk for neural tube defects (NTDs). FB(1) induces NTDs in inbred LM/Bc mice. FB(1) inhibits ceramide synthase in de novo sphingolipid biosynthesis, resulting in accumulation of sphinganine and sphinganine-1-phosphate (Sa1P). Sa1P functions as a ligand for a family of G protein-coupled S1P receptors. METHODS: Pregnant SWV and LM/Bc mice were treated with FB(1) (20 mg/kg/day intraperitoneally on embryonic day (ED) 7.5-8.5) or the known S1P receptor agonist FTY720 (10 mg/kg/day oral gavage on ED 6.5-8.5). LC/MS was used to detect sphingoid base-1-phosphates in maternal blood spots, plasma, and embryonic tissue. Strain-specific SWV and LM/Bc mouse embryonic fibroblasts (MEFs) and serum free mouse embryo (SFME) neural progenitor cells were treated with FB(1) (40 µM for 24 hr) and LC/MS was used to detect sphingoid base-1-phosphates. RESULTS: FTY720 induced NTDs in both the SWV and the LM/Bc strains of mice. Sphinganine-1-P (Sa1P) and FTY720-P were elevated in the blood spots and plasma of mice treated with FB(1) or FTY720, respectively. FTY720-P was elevated in ED 9.5 exencephalic embryos. Sa1P was elevated in SFME and MEF cells treated with FB(1), and Sa1P was higher in MEFs generated from the FB(1)-NTD-susceptible LM/Bc strain. CONCLUSIONS: Elevated sphingoid base-1-P after FB(1) or FTY720 suggest a potential role for these bioactive lipid ligands and activation of S1P receptor signaling pathways in the failure of neural tube closure after FB(1) or FTY720. Sa1P may represent a biomarker for FB(1)-NTD risk assessment.


Assuntos
Fumonisinas/efeitos adversos , Defeitos do Tubo Neural/induzido quimicamente , Propilenoglicóis/efeitos adversos , Esfingosina/análogos & derivados , Animais , Células Cultivadas , Embrião de Mamíferos , Feminino , Cloridrato de Fingolimode , Fumonisinas/farmacologia , Camundongos , Camundongos Endogâmicos , Modelos Biológicos , Defeitos do Tubo Neural/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/sangue , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Propilenoglicóis/farmacologia , Esfingosina/efeitos adversos , Esfingosina/sangue , Esfingosina/farmacologia , Regulação para Cima/efeitos dos fármacos
6.
PLoS One ; 6(7): e21858, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21779340

RESUMO

Variation in genetic background can significantly influence the phenotypic outcome of both disease and non-disease associated traits. Additionally, differences in temporal and strain specific gene expression can also contribute to phenotypes in the mammalian retina. This is the first report of microarray based cross-strain analysis of gene expression in the retina investigating genetic background effects. Microarray analyses were performed on retinas from the following mouse strains: C57BL6/J, AKR/J, CAST/EiJ, and NOD.NON-H2(-nb1) at embryonic day 18.5 (E18.5) and postnatal day 30.5 (P30.5). Over 3000 differentially expressed genes were identified between strains and developmental stages. Differential gene expression was confirmed by qRT-PCR, Western blot, and immunohistochemistry. Three major gene networks were identified that function to regulate retinal or photoreceptor development, visual perception, cellular transport, and signal transduction. Many of the genes in these networks are implicated in retinal diseases such as bradyopsia, night-blindness, and cone-rod dystrophy. Our analysis revealed strain specific variations in cone photoreceptor cell patterning and retinal function. This study highlights the substantial impact of genetic background on both development and function of the retina and the level of gene expression differences tolerated for normal retinal function. These strain specific genetic variations may also be present in other tissues. In addition, this study will provide valuable insight for the development of more accurate models for human retinal diseases.


Assuntos
Variação Genética/genética , Retina/metabolismo , Animais , Western Blotting , Imuno-Histoquímica , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Retina/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
PLoS One ; 6(7): e21926, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21779355

RESUMO

BACKGROUND: Progression of age-related macular degeneration has been linked to iron dysregulation and oxidative stress that induce apoptosis of neural retinal cells. Since both antioxidants and chelating agents have been reported to reduce the progression of retinal lesions associated with AMD in experimental animals, the present study evaluates the ability of multi-functional antioxidants containing functional groups that can independently chelate redox metals and quench free radicals to protect the retina against light-induced retinal degeneration, a rat model of dry atrophic AMD. METHODS/RESULTS: Proof of concept studies were conducted to evaluate the ability of 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8) to reduce retinal damage in 2-week dark adapted Wistar rats exposed to 1000 lx of light for 3 hours. Assessment of the oxidative stress markers 4- hydroxynonenal and nitrotyrosine modified proteins and Thioredoxin by ELISA and Western blots indicated that these compounds reduced the oxidative insult caused by light exposure. The beneficial antioxidant effects of these compounds in providing significant functional and structural protection were confirmed by electroretinography and quantitative histology of the retina. CONCLUSIONS/SIGNIFICANCE: The present study suggests that multi-functional compounds may be effective candidates for preventive therapy of AMD.


Assuntos
Antioxidantes/farmacologia , Luz/efeitos adversos , Retina/efeitos dos fármacos , Retina/efeitos da radiação , Sulfonamidas/farmacologia , Aldeídos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Eletrorretinografia , Ensaio de Imunoadsorção Enzimática , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Wistar , Tiorredoxinas/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
8.
PLoS One ; 6(3): e17494, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21408158

RESUMO

The majority of diseases in the retina are caused by genetic mutations affecting the development and function of photoreceptor cells. The transcriptional networks directing these processes are regulated by genes such as nuclear hormone receptors. The nuclear hormone receptor gene Rev-erb alpha/Nr1d1 has been widely studied for its role in the circadian cycle and cell metabolism, however its role in the retina is unknown. In order to understand the role of Rev-erb alpha/Nr1d1 in the retina, we evaluated the effects of loss of Nr1d1 to the developing retina and its co-regulation with the photoreceptor-specific nuclear receptor gene Nr2e3 in the developing and mature retina. Knock-down of Nr1d1 expression in the developing retina results in pan-retinal spotting and reduced retinal function by electroretinogram. Our studies show that NR1D1 protein is co-expressed with NR2E3 in the outer neuroblastic layer of the developing mouse retina. In the adult retina, NR1D1 is expressed in the ganglion cell layer and is co-expressed with NR2E3 in the outer nuclear layer, within rods and cones. Several genes co-targeted by NR2E3 and NR1D1 were identified that include: Nr2c1, Recoverin, Rgr, Rarres2, Pde8a, and Nupr1. We examined the cyclic expression of Nr1d1 and Nr2e3 over a twenty-four hour period and observed that both nuclear receptors cycle in a similar manner. Taken together, these studies reveal a novel role for Nr1d1, in conjunction with its cofactor Nr2e3, in regulating transcriptional networks critical for photoreceptor development and function.


Assuntos
Redes Reguladoras de Genes/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Nucleares Órfãos/metabolismo , Retina/metabolismo , Envelhecimento/metabolismo , Envelhecimento/efeitos da radiação , Animais , Contagem de Células , Elementos Facilitadores Genéticos/genética , Injeções , Luz , Transdução de Sinal Luminoso/genética , Transdução de Sinal Luminoso/efeitos da radiação , Camundongos , Especificidade de Órgãos/efeitos da radiação , Transporte Proteico/efeitos da radiação , RNA Interferente Pequeno/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/efeitos da radiação , Coloração e Rotulagem , Ativação Transcricional/genética , Ativação Transcricional/efeitos da radiação
9.
Invest Ophthalmol Vis Sci ; 51(6): 3115-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20042664

RESUMO

PURPOSE: Intraocular injection of linezolid, a synthetic oxazolidinone antibiotic, was performed in rabbits to assess its safety as a possible treatment for endophthalmitis. METHODS: Linezolid, 300 microg/0.1 mL, 200 microg/0.1 mL, or 100 microg/0.1 mL, was injected into the vitreous of the right eye of 12 rabbits. Balanced saline solution was injection into the left eye of each rabbit as a control. A standard electroretinogram (ERG) was obtained before injection and repeated 2 days and 1 and 4 weeks after injection. Intraocular pressure (IOP) was also measured after injection. After the experiment, the rabbits were euthanatized and the retinas were examined by light and electron microscopy. Differences between the two eyes in the ERGs, IOP, and histopathology were recorded. RESULTS: There were no statistically significant differences in the electroretinograms obtained between the linezolid-injected eyes and the control eyes. Histopathology showed no changes in the study eyes compared with the control eyes. CONCLUSIONS: Preservative-free linezolid is nontoxic to the retinas of rabbits when injected intravitreally, and this route can therefore be considered for the administration of linezolid in the treatment of endophthalmitis.


Assuntos
Acetamidas/toxicidade , Anti-Infecciosos/toxicidade , Oxazolidinonas/toxicidade , Retina/efeitos dos fármacos , Animais , Eletrorretinografia/efeitos dos fármacos , Injeções , Pressão Intraocular , Linezolida , Microscopia Eletrônica , Coelhos , Corpo Vítreo
10.
PLoS One ; 4(12): e8270, 2009 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-20011524

RESUMO

The weeble mutant mouse has a frame shift mutation in inositol polyphosphate 4-phosphatase type I (Inpp4a). The phenotype is characterized by an early onset cerebellar ataxia and neurodegeneration, especially apparent in the Purkinje cells. Purkinje cell loss is a common pathological finding in many human and mouse ataxic disorders. Here we show that in the Inpp4a(wbl) mutant, Purkinje cells are lost in a specific temporal and spatial pattern. Loss occurs early in postnatal development; however, prior to the appearance of climbing fibers in the developing molecular layer, the mutant has a normal complement of Purkinje cells and they are properly positioned. Degeneration and reactive gliosis are present at postnatal day 5 and progress rapidly in a defined pattern of patches; however, Inpp4a is expressed uniformly across Purkinje cells. In late stage mutants, patches of surviving Purkinje cells appear remarkably normal with the exception that the climbing fibers have been excessively eliminated. Surviving Purkinje cells express Eaat4, a glutamate transporter that is differentially expressed in subsets of Purkinje cells during development and into adult stages. Prior to Purkinje cell loss, reactive gliosis and dendritic atrophy can be seen in Eaat4 negative stripes. Our data suggest that Purkinje cell loss in the Inpp4a(wbl) mutant is due to glutamate excitotoxicity initiated by the climbing fiber, and that Eaat4 may exert a protective effect.


Assuntos
Cerebelo/enzimologia , Cerebelo/patologia , Citoproteção , Transportador 4 de Aminoácido Excitatório/metabolismo , Neurônios/enzimologia , Neurônios/patologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Camundongos , Camundongos Mutantes Neurológicos , Fenótipo , Células de Purkinje/enzimologia , Células de Purkinje/patologia , Análise de Sobrevida , Fatores de Tempo , Síndrome de Emaciação/enzimologia , Síndrome de Emaciação/patologia , Desmame
11.
Exp Eye Res ; 89(3): 365-72, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19379737

RESUMO

The retinal transcription factor Nr2e3 plays a key role in photoreceptor development and function. In this study we examine gene expression in the retina of Nr2e3(rd7/rd7) mutants with respect to wild-type control mice, to identify genes that are misregulated and hence potentially function in the Nr2e3 transcriptional network. Quantitative candidate gene real time PCR and subtractive hybridization approaches were used to identify transcripts that were misregulated in Nr2e3(rd7/rd7) mice. Chromatin immunoprecipitation assays were then used to determine which of the misregulated transcripts were direct targets of NR2E3. We identified 24 potential targets of NR2E3. In the developing retina, NR2E3 targets transcription factors such as Ror1, Rorg, and the nuclear hormone receptors Nr1d1 and Nr2c1. In the mature retina NR2E3 targets several genes including the rod specific gene Gnb1 and cone specific genes blue opsin, and two of the cone transducin subunits, Gnat2 and Gnb3. In addition, we identified 5 novel transcripts that are targeted by NR2E3. While mislocalization of proteins between rods and cones was not observed, we did observe diminished concentration of GNB1 protein in adult Nr2e3(rd7/rd7) retinas. These studies identified novel transcriptional pathways that are potentially targeted by Nr2e3 in the retina and specifically demonstrate a novel role for NR2E3 in regulating genes involved in phototransduction.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Transdução de Sinal Luminoso/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Degeneração Retiniana/genética , Animais , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Camundongos , Camundongos Mutantes , Receptores Nucleares Órfãos , Retina/crescimento & desenvolvimento , Retina/metabolismo , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Fatores de Transcrição/fisiologia
12.
Mamm Genome ; 19(9): 623-33, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18763016

RESUMO

The nmf193 mutant was generated by a large-scale ENU mutagenesis screen and originally described as having a dominantly inherited phenotype characterized by fundus abnormalities. We determined that nmf193 mice exhibit outer-segment defects and progressive retinal degeneration. Clinical examination revealed retinal spotting apparent at 6 weeks of age. Histologic analysis of homozygous mutant mice at 6 weeks indicated an absence of outer segments (OS) and a 50% reduction of photoreceptor cells which progressed to complete loss of photoreceptors by 10 months. Mice heterozygous for the nmf193 mutation had a less severe phenotype of shortened outer segments at 2 months with progressive loss of photoreceptor cells to 50% by 10 months. A positional cloning approach using a DNA pooling strategy was performed to identify the causative mutation in nmf193 mice. The nmf193 mutation was linked to chromosome 17 and fine mapped to an interval containing the peripherin/rds (Prph2) gene. Mutation analysis identified a single base change in Prph2 that causes aberrant splicing between exons 1 and 2. Interestingly, a comparative histologic analysis demonstrated that Prph2 ( nmf193/+ ) mutants have similar photoreceptor degeneration to that of Nr2e3 ( rd7/rd7 ). We show that Prph2 mRNA and protein levels are reduced in the Nr2e3 ( rd7/rd7 ) mutant compared to control littermates. Chromatin immunoprecipitation analysis shows that Prph2 is a direct target of NR2E3. In addition, the downregulation of Prph2 gene expression is similar in both the Nr2e3 ( rd7/rd7 ) and Prph2 ( nmf193/+ ) mutants, suggesting that the reduction of Prph2 may contribute to the degenerative pathology seen in Nr2e3 ( rd7/rd7 ).


Assuntos
Proteínas de Filamentos Intermediários/genética , Glicoproteínas de Membrana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Receptores Citoplasmáticos e Nucleares/genética , Degeneração Retiniana/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA/genética , Etilnitrosoureia/toxicidade , Feminino , Heterozigoto , Homozigoto , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Receptores Nucleares Órfãos , Periferinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Segmento Externo da Célula Bastonete/patologia , Segmento Externo da Célula Bastonete/fisiologia
13.
Mamm Genome ; 18(11): 749-56, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17934773

RESUMO

The identification of novel mutant alleles is important for understanding critical functional domains of a protein and establishing genotype:phenotype correlations. The recoil wobbler (rcw) allelic series of spontaneous ataxic mutants and the ENU-induced mutant nmf373 genetically mapped to a shared region of chromosome 10. Their mutant phenotypes are strikingly similar; all have an ataxic phenotype that is recessive, early-onset, and is not associated with neurodegeneration. In this study we used complementation tests to show that these series of mutants are allelic to a knockout mutant of Grm1. Subsequently, a duplication of exon 4 and three missense mutations were identified in Grm1: I160T, E292D, and G337E. All mutations occurred within the ligand-binding region and changed conserved amino acids. In the rcw mutant, the Grm1 gene is expressed and the protein product is properly localized to the molecular layer of the cerebellar cortex. Grm1 is responsible for the generation of inositol 1,4,5-trisphosphate (IP(3)). The inositol second messenger system is the central mechanism for calcium release from intracellular stores in cerebellar Purkinje cells. Several of the genes involved in this pathway are mutated in mouse ataxic disorders. The novel rcw mutants represent a resource that will have utility for further studies of inositol second-messenger-system defects in neurogenetic disorders.


Assuntos
Mutação , Receptores de Glutamato Metabotrópico/genética , Alelos , Sequência de Aminoácidos , Animais , Ataxia/genética , Ataxia/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Sinalização do Cálcio , Córtex Cerebelar/metabolismo , Mapeamento Cromossômico , DNA/genética , Éxons , Feminino , Teste de Complementação Genética , Inositol 1,4,5-Trifosfato/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação de Sentido Incorreto , Fenótipo , Células de Purkinje/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sistemas do Segundo Mensageiro , Homologia de Sequência de Aminoácidos
14.
Neurogenetics ; 8(1): 1-10, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17102983

RESUMO

The soluble N-ethylmaleimide sensitive factor attachment receptors are a large family of membrane-associated proteins that are critical for Ca(2+)-mediated synaptic vesicle release. This family includes the VAMP, synaptosomal-associated protein, and syntaxin proteins. In this report, we describe a mutation in vesicle-associated membrane protein 1(VAMP1)/synaptobrevin in the mouse neurological mutant lethal-wasting (lew). The lethal-wasting mutant phenotype is characterized by a general lack of movement and wasting, eventually leading to death before weaning. Mutants are visibly immobile and lay on their side by postnatal day 10 (P10). Before this stage, mutants can be identified by a failure to attempt to right themselves. Affected mice die on average at P15. We used a positional cloning strategy to identify the mutation associated with this neurological phenotype. Lethal wasting had previously been linked to chromosome 6. We further narrowed the genetic disease interval and selected a small number of candidate genes for mutation screening. Genes were evaluated by quantitative reverse transcription-polymerase chain reaction (RT-PCR) to detect differences in their expression levels between control and mutant brain ribonucleic acid (RNA) samples. VAMP1 mRNA was found to be significantly downregulated in the lethal-wasting brain compared to wild-type littermates. Subsequently, a nonsense mutation was identified in the coding region of the gene. This mutation is predicted to truncate approximately half of the protein; however, Western blot analysis showed that no protein is detectable in the mutant. VAMP1 is selectively expressed in the retina and in discrete areas of the brain including the zona incerta and rostral periolivary region, although no gross histological abnormalities were observed in these tissues. Taken together, these data indicate that VAMP1 has a vital role in a subset of central nervous system tissues.


Assuntos
Encéfalo/patologia , Códon sem Sentido , Doenças do Sistema Nervoso/genética , Proteínas R-SNARE/genética , Proteína 1 Associada à Membrana da Vesícula/genética , Síndrome de Emaciação/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Genótipo , Camundongos , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Doenças do Sistema Nervoso/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome de Emaciação/mortalidade , Síndrome de Emaciação/patologia
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