RESUMO
Direct laser writing of surface waveguides with ultrashort pulses is a crucial achievement towards all-laser manufacturing of photonic integrated circuits sensitive to their environment. In this Letter, few-cycle laser pulses (with a sub-10 fs duration) are used to produce subsurface waveguides in a non-doped, non-coated fused-silica substrate. The fabrication technique relies on laser-induced microdensification below the threshold for nanopore formation. The optical losses of the fabricated waveguides are governed by the optical properties of the superstrate. We have measured losses ranging from less than 0.1 dB/mm (air superstrate) up to 2.8 dB/mm when immersion oil is applied on top of the waveguide.
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We present experimental data and a complete theoretical description of the magneto-optical contributions to the complex refractive index in the extreme ultraviolet (XUV) range covering the 3p resonances of Fe, Co, and Ni. The direct comparison of the two allows us to conclude that many-body corrections to the ground state and local field effects are crucial for an accurate description of M-edge spectra. Our results are relevant for investigation of static magnetization, via XUV spectroscopy of multielement systems, as well as the dynamics of magnetization, as needed in the study of femtomagnetism and spintronics.
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Features of Costello Syndrome, a systemic disorder caused by germline mutations in the proto-oncogene HRAS from the RAS/MAPK pathway, include failure-to-thrive, short stature, coarse facial features, cardiac defects including hypertrophic cardiomyopathy, intellectual disability, and predisposition to neoplasia. Two unrelated boys with Costello syndrome and an HRAS mutation (p.Gly13Cys) are presented with their ophthalmologic findings. Both had early symptoms of nystagmus, photophobia, and vision abnormalities. Fundus examination findings of retinal dystrophy were present at age 3 years. Both boys have abnormal electroretinograms with reduced or undetectable rod responses along with reduced cone responses consistent with rod-cone dystrophy. Our observations suggest that early ophthalmic examination and re-evaluations are indicated in children with Costello syndrome.
Assuntos
Anormalidades Múltiplas/genética , Síndrome de Costello/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Distrofias Retinianas/genética , Anormalidades Múltiplas/fisiopatologia , Adulto , Criança , Síndrome de Costello/complicações , Síndrome de Costello/fisiopatologia , Genótipo , Mutação em Linhagem Germinativa , Humanos , Masculino , Fenótipo , Proto-Oncogene Mas , Distrofias Retinianas/complicações , Distrofias Retinianas/fisiopatologiaRESUMO
Ageing of the neuromuscular system in elderhood ingravescently contributes to slowness, weakness, falling and death, often accompanied by numbness and pain. This article is to put in perspective examples from a half-century of personal and team neuromuscular histochemical-pathological and clinical-pathological research, including a number of lucky and instructive accomplishments identifying new treatments and new diseases. A major focus currently is on some important, still enigmatic, aspects of the ageing neuromuscular system. It is also includes some of the newest references of others on various closely-related aspects of this ageing system. The article may help guide others in their molecular-based endeavors to identify paths leading to discovering new treatments and new pathogenic aspects. These are certainly needed - our ageing and unsteady constituents are steadily increasing. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.
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Envelhecimento , Doenças Neuromusculares , Envelhecimento/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Humanos , Doenças Neuromusculares/diagnóstico , Doenças Neuromusculares/genética , Doenças Neuromusculares/metabolismo , Doenças Neuromusculares/patologia , Doenças Neuromusculares/terapiaRESUMO
Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease in which aging appears to be a key risk factor. In this review we focus on several cellular molecular mechanisms responsible for multiprotein aggregation and accumulations within s-IBM muscle fibers, and their possible consequences. Those include mechanisms leading to: a) accumulation in the form of aggregates within the muscle fibers, of several proteins, including amyloid-ß42 and its oligomers, and phosphorylated tau in the form of paired helical filaments, and we consider their putative detrimental influence; and b) protein misfolding and aggregation, including evidence of abnormal myoproteostasis, such as increased protein transcription, inadequate protein disposal, and abnormal posttranslational modifications of proteins. Pathogenic importance of our recently demonstrated abnormal mitophagy is also discussed. The intriguing phenotypic similarities between s-IBM muscle fibers and the brains of Alzheimer and Parkinson's disease patients, the two most common neurodegenerative diseases associated with aging, are also discussed. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.
Assuntos
Envelhecimento , Encéfalo , Fibras Musculares Esqueléticas , Miosite de Corpos de Inclusão , Agregação Patológica de Proteínas , Deficiências na Proteostase , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Humanos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fragmentos de Peptídeos , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/mortalidadeRESUMO
Sporadic inclusion-body myositis (s-IBM) is a severe, progressive muscle disease for which there is no enduring treatment. Pathologically characteristic are vacuolated muscle fibers having: accumulations of multi-protein aggregates, including amyloid-ß(Aß) 42 and its toxic oligomers; increased γ-secretase activity; and impaired autophagy. Cultured human muscle fibers with experimentally-impaired autophagy recapitulate some of the s-IBM muscle abnormalities, including vacuolization and decreased activity of lysosomal enzymes, accompanied by increased Aß42, Aß42 oligomers, and increased γ-secretase activity. Sodium phenylbutyrate (NaPB) is an orally bioavailable small molecule approved by the FDA for treatment of urea-cycle disorders. Here we describe that NaPB treatment reverses lysosomal dysfunction in an in vitro model of inclusion-body myositis, involving cultured human muscle fibers. NaPB treatment improved lysosomal activity, decreased Aß42 and its oligomers, decreased γ-secretase activity, and virtually prevented muscle-fiber vacuolization. Accordingly, NaPB might be considered a potential treatment of s-IBM patients.
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Peptídeos beta-Amiloides/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Tiazinas/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Análise de Variância , Catepsina D/metabolismo , Técnicas de Cultura de Células , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Compostos de Epóxi/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transfecção , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.
Assuntos
Corantes Fluorescentes/química , Proteínas/química , Tiofenos/química , Proteínas Adaptadoras de Transdução de Sinal/análise , Proteínas Adaptadoras de Transdução de Sinal/química , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Técnicas Biossensoriais , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Ligantes , Microscopia de Fluorescência , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Proteínas/análise , Proteína Sequestossoma-1RESUMO
AIMS: Sporadic inclusion-body myositis (s-IBM) is an age-associated degenerative muscle disease. Characteristic features are muscle-fibre vacuolization and intramuscle-fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone-mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome-associated membrane protein type 2A (LAMP2A) and the heat-shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge. METHODS: We studied CMA components by immunocytochemistry, immunoblots, real-time PCR and immunoprecipitation in: (a) 16 s-IBM, nine aged-matched normal and nine disease control muscle biopsies; and (b) cultured human muscle fibres (CHMFs) with experimentally inhibited activities of either the 26S proteasome or autophagy. RESULTS: Compared with age-matched controls, in s-IBM muscle, LAMP2A and Hsc70 were on a given transverse section accumulated as aggregates in approximately 5% of muscle fibres, where they (a) colocalized with each other and α-synuclein (α-syn), a CMA-targeted protein; and (b) were bound to each other and to α-syn by immunoprecipitation. By immunoblots, LAMP2A was increased sevenfold P < 0.001 and Hsc70 2.6-fold P < 0.05. LAMP2A mRNA was increased 4.4-fold P < 0.001 and Hsc70 mRNA 1.9-fold P < 0.05. In CHMFs inhibition of either the 26S proteasome or autophagy induced CMA, evidenced by a significant increase of both LAMP2A and Hsc70. CONCLUSIONS: Our study demonstrates, for the first time, up-regulation of CMA components in s-IBM muscle, and it provides further evidence that altered protein degradation is likely an important pathogenic aspect in s-IBM.
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Autofagia/fisiologia , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Lisossomos/metabolismo , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Miosite de Corpos de Inclusão/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/metabolismo , Regulação para Cima/fisiologia , alfa-Sinucleína/metabolismoRESUMO
Townes-Brocks syndrome (TBS, OMIM #107480) is a rare autosomal-dominant malformation syndrome with a combination of anal, renal, limb and ear anomalies. Cytogenetic findings suggested that the gene mutated in TBS maps to chromosome 16q12.1, where SALL1 (previously known as HSAL1), a human homologue of spalt (sal), is located. SAL is a developmental regulator in Drosophila melanogaster and is conserved throughout evolution. No phenotype has yet been attributed to mutations in vertebrate sal-like genes. The expression patterns of sal-like genes in mouse, Xenopus and the fish Medaka, and the finding that Medaka sal is regulated by Sonic hedgehog (Shh; ref. 11), prompted us to examine SALL1 as a TBS candidate gene. Here we demonstrate that SALL1 mutations cause TBS in a family with vertical transmission of TBS and in an unrelated family with a sporadic case of TBS. Both mutations are predicted to result in a prematurely terminated SALL1 protein lacking all putative DNA binding domains. TBS therefore represents another human developmental disorder caused by mutations in a putative C2H2 zinc-finger transcription factor.
Assuntos
Anormalidades Múltiplas/genética , Mutação , Fatores de Transcrição/genética , Dedos de Zinco/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Complementar , Orelha Externa/anormalidades , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Polidactilia/genética , Síndrome , Polegar/anormalidadesRESUMO
The muscle-fiber phenotype of sporadic inclusion-body myositis (s-IBM), the most common muscle disease associated with aging, shares several pathological abnormalities with Alzheimer disease (AD) brain, including accumulation of amyloid-ß 42 (Aß42) and its cytotoxic oligomers. The exact mechanisms leading to Aß42 production within s-IBM muscle fibers are not known. Aß42 and Aß40 are generated after the amyloid-precursor protein (AßPP) is cleaved by ß-secretase and the γ-secretase complex. Aß42 is considered more cytotoxic than Aß40, and it has a higher propensity to oligomerize, form amyloid fibrils, and aggregate. Recently, we have demonstrated in cultured human muscle fibers that experimental inhibition of lysosomal enzyme activities leads to Aß42 oligomerization. In s-IBM muscle, we here demonstrate prominent abnormalities of the γ-secretase complex, as evidenced by: a) increase of γ-secretase components, namely active presenilin 1, presenilin enhancer 2, nicastrin, and presence of its mature, glycosylated form; b) increase of mRNAs of these γ-secretase components; c) increase of γ-secretase activity; d) presence of an active form of a newly-discovered γ-secretase activating protein (GSAP); and e) increase of GSAP mRNA. Furthermore, we demonstrate that experimental inhibition of lysosomal autophagic enzymes in cultured human muscle fibers a) activates γ-secretase, and b) leads to posttranslational modifications of AßPP and increase of Aß42. Since autophagy is impaired in biopsied s-IBM muscle, the same mechanism might be responsible for its having increased γ-secretase activity and Aß42 production. Accordingly, improving lysosomal function might be a therapeutic strategy for s-IBM patients.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Músculo Esquelético/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/patologia , Polimiosite/metabolismo , Polimiosite/patologiaRESUMO
Previous studies showed that in the mouse mutant Lis1(GT/GT) gene trap integration in intron 2 of Lis1 gene leads to male infertility in homozygous Lis1(GT/GT) mice. We further analyzed this line and could confirm the suggested downregulation of a testis-specific Lis1 transcript in mutant animals in a quantitative manner. Moreover, we analyzed the gene trap mutation on different genetic backgrounds in incipient congenic animals and could exclude a genetic background effect. To gain further insights into the role and requirement of LIS1 in spermatogenesis, 3 transgenic lines were generated, that overexpress Lis1 under control of the testis-specific promoters hEF-1α, which is exclusively active in spermatogonial cells, PGK2, which is active in pachytene spermatocytes and following stages of spermatogenesis, and Tnp2 which is active in round spermatids and following stages of spermatogenesis, respectively. All 3 transgenic lines remained fertile and testis sections displayed no abnormalities. To overcome the infertility of Lis1(GT/GT) males, these transgenic Lis1-overexpressing animals were mated with Lis1(GT/GT) mice to generate 'rescued' Lis1(GT/GT)/Lis1(Tpos) males. 'Rescued' animals from all transgenic lines remained infertile, thus overexpression of Lis1 in different stages of spermatogenesis could not rescue the infertility phenotype of homozygous gene trap males.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/genética , Perfilação da Expressão Gênica , Proteínas Associadas aos Microtúbulos/genética , Espermatogênese/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Feminino , Fertilidade/genética , Imuno-Histoquímica , Infertilidade Masculina/genética , Rim/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Miocárdio/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatogônias/metabolismo , Testículo/citologia , Testículo/metabolismo , Fatores de TempoRESUMO
The hallmark pathologies of sporadic inclusion-body myositis (s-IBM) muscle fibers are autophagic vacuoles and accumulation of ubiquitin-positive multiprotein aggregates that contain amyloid-beta or phosphorylated tau in a beta-pleated sheet amyloid configuration. Endoplasmic reticulum stress (ERS) and 26S proteasome inhibition, also associated with s-IBM, putatively aggrandize the accumulation of misfolded proteins. However, autophagosomal-lysosomal pathway formation and function, indicated by autophagosome maturation, have not been previously analyzed in this system. Here we studied the autophagosomal-lysosomal pathway using 14 s-IBM and 30 disease control and normal control muscle biopsy samples and our cultured human muscle fibers in a microenvironment modified to resemble aspects of s-IBM pathology. We report for the first time that in s-IBM, lysosomal enzyme activities of cathepsin D and B were decreased 60% (P < 0.01) and 40% (P < 0.05), respectively. We also detected two indicators of increased autophagosome maturation, the presence of LC3-II and decreased mammalian target of rapamycin-mediated phosphorylation of p70S6 kinase. Moreover, in cultured human muscle fibers, ERS induction significantly decreased activities of cathepsins D and B, increased levels of LC3-II, decreased phosphorylation of p70S6 kinase, and decreased expression of VMA21, a chaperone for assembly of lysosomal V-ATPase. We conclude that in s-IBM muscle, decreased lysosomal proteolytic activity might enhance accumulation of misfolded proteins, despite increased maturation of autophagosomes, and that ERS is a possible cause of s-IBM-impaired lysosomal function. Thus, unblocking protein degradation in s-IBM muscle fibers may be a desirable therapeutic strategy.
Assuntos
Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Idoso , Western Blotting , Catepsina B/metabolismo , Catepsina D/metabolismo , Células Cultivadas , Retículo Endoplasmático/patologia , Humanos , Imuno-Histoquímica , Lisossomos/metabolismo , Lisossomos/patologia , Pessoa de Meia-Idade , Miosite de Corpos de Inclusão/patologiaRESUMO
Intra-muscle fiber accumulation of ubiquitinated protein aggregates containing several conformationally modified proteins, including amyloid-ß and phosphorylated tau, is characteristic of the pathologic phenotype of sporadic inclusion-body myositis (s-IBM), the most common progressive degenerative myopathy of older persons. Abnormalities of protein-degradation, involving both the 26S proteasome and autophagic-lysosomal pathways, were previously demonstrated in s-IBM muscle. NBR1 is a ubiquitin-binding scaffold protein importantly participating in autophagic degradation of ubiquitinated proteins. Whereas abnormalities of p62, a ubiquitin-binding protein, were previously described in s-IBM, abnormalities of NBR1 have not been reported in s-IBM. We have now identified in s-IBM muscle biopsies that NBR1, by: (a) immunohistochemistry, was strongly accumulated within s-IBM muscle-fiber aggregates, where it closely co-localized with p62, ubiquitin, and phosphorylated tau; (b) immunoblots, was increased threefold (p < 0.001); and (c) immunoprecipitation, was associated with p62 and LC3. By real-time PCR, NBR1 mRNA was increased twofold (p < 0.01). None of the various disease- and normal-control muscle biopsies had any NBR1 abnormality. In cultured human muscle fibers, NBR1 also physically associated with both p62 and LC3, and experimental inhibition of either the 26S proteasome or the lysosomal activity resulted in NBR1 increase. Our demonstration of NBR1 abnormalities in s-IBM provides further evidence that altered protein degradation pathways may be critically involved in the s-IBM pathogenesis. Accordingly, attempts to unblock defective protein degradation might be a therapeutic strategy for s-IBM patients.
Assuntos
Autofagia/fisiologia , Fibras Musculares Esqueléticas/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biópsia , Estudos de Casos e Controles , Células Cultivadas , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético , Miosite de Corpos de Inclusão/patologia , RNA Mensageiro/metabolismo , Proteína Sequestossoma-1RESUMO
Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Cloreto de Lítio/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Miosite de Corpos de Inclusão/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Precursor de Proteína beta-Amiloide/genética , Estudos de Casos e Controles , Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Imunoprecipitação/métodos , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/metabolismo , Estresse Oxidativo/fisiologia , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma , Isoformas de Proteínas/metabolismo , Treonina/metabolismo , Técnicas de Cultura de TecidosRESUMO
DNA microarray analysis was performed with mouse multipotent adult germline stem cells (maGSCs) and embryonic stem cells (ESCs) from different genetic backgrounds cultured under standard ESC-culture conditions and under differentiation-promoting conditions by the withdrawal of the leukemia inhibitory factor (LIF) and treatment with retinoic acid (RA). The analyzed undifferentiated cell lines are very similar based on their global gene expression pattern and show 97-99% identity dependent on the analyzed background. Only 621 genes are differentially expressed in cells derived from mouse 129SV-background and 72 genes show differences in expression in cells generated from transgenic Stra8-EGFP/Rosa26-LacZ-background. Both maGSCs and ESCs express the same genes involved in the regulation of pluripotency and even show no differences in the expression level of these genes. When comparing maGSCs with previously published signature genes of other pluripotent cell lines, we found that maGSCs shared a very similar gene expression pattern with embryonic germ cells (EGCs). Also after differentiation of maGSCs and ESCs the transcriptomes of the cell lines are nearly identical which suggests that both cell types differentiate spontaneously in a very similar way. This is the first study, at transcriptome level, to compare ESCs and a pluripotent cell line derived from an adult organism (maGSCs).
Assuntos
Células-Tronco Adultas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células-Tronco Multipotentes/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Camundongos , Regulação para CimaRESUMO
Accumulation of amyloid-ß (Aß) within muscle fibers has been considered an upstream step in the development of the s-IBM pathologic phenotype. Aß42, which is considered more cytotoxic than Aß40 and has a higher propensity to oligomerize, is preferentially increased in s-IBM muscle fibers. In Alzheimer disease (AD), low-molecular weight Aß oligomers and toxic oligomers, also referred to as "Aß-Derived Diffusible Ligands" (ADDLs), are considered strongly cytotoxic and proposed to play an important pathogenic role. ADDLs have been shown to be increased in AD brain. We now report for the first time that in s-IBM muscle biopsies Aß-dimer, -trimer, and -tetramer are identifiable by immunoblots. While all the s-IBM samples we studied had Aß-oligomers, their molecular weights and intensity varied between the patient samples. None of the control muscle biopsies had Aß oligomers. Dot-immunoblots using highly specific anti-ADDL monoclonal antibodies also showed highly increased ADDLs in all s-IBM biopsies studied, while controls were negative. By immunofluorescence, in some of the abnormal s-IBM muscle fibers ADDLs were accumulated in the form of plaque-like inclusions, and were often increased diffusely in very small fibers. Normal and disease-controls were negative. By gold-immuno-electron microscopy, ADDL-immunoreactivities were in close proximity to 6-10 nm amyloid-like fibrils, and also were immunodecorating amorphous and floccular material. In cultured human muscle fibers, we found that inhibition of autophagy led to the accumulation of Aß oligomers. This novel demonstration of Aß42 oligomers in s-IBM muscle biopsy provides additional evidence that intra-muscle fiber accumulation of Aß42 oligomers in s-IBM may contribute importantly to s-IBM pathogenic cascade.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Western Blotting , Humanos , Immunoblotting , Imuno-Histoquímica , Microscopia Imunoeletrônica , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/patologiaRESUMO
Sporadic inclusion-body myositis (s-IBM) is the only muscle disease in which accumulation of amyloid-beta (Abeta) in abnormal muscle fibers appears to play a key pathogenic role. Increased amyloid-beta precursor protein (AbetaPP) and Abeta accumulation have been reported to be upstream steps in the development of the s-IBM pathologic phenotype, based on cellular and animal models. Abeta is released from AbetaPP as a 40 or 42 aminoacid peptide. Abeta42 is considered more cytotoxic than Abeta40, and it has a higher propensity to aggregate and form amyloid fibrils. Using highly specific antibodies, we evaluated in s-IBM muscle biopsies intra-muscle fiber accumulation of Abeta40 and Abeta42-immunoreactive aggregates by light- and electron-microscopic immunocytochemistry, and quantified their amounts by ELISA. In s-IBM, 80-90% of the vacuolated muscle fibers and 5-20% of the non-vacuolated muscle fibers contained plaque-like Abeta42-immunoreactive inclusions, while only 69% of those fibers also contained Abeta40 deposits. By immuno-electronmicroscopy, Abeta42 was associated with 6-10 nm amyloid-like fibrils, small electron-dense floccular clumps and larger masses of amorphous material. Abeta40 was present only on small patches of floccular clumps and amorphous material; it was not associated with 6-10 nm amyloid fibrils. By ELISA, in s-IBM muscle biopsies Abeta42 was present in values 8.53-44.7 pg/ml, while Abeta40 was not detectable; normal age-matched control biopsies did not have any detectable Abeta42 or Abeta40. Thus, in s-IBM muscle fibers, Abeta42 is accumulated more than Abeta40. We suggest that Abeta42 oligomers and their cytotoxicity may play an important role in the s-IBM pathogenesis.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Miosite de Corpos de Inclusão/metabolismo , Miosite de Corpos de Inclusão/patologia , Fragmentos de Peptídeos/metabolismo , Biópsia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Humanos , Imuno-Histoquímica , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Microscopia Imunoeletrônica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/ultraestrutura , Miosite de Corpos de Inclusão/etiologiaRESUMO
p62, also known as sequestosome1, is a shuttle protein transporting polyubiquitinated proteins for both the proteasomal and lysosomal degradation. p62 is an integral component of inclusions in brains of various neurodegenerative disorders, including Alzheimer disease (AD) neurofibrillary tangles (NFTs) and Lewy bodies in Parkinson disease. In AD brain, the p62 localized in NFTs is associated with phosphorylated tau (p-tau). Sporadic inclusion-body myositis (s-IBM) is the most common progressive muscle disease associated with aging, and its muscle tissue has several phenotypic similarities to AD brain. Abnormal accumulation of intracellular multiprotein inclusions, containing p-tau in the form of paired helical filaments, amyloid-beta, and several other "Alzheimer-characteristic proteins", is a characteristic feature of the s-IBM muscle fiber phenotype. Diminished proteasomal and lysosomal protein degradation appear to play an important role in the formation of intra-muscle-fiber inclusions. We now report that: (1) in s-IBM muscle fibers, p62 protein is increased on both the protein and the mRNA levels, and it is strongly accumulated within, and as a dense peripheral shell surrounding, p-tau containing inclusions, by both the light- and electron-microscopy. Accordingly, our studies provide a new, reliable, and simple molecular marker of p-tau inclusions in s-IBM muscle fibers. The prominent p62 immunohistochemical positivity and pattern diagnostically distinguish s-IBM from polymyositis and dermatomyositis. (2) In normal cultured human muscle fibers, experimental inhibition of either proteasomal or lysosomal protein degradation caused substantial increase of p62, suggesting that similar in vivo mechanisms might contribute to the p62 increase in s-IBM muscle fibers.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Miosite de Corpos de Inclusão/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Western Blotting , Células Cultivadas , Dermatomiosite/diagnóstico , Diagnóstico Diferencial , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Lisossomos/metabolismo , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/citologia , Miosite de Corpos de Inclusão/diagnóstico , Miosite de Corpos de Inclusão/genética , Fosforilação , Polimiosite/diagnóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Sequestossoma-1 , Proteínas tau/metabolismoRESUMO
Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.
Assuntos
Creatina Quinase/biossíntese , Isoenzimas/biossíntese , Músculos/enzimologia , Fosforilases/biossíntese , Animais , Células Cultivadas , Creatina Quinase/genética , Regulação da Expressão Gênica , Humanos , Isoenzimas/genética , Músculos/inervação , Fosforilases/genética , Ratos , Medula EspinalRESUMO
In 29 acutely psychotic patients (mostly schizophrenic), histochemical abnormalities of a myopathic type were demonstrated in skeletal muscle biopsies from 13 and were generally correlated with elevation of the "muscle" type isoenzymes of creatine phosphokinase in the patients' serum. The incidence was much higher than found in normal controls, hospitalized neurotic psychiatric patients, or parents of acutely psychotic patients. A diazo-coupling type of "alka-line phosphatase" reaction was particularly useful in identifying abnormal muscle fibers.