RESUMEN
Clonal expansion sets the stage for cancer genesis by allowing for the accumulation of molecular alterations. Although genetic mutations such as Tet2 that induce clonal expansion and malignancy have been identified, these mutations are also frequently found in healthy individuals. Here, we tracked preleukemic clonal expansion using genetic barcoding in an inducible Tet2 knockout mouse model and found that only a small fraction of hematopoietic stem cells (HSCs) expanded excessively upon Tet2 knockout. These overexpanded HSCs expressed significantly lower levels of genes associated with leukemia and RNA splicing than nonoverexpanded Tet2 knockout HSCs. Knocking down Rbm25, an identified RNA splicing factor, accelerated the expansion of Tet2-knockout hematopoietic cells in vitro and in vivo. Our data suggest that mutations of an epigenetic factor Tet2 induce variability in the expression of an RNA splicing factor Rbm25, which subsequently drives heterogeneous preleukemic clonal expansion. This heterogeneous clonal expansion could contribute to the variable disease risks across individuals.
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Leucemia , Neoplasias , Factores de Empalme de ARN , Animales , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , ARN , Factores de Empalme de ARN/metabolismoRESUMEN
The objective of this study was to determine the clinical applicability of maternal intrapelvic area (PA) and selected morphometric parameters that can be measured before parturition in predicting dystocia in dairy heifers. The measurements were performed in 374 late-gestation Holstein-Friesian heifers. Inner pelvic height and width were measured using a pelvimeter, and PA was calculated. The heifers were monitored continuously around the time of calving, and calving difficulty was categorized as: unassisted calving (UC), slight assistance (SA), considerable difficulty (CD) and veterinary assistance (VA). Calving performance was analysed with the χ2 test, and the effect of body dimensions on the course of parturition was evaluated by one-way analysis of variance. Dystocia (CD + VA) was predicted with the use of the classification tree method. Dystocia accounted for 29.14% of all deliveries. The percentages of stillbirths and retained placenta increased (p < .01) with increasing calving difficulty. Average PA immediately before parturition was smaller (p < .01) in group VA (223.2 cm2 ) than in group UC (253.3 cm2 ). According to the classification tree, dystocia may occur (74.07% odds) in heifers with PA < 254.2 cm2 and a rump angle <5.68° before parturition. Measurements of heifer's cannon circumference and sire's body size improve the accuracy of dystocia prediction.
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Enfermedades de los Bovinos , Distocia , Embarazo , Animales , Bovinos , Femenino , Distocia/veterinaria , Parto , Pelvis , Peso al NacerRESUMEN
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.
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Envejecimiento , Encéfalo , Fibras Musculares Esqueléticas , Miositis por Cuerpos de Inclusión , Agregación Patológica de Proteínas , Deficiencias en la Proteostasis , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides , Animales , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Fragmentos de Péptidos , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/mortalidadRESUMEN
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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Péptidos beta-Amiloides/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Tiazinas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Análisis de Varianza , Catepsina D/metabolismo , Técnicas de Cultivo de Célula , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Compuestos Epoxi/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Transfección , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Individual hematopoietic stem cells (HSCs) produce different amounts of blood cells upon transplantation. Taking advantage of the intercellular variation, we developed an experimental and bioinformatic approach to evaluating the quantitative association between gene expression and blood cell production across individual HSCs. We found that most genes associated with blood production exhibit the association only at some levels of blood production. By mapping gene expression with blood production, we identified four distinct patterns of their quantitative association. Some genes consistently correlate with blood production over a range of levels or across all levels, and these genes are found to regulate lymphoid but not myeloid production. Other genes exhibit one or more clear peaks of association. Genes with overlapping peaks are found to be coexpressed in other tissues and share similar molecular functions and regulatory motifs. By dissecting intercellular variations, our findings revealed four quantitative association patterns that reflect distinct dose-response molecular mechanisms modulating the blood cell production of HSCs.
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Células Sanguíneas , Células Madre Hematopoyéticas , Ratones , Animales , Células Madre Hematopoyéticas/metabolismo , Expresión Génica , Diferenciación CelularRESUMEN
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.
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Colorantes Fluorescentes/química , Proteínas/química , Tiofenos/química , Proteínas Adaptadoras Transductoras de Señales/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Técnicas Biosensibles , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Ligandos , Microscopía Fluorescente , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Proteínas/análisis , Proteína Sequestosoma-1RESUMEN
After transplantation, hematopoietic stem cells (HSCs) sustain blood cell regeneration throughout the patient's life. Recent studies suggest that several types of mature blood cells provide feedback signals to regulate HSC fate. However, the potential feedback effect of hematopoietic progenitor cells has not been characterized to date. The present investigation demonstrated that multipotent progenitors (MPPs) promoted T cell production of HSCs when both cell types were cotransplanted in mice. Using genetic barcodes to track individual HSCs in mice, we found that the increased T cell production by HSCs was associated with the combined effects of altered lineage bias and clonal expansion during HSC differentiation. We showed that MPP and HSC co-transplantation promoted the multilineage differentiation of HSCs in the short term while preserving lymphoid-specialized HSC differentiation in the long term. Our findings indicate that MPPs derived from HSCs regulate the fate of HSCs after bone marrow transplantation.
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Células Madre Hematopoyéticas , Células Madre Multipotentes , Animales , Ratones , Diferenciación Celular/genética , Linaje de la Célula/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Multipotentes/metabolismo , Linfocitos TRESUMEN
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.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Músculo Esquelético/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Humanos , Persona de Mediana Edad , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/patología , Polimiositis/metabolismo , Polimiositis/patologíaRESUMEN
Insect farming is growing in popularity, and in addition to insect meal, it generates waste products such as exuviae and frass, which can be recycled in agriculture. The aim of this incubation experiment was to evaluate the effect of Tenebrio molitor L. frass on selected chemical and biological properties of deacidified peat, which is widely used in horticulture. The optimal rate of frass fertilizer in peat for growing vegetables and ornamental plants was determined, with special emphasis on mineral nitrogen levels. Peat was fertilized with five nitrogen rates, 0, 50, 100, 200, and 400 mg dm-3, and supplied with frass or urea. The study demonstrated that frass can be used as organic fertilizer. An increase in the nitrogen rate significantly increased mineral nitrogen content and electrical conductivity and decreased Ca content in peat. Both frass and urea increased the ammonification rate at the beginning of incubation and the nitrification rate from the second week of the experiment. Higher frass rates (5 and 10 g dm-3) increased the content of plant-available nutrients (nitrogen, phosphorus, magnesium, potassium, and sodium) in peat as well as the abundance of microorganisms supporting organic matter mineralization. Unlike frass, urea increased the counts of nitrogen-fixing bacteria in peat.
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Suelo , Tenebrio , Animales , Suelo/química , Fertilizantes , Horticultura , Minerales , Plantas , Urea , NitrógenoRESUMEN
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.
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Autofagia/fisiología , Retículo Endoplásmico/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Anciano , Western Blotting , Catepsina B/metabolismo , Catepsina D/metabolismo , Células Cultivadas , Retículo Endoplásmico/patología , Humanos , Inmunohistoquímica , Lisosomas/metabolismo , Lisosomas/patología , Persona de Mediana Edad , Miositis por Cuerpos de Inclusión/patologíaRESUMEN
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.
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Autofagia/fisiología , Fibras Musculares Esqueléticas/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anciano , Anciano de 80 o más Años , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Fibras Musculares Esqueléticas/patología , Músculo Esquelético , Miositis por Cuerpos de Inclusión/patología , ARN Mensajero/metabolismo , Proteína Sequestosoma-1RESUMEN
Cellular heterogeneity is a major cause of treatment resistance in cancer. Despite recent advances in single-cell genomic and transcriptomic sequencing, it remains difficult to relate measured molecular profiles to the cellular activities underlying cancer. Here, we present an integrated experimental system that connects single cell gene expression to heterogeneous cancer cell growth, metastasis, and treatment response. Our system integrates single cell transcriptome profiling with DNA barcode based clonal tracking in patient-derived xenograft models. We show that leukemia cells exhibiting unique gene expression respond to different chemotherapies in distinct but consistent manners across multiple mice. In addition, we uncover a form of leukemia expansion that is spatially confined to the bone marrow of single anatomical sites and driven by cells with distinct gene expression. Our integrated experimental system can interrogate the molecular and cellular basis of the intratumoral heterogeneity underlying disease progression and treatment resistance.
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Análisis de la Célula Individual/métodos , Transcriptoma/genética , Animales , Adhesión Celular/genética , Adhesión Celular/fisiología , Células Cultivadas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Código de Barras del ADN Taxonómico , Humanos , Ratones , Análisis de Secuencia de ARNRESUMEN
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.
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Precursor de Proteína beta-Amiloide/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Cloruro de Litio/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Miositis por Cuerpos de Inclusión/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Anciano , Anciano de 80 o más Años , Precursor de Proteína beta-Amiloide/genética , Estudios de Casos y Controles , Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Glucógeno Sintasa Quinasa 3 beta , Humanos , Inmunoprecipitación/métodos , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/metabolismo , Estrés Oxidativo/fisiología , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Inhibidores de Proteasoma , Isoformas de Proteínas/metabolismo , Treonina/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
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.
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Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Western Blotting , Humanos , Immunoblotting , Inmunohistoquímica , Microscopía Inmunoelectrónica , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/patologíaRESUMEN
Embedded viral barcoding in combination with high-throughput sequencing is a powerful technology with which to track single-cell clones. It can provide clonal-level insights into cellular proliferation, development, differentiation, migration, and treatment efficacy. Here, we present a detailed protocol for a viral barcoding procedure that includes the creation of barcode libraries, the viral delivery of barcodes, the recovery of barcodes, and the computational analysis of barcode sequencing data. The entire procedure can be completed within a few weeks. This barcoding method requires cells to be susceptible to viral transduction. It provides high sensitivity and throughput, and enables precise quantification of cellular progeny. It is cost efficient and does not require any advanced skills. It can also be easily adapted to many types of applications, including both in vitro and in vivo experiments.
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Rastreo Celular/métodos , Células Clonales/citología , Código de Barras del ADN Taxonómico/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Proliferación Celular/genética , ADN/genética , Vectores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , RatonesRESUMEN
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.
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Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/metabolismo , Miositis por Cuerpos de Inclusión/patología , Fragmentos de Péptidos/metabolismo , Biopsia , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Microscopía Inmunoelectrónica , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/ultraestructura , Miositis por Cuerpos de Inclusión/etiologíaRESUMEN
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.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Miositis por Cuerpos de Inclusión/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Anciano , Anciano de 80 o más Años , Biomarcadores , Western Blotting , Células Cultivadas , Dermatomiositis/diagnóstico , Diagnóstico Diferencial , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Lisosomas/metabolismo , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Fibras Musculares Esqueléticas/citología , Miositis por Cuerpos de Inclusión/diagnóstico , Miositis por Cuerpos de Inclusión/genética , Fosforilación , Polimiositis/diagnóstico , Complejo de la Endopetidasa Proteasomal/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Sequestosoma-1 , Proteínas tau/metabolismoRESUMEN
BACKGROUND: The rate of lipogenesis and gene expression of lipogenic enzymes in white adipose tissue (WAT) of rats decreases with age. Previously, we showed the inverse relationship between serum leptin concentration and lipogenic enzymes activities in WAT of ageing rats. Based on those results, we postulated that leptin could play some role in the downregulation of lipogenic enzyme genes expression in WAT of old rats. OBJECTIVE: To further test this hypothesis, in the present paper we examined the effect of surgical removal of perirenal and epididymal WAT on serum leptin concentration and lipogenic enzymes activities in remnant adipose tissue of old rats. METHODS: One-year-old rats, which display a high serum leptin concentration and low lipogenic enzyme activities in WAT, were subjected to surgical removal of the perirenal and epididymal WAT. Two months after the surgery, the serum leptin concentration and lipogenic enzyme activities in remnant WAT were compared to the serum leptin concentration and lipogenic enzyme activities measured in adipose tissue collected during the surgery. RESULTS: WAT removal resulted in an approximately 6-fold decrease of serum leptin concentration and about a 2-fold increase of lipogenic enzyme activities in remnant adipose tissue. CONCLUSIONS: These results indicate that interventions designed to reduce abdominal adipose tissue mass can change the lipogenic activity of remnant adipose tissue. Additionally, leptin could be one of the factors contributing to the downregulation of the genes expression of lipogenic enzymes during ageing in rats.
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Tejido Adiposo Blanco/enzimología , Envejecimiento/sangre , Envejecimiento/metabolismo , Leptina/sangre , Lipogénesis/fisiología , Grasa Abdominal/enzimología , Grasa Abdominal/fisiología , Grasa Abdominal/cirugía , Tejido Adiposo Blanco/fisiología , Tejido Adiposo Blanco/cirugía , Envejecimiento/genética , Animales , Glucemia/metabolismo , Regulación hacia Abajo , Epidídimo/fisiología , Epidídimo/cirugía , Insulina/sangre , Riñón/fisiología , Riñón/cirugía , Lipogénesis/genética , Masculino , Modelos Biológicos , Ratas , Ratas WistarRESUMEN
Sporadic inclusion-body myositis (s-IBM) is the most common muscle disease of older persons. The muscle-fiber molecular phenotype exhibits similarities to both Alzheimer-disease (AD) and Parkinson-disease (PD) brains, including accumulations of amyloid-beta, phosphorylated tau, alpha-synuclein, and parkin, as well as evidence of oxidative stress and mitochondrial abnormalities. Early-onset autosomal-recessive PD can be caused by mutations in the DJ-1 gene, leading to its inactivation. DJ-1 has antioxidative and mitochondrial-protective properties. In AD and PD brains, DJ-1 is increased and oxidized. We studied DJ-1 in 17 s-IBM and 18 disease-control and normal muscle biopsies by: (1) immunoblots of muscle homogenates and mitochondrial fractions; (2) real-time PCR; (3) oxyblots evaluating DJ-1 oxidation; (4) light- and electron-microscopic immunocytochemistry. Compared to controls, in s-IBM muscle fibers DJ-1 was: (a) increased in the soluble fraction, monomer 2-fold (P = 0.01), and dimer 2.8-fold (P = 0.004); (b) increased in the mitochondrial fraction; (c) highly oxidized; and (d) aggregated in about 15% of the abnormal muscle fibers. DJ-1 mRNA was increased 3.5-fold (P = 0.034). Accordingly, DJ-1 might play a role in human muscle disease, and thus not be limited to human CNS degenerations. In s-IBM muscle fibers, DJ-1 could be protecting these fibers against oxidative stress, including protection of mitochondria.