Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 19 de 19
Filter
Add more filters










Publication year range
1.
J Immunol ; 197(6): 2269-79, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27511731

ABSTRACT

ESET/SETDB1, one of the major histone methyltransferases, catalyzes histone 3 lysine 9 (H3K9) trimethylation. ESET is critical for suppressing expression of retroviral elements in embryonic stem cells; however, its role in the immune system is not known. We found that thymocyte-specific deletion of ESET caused impaired T cell development, with CD8 lineage cells being most severely affected. Increased apoptosis of CD8 single-positive cells was observed, and TCR-induced ERK activation was severely inhibited in ESET(-/-) thymocytes. Genome-wide comprehensive analysis of mRNA expression and H3K9 trimethylation revealed that ESET regulates expression of numerous genes in thymocytes. Among them, FcγRIIB, whose signaling can inhibit ERK activation, was strongly and ectopically expressed in ESET(-/-) thymocytes. Indeed, genetic depletion of FcγRIIB in ESET(-/-) thymocytes rescued impaired ERK activation and partially restored defective positive selection in ESET(-/-) mice. Therefore, impaired T cell development in ESET(-/-) mice is partly due to the aberrant expression of FcγRIIB. Collectively, to our knowledge, we identify ESET as the first trimethylated H3K9 histone methyltransferase playing a crucial role in T cell development.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Gene Expression Regulation , Histone-Lysine N-Methyltransferase/metabolism , Animals , Apoptosis , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Differentiation/genetics , Embryonic Stem Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/immunology , Extracellular Signal-Regulated MAP Kinases/metabolism , Genome , Histone-Lysine N-Methyltransferase/deficiency , Histones/metabolism , Lysine/metabolism , Methylation , Mice , Promoter Regions, Genetic , Receptors, IgG/genetics , Receptors, IgG/metabolism , Thymocytes/immunology , Thymocytes/physiology
2.
Mol Genet Metab Rep ; 4: 25-9, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26937406

ABSTRACT

Hypomyelination in developing brain is often accompanied by congenital metabolic disorders. Menkes kinky hair disease is an X-linked neurodegenerative disease of impaired copper transport, resulting from a mutation of the Menkes disease gene, a transmembrane copper-transporting p-type ATPase gene (ATP7A). In a macular mutant mouse model, the murine ortholog of Menkes gene (mottled gene) is mutated, and widespread neurodegeneration and subsequent death are observed. Although some biochemical analysis of myelin protein in macular mouse has been reported, detailed histological study of myelination in this mouse model is currently lacking. Since myelin abnormality is one of the neuropathologic findings of human Menkes disease, in this study early myelination in macular mouse brain was evaluated by immunohistochemistry. Two-week-old macular mice and normal littermates were perfused with 4% paraformaldehyde. Immunohistochemical staining of paraffin embedded and vibratome sections was performed using antibodies against either CNPase, cleaved caspase-3 or O4 (marker of immature oligodendrocytes). This staining showed that cerebral myelination in macular mouse was generally hypoplastic and that hypomyelination was remarkable in internal capsule, corpus callosum, and cingulate cortex. In addition, an increased number of cleaved caspase-3 positive cells were observed in corpus callosum and internal capsule. Copper deficiency induced by low copper diet has been reported to induce oligodendrocyte dysfunction and leads to hypomyelination in this mouse model. Taken together, hypomyelination observed in this study in a mouse model of Menkes disease is assumed to be induced by increased apoptosis of immature oligodendrocytes in developing cerebrum, through deficient intracellular copper metabolism.

3.
J Natl Cancer Inst ; 104(22): 1750-64, 2012 Nov 21.
Article in English | MEDLINE | ID: mdl-23150719

ABSTRACT

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome is a hereditary hamartoma syndrome that predisposes patients to develop hair follicle tumors, lung cysts, and kidney cancer. Genetic studies of BHD patients have uncovered the causative gene, FLCN, but its function is incompletely understood. METHODS: Mice with conditional alleles of FLCN and/or peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A), a transcriptional coactivator that regulates mitochondrial biogenesis, were crossbred with mice harboring either muscle creatine kinase (CKM) -Cre or myogenin (MYOG) -Cre transgenes to knock out FLCN and/or PPARGC1A in muscle, or cadherin 16 (CDH16)- Cre transgenes to knock out FLCN and/or PPARGC1A in kidney. Real-time polymerase chain reaction, immunoblotting, electron microscopy, and metabolic profiling assay were performed to evaluate mitochondrial biogenesis and function in muscle. Immunoblotting, electron microscopy, and histological analysis were used to investigate expression and the pathological role of PPARGC1A in FLCN-deficient kidney. Real-time polymerase chain reaction, oxygen consumption measurement, and flow cytometry were carried out using a FLCN-null kidney cancer cell line. All statistical analyses were two-sided. RESULTS: Muscle-targeted FLCN knockout mice underwent a pronounced metabolic shift toward oxidative phosphorylation, including increased mitochondrial biogenesis (FLCN ( f/f ) vs FLCN ( f/f ) /CKM-Cre: % mitochondrial area mean = 7.8% vs 17.8%; difference = 10.0%; 95% confidence interval = 5.7% to 14.3%; P < .001), and the observed increase in mitochondrial biogenesis was PPARGC1A dependent. Reconstitution of FLCN-null kidney cancer cells with wild-type FLCN suppressed mitochondrial metabolism and PPARGC1A expression. Kidney-targeted PPARGC1A inactivation partially rescued the enlarged kidney phenotype and abrogated the hyperplastic cells observed in the FLCN-deficient kidney. CONCLUSION: FLCN deficiency and subsequent increased PPARGC1A expression result in increased mitochondrial function and oxidative metabolism as the source of cellular energy, which may give FLCN-null kidney cells a growth advantage and drive hyperplastic transformation.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Kidney Neoplasms/metabolism , Kidney Neoplasms/pathology , Muscles/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Birt-Hogg-Dube Syndrome/genetics , Blotting, Western , Cell Line, Tumor , Cell Transformation, Neoplastic/genetics , Flow Cytometry , Fluorescent Antibody Technique , Humans , Mice , Mice, Knockout , Microscopy, Electron , Mitochondria/metabolism , Mitochondrial Turnover , Muscles/pathology , Oxidation-Reduction , Oxygen Consumption , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Phosphorylation , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/genetics , Real-Time Polymerase Chain Reaction , Trans-Activators/metabolism , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics
4.
PLoS One ; 5(12): e15239, 2010 Dec 13.
Article in English | MEDLINE | ID: mdl-21179212

ABSTRACT

PGC-1α is a transcriptional co-activator that plays a central role in the regulation of energy metabolism. Our interest in this protein was driven by its ability to promote muscle remodeling. Conversion from fast glycolytic to slow oxidative fibers seemed a promising therapeutic approach in Pompe disease, a severe myopathy caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA) which is responsible for the degradation of glycogen. The recently approved enzyme replacement therapy (ERT) has only a partial effect in skeletal muscle. In our Pompe mouse model (KO), the poor muscle response is seen in fast but not in slow muscle and is associated with massive accumulation of autophagic debris and ineffective autophagy. In an attempt to turn the therapy-resistant fibers into fibers amenable to therapy, we made transgenic KO mice expressing PGC-1α in muscle (tgKO). The successful switch from fast to slow fibers prevented the formation of autophagic buildup in the converted fibers, but PGC-1α failed to improve the clearance of glycogen by ERT. This outcome is likely explained by an unexpected dramatic increase in muscle glycogen load to levels much closer to those observed in patients, in particular infants, with the disease. We have also found a remarkable rise in the number of lysosomes and autophagosomes in the tgKO compared to the KO. These data point to the role of PGC-1α in muscle glucose metabolism and its possible role as a master regulator for organelle biogenesis - not only for mitochondria but also for lysosomes and autophagosomes. These findings may have implications for therapy of lysosomal diseases and other disorders with altered autophagy.


Subject(s)
Glycogen Storage Disease Type II/metabolism , Lysosomes/metabolism , Muscle, Skeletal/metabolism , Trans-Activators/genetics , Trans-Activators/physiology , Animals , Autophagy , Disease Models, Animal , Glucose/metabolism , Glycogen/metabolism , Golgi Apparatus/metabolism , Humans , Mice , Mice, Knockout , Mice, Transgenic , Muscles/cytology , Muscles/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Transcription Factors
5.
Autophagy ; 6(8): 1078-89, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20861693

ABSTRACT

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50­60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.


Subject(s)
Autophagy , Enzyme Replacement Therapy , Glycogen Storage Disease Type II/therapy , alpha-Glucosidases/therapeutic use , Animals , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Disease Models, Animal , Glycogen/metabolism , Glycogen Storage Disease Type II/pathology , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Integrases/metabolism , Mice , Muscle Fibers, Fast-Twitch/enzymology , Muscle Fibers, Fast-Twitch/pathology , Muscle Fibers, Fast-Twitch/ultrastructure , Myosin Light Chains/metabolism , Phosphorylation , Ubiquitin/metabolism , alpha-Glucosidases/deficiency , alpha-Glucosidases/metabolism
6.
Hum Mol Genet ; 19(4): 684-96, 2010 Feb 15.
Article in English | MEDLINE | ID: mdl-19959526

ABSTRACT

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.


Subject(s)
Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/physiopathology , Glycogen/biosynthesis , Muscle, Skeletal/physiopathology , Animals , Disease Models, Animal , Female , Glucose/metabolism , Glycogen Storage Disease Type II/enzymology , Glycogen Storage Disease Type II/therapy , Glycogen Synthase/genetics , Glycogen Synthase/metabolism , Humans , Lysosomes/genetics , Lysosomes/metabolism , Male , Mice , Mice, Knockout , Muscle, Skeletal/metabolism , alpha-Glucosidases/genetics , alpha-Glucosidases/metabolism
7.
Autophagy ; 5(5): 729-31, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19571661

ABSTRACT

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology-massive accumulation of autophagic debris-that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations-a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen containing lysosomes-raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.


Subject(s)
Glycogen Storage Disease Type II/pathology , Animals , Disease Models, Animal , Humans , Lysosomes/pathology , Mice , Muscle Fibers, Skeletal/pathology
8.
Mol Genet Metab ; 96(4): 208-17, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19167256

ABSTRACT

Lysosomes filled with glycogen are a major pathologic feature of Pompe disease, a fatal myopathy and cardiomyopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase (GAA). To facilitate studies germane to this genetic disorder, we developed two in vitro Pompe models: myotubes derived from cultured primary myoblasts isolated from Pompe (GAA KO) mice, and myotubes derived from primary myoblasts of the same genotype that had been transduced with cyclin-dependent kinase 4 (CDK4). This latter model is endowed with extended proliferative capacity. Both models showed extremely large alkalinized, glycogen-filled lysosomes as well as impaired trafficking to lysosomes. Although both Pompe tissue culture models were derived from fast muscles and were fast myosin positive, they strongly resemble slow fibers in terms of their pathologic phenotype and their response to therapy with recombinant human GAA (rhGAA). Autophagic buildup, a hallmark of Pompe disease in fast muscle fibers, was absent, but basal autophagy was functional. To evaluate substrate deprivation as a strategy to prevent the accumulation of lysosomal glycogen, we knocked down Atg7, a gene essential for autophagosome formation, via siRNA, but we observed no effect on the extent of glycogen accumulation, thus confirming our recent observation in autophagy-deficient Pompe mice [N. Raben, V. Hill, L. Shea, S. Takikita, R. Baum, N. Mizushima, E. Ralston, P. Plotz, Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease, Hum. Mol. Genet. 17 (2008) 3897-3908] that macroautophagy is not the major route of glycogen transport to lysosomes. The in vitro Pompe models should be useful in addressing fundamental questions regarding the pathway of glycogen to the lysosomes and testing panels of small molecules that could affect glycogen biosynthesis or speed delivery of the replacement enzyme to affected lysosomes.


Subject(s)
Glycogen Storage Disease Type II/pathology , Glycogen Storage Disease Type II/therapy , Muscle Cells/pathology , Animals , Autophagy , Autophagy-Related Protein 7 , Cathepsin B/metabolism , Cathepsin D/metabolism , Cells, Cultured , Cyclin-Dependent Kinase 4/metabolism , Disease Models, Animal , Endocytosis , Humans , Hydrogen-Ion Concentration , Lysosomes/pathology , Mice , Microtubule-Associated Proteins/metabolism , Muscle Fibers, Skeletal/enzymology , Muscle Fibers, Skeletal/pathology , Myoblasts/enzymology , Myoblasts/pathology , RNA, Small Interfering/metabolism , Transduction, Genetic , alpha-Glucosidases/deficiency , alpha-Glucosidases/metabolism
9.
Autophagy ; 5(1): 111-3, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19001870

ABSTRACT

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, which results from a deficiency of the glycogen-degrading lysosomal hydrolase acid-alpha glucosidase (GAA). Skeletal and cardiac muscle are most severely affected by the progressive expansion of glycogen-filled lysosomes. In both humans and an animal model of the disease (GAA KO), skeletal muscle pathology also involves massive accumulation of autophagic vesicles and autophagic buildup in the core of myofibers, suggesting an induction of autophagy. Only when we suppressed autophagy in the skeletal muscle of the GAA KO mice did we realize that the excess of autophagy manifests as a functional deficiency. This failure of productive autophagy is responsible for the accumulation of potentially toxic aggregate-prone ubiquitinated proteins, which likely cause profound muscle damage in Pompe mice. Also, by generating muscle-specific autophagy-deficient wild-type mice, we were able to analyze the role of autophagy in healthy skeletal muscle.


Subject(s)
Autophagy , Glycogen Storage Disease Type II/pathology , Muscle, Skeletal/pathology , Animals , Glycogen Storage Disease Type II/enzymology , Humans , Mice , Mice, Knockout , Muscle, Skeletal/enzymology , Muscle, Skeletal/ultrastructure , Organ Specificity , alpha-Glucosidases/deficiency , alpha-Glucosidases/metabolism
10.
Hum Mol Genet ; 17(24): 3897-908, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-18782848

ABSTRACT

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.


Subject(s)
Autophagy , Glycogen Storage Disease Type II/pathology , Muscle, Skeletal/pathology , Muscular Diseases/etiology , Muscular Diseases/pathology , Proteins/adverse effects , Proteins/metabolism , Ubiquitination , Animals , Autophagy/genetics , Female , Glycogen Storage Disease Type II/enzymology , Glycogen Storage Disease Type II/genetics , Male , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/enzymology , Muscle, Skeletal/metabolism , Muscular Diseases/metabolism , Proteins/genetics , Ubiquitination/genetics , alpha-Glucosidases/deficiency , alpha-Glucosidases/genetics
11.
Hum Mol Genet ; 17(24): 3876-86, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-18782850

ABSTRACT

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.


Subject(s)
Genetic Therapy , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/therapy , Glycogen/biosynthesis , Glycogen/genetics , RNA Interference/physiology , Animals , Animals, Newborn , Cell Line , Dependovirus/genetics , Genetic Vectors/administration & dosage , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/genetics , Glycogen Storage Disease Type II/enzymology , Glycogen Synthase/antagonists & inhibitors , Glycogen Synthase/genetics , Glycoproteins/antagonists & inhibitors , Glycoproteins/genetics , Humans , Mice , Mice, Knockout
12.
Autophagy ; 3(6): 546-52, 2007.
Article in English | MEDLINE | ID: mdl-17592248

ABSTRACT

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.


Subject(s)
Autophagy/physiology , Glycogen Storage Disease Type II/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/metabolism , Adolescent , Adult , Autophagy/genetics , Biomarkers/metabolism , Cell Line, Transformed , Cell Transformation, Viral , Child , Glycogen Storage Disease Type II/genetics , Glycogen Storage Disease Type II/pathology , Heterozygote , Histocytochemistry , Humans , Lysosomal-Associated Membrane Protein 2 , Lysosomal Membrane Proteins/metabolism , Lysosomes/metabolism , Microscopy, Confocal , Microtubule-Associated Proteins/metabolism , Middle Aged , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/pathology , Myoblasts/metabolism
13.
J Neurochem ; 97(3): 641-51, 2006 May.
Article in English | MEDLINE | ID: mdl-16515539

ABSTRACT

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dually functional protein, acting both as a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. L-PGDS is expressed in oligodendrocytes (OLs) in the central nervous system and is up-regulated in OLs of the twitcher mouse, a model of globoid cell leukodystrophy (Krabbe's disease). We investigated whether up-regulation of L-PGDS is either unique to Krabbe's disease or is a more generalized phenomenon in lysosomal storage disorders (LSDs), using LSD mouse models of Tay-Sachs disease, Sandhoff disease, GM1 gangliosidosis and Niemann-Pick type C1 disease. Quantitative RT-PCR revealed that L-PGDS mRNA was up-regulated in the brains of all these mouse models. In addition, strong L-PGDS immunoreactivity was observed in OLs, but not in either astrocytes or microglia in these models. Thus, up-regulation of L-PGDS appears to be a common response of OLs in LSDs. Moreover, surface plasmon resonance analyses revealed that L-PGDS binds GM1 and GM2 gangliosides, accumulated in neurons in the course of LSD, with high affinities (KD = 65 and 210 nm, respectively). This suggests that L-PGDS may play a role in scavenging harmful lipophilic substrates in LSD.


Subject(s)
Gangliosides/metabolism , Intramolecular Oxidoreductases/metabolism , Lysosomal Storage Diseases/metabolism , Lysosomal Storage Diseases/pathology , Oligodendroglia/metabolism , Up-Regulation/physiology , Animals , Disease Models, Animal , Dose-Response Relationship, Drug , Gangliosides/classification , Immunohistochemistry/methods , In Situ Hybridization/methods , Intracellular Signaling Peptides and Proteins , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/pharmacokinetics , Lectins , Lipocalins , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Niemann-Pick C1 Protein , Oligodendroglia/drug effects , Proteins/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Surface Plasmon Resonance/methods , Time Factors , beta-Galactosidase/deficiency , beta-N-Acetylhexosaminidases/classification , beta-N-Acetylhexosaminidases/deficiency
14.
Congenit Anom (Kyoto) ; 45(1): 9-13, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15737125

ABSTRACT

The study presented here investigated the pathogenetic relationship among different types of neuronal migration disorders occurring simultaneously in the brain using an experimental model induced by ibotenate in hamsters. In the cerebral cortex, abnormal neuronal arrangement was induced 1 day after ibotenate injection. This brain lesion resulted in microgyria in the rostral portion, focal subcortical heterotopia in the mid-portion, and focal subependymal heterotopia in the caudal portion in the same specimen. Vimentin-immunoreactive radial glial fibers were lacking in the area of disorganized neuronal arrangement, but were detected around the microgyria and the intermediate zone surrounding focal subcortical heterotopia. The focal subependymal heterotopia did not include radial glial elements. Glial fibrillary acidic protein (GFAP)-positive glial reaction was weak in these cortical lesions. We suggest that the occurrence of each type of migration disorder depends on the depth of the cortical lesion, that is, the production of microgyria, focal subcortical heterotopia and focal subependymal heterotopia are closely related to the lesions including the cortical plate, subplate and ventricular zone, respectively.


Subject(s)
Cerebral Cortex/drug effects , Cerebral Cortex/pathology , Ibotenic Acid/toxicity , Animals , Animals, Newborn , Cerebral Cortex/injuries , Cerebral Cortex/metabolism , Cricetinae , Glial Fibrillary Acidic Protein/metabolism , Ibotenic Acid/administration & dosage , Neuroglia/drug effects , Neuroglia/pathology , Neurons/drug effects , Neurons/pathology , Receptors, N-Methyl-D-Aspartate/agonists , Vimentin/metabolism
15.
J Neuropathol Exp Neurol ; 63(7): 721-34, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15290897

ABSTRACT

The Twitcher mouse (twi/twi) has been widely used as an animal model of globoid cell leukodystrophy (GLD; Krabbe disease), a hereditary leukodystrophy due to genetic galactosylceramidase deficiency. Recently, we generated a new mouse model of late-onset, chronic GLD (SAP-A-/- mice) by introducing a mutation (C106F) in the saposin A domain of the sphingolipid activator protein gene. Comparative study of SAP-A-/- and twi/twi mice revealed delay in the onset of neurological symptoms in SAP-A-/- mice (90 days vs 20 to 25 days), milder symptoms, and prolonged average survival (134.4 +/- 29.1 days vs 47.5 +/- 3.9 days). However, in both, the earliest sites of demyelination and macrophage infiltration were in regions of the 8th nerve and the spinal tract of the 5th nerve and spinal-cord, where macrophages could be detected as early as day 30 in asymptomatic SAP-A-/- mice. Furthermore, spacio-temporal pattern of demyelination/macrophage infiltration and the extent of neuropathology at the terminal stage are closely similar in both. These results suggest that peripheral macrophages are readily accessible in these sites and participate in the demyelinating process in the central nervous system.


Subject(s)
Central Nervous System/pathology , Galactosylceramidase/deficiency , Glycoproteins/deficiency , Leukodystrophy, Globoid Cell/pathology , Nerve Degeneration/pathology , Age of Onset , Animals , Astrocytes/metabolism , Astrocytes/pathology , Blood-Brain Barrier/physiology , Central Nervous System/metabolism , Central Nervous System/physiopathology , Chemotaxis, Leukocyte/genetics , Demyelinating Diseases/genetics , Demyelinating Diseases/pathology , Demyelinating Diseases/physiopathology , Disease Models, Animal , Female , Glycoproteins/genetics , Glycoproteins/metabolism , Leukodystrophy, Globoid Cell/genetics , Leukodystrophy, Globoid Cell/physiopathology , Macrophages/metabolism , Male , Mice , Mice, Knockout , Mice, Neurologic Mutants , Nerve Degeneration/metabolism , Oligodendroglia/metabolism , Oligodendroglia/pathology , Protein Structure, Tertiary/genetics , Saposins , Sphingolipid Activator Proteins , Survival Rate
16.
J Neuropathol Exp Neurol ; 63(6): 660-73, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15217094

ABSTRACT

Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral lipid storage disease caused by a loss of NPCI function, which results in perturbation of intracellular cholesterol transport. In BALB/c npc(nih) mice, the murine ortholog of NPCI gene is mutated. In NPC mouse, hypomyelination is conspicuous in the cerebral white matter and corpus callosum in addition to neuronal storage. However, the pathogenesis on hypomyelination is not well elucidated. We hypothesized that the hypomyelination in NPC mice resulted from either defective differentiation of oligodendrocyte lineage cells or a failure of proper axon-glial interaction. Myelin basic protein immunohistochemistry disclosed severe hypomyelination of cerebral cortex as well. NG2- or O4-positive progenitor cells and premyelinating oligodendrocytes (OLs) were abundant. However, pi-glutathione-S-transferase-positive mature OLs were considerably reduced. In hypomyelinated white matter, strong immunoreactivity of polysialylated-neural cell adhesion molecule, a negative regulator of myelination, was observed in axons. Given the fact that neuro-axonal degeneration has been observed in NPC mouse as early as 9 days of age prior to the commencement of myelination in the corpus callosum and that axonal signals are essential for the proper myelination, subtle axonal injury might be contributing to the pathogenesis of disturbed myelination in the NPC mouse.


Subject(s)
Nerve Fibers, Myelinated/pathology , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/pathology , Oligodendroglia/pathology , Animals , Animals, Newborn , Mice , Mice, Inbred BALB C , Mice, Neurologic Mutants , Nerve Fibers, Myelinated/ultrastructure , Oligodendroglia/ultrastructure
17.
Neurobiol Dis ; 16(1): 98-109, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15207267

ABSTRACT

The twitcher mouse is an authentic murine model of a genetic demyelinating disease globoid cell leukodystrophy. Allogeneic bone marrow transplantation (BMT) in twitcher mice resulted in the clinicopathological improvement. Thus, using green fluorescent protein (GFP) transgenic mice as the donor, we investigated the behavior and fate of the donor cells and the possibility of transdifferentiation of the donor cells into neuroglial cells in the chimeric twitcher mice. GFP(+) cells were found throughout the brain, most conspicuous in the areas of demyelination. The donor GFP(+) cells expressed RCA-1, a marker for microglia/macrophages but were never exceed 70% of the entire population of the RCA-1(+) microglia/macrophages. There was no convincing evidence that GFP(+) donor cells expressed markers for neurons, astrocytes, or oligodendrocytes. We concluded BMT is therapeutic for this model. However, this effect is not mediated by donor cells transdifferentiation in the brain.


Subject(s)
Bone Marrow Transplantation/methods , Demyelinating Diseases/surgery , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cells/chemistry , Luminescent Proteins/biosynthesis , Animals , Bone Marrow Transplantation/pathology , Demyelinating Diseases/genetics , Demyelinating Diseases/pathology , Female , Genotype , Green Fluorescent Proteins , Hematopoietic Stem Cells/pathology , Liver/chemistry , Liver/cytology , Luminescent Proteins/genetics , Lung/chemistry , Lung/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Neurologic Mutants , Mice, Transgenic
18.
Am J Pathol ; 163(1): 277-86, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12819032

ABSTRACT

We previously reported that the platelet-derived growth factor B-chain (PDGF-B)/PDGF receptor (PDGFR) axis is involved in tubular regeneration after ischemia/reperfusion injury of the kidney. In the present study, we examined the activation of Src tyrosine kinase, a crucially important signaling molecule for PDGFR, and assessed the role of Src in PDGF-B-dependent renal tubular regeneration afterischemia/reperfusion injury. Immunoblot using clone 28, a monoclonal antibody specific for the active form of Src kinases, demonstrated increased active Src expression in the injured rat kidney 6 hours after reperfusion with peak activation at 12 hours. In vitro kinase assay confirmed increased Src activity that concurred with PDGFR-beta activation as detected by the increment of receptor-phosphorylated tyrosine. Immunohistochemistry using clone 28 demonstrated that active Src was preferentially expressed in the S3 segment of the proximal tubule in reperfused kidney, where it is not normally expressed. This enhanced expression of active Src was co-localized with the increased PDGFR expression in the tubular cells that were undergoing cell proliferation cycle. Trapidil administration suppressed Src and PDGFR-beta activation in the reperfused kidney and resulted in deteriorated renal function. These findings suggest that active Src participates in PDGF-B-dependent regeneration of tubular cells from acute ischemic injury.


Subject(s)
Kidney Tubules/physiology , Proto-Oncogene Proteins c-sis/metabolism , Regeneration/physiology , Reperfusion Injury , src-Family Kinases/metabolism , Animals , Creatinine/blood , Enzyme Activation , Humans , Immunohistochemistry , Kidney Tubules/cytology , Kidney Tubules/pathology , Male , Platelet Aggregation Inhibitors/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Platelet-Derived Growth Factor beta/metabolism , Trapidil/metabolism
19.
Acta Neuropathol ; 103(4): 356-62, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11904755

ABSTRACT

Neuronal cell death in the brain of macular mutant mouse, a model of copper metabolism abnormality, has features of both apoptosis and necrosis. Apoptotic cells were morphologically identified by the terminal deoxynucleotidyl transferase nick end-labeling (TUNEL) method and electron microscopy. Numerous TUNEL-positive cells were identified in the cerebral cortex, hippocampus and thalamus of the hemizygotes after postnatal day 11. Ultramicroscopic studies confirmed that a number of cells had apoptotic features characterized by condensation and segregation of the nuclei. Furthermore, genomic DNA gel electrophoresis revealed a laddering pattern in the hemizygous brain. Starvation, which produced a low body weight in normal mice similar to that seen in the hemizygotes, did not result in an increase of TUNEL-positive cells. We also found that there was no increase of apoptotic cells in the brains of heterozygotes and copper-supplemented hemizygotes. Immunocytochemical analysis revealed that the distribution of copper/zinc superoxide dismutase-containing cells differed from that of TUNEL-positive cells. These findings suggest that copper deficiency is a key factor triggering apoptosis in the brain of macular mutant mouse through a mechanism different from suppression of antioxidant action of the dismutase. The improved survival period of the copper-supplemented hemizygotes may be attributed, in part, to inhibition of excessive neuronal apoptosis identified in the late stage of the disease.


Subject(s)
Apoptosis , Brain/physiopathology , Menkes Kinky Hair Syndrome/genetics , Menkes Kinky Hair Syndrome/physiopathology , Mice, Mutant Strains/physiology , Animals , Apoptosis/drug effects , Brain/ultrastructure , Copper/pharmacology , DNA/genetics , Electrophoresis, Agar Gel , Heterozygote , Immunohistochemistry , In Situ Nick-End Labeling , Menkes Kinky Hair Syndrome/pathology , Mice , Reference Values , Superoxide Dismutase/metabolism
SELECTION OF CITATIONS
SEARCH DETAIL
...