BACKGROUNDBirt-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.METHODSMice 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.RESULTSMuscle-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.CONCLUSIONFLCN 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.

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.

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 50–60%. 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.

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.

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.†From To a Mouse, Robert Burns, 1785

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.

Filovirus infection of target cells leads to the formation of virally induced cytoplasmic inclusions that contain viral nucleocapsids at different stages of maturation. While the role of the inclusions has been unclear since the identification of Marburg and Ebola viruses, it recently became clear that the inclusions are the sites of viral replication, nucleocapsid formation and maturation. Live cell imaging analyses revealed that mature nucleocapsids are transported from inclusions to the filopodia, which represent the major budding sites. Moreover, inclusions recruit cellular proteins that have been shown to support the transport of nucleocapsids. For example, the tumor susceptibility gene 101 protein (Tsg101) interacts with a late domain motif in the nucleocapsid protein NP and recruits the actin-nucleation factor IQGAP1. Complexes of nucleocapsids together with Tsg101 and IQGAP1 are then co-transported along actin filaments. We detected additional proteins (Alix, Nedd4 and the AAA-type ATPase VPS4) of the endosomal sorting complex required for transport (ESCRT) that are recruited into inclusions. Together, the results suggest that nucleocapsids recruit the machinery that enhances viral budding at the plasma membrane. Furthermore, we identified Lamp1 as a marker of the late endosomal compartment in inclusions, while ER, Golgi, TGN and early endosomal markers were absent. In addition, we observed that LC3, a marker of autophagosomal membranes, was present in inclusions. The 3D structures of inclusions show an intricate structure that seems to accommodate an intimate cooperation between cellular and viral components with the intention to support viral transport and budding.

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 α-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.

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.

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.

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, glycogen accumulates in multiple tissues, but clinical manifestations are mainly due to skeletal and cardiac muscle involvement. A major advance has been the development of enzyme replacement therapy (ERT), which recently became available for Pompe patients. Based on clinical and pre-clinical studies, the effective clearance of skeletal muscle glycogen appears to be more difficult than anticipated. Skeletal muscle destruction and resistance to therapy remain unsolved problems. We have found that the cellular pathology in Pompe disease spreads to affect both the endocytic and autophagic pathways, leading to excessive autophagic buildup in therapy resistant muscle fibers of knockout mice. Furthermore, the autophagic buildup had a profound effect on the trafficking and processing of the therapeutic enzyme along the endocytic pathway. These findings may explain why ERT often falls short of reversing the disease process, and point to new avenues for the development of pharmacological intervention.

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.

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.

Thyrotropin-releasing hormone (TRH) has been successfully used for treating children with neurologic disorders including epilepsy. The effectiveness of TRH and a TRH analog has been reported in West syndrome, Lennox-Gastaut syndrome, and early infantile epileptic encephalopathy that were intractable to anticonvulsants and adrenocorticotrophic hormone (ACTH). However, the peptide has not been widely studied as a treatment of intractable epilepsy outside Japan. TRH is safe in children and effective in some cases of West syndrome and Lennox-Gastaut syndrome. TRH is considered as a possible new strategy for treating West syndrome and Lennox-Gastaut syndrome prior to ACTH therapy, especially for the patient with an infection, immunosuppression, or severe organic lesions in the brain. The mechanisms of its antiepileptic action may differ from those of other antiepileptic drugs. One possibility is that TRH may act as an antiepileptic through a kynurenine mechanism, considering that kynurenic acid acts as an antagonist on the N-methyl-D-aspartate receptor complex.

The neuroadapted Kilham strain of the mumps virus produces lethal encephalitis in newborn hamsters after intracerebral inoculation. The pathogenesis of this encephalitis is not fully understood, but recently, apoptosis and associated cytokine production have been recognized to be major pathologic mechanisms by which viruses cause injury to neuronal host cells. To analyze the main factors producing brain injury in this viral encephalitis, the following questions were investigated: (1) does the virus induce neuronal apoptosis and (2) does expression of cytokines regulate the induction of neuronal apoptosis? Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) was used as a marker of neuronal apoptosis and TUNEL-positive neurons were widespread in the infected cerebral cortex. DNA fragmentation yielding DNA ladders characteristic of apoptosis was also observed in infected hamster brain tissue. Apoptotic cells in infected brains were observed after the appearance of inflammatory changes. Overexpression of IL-1 beta, but not TNF-alpha or Fas-L, was clearly detected in infected brains, as determined by Western blot and RT-PCR. Immunohistochemistry revealed a striking correlation between IL-1 beta expression and neuronal apoptosis. Injection of recombinant IL-1 beta into normal hamster brain resulted in neuronal apoptosis in cerebral cortex. On the other hand, neutralizing IL-1 beta antibodies decreased the number of cells undergoing apoptosis in infected hamster brains and subsequent death. We conclude that the fatal encephalitis induced by the Kilham strain of the mumps virus is mediated by immunopathological processes and that overexpression of IL-1 beta, which mediates the induction of neuronal apoptosis, may play a major role in these processes.

The pathogenesis of cleft formation in schizencephaly was analyzed by examining the brain lesions produced by the infection of the Kilham strain of mumps virus during the period of neuronal migration in hamsters. Mumps virus antigen was detected in the neuroepithelial cells within the ventricular zone, the choroid plexus in the lateral ventricles, and vimentin-immunoreactive radial glial fibers. The main pathological findings were cerebral hemorrhage, neuronal necrosis, microsulci on the cerebral cortex and cleft formation through the entire thickness of the cerebral mantle. The clefts seen in these experiments were lined by embryonal elements such as neuroepithelial cells and germinal cells. Based on these results and the original definition by Yakovlev and Wadsworth, the following two conclusions were suggested. First, the mumps virus localized to the neuroepithelial cells within the ventricular zone and the radial glial fibers may induce a destructive process and subsequent anomalous neuronal migration, resulting in cleft formation. Second, the formation of the ventricular cleft extending to the pial surface, which should be complete before the cortical infolding appears, is necessary in order to produce the characteristic cleft in schizencephaly which is associated with the pial-ependymal seam.

In order to elucidate the relationship between virus neurotropism and neuronal maturity, two experiments were performed. First, mumps virus infectivity was compared among the different developmental stages of hamster brains inoculated with mumps virus by examining the immunohistochemical distribution of mumps virus antigen. Second, brain lesions resulting from mumps virus infection during the period of neuronal migration were histologically and ultrastructurally analyzed. Three groups of Syrian hamsters, Group E12 (fetuses on the 12th day of gestation), and Groups P2 and P30 (2 and 30 days old, respectively), were injected with mumps virus intraplacentally or intracerebrally. In Group P30, mumps virus antigen was observed specifically in ependymal cells and the choroid plexus. In addition to these areas, in Group P2, some neurons in layers II and III of the cerebral cortex also showed virus antigen immunoreactivity. In Group E12, mumps virus antigen accumulated primarily in the neuroepithelial cells within the ventricular zone. Neither specific intranuclear changes related to viral replication nor the formation of complete virions and nucleocapsids was observed. We conclude that mumps virus neurotropism to hamster brains is dependent on the degree of neuronal maturity and that mumps virus can induce an abortive infection and resultant neuronal cell necrosis in the immature developing hamster brain.