Jin-Song Shen

Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of alpha-galactosidase A resulting in globotriaosylceramide (Gb(3)) storage in cells. The pathogenic role of Gb(3) in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb(3) in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb(3)-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb(3) also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb(3) by treatment of the cells with an inhibitor of glycosphingolipid synthase or alpha-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb(3). This study provided direct evidence that excess intracellular Gb(3) induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patients plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

Brain transplantation of genetically engineered human neural stem cells globally corrects brain lesions in the mucopolysaccharidosis type VII mouse

In the present study, we investigated the feasibility of using human neural stem cells (NSCs) in the treatment of diffuse central nervous system (CNS) alterations in a murine model of mucopolysaccharidosis VII (MPS VII), a lysosomal storage disease caused by a genetic defect in the β‐glucuronidase gene. An immortalized NSC line derived from human fetal telencephalon was genetically engineered to overexpress β‐glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mouse. Transplanted human NSCs were found to integrate and migrate in the host brain and to produce large amount of β‐glucuronidase. Brain contents of the substrates of β‐glucuronidase were reduced to nearly normal levels, and widespread clearing of lysosomal storage was observed in the MPS VII mouse brain at 25 days posttransplantation. The number of engrafted cells decreased markedly after the transplantation, and it appears that the major cause of the cell death was not the immune response of the host but apoptotic cell death of grafted human NSCs. Results showed that human NSCs would serve as a useful gene transfer vehicle for the treatment of diffuse CNS lesions in human lysosomal storage diseases and are potentially applicable in the treatment of patients suffering from neurological disorders.

Adenoviral gene transfer of GDNF, BDNF and TGFβ2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line‐derived neurotrophic factor (GDNF), brain‐derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin‐1 (CT1), insulin‐like growth factor‐1 (IGF1), and transforming growth factor‐β2 (TGFβ2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFβ2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFβ2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFβ2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Intraventricular administration of recombinant adenovirus to neonatal twitcher mouse leads to clinicopathological improvements

Twitcher mouse is a murine model of human globoid cell leukodystrophy (Krabbe disease), which is characterized by a genetic deficiency in galactocerebrosidase (GALC) activity. The nervous system is affected early and severely by demyelination in the white matter. So far, there is no effective treatment for Krabbe disease except bone marrow transplantation (BMT). However, BMT has inherent limitations such as unavailability of donors and graft-versus-host disease. In this study, we injected recombinant adenovirus encoding GALC into the lateral ventricle of twitcher mice at postnatal day 0 (PND 0) and the therapeutic effects were evaluated. Our results showed slight, but significant improvements in motor functions, body weight and twitching and a prolonged life span. In brain, GALC activity was increased to 15% that of normal littermates and psychosine concentration was decreased to 55% that of untreated twitcher mice at PND 15. The number of PAS-positive globoid cells in brain stem was also reduced significantly at PND 35. In contrast, when adenoviruses were injected to the twitcher mice at PND 15, almost no improvements were observed. These results demonstrate that the timing of treatment may be of great importance in Krabbe disease.

Induced pluripotent stem cells derived from mouse models of lysosomal storage disorders

Most lysosomal storage diseases (LSDs) are life-threatening genetic diseases. The pathogenesis of these diseases is poorly understood. Induced pluripotent stem (iPS) cell technology offers new opportunities for both mechanistic studies and development of stem cell– based therapies. Here we report the generation of disease-specific iPS cells from mouse models of Fabry disease, globoid cell leukodystrophy (GLD), and mucopolysaccharidosis VII (MPSVII). These mouse model–derived iPS cells showed defects in disease-specific enzyme activities and significant accumulation of substrates for these enzymes. In the lineage-directed differentiation studies, Fabry-iPS and GLD-iPS cells were efficiently differentiated into disease-relevant cell types, such as cardiomyocytes and neural stem cells, which might be useful in mechanistic and therapeutic studies. Notably, MPSVII-iPS cells demonstrated a markedly impaired ability to form embryoid bodies (EBs) in vitro. MPSVII-EBs exibited elevated levels of hyaluronan and its receptor CD44, and markedly reduced expression levels of E-cadherin and cell-proliferating marker. Partial correction of enzyme deficiency in MSPVII-iPS cells led to improved EB formation and reversal of aberrant protein expression. These data indicate a potential mechanism for the partial lethality of MPSVII mice in utero, and suggest a possible abnormality of embryonic development in MPSVII patients. Thus, our study demonstrates the unique promise of iPS cells for studying the pathogenesis and treatment of LSDs.

Treatment of lysosomal storage disorders: Cell therapy and gene therapy

Summary: Most lysosomal storage diseases have central nervous system (CNS) involvement. No effective treatment is available at present. We investigated the usefulness of brain-directed gene therapy and cell therapy using mouse models of lysosomal storage diseases. For gene therapy to the CNS, a recombinant adenovirus encoding β-galactocerebrosidase gene was injected into the cerebral ventricle of neonatal twitcher mice, a murine model of Krabbe disease. Improvements in neurological symptoms and a prolonged lifespan were observed. Brain activity of β-galactocerebrosidase was increased significantly and the concentration of a cytotoxic metabolite, psychosine, was decreased. Pathological observations of the brain were also improved in treated twitcher mice. For cell therapy to the CNS, a neural stem cell line derived from human fetal brain was genetically engineered to overexpress β-glucuronidase and transplanted into the cerebral ventricles of neonatal MPS VII mice, a model of β-glucuronidase deficiency. Transplanted human neural stem cells were found to integrate and migrate in the host brain and to produce large amounts of β-glucuronidase. Brain contents of the substrate of β-glucuronidase were reduced and widespread clearing of lysosomal storage was observed in treated MPS VII mice. These data suggest that brain-directed gene/cell therapy may be useful in the treatment of neurological alterations in lysosomal storage diseases.

Brain transplantation of genetically modified bone marrow stromal cells corrects CNS pathology and cognitive function in MPS VII mice

Current therapies for lysosomal storage diseases (LSDs), enzyme replacement therapy and bone marrow transplantation are effective for visceral organ pathology of LSD, but their effectiveness for brain involvement in LSDs is still a subject of controversy. As an alternative approach, we transplanted genetically modified bone marrow stromal (BMS) cells to lateral ventricle of newborn mucopolysaccharidosis VII (MPS VII) mice. MPS VII is one of LSDs and caused by deficiency of beta-glucuronidase (GUSB), resulting in accumulation of glycosaminoglycans (GAGs) in brain. At 2 weeks after transplantation, the GUSB enzyme-positive cells were identified in olfactory bulb, striatum and cerebral cortex, and the enzymatic activities in various brain areas increased. The GAGs contents in brain were reduced to near normal level at 4 weeks after transplantation. Although GUSB activity declined to homozygous level after 8 weeks, the reduction of GAGs persisted for 16 weeks. Microscopic examination indicated that the lysosomal distention was not found in treated animal brain. Cognitive function in MPS VII animals as evaluated by Morris Water Maze test in treated mice showed a marked improvement over nontreated animals. Brain transplantation of genetically modified BMS cells appears to be a promising approach to treat diffuse CNS involvement of LSDs.

Widespread gene transduction to the central nervous system by adenovirus in utero: implication for prenatal gene therapy to brain involvement of lysosomal storage disease

In some lysosomal storage diseases, considerable alterations of the central nervous system (CNS) occur prior to birth and neurodegeneration progresses rapidly soon after birth causing early death in patients. No effective treatment is available after birth. Treatment may need to be initiated before birth to prevent the onset or progression of neurological changes and thereby irreversible brain damage. The aim of this study is to investigate the feasibility and effectiveness of brain‐directed prenatal gene therapy for lysosomal storage diseases.

Establishment and characterization of spontaneously immortalized Schwann cells from murine model of globoid cell leukodystrophy (twitcher)

The twitcher mouse is a murine model of human globoid cell leukodystrophy (GLD; Krabbe disease) caused by a genetic defect in the activity of galactosylceramidase (GALC). An accumulation of cytotoxic metabolite, galactosylsphingosine (psychosine), in myelin forming cells (oligodendrocytes and Schwann cells) of the twitcher mouse as well as patients with GLD has been suggested to cause dysfunction of these cells and subsequent demyelination in the central and peripheral nervous system. To investigate further the cellular pathomechanism of GLD, we established spontaneously immortalized Schwann cell lines from the twitcher mouse. Long‐term cultures of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of 3‐week‐old twitcher mice were maintained for 6 months, and spontaneously developed colonies were expanded further and characterized. One of the cell lines, designated TwS1, showed distinct Schwann cell phenotypes, was passaged twice a week and maintained for over 10 months without phenotypic alterations. The TwS1 cells had a nonsense mutation in the GALC genome, and showed markedly reduced GALC activity and elevated psychosine levels. Ultrastructurally, varieties of cytoplasmic inclusions were demonstrated in TwS1 cells. When TwS1 cells were infected with a retrovirus vector encoding GALC, GALC activity was markedly increased and psychosine levels were significantly decreased. These immortalized Schwann cells can be useful in studies on the nervous system lesions in GLD.

Neonatal gene transfer using lentiviral vector for murine Pompe disease: long-term expression and glycogen reduction

Pompe disease results from the deficiency of the lysosomal enzyme acid α-glucosidase (GAA), leading to accumulated glycogen in the heart and the skeletal muscles, which causes cardiomyopathy and muscle weakness. In this study, we tested the feasibility of gene therapy for Pompe disease using a lentivirus vector (LV). Newborn GAA knockout mice were treated with intravenous injection of LV encoding human GAA (hGAA) through the facial superficial temporal vein. The transgene expression in the tissues was analyzed up to 24 weeks after treatment. Our results showed that the recombinant LV was efficient not only in increasing the GAA activity in tissues but also in decreasing their glycogen content. The examination of histological sections showed clearence of the glycogen storage in skeletal and cardiac muscles 16 and 24 weeks after a single vector injection. Levels of expressed hGAA could be detected in serum of treated animals until 24 weeks. No significant immune reaction to transgene was detected in most treated animals. Therefore, we show that LV-mediated delivery system was effective in correcting the biochemical abnormalities and that this gene transfer system might be suitable for further studies on delivering GAA to Pompe disease mouse models.