Kazuhiko Watabe

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line-derived neurotrophic factor

Glial cell line‐derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat spinal motoneurons. The seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed, and AxCAhGDNF, AxCALacZ (adenovirus encoding β‐galactosidase gene) or PBS was inoculated in C7 vertebral foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of β‐galactosidase activity in lesioned spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned spinal motoneurons and expression of virus‐induced human GDNF mRNA transcripts in the lesioned spinal cord tissue. Nissl‐stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned ventral horn motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus‐mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with spinal cord injury and motor neuron diseases. J. Neurosci. Res. 60:511–519, 2000

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.

Skeletal muscle regeneration after insulin-like growth factor I gene transfer by recombinant Sendai virus vector.

We scrutinized the applicability and efficacy of Sendai virus (SeV) vectors expressing either LacZ or human insulin-like growth factor-I (hIGF-I) in gene transfer into skeletal muscle. Seven days after the intramuscular injection of LacZ/SeV X-gal labeled myofibers were demonstrated in rat anterior tibialis muscle with/without bupivacaine treatment and the transgene expression persisted up to 1 month after injection. Recombinant hIGF-I was detected as a major protein species in culture supernatants of a neonatal rat myoblast cell line L6 and thus induced the cells to undergo myogenetic differentiation. The introduction of hIGF-I/SeV into the muscle showed a significant increase in regenerating and split myofibers which were indicative of hypertrophy, and also an increase in the total number of myofibers, in comparison to that seen in the LacZ/SeV-treated control muscle. These results demonstrate that SeV achieves high-level transgene expression in skeletal muscle, and that hIGF-I gene transfer using SeV vector may therefore have great potential in the treatment of neuromuscular disorders.

Neuroprotective effect of sonic hedgehog up-regulated in Schwann cells following sciatic nerve injury.

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush‐injured adult rat sciatic nerves. The Shh expression was up‐regulated in Schwann cells adjacent to the injured site. The time‐course analyses of various neurotrophic factors revealed the up‐regulation of Shh mRNA followed by that of brain‐derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co‐transfected with Shh and its receptor Smoothened. These findings imply that the up‐regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.

Transplantation of human sympathetic neurons and adrenal chromaffin cells into parkinsonian monkeys: no reversal of clinical symptoms

Cultured human fetal sympathetic ganglion explants or adrenal chromaffin cell aggregates were implanted into the left striatum of monkeys whose left nigrostriatal pathway had been lesioned with the neurotoxin MPTP. There was no clinical reversal of parkinsonian symptoms and PET scans did not show increased striatal fluorodopa uptake from pre-implant levels. At sacrifice, left striatal contents of dopamine were not statistically different from MPTP-treated but non-implanted controls. Histological examinations revealed pockets of extrinsic cells which were found at the end of needle tracks. There was no evidence of immune rejection. The extrinsic cells did not stain for tyrosine hydroxylase or neurofilament, suggesting that they were not dopaminergic neurons. The failure to reverse clinical parkinsonian symptoms highlights the stage of infancy of neural implantation in Parkinsons disease.

Postischemic administration of Sendai virus vector carrying neurotrophic factor genes prevents delayed neuronal death in gerbils

Sendai virus (SeV) vector-mediated gene delivery of glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) prevented the delayed neuronal death induced by transient global ischemia in gerbils, even when the vector was administered several hours after ischemia. Intraventricular administration of SeV vector directed high-level expression of the vector-encoded neurotrophic factor genes, which are potent candidates for the treatment of neurodegenerative diseases. After occlusion of the bilateral carotid arteries of gerbils, SeV vector carrying GDNF (SeV/GDNF), NGF (SeV/NGF), brain-derived neurotrophic factor (SeV/BDNF), insulin-like growth factor-1 (SeV/IGF-1) or vascular endothelial growth factor (SeV/VEGF) was injected into the lateral ventricle. Administration of SeV/GDNF, SeV/NGF or SeV/BDNF 30 min after the ischemic insult effectively prevented the delayed neuronal death of the hippocampal CA1 pyramidal neurons. Furthermore, the administration of SeV/GDNF or SeV/NGF as late as 4 or 6 h after the ischemic insult also prevented the death of these neurons. These results indicate that SeV vector-mediated gene transfer of neurotrophic factors has high therapeutic potency for preventing the delayed neuronal death induced by transient global ischemia, and provides an approach for gene therapy of stroke.

Establishment of an Improved Mouse Model for Infantile Neuroaxonal Dystrophy That Shows Early Disease Onset and Bears a Point Mutation in Pla2g6

Calcium-independent group VIA phospholipase A(2) (iPLA(2)beta), encoded by PLA2G6, has been shown to be involved in various physiological and pathological processes, including immunity, cell death, and cell membrane homeostasis. Mutations in the PLA2G6 gene have been recently identified in patients with infantile neuroaxonal dystrophy (INAD). Subsequently, it was reported that similar neurological impairment occurs in gene-targeted mice with a null mutation of iPLA(2)beta, whose disease onset became apparent approximately 1 to 2 years after birth. Here, we report the establishment of an improved mouse model for INAD that bears a point mutation in the ankyrin repeat domain of Pla2g6 generated by N-ethyl-N-nitrosourea mutagenesis. These mutant mice developed severe motor dysfunction, including abnormal gait and poor performance in the hanging grip test, as early as 7 to 8 weeks of age, in a manner following Mendelian law. Neuropathological examination revealed widespread formation of spheroids containing tubulovesicular membranes similar to human INAD. Molecular and biochemical analysis revealed that the mutant mice expressed Pla2g6 mRNA and protein, but the mutated Pla2g6 protein had no glycerophospholipid-catalyzing enzyme activity. Because of the significantly early onset of the disease, this mouse mutant (Pla2g6-inad) could be highly useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration.

High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32

We investigated the polyol pathway activity and the gene expression profiles in immortalized adult mouse Schwann cells (IMS32) under normal (5.6 mm) and high (30 and 56 mm) glucose conditions for 7–14 days in culture. Messenger RNA and the protein expression of aldose reductase (AR) and the intracellular sorbitol and fructose contents were up‐regulated in IMS32 under high glucose conditions compared with normal glucose conditions. By employing DNA microarray and subsequent RT–PCR/northern blot analyses, we observed significant up‐regulation of the mRNA expressions for serum amyloid A3 (SAA3), angiopoietin‐like 4 (ANGPTL4) and ecotropic viral integration site 3 (Evi3), and the down‐regulation of aldehyde reductase (AKR1A4) mRNA expression in the cells under high glucose (30 mm) conditions. The application of an AR inhibitor, SNK‐860, to the high glucose medium ameliorated the increased sorbitol and fructose contents and the reduced AKR1A4 mRNA expression, while it had no effect on mRNA expressions for SAA3, ANGPTL4 or Evi3. Considering that the exposure to the high glucose (≥ 30 mm) conditions mimicking hyperglycaemia in vivo accelerated the polyol pathway in IMS32, but not in other previously reported Schwann cells, the culture system of IMS32 under those conditions may provide novel findings about the polyol pathway‐related abnormalities in diabetic neuropathy.

Adenoviral GDNF gene transfer enhances neurofunctional recovery after recurrent laryngeal nerve injury

To assess the possibility of gene therapy for recurrent laryngeal nerve (RLN) injury, we examined functional and histological recovery after glial cell line-derived neurotrophic factor (GDNF) gene transfer in a rat RLN crush model. Adenoviral vector encoding β-galactosidase gene (AxCALacZ) or human GDNF gene (AxCAhGDNF) was injected into the crush site of the RLN. Neurons in the nucleus ambiguus on the crushed side were labeled with X-gal or GDNF immnohistochemistry after AxCALacZ or AxCAhGDNF injection. Reverse transcription-polymerase chain reaction analysis revealed expression of human GDNF mRNA transcripts in brainstem tissue containing the nucleus ambiguus on the crushed side after AxCAhGDNF injection. Animals injected with AxCAhGDNF displayed significantly improved motor nerve conduction velocity of the RLN and recovery rate of vocal fold movement at 2 and 4 weeks after treatment as compared to controls. AxCAhGDNF-injected animals showed a significantly larger axonal diameter and improved remyelination in crushed RLN as compared to controls. Adenoviral GDNF gene transfer may thus promote laryngeal function recovery after RLN injury. Inoculation of adenoviral vector containing the GDNF gene at the site of damage soon after nerve injury may assist patients with laryngeal paralysis caused by nerve injury during head and neck surgery.