We describe a Japanese patient with familial amyotrophic lateral sclerosis (ALS) and a p.K510M mutation in the fused in sarcoma gene (FUS). The patient's condition was characterized clinically by an early onset and rapid progression. The patient eventually required mechanical ventilation and progressed to the totally locked-in state. Neuropathologically, multiple system degeneration with many FUS-immunoreactive structures was observed. The involvement of the globus pallidus, subthalamic nucleus, substantia nigra, cerebellar efferent system, and both upper and lower motor neurons in the present patient was comparable to that described for ALS patients with different mutations in FUS, all of whom progressed to the totally locked-in state. However, the patient also exhibited degeneration of the cerebellar afferent system and posterior column. Furthermore, the appearance of non-compact FUS-immunoreactive neuronal cytoplasmic inclusions and many FUS-immunoreactive glial cytoplasmic inclusions were unique to the present patient. These features suggest that the morphological characteristics of the FUS-immunoreactive structures and distribution of the lesions vary with the diversity of mutations in FUS.
Previously we found that, after temporary cerebral ischemia, microvasculogenic secondary focal cerebral cortical ischemia occurred, caused by microvascular obstruction due to compression by swollen astrocytic end-feet, resulting in focal infarction. Herein, we examined whether mannitol infusion immediately after restoration of blood flow could protect the cerebral cortex against the development of such an infarction. If so, the infusion of mannitol might improve the results of vascular reperfusion therapy. We selected stroke-positive animals during the first 10?min after left carotid occlusion performed twice with a 5-h interval, and allocated them into four groups: sham-operated control, no-treatment, mannitol-infusion, and saline-infusion groups. Light- and electron-microscopic studies were performed on cerebral cortices of coronal sections prepared at the chiasmatic level, where the focal infarction develops abruptly in the area where disseminated selective neuronal necrosis is maturing. Measurements were performed to determine the following: (A) infarct size in HE-stained specimens from all groups at 72 and 120 h after return of blood flow; (B) number of carbon-black-suspension-perfused microvessels in the control and at 0.5, 3, 5, 8, 12 and 24 h in the no-treatment and mannitol-infusion groups; (C) area of astrocytic end-feet; and (D) number of mitochondria in the astrocytic end-feet in electron microscopic pictures taken at 5?h. The average decimal fraction area ratio of infarct size in the mannitol group was significantly reduced at 72 and 120?h, associated with an increased decimal fraction number ratio of carbon-black-suspension-perfused microvessels at 3, 5 and 8?h, and a marked reduction in the size of the end-feet at 5 h. Mannitol infusion performed immediately after restitution of blood flow following temporary cerebral ischemia remarkably reduced the size of the cerebral cortical focal infarction by decreasing the swelling of the end-feet, thus preventing the microvascular compression and stasis and thereby microvasculogenic secondary focal cerebral ischemia.
We have established spontaneously immortalized Schwann cell lines from adult ICR mice [IMS32] and Fischer344 rats [IFRS1]. IMS32 cells display distinct Schwann cell phenotypes such as a spindle-shaped morphology and the expression of glial cell markers (e.g. S100, glial fibrillary acidic protein (GFAP), p75 low-affinity neurotrophin receptor (p75(NTR))) and neurotrophic factors. In addition, conditioned medium obtained from IMS32 cells enhances neurite elongation of PC12 cells and mouse dorsal root ganglion (DRG) neurons. IMS32 cells have been utilized to investigate the action mechanisms of various molecules that accelerate peripheral nerve regeneration (e.g. ciliary neurotrophic factor, sonic hedgehog, galectin-1). Like IMS32 cells, IFRS1 cells retain the characteristic features of mature Schwann cells as described above. Furthermore, IFRS1 cells have been shown to myelinate neurites in coculture with adult rat DRG neurons and PC12 cells. Our current investigation with IFRS1 cells focuses on the molecular mechanisms of myelination-inducible factors, such as soluble neuregulin-1 type III and exendin-4. These Schwann cell lines can be valuable tools for exploring neuron-Schwann cell interactions, pathobiology of axonal degeneration and regeneration in the peripheral nervous system, and novel therapeutic approaches against neurological disorders in patients with relevant diseases.
Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding transactivation response (TAR) DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. In the present study, we produced recombinant adenovirus vectors encoding wild type and mutant TDP-43 and FUS, and those encoding short hairpin RNAs (shRNAs) for proteasome (PSMC1), autophagy (ATG5), and endosome (VPS24) systems to investigate whether the coupled gene transductions in motoneurons by these adenoviruses elicit ALS pathology. Cultured neurons, astrocytes and oligodendrocytes differentiated from adult rat neural stem cells and motoneurons derived from mouse embryonic stem cells were successfully infected with these adenoviruses showing cytoplasmic aggregate formation. When these adenoviruses were injected into the facial nerves of adult rats, exogenous TDP-43 and FUS proteins were strongly expressed in facial motoneurons by a retrograde axonal transport of the adenoviruses. Co-infections of adenovirus encoding shRNA for PSMC1, ATG5 or VPS24 with TDP-43 or FUS adenovirus enhanced cytoplasmic aggregate formation in facial motoneurons, suggesting that impairment of protein degradation pathways accelerates formation of TDP-43 and FUS-positive aggregates in ALS.
The aim of myocardial tissue engineering is to repair or regenerate damaged myocardium with engineered cardiac tissue. However, this strategy has been hampered by lack of functional integration of grafts with native myocardium. Autonomic innervation may be crucial for grafts to function properly with host myocardium. In this study, we explored the feasibility of in vivo induction of autonomic innervation to engineered myocardial tissue using genetic modulation by adenovirus encoding glial cell line derived neurotrophic factor (GDNF). GFP-transgene (control group) or GDNF overexpressing (GDNF group) engineered cardiomyocyte sheets were transplanted on cryoinjured hearts in rats. Nerve fibers in the grafts were examined by immunohistochemistry at 1, 2, and 4 weeks postoperatively. Growth associated protein-43 positive growing nerves and tyrosine hydroxylase positive sympathetic nerves were first detected in the grafts at 2 weeks postoperatively in control group and 1 week in GDNF group. The densities of growing nerve and sympathetic nerve in grafts were significantly increased in GDNF group. No choline acetyltransferase immunopositive parasympathetic nerves were observed in grafts. In conclusion, sympathetic innervation could be effectively induced into engrafted engineered cardiomyocyte sheets using GDNF.
Astrocytes support neuronal functions by regulating the extracellular ion homeostasis and levels of neurotransmitters, and by providing fuel such as lactate to the neurons via their processes (APs). After two 10-min unilateral carotid occlusions with a 5-h interval in gerbils, we investigated maturing disseminated selective neuronal necrosis (DSNN) on the coronal surface sectioned at the infundibular level. We chronologically counted the normal appearing, degenerated, and dead neurons and astrocytes in the cerebral cortex; observed the ultrastructure of APs, and counted the number of their cut-ends and mitochondria in the neuropil; determined the percentage volume of APs according to Weibels point-counting method; compared the number of cut-ends and mitochondria and percentage volume of APs around the astrocytes and around the normal-appearing, degenerated, and dead -neurons. Heterogeneous degeneration of APs was concluded to be closely associated with the maturation of DSNN.Using the same model, at the coronally sectioned surface on the chiasmatic level, we investigated the mechanism of development of focal infarction in the maturing DSNN. Same as in the above study, we chronologically counted various neurons and astrocytes; observed and measured the area of the ultrastructure of astrocytic end-feet; counted the number of carbon-black-suspension-perfused microvessels. We concluded that after temporary cerebral ischemia, secondary focal ischemia was induced by microvascular obstruction compressed by swollen astrocytic end-feet, resulting in delayed focal infarction.
Galectin-1 (GAL-1), a member of a family of ?-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.
Molecular signaling of cardiac autonomic innervation is an unresolved issue. Here, we show that glial cell line-derived neurotrophic factor (GDNF) promotes cardiac sympathetic innervation in vitro and in vivo. In vitro, ventricular myocytes (VMs) and sympathetic neurons (SNs) isolated from neonatal rat ventricles and superior cervical ganglia were cultured at a close distance. Then, morphological and functional coupling between SNs and VMs was assessed in response to GDNF (10 ng/ml) or nerve growth factor (50 ng/ml). As a result, fractions of neurofilament-M-positive axons and synapsin-I-positive area over the surface of VMs were markedly increased with GDNF by 9-fold and 25-fold, respectively, compared to control without neurotrophic factors. Pre- and post-synaptic stimulation of ?1-adrenergic receptors (BAR) with nicotine and noradrenaline, respectively, resulted in an increase of the spontaneous beating rate of VMs co-cultured with SNs in the presence of GDNF. GDNF overexpressing VMs by adenovirus vector (AdGDNF-VMs) attracted more axons from SNs compared with mock-transfected VMs. In vivo, axon outgrowth toward the denervated myocardium in adult rat hearts after cryoinjury was also enhanced significantly by adenovirus-mediated GDNF overexpression. GDNF acts as a potent chemoattractant for sympathetic innervation of ventricular myocytes, and is a promising molecular target for regulation of cardiac function in diseased hearts.
We have established spontaneously immortalized Schwann cell lines from normal adult mice and rats and murine disease models. One of the normal mouse cell lines, IMS32, possesses some biological properties of mature Schwann cells and high proliferative activities. The IMS32 cells under hyperglycemic and/or hyperlipidemic conditions have been utilized to investigate the pathogenesis of diabetic neuropathy, especially the polyol pathway hyperactivity, glycation, increased oxidative stress, and reduced synthesis of neurotrophic factors. In addition to the mouse cell lines, our current study focuses on the characterization of a normal rat cell line, IFRS1, under normal and high glucose conditions. These Schwann cell lines can be valuable tools for exploring the detailed mechanisms leading to diabetic neuropathy and novel therapeutic approaches against that condition.
Amyotrophic lateral sclerosis (ALS) shows peculiar abnormalities of the autonomic nervous system, including sympathetic hyperactivity, which might result in sudden death. In general, the sympathetic hyperactivity could be caused by disruption of vagal inhibition. Our objective was to evaluate the vagus nerve morphometrically in autopsy cases of ALS with sympathetic hyperactivity and circulatory collapse (CC). We investigated 10 autopsied ALS patients, six of whom had exhibited autonomic storms or CC. We also examined 10 patients without ALS as controls, and one patient with Guillain-Barré syndrome (GBS) who died from CC, for comparison. After obtaining the visceral branch of the left vagus nerve at necropsy, we analyzed the density of the myelinated and unmyelinated fibers, and the fiber diameter distribution for each fiber. Results showed that the densities of both myelinated and unmyelinated fibers in ALS patients with or without CC were not significantly different from those in control patients. In contrast, the GBS patient showed marked reduction in the whole myelinated and large unmyelinated fiber density. In conclusion, the autonomic storms or CC due to sympathetic hyperactivity in ALS could not be ascribed to the deafferentation of the baroreflex, and more central neural pathophysiology should be investigated.
We established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle-shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75(NTR) ), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75(NTR) , truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N-cadherin), myelin proteins [P0, PMP22, and myelin-associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin-1 receptors (ErbB2 and ErbB3), and an orphan G protein-coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration.
Our objective was to describe cases of hyperosmolar hyperglycemic state (HHS) in advanced amyotrophic lateral sclerosis (ALS) patients and discuss its pathophysiology. Five ventilator-dependent patients with ALS, with no previous history of diabetes, showed development of marked hyperglycemia (plasma glucose levels of 755-1544 mg/dl) after preceding infectious episodes. All patients had severe generalized muscle wasting and tetraplegia. The initial manifestations of HHS were fever, drowsiness, or polyuria. Hydration and intravenous insulin therapy were markedly effective, resulting in favorable recovery without the necessity of chronic medication for hyperglycemia in all cases. Seventy-five grams oral glucose tolerance tests performed via feeding tubes in two patients after the successful treatment of HHS revealed increased insulin resistance and diminished early-phase insulin secretion with preserved total insulin secretion. In conclusion, a marked loss of skeletal muscle, the largest glucose consumer of the human body, with background abnormality of early-phase insulin secretion, might be a causative factor of HHS in advanced ALS.
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.
We have constructed a chitosan nonwoven nanofiber mesh tube consisting of oriented fibers by the electrospinning method. The efficacy of oriented nanofibers on Schwann cell alignment and positive effect of this tube on peripheral nerve regeneration were confirmed. The physical properties of the chitosan nanofiber mesh sheets prepared by electrospinning with or without fiber orientation were characterized. Then, immortalized Schwann cells were cultured on these sheets. Furthermore, the chitosan nanofiber mesh tubes with or without orientation, and bilayered chitosan mesh tube with an inner layer of oriented nanofibers and an outer layer of randomized nanofibers were bridgegrafted into rat sciatic nerve defect. As a result of fiber orientation, the tensile strength along the axis of the sheet increased. Because Schwann cells aligned along the nanofibers, oriented fibrous sheets could exhibit a Schwann cell column. Functional recovery and electrophysiological recovery occurred in time in the oriented group as well as in the bilayered group, and approximately matched those in the isograft. Furthermore, histological analysis revealed that the sprouting of myelinated axons occurred vigorously followed by axonal maturation in the isograft, oriented, and bilayered group in the order. The oriented chitosan nanofiber mesh tube may be a promising substitute for autogenous nerve graft.
Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 ?g/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 ?g/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.
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