Human transthyretin gene expression is markedly increased in repair Schwann cells in an in vitro model of hereditary transthyretin amyloidosis.

Hereditary transthyretin (TTR) amyloidosis (ATTRv) is characterized by TTR amyloid deposition in the peripheral nervous system. It remains unknown why variant TTR preferentially deposits in the peripheral nerves and dorsal root ganglia. We previously detected low levels of TTR expression in Schwann cells and established an immortalized Schwann cell line, TgS1, derived from a mouse model of ATTRv amyloidosis expressing the variant TTR gene. In the present study, the expression of TTR and Schwann cell marker genes was investigated in TgS1 cells by quantitative RT-PCR. TTR gene expression was markedly upregulated in TgS1 cells incubated in non-growth medium-Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The expression levels of c-Jun, Gdnf and Sox2 were increased, while Mpz was downregulated, suggesting that TgS1 cells exhibit a repair Schwann cell-like phenotype in the non-growth medium. Western blot analysis revealed that TTR protein was produced and secreted by the TgS1 cells. Furthermore, downregulation of Hsf1 with siRNA induced TTR aggregates in the TgS1 cells. These findings indicate that TTR expression is markedly increased in repair Schwann cells, likely to promote axonal regeneration. Therefore, aged dysfunctional repair Schwann cells may cause the deposition of variant TTR aggregates in the nerves of patients with ATTRv.

The Protective Effect of Edaravone on TDP-43 Plus Oxidative Stress-Induced Neurotoxicity in Neuronal Cells: Analysis of Its Neuroprotective Mechanisms Using RNA Sequencing.

Edaravone is a free-radical scavenger drug that was recently approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. A pathological hallmark of ALS is the accumulation of ubiquitinated or phosphorylated aggregates of the 43-kDa transactive response DNA binding protein (TDP-43) within the cytoplasm of motor neurons. This study revealed the efficacy of edaravone in preventing neuronal cell death in a TDP-43 proteinopathy model and analyzed the molecular changes associated with the neuroprotection. The viability of the neuronal cells expressing TDP-43 was reduced by oxidative stress, and edaravone (≥10 µmol/L) protected in a concentration-dependent manner against the neurotoxic insult. Differential gene expression analysis revealed changes among pathways related to nuclear erythroid 2-related-factor (Nrf2)-mediated oxidative stress response in cells expressing TDP-43. In edaravone-treated cells expressing TDP-43, significant changes in gene expression were also identified among Nrf2-oxidative response, unfolded protein response, and autophagy pathways. In addition, the expression of genes belonging to phosphatidylinositol metabolism pathways was modified. These findings suggest that the neuroprotective effect of edaravone involves the prevention of TDP-43 misfolding and enhanced clearance of pathological TDP-43 in TDP-43 proteinopathy.

Praja1 RING-finger E3 ubiquitin ligase is a common suppressor of neurodegenerative disease-associated protein aggregation.

The formation of misfolded protein aggregates is one of the pathological hallmarks of neurodegenerative diseases. We have previously demonstrated the cytoplasmic aggregate formation of adenovirally expressed transactivation response DNA-binding protein of 43 kDa (TDP-43), the main constituent of neuronal cytoplasmic aggregates in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), in cultured neuronal cells under the condition of proteasome inhibition. The TDP-43 aggregate formation was markedly suppressed by co-infection of adenoviruses expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response, and Praja1 RING-finger E3 ubiquitin ligase (PJA1) located downstream of the HSF1 pathway. In the present study, we examined other reportedly known E3 ubiquitin ligases for TDP-43, i.e. Parkin, RNF112 and RNF220, but failed to find their suppressive effects on neuronal cytoplasmic TDP-43 aggregate formation, although they all bind to TDP-43 as verified by co-immunoprecipitation. In contrast, PJA1 also binds to adenovirally expressed wild-type and mutated fused in sarcoma, superoxide dismutase 1, α-synuclein and ataxin-3, and huntingtin polyglutamine proteins in neuronal cultures and suppressed the aggregate formation of these proteins. These results suggest that PJA1 is a common sensing factor for aggregate-prone proteins to counteract their aggregation propensity, and could be a potential therapeutic target for neurodegenerative diseases that include ALS, FTLD, Parkinson's disease and polyglutamine diseases.

A low amyloidogenic E61K transthyretin mutation may cause familial amyloid polyneuropathy.

Patients with transthyretin (TTR)-type familial amyloid polyneuropathy (FAP) typically exhibit sensory dominant polyneuropathy and autonomic neuropathy. However, the molecular pathogenesis of the neuropathy remains unclear. In this study, we characterize the features of FAP TTR the substitution of lysine for glutamic acid at position 61 (E61K). This FAP was late-onset, with sensory dominant polyneuropathy, autonomic neuropathy, and cardiac amyloidosis. Interestingly, no amyloid deposits were found in the endoneurium of the four nerve specimens examined. Therefore, we examined the amyloidogenic properties of E61K TTR in vitro. Recombinant wild-type TTR, the substitution of methionine for valine at position 30 (V30M) TTR, and E61K TTR proteins were incubated at 37°C for 72 hr, and amyloid fibril formation was assessed using the thioflavin-T binding assay. Amyloid fibril formation by E61K TTR was less than that by V30M TTR, and similar to that by wild-type TTR. E61K TTR did not have an inhibitory effect on neurite outgrowth from adult rat dorsal root ganglion (DRG) neurons, but V30M TTR did. Furthermore, we studied the sural nerve of our patient by terminal deoxynucleotidyl transferase dUTP nick end labeling and electron microscopy. A number of apoptotic cells were observed in the endoneurium of the nerve by transferase dUTP nick end labeling. Chromatin condensation was confirmed in the nucleus of non-myelinating Schwann cells by electron microscopy. These findings suggest that E61K TTR is low amyloidogenic, in vitro and in vivo. However, TTR aggregates and amyloid fibrils in the DRG may cause sensory impairments in FAP because the DRG has no blood-nerve barrier. Moreover, Schwann cell apoptosis may contribute to the neurodegeneration.

Astrocytes release glutamate via cystine/glutamate antiporter upregulated in response to increased oxidative stress related to sporadic amyotrophic lateral sclerosis.

A vast body of evidence implicates increased oxidative stress and extracellular glutamate accumulation in the pathomechanism of sporadic amyotrophic lateral sclerosis (ALS). Cystine/glutamate antiporter (xCT) carries extracellular cystine uptake and intracellular glutamate release (cystine/glutamate exchange) in the presence of oxidative stress. The aim of the present study was to determine the involvement of xCT in ALS. Immunohistochemical observations in the spinal cord sections demonstrated that xCT was mainly expressed in astrocytes, with staining more intense in 12 sporadic ALS patients as compared to 12 age-matched control individuals. Western blot and densitometric analyses of the spinal cord samples revealed that the relative value of xCT/ß-actin optical density ratio was significantly higher in the ALS group as compared to the control group. Next, we conducted cell culture experiments using a human astrocytoma-derived cell line (1321N1) and a mouse motor neuron/neuroblastoma hybrid cell line (NSC34). In 1321N1 cells, the normalized xCT expression levels in cell lysates were significantly increased by H2 O2 treatment. Glutamate concentrations in 1321 N1 cell culture-conditioned media were significantly elevated by H2 O2 treatment, and the H2 O2 -driven elevations were completely canceled by the xCT inhibitor erastin pretreatment. In motor neuron-differentiated NSC34 cells (NSC34d cells), both the normalized xCT expression levels in the cell lysates and glutamate concentrations in the cell-conditioned media were constant with or without H2 O2 treatment. The present results provide in vivo and in vitro evidence that astrocytes upregulate xCT expression to release glutamate in response to increased oxidative stress associated with ALS, contributing to extracellular glutamate accumulation.

Praja1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation.

Transactivation response DNA-binding protein of 43 kDa (TDP-43) is a major constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously shown neuronal cytoplasmic aggregate formation induced by recombinant adenoviruses expressing human wild-type and C-terminal fragment (CTF) TDP-43 under the condition of proteasome inhibition in vitro and in vivo. In the present study, we demonstrated that the formation of the adenoviral TDP-43 aggregates was markedly suppressed in rat neural stem cell-derived neuronal cells by co-infection of an adenovirus expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response. We performed DNA microarray analysis and searched several candidate molecules, located downstream of HSF1, which counteract TDP-43 aggregate formation. Among these, we identified Praja 1 RING-finger E3 ubiquitin ligase (PJA1) as a suppressor of phosphorylation and aggregate formation of TDP-43. Co-immunoprecipitation assay revealed that PJA1 binds to CTF TDP-43 and the E2-conjugating enzyme UBE2E3. PJA1 also suppressed formation of cytoplasmic phosphorylated TDP-43 aggregates in mouse facial motor neurons in vivo. Furthermore, phosphorylated TDP-43 aggregates were detected in PJA1-immunoreactive human ALS motor neurons. These results indicate that PJA1 is one of the principal E3 ubiquitin ligases for TDP-43 to counteract its aggregation propensity and could be a potential therapeutic target for ALS and FTLD.

Soluble iron accumulation induces microglial glutamate release in the spinal cord of sporadic amyotrophic lateral sclerosis.

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) demonstrated iron accumulation in the spinal cord and increased glutamate concentration in the cerebrospinal fluid. To clarify the relationship between the two phenomena, we first performed quantitative and morphological analyses of substances related to iron and glutamate metabolism using spinal cords obtained at autopsy from 12 SALS patients and 12 age-matched control subjects. Soluble iron content determined by the Ferrozine method as well as ferritin (Ft) and glutaminase C (GLS-C) expression levels on Western blots were significantly higher in the SALS group than in the control group, while ferroportin (FPN) levels on Western blots were significantly reduced in the SALS group as compared to the control group. There was no significant difference in aconitase 1 (ACO1) and tumor necrosis factor-alpha (TNFα)-converting enzyme (TACE) levels on Western blots between the two groups. Immunohistochemically, Ft, ACO1, TACE, TNFα, and GLS-C were proven to be selectively expressed in microglia. Immunoreactivities for FPN and hepcidin were localized in neuronal and glial cells. Based on these observations, it is predicted that soluble iron may stimulate microglial glutamate release. To address this issue, cell culture experiments were carried out on a microglial cell line (BV-2). Treatment of BV-2 cells with ferric ammonium citrate (FAC) brought about significant increases in intracellular soluble iron and Ft expression levels and conditioned medium glutamate and TNFα concentrations. Glutamate concentration was also significantly increased in conditioned media of TNFα-treated BV-2 cells. While the FAC-driven increases in glutamate and TNFα release were completely canceled by pretreatment with ACO1 and TACE inhibitors, respectively, the TNFα-driven increase in glutamate release was completely canceled by GLS-C inhibitor pretreatment. Moreover, treatment of BV-2 cells with hepcidin resulted in a significant reduction in FPN expression levels on Western blots of the intracellular total protein extracts. The present results provide in vivo and in vitro evidence that microglial glutamate release in SALS spinal cords is enhanced by intracellular soluble iron accumulation-induced activation of ACO1 and TACE and by increased extracellular TNFα-stimulated GLS-C upregulation, and suggest a positive feedback mechanism to maintain increased intracellular soluble iron levels, involving TNFα, hepcidin, and FPN.

SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes.

Small integral membrane protein of the lysosome/late endosome (SIMPLE) is a 161-amino acid cellular protein that contains a characteristic C-terminal domain known as the SIMPLE-like domain (SLD), which is well conserved among species. Several studies have demonstrated that SIMPLE localizes to the trans-Golgi network (TGN), early endosomes, lysosomes, multivesicular bodies, aggresomes and the plasma membrane. However, the amino acid regions responsible for its subcellular localization have not yet been identified. The SLD resembles the FYVE domain, which binds phosphatidylinositol (3)-phosphate (PI3P) and determines the subcellular localization of FYVE domain-containing proteins. In the present study, we have found that SIMPLE binds specifically to PI4P through its SLD. SIMPLE co-localized with PI4P and Rab11, a marker for recycling endosomes (REs, organelles enriched in PI4P) in both the IMS32 mouse Schwann cell line and Hela cells. Sucrose density-gradient centrifugation revealed that SIMPLE co-fractionated with syntaxin-6 (a TGN marker) and Rab11. We have also found that SIMPLE knockdown impeded recycling of transferrin and of transferrin receptor. Our overall results indicate that SIMPLE may regulate protein trafficking physiologically by localizing to the TGN and/or REs by binding PI4P.

A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism.

The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long-term cultures of AR-deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real-time RT-PCR analyses revealed significantly down-regulated mRNA expression of polyol pathway-related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up-regulated mRNA expression of aldo-keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3-deoxyglucosone, methylglyoxal, and 4-hydroxynonenal) significantly up-regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR.

L-leucine and SPNS1 coordinately ameliorate dysfunction of autophagy in mouse and human Niemann-Pick type C disease.

Lysosomal storage disorders are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. 573C10 is a Schwann cell line derived from a mouse model of Niemann-Pick type C disease-1, NPC (-/-). Under serum-starved conditions, NPC (-/-) cells manifested impaired autophagy accompanied by an increase in the amount of p62 and lysosome enlargement. Addition of L-leucine to serum-starved NPC (-/-) cells ameliorated the enlargement of lysosomes and the p62 accumulation. Similar autophagy defects were observed in NPC (-/-) cells even without serum starvation upon the knockdown of Spinster-like 1 (SPNS1), a putative transporter protein thought to function in lysosomal recycling. Conversely, SPNS1 overexpression impeded the enlargement of lysosomes, p62 accumulation and mislocalization of the phosphorylated form of the mechanistic Target of rapamycin in NPC (-/-) cells. In addition, we found a reduction in endogenous SPNS1 expression in fibroblasts derived from NPC-1 patients compared with normal fibroblasts. We propose that SPNS1-dependent L-leucine export across the lysosomal membrane is a key step for triggering autophagy, and that this mechanism is impaired in NPC-1.

Myelinating cocultures of rodent stem cell line-derived neurons and immortalized Schwann cells.

Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.

Formation and spreading of TDP-43 aggregates in cultured neuronal and glial cells demonstrated by time-lapse imaging.

TAR DNA-binding protein 43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We have previously demonstrated that adenovirus-transduced artificial TDP-43 cytoplasmic aggregates formation is enhanced by proteasome inhibition in vitro and in vivo. However, the relationship between cytoplasmic aggregate formation and cell death remains unclear. In the present study, rat neural stem cell lines stably transfected with EGFP- or Sirius-expression vectors under the control of tubulin beta III, glial fibrillary acidic protein, or 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter were differentiated into neurons, astrocytes, and oligodendrocytes, respectively, in the presence of retinoic acid. The differentiated cells were then transduced with adenoviruses expressing DsRed-tagged human wild type and C-terminal fragment TDP-43 under the condition of proteasome inhibition. Time-lapse imaging analyses revealed growing cytoplasmic aggregates in the transduced neuronal and glial cells, followed by collapse of the cell. The aggregates remained insoluble in culture media, consisted of sarkosyl-insoluble granular materials, and contained phosphorylated TDP-43. Moreover, the released aggregates were incorporated into neighboring neuronal cells, suggesting cell-to-cell spreading. The present study provides a novel tool for analyzing the detailed molecular mechanisms of TDP-43 proteinopathy in vitro.

Clinicopathological characteristics of patients with amyotrophic lateral sclerosis resulting in a totally locked-in state (communication Stage V).

In the present study, we performed a comprehensive analysis to clarify the clinicopathological characteristics of patients with amyotrophic lateral sclerosis (ALS) that had progressed to result in a totally locked-in state (communication Stage V), in which all voluntary movements are lost and communication is impossible. In 11 patients, six had phosphorylated TAR DNA-binding protein 43 (pTDP-43)-immunoreactive (ir) neuronal cytoplasmic inclusions (NCI), two had fused in sarcoma (FUS)-ir NCI, and three had copper/zinc superoxide dismutase (SOD1)-ir NCI. The time from ALS onset to the need for tracheostomy invasive ventilation was less than 24 months in ten patients. Regardless of accumulated protein, all the patients showed common lesions in the pallido-nigro-luysian system, brainstem reticular formation, and cerebellar efferent system, in addition to motor neurons. In patients with pTDP-43-ir NCI, patients with NCI in the hippocampal dentate granule neurons (DG) showed a neuronal loss in the cerebral cortex, and patients without NCI in DG showed a preserved cerebral cortex. By contrast, in patients with FUS-ir NCI, patients with NCI in DG showed a preserved cerebral cortex and patients without NCI in DG showed marked cerebral degeneration. The cerebral cortex of patients with SOD1-ir NCI was preserved. Together, these findings suggest that lesions of the cerebrum are probably not necessary for progression to Stage V. In conclusion, patients with ALS that had progressed to result in communication Stage V showed rapidly-progressed symptoms, and their common lesions could cause the manifestations of communication Stage V.

Quantitative analysis of the features of fasciculation potentials and their relation with muscle strength in amyotrophic lateral sclerosis.

This study aimed to quantitatively analyze fasciculation potentials (FPs) and to investigate their relationship with muscle strength in amyotrophic lateral sclerosis (ALS). Fifty-one patients with sporadic ALS or progressive muscular atrophy (25 men, 26 women, mean age of 68 years) underwent needle EMG. We determined the duration, phase number, and amplitude of FPs from three muscles (upper trapezius, biceps brachii, and tibialis anterior) and examined their relations with muscle strength. In total, 878 FPs were analyzed. FP duration displayed a significant negative relation with the strength of all three muscles; the weaker muscles showed longer durations of FPs than the muscles with normal strength. The amplitude and phase number were not related with muscle strength, but there were significant correlations between the duration and amplitude of FPs in the trapezius and tibialis anterior muscles. The longer duration of FPs in muscles with weak strength suggests that the morphological changes of FPs were caused by temporal dispersion through progressively degenerating and/or immature reinnervating motor branches, and were observed uniformly in different muscles along with disease progression.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone-induced schwannopathy.

Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10 µm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

Predictive factors for prognosis following unsedated percutaneous endoscopic gastrostomy in ALS patients.

INTRODUCTION: This study aimed to determine the prognostic factors and the values that predict survival after percutaneous endoscopic gastrostomy (PEG) tube placement in patients with amyotrophic lateral sclerosis (ALS). METHODS: We retrospectively analyzed the correlations for 97 consecutive patients with ALS between clinical parameters and survival following PEG tube placement using the log-rank test and Cox proportional-hazards models. RESULTS: The log-rank test showed that an arterial carbon dioxide pressure (PaCO2 ) of ≤ 40 mmHg (P = 0.0054), a forced vital capacity (FVC) of ≥ 38% of predicted (P = 0.0003), and bulbar-onset (P = 0.0121) were significantly associated with better post-PEG survival. Multivariate analysis showed that the FVC and PaCO2 were associated with better post-PEG survival (P = 0.0081 and P = 0.0265, respectively). CONCLUSIONS: PEG tube placement in ALS is recommended when FVC is ≥ 38% of predicted and when PaCO2 is normal. Muscle Nerve 54: 277-283, 2016.

Predictors of impaired communication in amyotrophic lateral sclerosis patients with tracheostomy-invasive ventilation.

Predictors of communication impairment in patients with amyotrophic lateral sclerosis (ALS) using tracheostomy-invasive ventilation (TIV) were investigated. Seventy-six ALS patients using TIV were enrolled and classified into three subgroups of communication ability: patients who could communicate with communication devices (Stage I), patients who had difficulty with communication (Stage II, III, or IV), and patients who could not communicate by any means (Stage V). Predictors of communication impairment were analysed by the Cox proportional hazard model. Results demonstrated that there were no significant differences in disease duration between subgroups. Within 24 months after disease onset, patients who needed TIV and tube feeding, developed oculomotor impairment or became totally quadriplegic and progressed from Stage I to II and V significantly earlier. Multivariate analyses revealed that within 24 months from onset, the need for TIV and progression to total quadriplegia were significant events in patients who progressed to Stage II, whereas the development of oculomotor limitation was significant in patients who progressed to Stage V. In conclusion, TIV, impaired oculomotor movement and total quadriplegia are predictors of severe communication impairment. Rapid disease progression might indicate future communication impairment after the use of TIV. We highly recommend early detection of impaired communication and identification of the best methods of communication.

Schwann cells contribute to neurodegeneration in transthyretin amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is one of the transthyretin (TTR) amyloidoses characterized by extracellular amyloid deposits and peripheral nerve involvement. Recently, we found significant expression of the TTR gene in Schwann cells of the peripheral nervous system. We hypothesized that local expression of variant TTR in Schwann cells may contribute to neurodegeneration in FAP. Schwann cells derived from the dorsal root ganglia (DRG) of transgenic mice expressing variant human TTR in a mouse null background were cultured long term to obtain spontaneously immortalized cell lines. We established an immortalized Schwann cell line, TgS1, derived from the transgenic mice. TgS1 cells synthesized variant TTR and secreted it into the medium. As sensory neuropathy usually arises early in FAP, we examined the effect of the conditioned medium derived from TgS1 cells on neurite outgrowth from DRG sensory neurons. Conditioned medium derived from TgS1 cells inhibited neurite outgrowth from the sensory neurons. TTR deposition in the DRG of aged transgenic mice was investigated by immunohistochemistry. TTR aggregates were observed in the cytoplasm of Schwann cells and satellite cells. Proteasome inhibition induced TTR aggregates as aggresomes in TgS1 cells. In conclusion, local variant TTR gene expression in Schwann cells might trigger neurodegeneration in FAP. We established a spontaneously immortalized Schwann cell line derived from familial amyloidotic polyneuropathy transgenic mice. Conditioned medium from the cells contained variant transthyretin (TTR), and inhibited neurite outgrowth of neurons. TTR aggregates were observed in the Schwann cells and satellite cells of aged mice. Proteasome inhibition induced TTR aggregates as aggresomes in the cultured cells. These results support the hypothesis that Schwann cells contribute to neurodegeneration in familial amyloidotic polyneuropathy (FAP).

Upregulation of galectin-3 in immortalized Schwann cells IFRS1 under diabetic conditions.

A spontaneously immortalized adult Fischer rat Schwann cell line IFRS1 retains the characteristic features of normal Schwann cells, and can be a useful tool for the study of diabetic neuropathy. In the present study, we examined the effects of high glucose and 3-deoxyglucosone (3-DG) on the viability and the protein expression of advanced glycation endproducts (AGE)-binding proteins, such as galectin-3 (GAL-3) and receptor for AGE (RAGE) in IFRS1 cells. Exposure to 30mM of glucose or 0.2mM of 3-DG for 7 days failed to impair the IFRS1 cell viability, but significantly upregulated the expression of GAL-3. The same exposure tended to increase the expression of RAGE, but the changes were not significant. The high glucose-induced upregulation of GAL-3 was attenuated by cotreatment with 0.2mM of an anti-glycated agent aminoguanidine or 20nM of an anti-oxidant trans-resveratrol. In addition, treatment of IFRS1 cells with 1µg/ml of recombinant GAL-3 for 48h resulted in the upregulation of B-cell lymphoma 2 (Bcl-2) and the downregulation of 4-hydroxynonenal (4HNE). These findings suggest the involvement of GAL-3 in the glycation and oxidative stress under diabetic conditions and its cytoprotective role in Schwann cells.

A Japanese patient with familial ALS and a p.K510M mutation in the gene for FUS (FUS) resulting in the totally locked-in state.

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.