Motor Neuron-like NSC-34 Cells Research Articles

Highly Efficient Conversion of Motor Neuron-Like NSC-34 Cells into Functional Motor Neurons by Prostaglandin E2.

Motor neuron diseases are a group of progressive neurological disorders that degenerate motor neurons. The neuroblastoma × spinal cord hybrid cell line NSC-34 is widely used as an experimental model in studies of motor neuron diseases. However, the differentiation efficiency of NSC-34 cells to neurons is not always sufficient. We have found that prostaglandin E2 (PGE2) induces morphological differentiation in NSC-34 cells. The present study investigated the functional properties of PGE2-differentiated NSC-34 cells. Retinoic acid (RA), a widely-used agent inducing cell differentiation, facilitated neuritogenesis, which peaked on day 7, whereas PGE2-induced neuritogenesis took only 2 days to reach the same level. Whole-cell patch-clamp recordings showed that the current threshold of PGE2-treated cell action potentials was lower than that of RA-treated cells. PGE2 and RA increased the protein expression levels of neuronal differentiation markers, microtubule-associated protein 2c and synaptophysin, and to the same extent, motor neuron-specific markers HB9 and Islet-1. On the other hand, protein levels of choline acetyltransferase and basal release of acetylcholine in PGE2-treated cells were higher than in RA-treated cells. These results suggest that PGE2 is a rapid and efficient differentiation-inducing factor for the preparation of functionally mature motor neurons from NSC-34 cells.

The Molecular Mechanisms Underlying Prostaglandin D2-Induced Neuritogenesis in Motor Neuron-Like NSC-34 Cells.

Prostaglandins are a group of physiologically active lipid compounds derived from arachidonic acid. Our previous study has found that prostaglandin E2 promotes neurite outgrowth in NSC-34 cells, which are a model for motor neuron development. However, the effects of other prostaglandins on neuronal differentiation are poorly understood. The present study investigated the effect of prostaglandin D2 (PGD2) on neuritogenesis in NSC-34 cells. Exposure to PGD2 resulted in increased percentages of neurite-bearing cells and neurite length. Although D-prostanoid receptor (DP) 1 and DP2 were dominantly expressed in the cells, BW245C (a DP1 agonist) and 15(R)-15-methyl PGD2 (a DP2 agonist) had no effect on neurite outgrowth. Enzyme-linked immunosorbent assay demonstrated that PGD2 was converted to 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) under cell-free conditions. Exogenously applied 15d-PGJ2 mimicked the effect of PGD2 on neurite outgrowth. GW9662, a peroxisome proliferator-activated receptor–gamma (PPARγ) antagonist, suppressed PGD2-induced neurite outgrowth. Moreover, PGD2 and 15d-PGJ2 increased the protein expression of Islet-1 (the earliest marker of developing motor neurons), and these increases were suppressed by co-treatment with GW9662. These results suggest that PGD2 induces neuritogenesis in NSC-34 cells and that PGD2-induced neurite outgrowth was mediated by the activation of PPARγ through the metabolite 15d-PGJ2.

Ibudilast enhances the clearance of SOD1 and TDP-43 aggregates through TFEB-mediated autophagy and lysosomal biogenesis: The new molecular mechanism of ibudilast and its implication for neuroprotective therapy

A key feature of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders including Alzheimer disease (AD), Parkinson disease (PD) and Huntington’s disease (HD) is abnormal aggregation and deposition of misfolded proteins. Previous studies have shown that autophagy plays an important role in the clearance of disease-linked protein aggregates. In the current study, we report that ibudilast, which is a non-selective inhibitor of phosphodiesterases (PDEs) and an anti-inflammation drug, can induce autophagy and lysosomal biogenesis through mammalian target of rapamycin complex 1 - transcription factor EB (mTORC1-TFEB) signaling. We have found that ibudilast significantly enhances the clearance of disease-linked TAR DNA binding protein (TDP-43) and superoxide dismutase 1 (SOD1) protein aggregates in transfected cellular models carrying corresponding gene mutations. The mechanistic study revealed that ibudilast could markedly enhance TFEB nuclear translocation and increase the autolysosomes by inhibiting mTORC1 activity. We have also demonstrated that ibudilast could protect TDP-43-induced cytotoxicity in motor neuron-like NSC-34 cells. Collectively, our study identifies ibudilast as an autophagy enhancer and provides insights into the molecular basis of ibudilast for the potential treatment of several neurodegenerative disorders.

Generation of Cellular Reactive Oxygen Species by Activation of the EP2 Receptor Contributes to Prostaglandin E2-Induced Cytotoxicity in Motor Neuron-Like NSC-34 Cells.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease characterized by progressive degeneration of motor neurons in the central nervous system. Prostaglandin E2 (PGE2) plays a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. We have shown previously that PGE2 directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, a motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) and examined the effects of N-acetylcysteine (NAC), a cell-permeable antioxidant, on PGE2-induced cell death in differentiated NSC-34 cells. Dichlorofluorescein (DCF) fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by a selective EP2 antagonist (PF-04418948) but not a selective EP3 antagonist (L-798,106). Although an EP2-selective agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the level of intracellular ROS in these cells. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with NAC in a concentration-dependent manner. Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Moreover, dibutyryl-cAMP treatment of differentiated NSC-34 cells caused intracellular ROS generation and cell death. Our data reveal the existence of a PGE2-EP2 signaling-dependent intracellular ROS generation pathway, with subsequent activation of the caspase-3 cascade, in differentiated NSC-34 cells, suggesting that PGE2 is likely a key molecule linking inflammation to oxidative stress in motor neuron-like NSC-34 cells.

Induction of reactive oxygen species by activation of the EP2 receptor contributes to prostaglandin E2-induced cytotoxicity in motor neuron-like NSC-34 cells

Induction of reactive oxygen species by activation of the EP2 receptor contributes to prostaglandin E2-induced cytotoxicity in motor neuron-like NSC-34 cells

Wild-type Cu/Zn-superoxide dismutase is misfolded in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis

BackgroundA subset of familial forms of amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene coding Cu/Zn-superoxide dismutase (SOD1). Mutant SOD1 proteins are susceptible to misfolding and abnormally accumulated in spinal cord, which is most severely affected in ALS. It, however, remains quite controversial whether misfolding of wild-type SOD1 is involved in more prevalent sporadic ALS (sALS) cases without SOD1 mutations.MethodsCerebrospinal fluid (CSF) from patients including sALS as well as several other neurodegenerative diseases and non-neurodegenerative diseases was examined with an immunoprecipitation assay and a sandwich ELISA using antibodies specifically recognizing misfolded SOD1.ResultsWe found that wild-type SOD1 was misfolded in CSF from all sALS cases examined in this study. The misfolded SOD1 was also detected in CSF from a subset of Parkinson’s disease and progressive supranuclear palsy, albeit with smaller amounts than those in sALS. Furthermore, the CSF samples containing the misfolded SOD1 exhibited significant toxicity toward motor neuron-like NSC-34 cells, which was ameliorated by removal of the misfolded wild-type SOD1 with immunoprecipitation.ConclusionsTaken together, we propose that misfolding of wild-type SOD1 in CSF is a common pathological process of ALS cases regardless of SOD1 mutations.

The Novel Direct Modulatory Effects of Perampanel, an Antagonist of AMPA Receptors, on Voltage-Gated Sodium and M-type Potassium Currents.

Perampanel (PER) is a selective blocker of AMPA receptors showing efficacy in treating various epileptic disorders including brain tumor-related epilepsy and also potential in treating motor neuron disease. However, besides its inhibition of AMPA-induced currents, whether PER has any other direct ionic effects in different types of neurons remains largely unknown. We investigated the effects of PER and related compounds on ionic currents in different types of cells, including hippocampal mHippoE-14 neurons, motor neuron-like NSC-34 cells and U87 glioma cells. We found that PER differentially and effectively suppressed the amplitude of voltage-gated Na+ currents (INa) in mHippoE-14 cells. The IC50 values required to inhibit peak and late INa were 4.12 and 0.78 μM, respectively. PER attenuated tefluthrin-induced increases in both amplitude and deactivating time constant of INa. Importantly, PER also inhibited the amplitude of M-type K+ currents (IK(M)) with an IC50 value of 0.92 μM. The suppression of IK(M) was attenuated by the addition of flupirtine or ZnCl2 but not by L-quisqualic acid or sorafenib. Meanwhile, in cell-attached configuration, PER (3 μM) decreased the activity of M-type K+ channels with no change in single-channel conductance but shifting the activation curve along the voltage axis in a rightward direction. Supportively, PER suppressed IK(M) in NSC-34 cells and INa in U87 glioma cells. The inhibitory effects of PER on both INa and IK(M), independent of its antagonistic effect on AMPA receptors, may be responsible for its wide-spectrum of effects observed in neurological clinical practice.

A VDAC1-Derived N-Terminal Peptide Inhibits Mutant SOD1-VDAC1 Interactions and Toxicity in the SOD1 Model of ALS.

Mutations in superoxide dismutase (SOD1) are the second most common cause of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the death of motor neurons in the brain and spinal cord. SOD1 neurotoxicity has been attributed to aberrant accumulation of misfolded SOD1, which in its soluble form binds to intracellular organelles, such as mitochondria and ER, disrupting their functions. Here, we demonstrate that mutant SOD1 binds specifically to the N-terminal domain of the voltage-dependent anion channel (VDAC1), an outer mitochondrial membrane protein controlling cell energy, metabolic and survival pathways. Mutant SOD1G93A and SOD1G85R, but not wild type SOD1, directly interact with VDAC1 and reduce its channel conductance. No such interaction with N-terminal-truncated VDAC1 occurs. Moreover, a VDAC1-derived N-terminal peptide inhibited mutant SOD1-induced toxicity. Incubation of motor neuron-like NSC-34 cells expressing mutant SOD1 or mouse embryonic stem cell-derived motor neurons with different VDAC1 N-terminal peptides resulted in enhanced cell survival. Taken together, our results establish a direct link between mutant SOD1 toxicity and the VDAC1 N-terminal domain and suggest that VDAC1 N-terminal peptides targeting mutant SOD1 provide potential new therapeutic strategies for ALS.

Naringenin, a dietary flavanone, enhances insulin-like growth factor 1 receptor-mediated antioxidant defense and attenuates methylglyoxal-induced neurite damage and apoptotic death

ABSTRACT Objectives: Recent studies revealed the neuroprotective effects of naringenin (NGEN), a common dietary bioflavonoid contained in citrus fruits. However, there are limited data on its protection against methylglyoxal (MG), the most potent precursor of advanced glycation end-products. The present study was to investigate the protection of NGEN on MG-induced neurotoxicity and the involvement of insulin-like growth factor 1 receptor (IGF-1R) signaling. Methods: NSC34 motor neuron-like cells was used. Cell viability was measured by MTT assay. Protein expressions were analyzed by western blots. Morphological changes of neurites were observed by an inverted microscope. Reactive oxygen species (ROS) production and apoptotic cell numbers were measured by flow cytometer. Glutathione (GSH) level and superoxide dismutase (SOD) activity were measured by ELISA. Results: >NGEN attenuated ROS production and increased GSH level, SOD activity and nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear expression in MG-treated NSC34 cells. NGEN also increased neurite length and enhanced IGF-1R and p-Akt in MG-treated NSC34 cells. Furthermore, NGEN attenuated MG-induced apoptotic death accompanied with down-regulation of cleaved-poly (ADP-ribose) polymerase (PARP) and up-regulation of B-cell lymphoma-2 (Bcl-2). However, AG1024, an IGF-1R antagonist, attenuated the anti-oxidative and anti-apoptotic effects of NGEN in MG-treated cells. Discussion: The present results demonstrated that NGEN decreased neuronal apoptosis and improved antioxidant defense in MG-treated NSC34 cells. Moreover, IGF-1R-mediated antioxidant defense plays an important role in this protective mechanism. These findings suggest the potential benefits of NGEN on the prevention of MG-induced or diabetes/hyperglycemia-related neurotoxicity. In vivo studies are needed for further confirmation on NGEN-mediated neuroprotection.

Diallyl Trisulfide Protects Motor Neurons from the Neurotoxic Protein TDP-43 via Activating Lysosomal Degradation and the Antioxidant Response.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disease for which only limited effective therapeutics are available. Currently, TAR DNA-binding protein 43 (TDP-43) is recognized as a pathological and biochemical marker for ALS. Increases in the levels of aggregated or mislocalized forms of TDP-43 might result in ALS pathology. Therefore, clearance pathways for intracellular protein aggregates have been suggested as potential therapeutic targets for the treatment of ALS. Here we report that treatment of motor neuron-like NSC34 cells overexpressing TDP-43 with diallyl trisulfide (DATS) induced neuronal autophagy and lysosomal clearance of TDP-43 and C-terminal TDP-43 fragments. We also observed that the antioxidant transcription factor NF-E2-related factor 2 (Nrf2) was accumulated in the nucleus and the expression of the antioxidant enzymes heme oxygenase1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) was increased. Consequently, DATS suppressed the increase in the levels of reactive oxygen species induced by TDP-43 expression. This study extends the findings of prior reports indicating that lower doses of DATS mediate cell survival in part by inducing autophagy and activating the Nrf2/antioxidant response element pathway.

CHP1 reduction ameliorates spinal muscular atrophy pathology by restoring calcineurin activity and endocytosis.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZATM, has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular junction size, impaired maturity of neuromuscular junctions and smaller muscle fibre size compared to low-dose SMN antisense oligonucleotide alone. In NSC34 cells, Chp1 knockdown tripled macropinocytosis whereas clathrin-mediated endocytosis remained unaffected. Importantly, Chp1 knockdown restored macropinocytosis in Smn-depleted cells by elevating calcineurin phosphatase activity. CHP1 is an inhibitor of calcineurin, which collectively dephosphorylates proteins involved in endocytosis, and is therefore crucial in synaptic vesicle endocytosis. Indeed, we found marked hyperphosphorylation of dynamin 1 in SMA motor neurons, which was restored to control level by the heterozygous Chp1 mutant allele. Taken together, we show that CHP1 is a novel SMA modifier that directly interacts with PLS3, and that CHP1 reduction ameliorates SMA pathology by counteracting impaired endocytosis. Most importantly, we demonstrate that CHP1 reduction is a promising SMN-independent therapeutic target for a combinatorial SMA therapy.

SIRT1 activation with neuroheal is neuroprotective but SIRT2 inhibition with AK7 is detrimental for disconnected motoneurons

Sirtuin 1 (SIRT1) activity is neuroprotective, and we have recently demonstrated its role in the retrograde degenerative process in motoneurons (MNs) in the spinal cord of rats after peripheral nerve root avulsion (RA) injury. SIRT2 has been suggested to exert effects opposite those of SIRT1; however, its roles in neurodegeneration and neuron response after nerve injury remain unclear. Here we compared the neuroprotective potentials of SIRT1 activation and SIRT2 inhibition in a mouse model of hypoglossal nerve axotomy. This injury induced a reduction of around half MN population within the hypoglossal nucleus by a non-apoptotic neurodegenerative process triggered by endoplasmic reticulum (ER) stress that resulted in activation of the unfolded protein response mediated by IRE1α and XBP1 by 21 days post injury. Both SIRT1 activation with NeuroHeal and SIRT2 inhibition with AK7 protected NSC-34 motor neuron-like cells against ER stress in vitro. In agreement with the in vitro results, NeuroHeal treatment or SIRT1 overexpression was neuroprotective of axotomized hypoglossal MNs in a transgenic mouse model. In contrast, AK7 treatment or SIRT2 genetic depletion in mice inhibited damaged MN survival. To resolve the in vitro/in vivo discrepancies, we used an organotypic spinal cord culture system that preserves glial cells. In this system, AK7 treatment of ER-stressed organotypic cultures was detrimental for MNs and increased microglial nuclear factor-κB and the consequent transcription of cytotoxic pro-inflammatory factors similarly. The results highlight the importance of glial cells in determining the neuroprotective impact of any treatment.

Stimulatory Action of Telmisartan, an Antagonist of Angiotensin II Receptor, on Voltage-Gated Na⁺ Current: Experimental and Theoretical Studies.

Telmisartan (Tel) is recognized as a non-peptide blocker of AT1R. Whether this agent has any direct effects on ion currents remains unexplored. In whole-cell current recordings, addition of Tel increased the peak amplitude of voltage-gated Na⁺ (NaV) current (INa) accompanied by the increased time constant of INa inactivation in differentiated NSC-34 motor neuron-like cells. Tel-stimulated INa in these cells is unlinked to either blockade of AT1R or activation of peroxisome proliferator-activated receptor gamma (PPAR-γ). In order to explore how this compound affects the amplitude and kinetics of INa in neurons, a Hodgkin-Huxley-based (HH-based) model designed to mimic effect of Tel on the functional activities of neurons was computationally created in this study. In this framework, the parameter for h inactivation gating variable, which was changed in a stepwise fashion, was implemented to predict changes in membrane potentials (V) as a function of maximal Na⁺ (GNa), K⁺ conductance (GK), or both. As inactivation time course of INa was increased, the bifurcation point of V versus GNa became lower, and the range between subcritical and supercritical values at the bifurcation of V versus GK correspondingly became larger. During a slowing in INa inactivation, the critical boundary between GNa and GK was shifted towards the left. Simulation studies demonstrated that progressive slowing in the inactivation time course of INa resulted in unanticipated increase of neuronal excitability by mimicking the effect of Tel in neuronal cells. Collectively, Tel can directly interact with the NaV channel to increase peak INa as well as to slow INa inactivation. It is thus highly likely that the effects of Tel or its structurally similar drugs could be another intriguing mechanism underlying their pharmacological actions in neurons or neuroendocrine cells occurring in vivo.

Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1.

Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5-7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12-16h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50µg/mL), BDNF (10ng/mL), and ciliary neurotrophic factor (10ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the βIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot-Marie-Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.

MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)—a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties—inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.

Pathogenic role of EP2 receptors up-regulation in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice

Pathogenic role of EP2 receptors up-regulation in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice

Evidence of Decreased Activity in Intermediate-Conductance Calcium-Activated Potassium Channels During Retinoic Acid–Induced Differentiation in Motor Neuron–Like NSC-34 Cells

Background/Aims: Intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca; K_Ca3.1 or KCNN4) channels affect the behaviors of central neurons including motor neurons. The mechanism through which neuronal differentiation is related to the activity of these channels remains largely unclear. Methods: By using various molecular biology tools and electrophysiological measurements, we investigated possible changes in the activity of IK_Ca channels in a retinoic acid (RA)-induced differentiation process in motor neuron-like NSC-34 cells. Results: The protein and messenger RNA expression of K_Ca3.1 substantially diminished as NSC-34 cells were differentiated with low serum (1%) and 1 µM RA. In whole-cell current recordings, the density of delayed-rectifier K⁺ currents obtained from differentiated cells was elevated. However, the density of a ramp pulse-elicited K⁺ current that was sensitive to blockage by 1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole (TRAM-34)—an inhibitor of IK_Ca channels—was significantly higher in undifferentiated NSC-34 cells than in differentiated cells. In undifferentiated cells, the activity of IK_Ca channels was readily detected and the probability of channel openings was resistant to stimulation by diazoxide or suppression by verruculogen. Furthermore, this probability was increased by 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one or 9-phenanthrol and reduced by TRAM-34. The channel-opening probability decreased in RA-induced differentiated cells, whereas the single-channel conductance of IK_Ca channels did not differ between undifferentiated and differentiated cells. Moreover, the slow component of the mean closed time in these channels was significantly shorter in undifferentiated cells than in differentiated cells; however, the mean open time in the channel remained unchanged as cells were differentiated. Conclusion: RA-induced differentiation in neurons could exert a suppressive effect on the activity of IK_Ca channels.

The water extract of Liuwei dihuang possesses multi-protective properties on neurons and muscle tissue against deficiency of survival motor neuron protein

BackgroundDeficiency of survival motor neuron (SMN) protein, which is encoded by the SMN1 and SMN2 genes, induces widespread splicing defects mainly in spinal motor neurons, and leads to spinal muscular atrophy (SMA). Currently, there is no effective treatment for SMA. Liuwei dihuang (LWDH), a traditional Chinese herbal formula, possesses multiple therapeutic benefits against various diseases via modulation of the nervous, immune and endocrine systems. Previously, we demonstrated water extract of LWDH (LWDH-WE) protects dopaminergic neurons and improves motor activity in models of Parkinson's disease. PurposeThis study aimed to investigate the potential protection of LWDH-WE on SMN deficiency-induced neurodegeneration and muscle weakness. Study designThe effects of LWDH-WE on SMN deficiency-induced neurotoxicity and muscle atrophy were examined by using SMN-deficient NSC34 motor neuron-like cells and SMA-like mice, respectively. MethodsInducible SMN-knockdown NSC34 motor neuron-like cells were used to mimic SMN-deficient condition. Doxycycline (1 µg/ml) was used to induce SMN deficiency in stable NSC34 cell line carrying SMN-specific shRNA. SMAΔ7 mice were used as a severe type of SMA mouse model. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Apoptotic cells and neurite length were observed by inverted microscope. Protein expressions were examined by western blots. Muscle strength of animals was evaluated by hind-limb suspension test. ResultsLWDH-WE significantly increased SMN protein level, mitochondrial membrane potential and cell viability of SMN-deficient NSC34 cells. LWDH-WE attenuated SMN deficiency-induced down-regulation of B-cell lymphoma-2 (Bcl-2) and up-regulation of cytosolic cytochrome c and cleaved caspase-3. Moreover, LWDH-WE prevented SMN deficiency-induced inhibition of neurite outgrowth and activation of Ras homolog gene family, member A (RhoA)/ Rho-associated protein kinase (ROCK2)/ phospho-LIM kinase (p-LIMK)/ phospho-cofilin (p-cofilin) pathway. Furthermore, in SMA-like mice, LWDH-WE improved muscle strength and body weight accompanied with up-regulation of SMN protein in spinal cord, brain, and gastrocnemius muscle tissues. ConclusionThe present study demonstrated that LWDH-WE protects motor neurons against SMN deficiency-induced neurodegeneration, and it also improves the muscle strength of SMA-like mice, suggesting the potential benefits of LWDH-WE as a complementary prescription for SMN deficiency-related diseases.

Pathophysiological role of prostaglandin E2-induced up-regulation of the EP2 receptor in motor neuron-like NSC-34 cells and lumbar motor neurons in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective degeneration of motor neurons. The primary triggers for motor neuronal death are still unknown, but inflammation is considered to be an important factor contributing to the pathophysiology of ALS both clinically and in ALS models. Prostaglandin E2 (PGE2) and its corresponding four E-prostanoid receptors play a pivotal role in the degeneration of motor neurons in human and transgenic models of ALS. It has also been shown that PGE2-EP2 signaling in glial cells (astrocytes or microglia) promotes motor neuronal death in G93A mice. The present study was designed to investigate the levels of expression of EP receptors in the spinal motor neurons of ALS model mice and to examine whether PGE2 alters the expression of EP receptors in differentiated NSC-34 cells, a motor neuron-like cell line. Immunohistochemical staining demonstrated that EP2 and EP3 immunoreactivity was localized in NeuN-positive large cells showing the typical morphology of motor neurons in mice. Semi-quantitative analysis showed that the immunoreactivity of EP2 in motor neurons was significantly increased in the early symptomatic stage in ALS model mice. In contrast, the level of EP3 expression remained constant, irrespective of age. In differentiated NSC-34 cells, bath application of PGE2 resulted in a concentration-dependent decrease of MTT reduction. Although PGE2 had no effect on cell survival at concentrations of less than 10 μM, pretreatment with 10 μM PGE2 significantly up-regulated EP2 and concomitantly potentiated cell death induced by 30 μM PGE2. These results suggest that PGE2 is an important effector for induction of the EP2 subtype in differentiated NSC-34 cells, and that not only EP2 up-regulation in glial cells but also EP2 up-regulation in motor neurons plays a pivotal role in the vulnerability of motor neurons in ALS model mice.

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells

Assay to Measure Nucleocytoplasmic Transport in Real Time within Motor Neuron-like NSC-34 Cells