Motor Neuron-like NSC-34 Cells Research Articles

Neuroprotective effects of activated fibroblast growth factor receptor 1 via the suppression of p53 accumulation against poly-PR-mediated toxicity.

A GGGGCC hexanucleotide repeat expansion (HRE) within the C9orf72 gene is a major causative factor in amyotrophic lateral sclerosis (ALS). This aberrant HRE results in the generation of five distinct dipeptide repeat proteins (DPRs). Among the DPRs, poly-PR accumulates in the nucleus and exhibits particularly strong toxicity to motor and cortical neurons. Fibroblast growth factor receptor 1 (FGFR1) is known to promote neurogenesis and inhibit apoptosis in neurons. Nevertheless, there has been no previous report of its neuroprotective effects against poly-PR toxicity. The objective of this study was to investigate the neuroprotective effects of FGFR1 activation in poly-PR-expressing NSC34 motor neuron-like cells. RT-qPCR analysis in NSC34cells showed that Fgfr1 was the most highly expressed member of the Fgfr family in NSC34cells. The activation of FGFR1 by FGF2, a common ligand for all FGFRs, exerted neuroprotective effects against the toxicity of poly-PR. Additionally, FGFR1 activation was observed to enhance cell viability through the PI3K-AKT pathway, while the contribution of the MEK-ERK pathway was found to be limited. Furthermore, FGFR1 activation suppressed the accumulation of p53 protein and promoted its degradation through increased murine double minute 2 (MDM2), an E3 ubiquitin ligase that targets p53. The neuroprotective effects were attenuated by PD173074, a selective FGFR1 inhibitor or Nutlin-3a, an inhibitor of the p53-MDM2 interaction. Overall, these findings suggest that FGFR1 activation provides neuroprotection against poly-PR toxicity. Consequently, this study suggests the potential utility of FGFR1 activation as a therapeutic strategy for ALS.

Activated Fibroblast Growth Factor Receptor 1 Mitigated Poly-PR–Induced Oxidative Stress and Protein Translational Impairment

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron cell death. A GGGGCC hexanucleotide repeat expansion (HRE) within the chromosome 9 open reading frame 72 (C9orf72) gene is a major causative factor in ALS. This abnormal HRE triggers five types of dipeptide repeat protein (DPR), each composed of two alternating amino acid expressions. Among the DPRs, arginine-rich Poly-PR localizes predominantly to the nucleus, exerting particularly strong toxicity on motor and cortical neurons. Several mechanisms have been proposed for poly-PR-induced neurotoxicity. In this study, poly-PR-expressing NSC34 motor neuron-like cells showed an increase in oxidative stress. Fibroblast growth factor receptor 1 (FGFR1) is known to promote neurogenesis and inhibit apoptosis in neurons. However, its neuroprotective effects against DPR-induced toxicity have not been previously reported. Here, we demonstrated that FGFR1 activation reduced oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (NRF2) expression. Furthermore, we propose that the increase in NRF2 through FGFR1 activation may result from the alleviation of protein translation impairment. Overall, these findings suggest that FGFR1 activation provides neuroprotection against poly-PR toxicity and may represent a potential therapeutic strategy for ALS.

Empowering the NSC-34 cell line as a motor neuron model: cytosine arabinoside's action.

The NSC-34 cell line is a widely recognized motor neuron model and various neuronal differentiation protocols have been exploited. Under previously reported experimental conditions, only part of the cells resemble differentiated neurons; however, they do not exhibit extensive and time-prolonged neuritogenesis, and maintain their duplication capacity in culture. The aim of the present work was to facilitate long-term and more homogeneous neuronal differentiation in motor neuron-like NSC-34 cells. We found that the antimitotic drug cytosine arabinoside promoted robust and persistent neuronal differentiation in the entire cell population. Long and interconnecting neuronal processes with abundant growth cones were homogeneously induced and were durable for up to at least 6 weeks in culture. Moreover, cytosine arabinoside was permissive, dispensable, and mostly irreversible in priming NSC-34 cells for neurite initiation and regeneration after mechanical dislodgement. Finally, the expression of the cell proliferation antigen Ki67 was inhibited by cytosine arabinoside, whereas the expression levels of neuronal growth associated protein 43, vimentin, and motor neuron-specific p75, Islet2, homeobox 9 markers were upregulated, as confirmed by western blot and/or confocal immunofluorescence analysis. Overall, these findings support the use of NSC-34 cells as a motor neuron model for properly investigating neurodegenerative mechanisms and prospectively identifying neuroprotective strategies.

Understanding the Role of the SMN Complex Component GEMIN5 and Its Functional Relationship with Demethylase KDM6B in the Flunarizine-Mediated Neuroprotection of Motor Neuron Disease Spinal Muscular Atrophy.

Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.

Tanshinone IIA protects motor neuron-like NSC-34 cells against lipopolysaccharide-induced cell injury by the regulation of the lncRNA TCTN2/miR-125a-5p/DUSP1 axis

BackgroundTanshinone IIA (TSIIA) exerts a protective role in spinal cord injury (SCI). However, the mechanism of TSIIA activity in SCI remains to be elucidated. MethodsCell viability and apoptosis were gauged by CCK-8 assay and flow cytometry, respectively. The expression levels of lncRNA TCTN2, miR-125a-5p and DUSP1 were detected by qRT-PCR and western blot. Direct relationship between miR-125a-5p and TCTN2 or DUSP1 was verified by dual-luciferase reporter assay. ResultsIn mouse NSC-34 cells, LPS reduced the expression of TCTN2. TSIIA alleviated cell injury induced by LPS and increased TCTN2 expression in LPS-exposed NSC-34 cells. TCTN2 was a downstream mediator of TSIIA activity. TCTN2 targeted miR-125a-5p, and TCTN2 over-expression attenuated LPS-induced cell damage in NSC-34 cells by down-regulating miR-125a-5p. TCTN2 functioned as a post-transcriptional regulator of DUSP1 expression through miR-125a-5p. DUSP1 was a functional target of miR-125a-5p in controlling NSC-34 cell injury induced by LPS. TSIIA inhibited miR-125a-5p expression and increased the level of DUSP1 protein in LPS-exposed NSC-34 cells. ConclusionOur study establishes a novel mechanism, the TCTN2/miR-125a-5p/DUSP1 axis, at least in part, for the protective activity of TSIIA in cell injury induced by LPS.

CXCR2 Is Deregulated in ALS Spinal Cord and Its Activation Triggers Apoptosis in Motor Neuron-Like Cells Overexpressing hSOD1-G93A.

Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by progressive depletion of motor neurons (MNs). Recent evidence suggests a role in ALS pathology for the C-X-C motif chemokine receptor 2 (CXCR2), whose expression was found increased at both mRNA and protein level in cortical neurons of sporadic ALS patients. Previous findings also showed that the receptor inhibition is able to prevent iPSC-derived MNs degeneration in vitro and improve neuromuscular function in SOD1-G93A mice. Here, by performing transcriptional analysis and immunofluorescence studies, we detailed the increased expression and localization of CXCR2 and its main ligand CXCL8 in the human lumbar spinal cord of sporadic ALS patients. We further investigated the functional role of CXCR2/ligands axis in NSC-34 motor neuron-like cells expressing human wild-type (WT) or mutant (G93A) SOD1. A significant expression of CXCR2 was found in doxycycline-induced G93A-SOD1-expressing cells, but not in WT cells. In vitro assays showed CXCR2 activation by GROα and MIP2α, two murine endogenous ligands and functional homologs of CXCL8, reduces cellular viability and triggers apoptosis in a dose dependent manner, while treatment with reparixin, a non-competitive allosteric CXCR2 inhibitor, effectively counteracts GROα and MIP2α toxicity, significantly inhibiting the chemokine-induced cell death. Altogether, data further support a role of CXCR2 axis in ALS etiopathogenesis and confirm its pharmacological modulation as a candidate therapeutic strategy.

MicroRNA-23a-3p Is Upregulated in Plasma Exosomes of Bulbar-onset ALS Patients and Targets ERBB4

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease related to the progressive death of motor neurons. Understanding the pathogenesis of ALS continues to provide considerable challenges. Bulbar-onset ALS involves faster functional loss and shorter survival time than spinal cord-onset ALS. However, debate is ongoing regarding typical plasma miRNA changes in ALS patients with bulbar onset. Exosomal miRNAs have not yet been described as a tool for bulbar-onset ALS diagnosis or prognosis prediction. In this study, candidate exosomal miRNAs were identified by small RNA sequencing using samples from patients with bulbar-onset ALS and healthy controls. Potential pathogenic mechanisms were identified through enrichment analysis of target genes for differential miRNAs. Expression of miR-16-5p, miR-23a-3p, miR-22-3p, and miR-93-5p was significantly up-regulated in plasma exosomes from bulbar-onset ALS patients compared with healthy control subjects. Among them, miR-16-5p and miR-23a-3p were significantly lower in spinal-onset ALS patients than those with bulbar-onset. Furthermore, up-regulation of miR-23a-3p in motor neuron-like NSC-34 cells promoted apoptosis and inhibited cell viability. This miRNA was found to directly target ERBB4 and regulate the AKT/GSK3β pathway. Collectively, the above miRNAs and their targets are related to the development of bulbar-onset ALS. Our research indicates that miR-23a-3p might have an effect on motor neuron loss observed in bulbar-onset ALS and may be a novel target for the therapy of ALS in the future.

Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting both upper and lower motor neurons (MNs) with large unmet medical needs. Multiple pathological mechanisms are considered to contribute to the progression of ALS, including neuronal oxidative stress and mitochondrial dysfunction. Honokiol (HNK) has been reported to exert therapeutic effects in several neurologic disease models including ischemia stroke, Alzheimer's disease and Parkinson's disease. Here we found that honokiol also exhibited protective effects in ALS disease models both invitro and invivo. Honokiol improved the viability of NSC-34 motor neuron-like cells that expressed the mutant G93A SOD1 proteins (SOD1-G93A cells for short). Mechanistical studies revealed that honokiol alleviated cellular oxidative stress by enhancing glutathione (GSH) synthesis and activating the nuclear factor erythroid 2-related factor 2 (NRF2)-antioxidant response element (ARE) pathway. Also, honokiol improved both mitochondrial function and morphology via fine-tuning mitochondrial dynamics in SOD1-G93A cells. Importantly, honokiol extended the lifespan of the SOD1-G93A transgenic mice and improved the motor function. The improvement of antioxidant capacity and mitochondrial function was further confirmed in the spinal cord and gastrocnemius muscle in mice. Overall, honokiol showed promising preclinical potential as a multiple target drug for ALS treatment.

Cannabigerol Activates Cytoskeletal Remodeling via Wnt/PCP in NSC-34: An In Vitro Transcriptional Study

Cannabigerol (CBG) is a non-psychoactive phytocannabinoid present in the Cannabis sativa L. plant. In our study, CBG at the concentration of 10 µM was used to treat NSC-34 motor neuron-like cells. The aim of the study was to evaluate the effects of CBG on NSC-34 cells, using next-generation sequencing (NGS) technology. Analysis showed the activation of the WNT/planar cell polarity (PCP) pathway and Ephrin-Eph signaling. The results revealed that CBG increases the expression of genes associated with the onset process of cytoskeletal remodeling and axon guidance.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Increased expression of coronin-1a in amyotrophic lateral sclerosis: a potential diagnostic biomarker and therapeutic target.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. At present, no definite ALS biomarkers are available. In this study, exosomes from the plasma of patients with ALS and healthy controls were extracted, and differentially expressed exosomal proteins were compared. Among them, the expression of exosomal coronin-1a (CORO1A) was 5.3-fold higher than that in the controls. CORO1A increased with disease progression at a certain proportion in the plasma of patients with ALS and in the spinal cord of ALS mice. CORO1A was also overexpressed in NSC-34 motor neuron-like cells, and apoptosis, oxidative stress, and autophagic protein expression were evaluated. CORO1A overexpression resulted in increased apoptosis and oxidative stress, overactivated autophagy, and hindered the formation of autolysosomes. Moreover, CORO1A activated Ca2+-dependent phosphatase calcineurin, thereby blocking the fusion of autophagosomes and lysosomes. The inhibition of calcineurin activation by cyclosporin A reversed the damaged autolysosomes. In conclusion, the role of CORO1A in ALS pathogenesis was discovered, potentially affecting the disease onset and progression by blocking autophagic flux. Therefore, CORO1A might be a potential biomarker and therapeutic target for ALS.

Activation of Voltage-Gated Na+ Current by GV-58, a Known Activator of CaV Channels.

GV-58 ((2R)-2-[(6-{[(5-methylthiophen-2-yl)methyl]amino}-9-propyl-9H-purin-2-yl)amino]butan-1-ol) is recognized to be an activator of N- and P/Q-type Ca2+ currents. However, its modulatory actions on other types of ionic currents in electrically excitable cells remain largely unanswered. This study was undertaken to explore the possible modifications caused by GV-58 in ionic currents (e.g., voltage-gated Na+ current [INa], A-type K+ current [IK(A)], and erg-mediated K+ current [IK(erg)]) identified from pituitary GH3 lactotrophs. GH3 cell exposure to GV-58 enhanced the transient and late components of INa with varying potencies; consequently, the EC50 values of GV-58 required for its differential increase in peak and late INa in GH3 cells were estimated to be 8.9 and 2.6 μM, respectively. The INa in response to brief depolarizing pulse was respectively stimulated or suppressed by GV-58 or tetrodotoxin, but it failed to be altered by ω-conotoxin MVIID. Cell exposure to this compound increased the recovery of INa inactivation evoked by two-pulse protocol based on a geometrics progression; however, in its presence, there was a slowing in the inactivation rate of current decay evoked by a train of depolarizing pulses. The existence of GV-58 also resulted in an increase in the amplitude of ramp-induced resurgent and window INa. The presence of this compound inhibited IK(A) magnitude, accompanied by a shortening in inactivation time course of the current; however, it mildly decreased IK(erg). Under current-clamp conditions, GV-58 increased the frequency of spontaneous action potentials in GH3 cells. Moreover, in NSC-34 motor neuron-like cells, the presence of GV-58 not only raised INa amplitude but also reduced current inactivation. Taken together, the overall work provides a noticeable yet unidentified finding which implies that, in addition to its agonistic effect on Ca2+ currents, GV-58 may concertedly modify the amplitude and gating kinetics of INa in electrically excitable cells, hence modifiying functional activities in these cells.

A Single Amino Acid Substitution in Structural Protein VP2 Abrogates the Neurotropism of Enterovirus A-71 in Mice.

Enterovirus 71 (EV-A71) causes hand, foot, and mouth disease (HFMD) in children and has been associated with neurological complications. With no specific treatment and a monovalent vaccine limited to the Chinese market, HFMD remains a serious public health concern and an economic burden to affected societies. The molecular mechanisms underpinning EV-A71 neurovirulence have yet to be fully elucidated. In this work, we provide experimental evidence that a single amino acid substitution (I to K) at position 149 in structural protein VP2 of a non-mouse-adapted EV-A71 strain completely and specifically abrogated its infectivity in murine motor neuron-like NSC-34 cells. We showed that VP2 I149K mutant was impaired in murine SCARB2-mediated entry step but retained the ability to attach at the cell surface. In vivo, VP2 I149K mutant was fully attenuated in a symptomatic mouse model of progressive limb paralysis. While viral titers in limb muscles were comparable to mice infected with parental wild-type strain, significantly lower viral titers were measured in the spinal cord and brain, with minimal tissue damage, therefore indicating that VP2 I149K mutant is specifically impaired in its ability to invade the central nervous system (CNS). This study highlights the key role of amino acid at position 149 in VP2 in EV-A71 neurovirulence, and lends further support that the EF loop of VP2 represents a potential therapeutic target.

Rutin increases alpha-tubulin acetylation via histone deacetylase 6 inhibition.

Microtubules are dynamic cytoskeletal filaments composed of alpha- (α) and beta (β)-tubulin proteins. α-tubulin proteins are posttranslationally acetylated, and loss of acetylation is associated with axonal transport defects, a common alteration contributing to the pathomechanisms of several neurodegenerative diseases. Restoring α-tubulin acetylation by pharmacological inhibition of HDAC6, a primary α-tubulin deacetylase, can rescue impaired transport. Therefore, HDAC6 is considered a promising therapeutic target for neurodegenerative diseases, but currently, there is no clinically approved inhibitor for this purpose. In this study, using drug repurposing strategy, we aimed to identify compounds possessing HDAC6 inhibition activity and inducing α-tubulin acetylation. We systematically analyzed the FDA-approved library by utilizing virtual screening and consensus scoring approaches. Inhibition activities of promising compounds were tested using in vitro assays. Motor neuron-like NSC34 cells were treated with the candidate compounds, and α-tubulin acetylation levels were determined by Western blot. Our results demonstrated that rutin, a natural flavonoid, inhibits cellular HDAC6 activity without inducing any toxicity, and it significantly increases α-tubulin acetylation level in motor neuron-like cells.

Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons.

The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.

The Transcriptional Complex Sp1/KMT2A by Up-Regulating Restrictive Element 1 Silencing Transcription Factor Accelerates Methylmercury-Induced Cell Death in Motor Neuron-Like NSC34 Cells Overexpressing SOD1-G93A.

Methylmercury (MeHg) exposure has been related to amyotrophic lateral sclerosis (ALS) pathogenesis and molecular mechanisms of its neurotoxicity has been associated to an overexpression of the Restrictive Element 1 Silencing Transcription factor (REST). Herein, we evaluated the possibility that MeHg could accelerate neuronal death of the motor neuron-like NSC34 cells transiently overexpressing the human Cu2+/Zn2+superoxide dismutase 1 (SOD1) gene mutated at glycine 93 (SOD1-G93A). Indeed, SOD1-G93A cells exposed to 100 nM MeHg for 24 h showed a reduction in cell viability, as compared to cells transfected with empty vector or with unmutated SOD1 construct. Interestingly, cell survival reduction in SOD1-G93A cells was associated with an increase of REST mRNA and protein levels. Furthermore, MeHg increased the expression of the transcriptional factor Sp1 and promoted its binding to REST gene promoter sequence. Notably, Sp1 knockdown reverted MeHg-induced REST increase. Co-immunoprecipitation experiments demonstrated that Sp1 physically interacted with the epigenetic writer Lysine-Methyltransferase-2A (KMT2A). Moreover, knocking-down of KMT2A reduced MeHg-induced REST mRNA and protein increase in SOD1-G93A cells. Finally, we found that MeHg-induced REST up-regulation triggered necropoptotic cell death, monitored by RIPK1 increased protein expression. Interestingly, REST knockdown or treatment with the necroptosis inhibitor Necrostatin-1 (Nec) decelerated MeH-induced cell death in SOD1-G93A cells. Collectively, this study demonstrated that MeHg hastens necroptotic cell death in SOD1-G93A cells via Sp1/KMT2A complex, that by epigenetic mechanisms increases REST gene expression.

The Ryanodine receptor stabilizer S107 fails to support motor neuronal neuritogenesis in vitro

Calcium homeostasis is essential for neuronal cell survival/differentiation. Imbalance of the Ca2+ homeostasis due to excessive Ca2+ overload is essential for spinal cord injury (SCI). The overload resulted from Ca2+ flux across the plasma membrane and from internal Ca2+ store release (mitochondria, endoplasmic reticulum, ER). Inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) are involved in releasing Ca2+ from ER contributing to axonal degeneration following SCI. In turn, block of both receptors is axoprotective. The calstabin RyR subunit, stabilizing the channel in a state of reduced activity, prevents pathological Ca2+ release too.We investigated whether S107, a RyR-stabilizing compound (Rycal), is beneficial for survival and neuritogenesis of spinal cord motor neurons in vitro. We used a spinal cord slice model and the motor neuron-like NSC-34 cell line. Effects of S107 were tested by propidium iodide/fluorescein diacetate vital staining, mitotic index determination via BrdU-incorporation, and neurite sprouting parameters. Results showed that S107 (i) had no effect on gliosis resulting from slices preparation; (ii) had no effect on motor neuronal survival and proliferation; and (iii) impaired neurite sprouting, no matter whether it was a differentiation (NSC-34 cells) or regeneration (spinal cord slices) process. The results underline the need for a flexible Ca2+homeostasis provided by the ER for re-initiation of neuritogenesis.

Bisperoxovanadium promotes motor neuron survival in models of amyotrophic lateral sclerosis

The objective of our study was to test the therapeutic efficacy of a small molecule, bisperoxovanadium (bpV), in cell culture and animal models of the motor neuron (MN) disease, amyotrophic lateral sclerosis (ALS). A common cause of hereditary ALS, a mutation in the superoxide dismutase 1 protein (mSOD1G93A), is well studied and available models recapitulate human disease progression and MN loss. BpV is a known inhibitor of the phosphatase and tensin homolog, PTEN, and we have shown that bpV promotes MN survival in traumatic nervous system injury models. PTEN is well known as a negative regulator of phosphatidylinositol-3-kinase (PI3K)/Akt signaling, and this signaling pathway is linked to bpV's neuroprotective effects. We hypothesized that bpV treatment of both a mSOD1G93A MN cell line and mouse model ofALS would prevent MN degeneration and death. To test this hypothesis, we administered 400 μg/kg bpV intraperitoneally daily to mSOD1G93A mice between 70 and 90 days of age, which is a period of lumbar spinal cord MN death in these mice. After treatment, we collected tissue and examined immunolabeling of MNs in cryosections of the lumbar spinal cord and microglial reactivity, which can contribute to neuroinflammation and exacerbate MN loss. For our in vitro model, we cultured motor neuron-like NSC-34 cells transfected with a plasmid to overexpress mutant SOD1G93A, differentiated these cells for four days, and starved them in a serum-free medium for 24 hours both with and without bpV. Furthermore, we administered an inhibitor of PI3K/Akt signaling, LY294002, in the cell culture model to determine bpV's influence on PTEN and elucidate a potential mechanism of its action. We found that 20 days of bpV therapy significantly reduced lumbar ventral horn MN loss (p<0.05) but did not significantly influence microglial reactivity in mSOD1G93A mice. BpV improved neuron viability in vitro, and PI3K/Akt signaling inhibition reversed this effect (both p<0.05). In conclusion, our study indicates that bpV treatment could be a useful neuroprotective therapy for ALS.

Thermosensitive chitosan-based hydrogels supporting motor neuron-like NSC-34 cell differentiation.

Motor neuron diseases are neurodegenerative diseases that predominantly affect the neuromuscular system. To date, there are no valid therapeutic treatments for such diseases, and the classical experimental models fail in faithfully reproducing the pathological mechanisms behind them. In this regard, the use of three-dimensional (3D) culture systems, which more closely reproduce the native in vivo environment, can be a promising approach. Hydrogel-based systems are among the most used materials to reproduce the extracellular matrix, featuring an intrinsic similarity with its physiological characteristics. In this study, we developed a thermosensitive chitosan-based hydrogel combined with β-glycerophosphate (βGP) and sodium hydrogen carbonate (SHC), which give the system optimal mechanical properties and injectability, inducing the hydrogel sol-gel transition at 37 °C. An ad hoc protocol for the preparation of the hydrogel was established in order to obtain a highly homogeneous system, leading to reproducible physicochemical characteristics and easy cell encapsulation. All formulations supported the viability of a neuroblastoma/spinal cord hybrid cell line (NSC-34) beyond two weeks of culture and enabled cell differentiation towards a motor neuron-like morphology, characterized by the presence of extended neurites. Based on our results, these hydrogels represent excellent candidates for establishing 3D in vitro models of motor neuron diseases.

Upregulation of JHDM1D-AS1 alleviates neuroinflammation and neuronal injury via targeting miR-101-3p-DUSP1 in spinal cord after brachial plexus injury

BackgroundNeuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury. MethodsA total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1β-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1β, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay. ResultsWe found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3′UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1. ConclusionsJHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis.