HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA.

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

FNDC5/Irisin protects neurons through Caspase3 and Bax pathways.

Irisin is a glycosylated protein formed from the hydrolysis of fibronectin type III domain-containing protein 5 (FNDC5). Recent studies have demonstrated that FNDC5/Irisin is involved in the regulation of glucose and lipid metabolism, it can inhibit inflammation and have neuroprotective effects. However, the effect and mechanism of FNDC5/Irisin on motor neuron-like cell lines (NSC-34) have not been reported. In this study, we used lipopolysaccharide to construct cellular oxidative stress injury models and investigated the potential roles of FNDC5/Irisin on neurons by different cellular and molecular pathways. Taken together, our findings showed that FNDC5/Irisin can protect neurons, and this effect might be associated with Caspase3 and Bax pathways. These results laid the foundation for neuronal protection and clinical translation of FNDC5/Irisin therapy.

Signatures in in vitro infection of NSC-34 mouse neurons and their cell nucleus with Rickettsia helvetica.

BACKGROUND: Rickettsia helvetica, a spotted fever rickettsia, is transmitted to humans via ticks in Europe, North Africa, and Asia. The central nervous system is a crucial target for rickettsial diseases, which has been reported for 12 of the 31 species, of which R. helvetica is one. This study aimed, in an experimental model, to identify characteristics of R. helvetica infection in a mouse neuronal cell line, NSC-34. RESULTS: NSC-34, a fusion cell line of mouse motor spinal cord neurons and neuroblastoma cells, was used as a model. Propagation of R. helvetica in neurons was confirmed. Short actin tails were shown at the polar end of the bacteria, which makes it likely that they can move intracellularly, and even spread between cells. Another protein, Sca4, which with the cell adhesion protein vinculin enables the passage of the cell membrane, was expressed during infection. No significant increase in TNFα levels was seen in the infected neurons, which is of interest because TNFα protects the host cell from infection-induced apoptotic death which is crucial for host cell survival. The bacteria were also shown to invade and grow in the cell nucleus of the neuron. CONCLUSIONS: The findings suggest that a R. helvetica infection may be harmful to NSC-34 neurons under these in vitro conditions, but the full effects of the infection on the cell need to be studied further, also on human neurons, to also understand the possible significance of this infection in relation to pathogenetic mechanisms.

Involvement of CXCL10 in Neuronal Damage under the Condition of Spinal Cord Injury and the Potential Therapeutic Effect of Nrg1.

OBJECTIVE: Few studies have reported the direct effect of C-X-C motif chemokine ligand 10 (CXCL10) and Neuregulin 1 (Nrg1) on neurons after spinal cord injury (SCI). This study reports the role of CXCL10 in the regulation of neuronal damage after SCI and the potential therapeutic effect of Nrg1. METHODS: The expression level of CXCL10 and Nrg1 in SCI mice was analyzed in the Gene Expression Omnibus DataSets, followed by immunohistochemical confirmation using a mouse SCI model. HT22 cells and NSC34 cells were treated with CXCL10 and Nrg1, individually or in combination, and then assayed for cell viability. The percentage of wound closure was determined through the cell scratch injury model using HT22 and NSC34 cells. Potential molecular mechanisms were also tested in response to either the individual administration of CXCL10 and Nrg1 or a mixture of both molecules. RESULTS: CXCL10 expression was significantly increased in both young and old mice subjected to SCI, while Nrg1 expression was significantly decreased. CXCL10 induced a decrease in cell viability, which was partially reversed by Nrg1. CXCL10 failed to inhibit scratch healing in HT22 and NSC34 cells, while Nrg1 promoted scratch healing. At the molecular level, CXCL10-activated cleaved caspase 9 and cleaved caspase 3 were both inhibited by Nrg1 through pERK1/2 signaling in HT22 and NSC34 cells. CONCLUSIONS: CXCL10 is upregulated in SCI. Despite the negative effect on cell viability, CXCL10 failed to inhibit the scratch healing of HT22 and NSC34 cells. Nrg1 may protect neurons by partially antagonizing the effect of CXCL10.

The mechanism of the WNT5A and FZD4 receptor mediated WNT/ß-catenin pathway in the degeneration of ALS spinal cord motor neurons.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/ß-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/ß-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/ß-catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/ß-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

Present State and Future Perspectives of Prostaglandins as a Differentiation Factor in Motor Neurons.

Spinal motor neurons have the longest axons that innervate the skeletal muscles of the central nervous system. Motor neuron diseases caused by spinal motor neuron cell death are incurable due to the unique and irreplaceable nature of their neural circuits. Understanding the mechanisms of neurogenesis, neuritogenesis, and synaptogenesis in motor neurons will allow investigators to develop new in vitro models and regenerative therapies for motor neuron diseases. In particular, small molecules can directly reprogram and convert into neural stem cells and neurons, and promote neuron-like cell differentiation. Prostaglandins are known to have a role in the differentiation and tissue regeneration of several cell types and organs. However, the involvement of prostaglandins in the differentiation of motor neurons from neural stem cells is poorly understood. The general cell line used in research on motor neuron diseases is the mouse neuroblastoma and spinal motor neuron fusion cell line NSC-34. Recently, our laboratory reported that prostaglandin E2 and prostaglandin D2 enhanced the conversion of NSC-34 cells into motor neuron-like cells with neurite outgrowth. Moreover, we found that prostaglandin E2-differentiated NSC-34 cells had physiological and electrophysiological properties of mature motor neurons. In this review article, we provide contemporary evidence on the effects of prostaglandins, particularly prostaglandin E2 and prostaglandin D2, on differentiation and neural conversion. We also discuss the potential of prostaglandins as candidates for the development of new therapeutic drugs for motor neuron diseases.

Cannabinerol and NSC-34 Transcriptomic Analysis: Is the Dose Who Makes Neuronal Differentiation?

Cannabis sativa L. proved to be a source of several phytocompounds able to help patients facing different diseases. Moreover, these phytocompounds can help ameliorate general conditions and control certain unpleasant effects of diseases. Some cannabinoids, however, provided more benefits applicable to settings other than palliative care. Using the NSC-34 cell line, we evaluated the barely known phytocompound named cannabinerol (CBNR) at different doses, in order to understand its unique characteristics and the ones shared with other cannabinoids. The transcriptomic analysis suggests a possible ongoing neuronal differentiation, principally due to the activation of cannabinoid receptor 1 (CB1), to which the phosphorylation of serine-threonine protein kinase (Akt) followed, especially between 20 and 7.5 µM. The increase of Neurod1 and Map2 genes at 7.5 µM, accompanied by a decrease of Vim, as well as the increase of Syp at all the other doses, point toward the initiation of differentiation signals. Our preliminary results indicate CBNR as a promising candidate to be added to the list of cannabinoids with neuronal differentiation-enhancer properties. However, further studies are needed to confirm this initial insight.

The Amyotrophic Lateral Sclerosis M114T PFN1 Mutation Deregulates Alternative Autophagy Pathways and Mitochondrial Homeostasis.

Mutations in profilin 1 (PFN1) have been identified in rare familial cases of Amyotrophic Lateral Sclerosis (ALS). PFN1 is involved in multiple pathways that could intervene in ALS pathology. However, the specific pathogenic role of PFN1 mutations in ALS is still not fully understood. We hypothesized that PFN1 could play a role in regulating autophagy pathways and that PFN1 mutations could disrupt this function. We used patient cells (lymphoblasts) or tissue (post-mortem) carrying PFN1 mutations (M114T and E117G), and designed experimental models expressing wild-type or mutant PFN1 (cell lines and novel PFN1 mice established by lentiviral transgenesis) to study the effects of PFN1 mutations on autophagic pathway markers. We observed no accumulation of PFN1 in the spinal cord of one E117G mutation carrier. Moreover, in patient lymphoblasts and transfected cell lines, the M114T mutant PFN1 protein was unstable and deregulated the RAB9-mediated alternative autophagy pathway involved in the clearance of damaged mitochondria. In vivo, motor neurons expressing M114T mutant PFN1 showed mitochondrial abnormalities. Our results demonstrate that the M114T PFN1 mutation is more deleterious than the E117G variant in patient cells and experimental models and suggest a role for the RAB9-dependent autophagic pathway in ALS.

Cannabidiol Promotes Neuronal Differentiation Using Akt and Erk Pathways Triggered by Cb1 Signaling.

Recently, the scientific community has started to focus on the neurogenic potential of cannabinoids. The phytocompound cannabidiol (CBD) shows different mechanism of signaling on cannabinoid receptor 1 (CB1), depending on its concentration. In this study, we investigated if CBD may induce in vitro neuronal differentiation after treatment at 5 µM and 10 µM. For this purpose, we decided to use the spinal cord × neuroblastoma hybrid cell line (NSC-34) because of its proliferative and undifferentiated state. The messenger RNAs (mRNAs) expression profiles were tested using high-throughput sequencing technology and Western blot assay was used to determine the number of main proteins in different pathways. Interestingly, the treatment shows different genes associated with neurodifferentiation statistically significant, such as Rbfox3, Tubb3, Pax6 and Eno2. The CB1 signaling pathway is responsible for neuronal differentiation at 10 µM, as suggested by the presence of p-ERK and p-AKT, but not at 5 µM. A new correlation between CBD, neurodifferentiation and retinoic acid receptor-related orphan receptors (RORs) has been observed.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.

Could the Combination of Two Non-Psychotropic Cannabinoids Counteract Neuroinflammation? Effectiveness of Cannabidiol Associated with Cannabigerol.

Background and Objectives: Neuroinflammation is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this study, we investigate the anti-inflammatory, anti-oxidant, and anti-apoptotic properties of two non-psychoactive phytocannabinoids, cannabigerol (CBG) and cannabidiol (CBD). Materials and Methods: The motoneuron-like cell line NSC-34 differentiated by serum deprivation and with the additional treatment of all-trans retinoic acid (RA) is a valid model to investigate molecular events linked to neurodegeneration in ALS. Results: Pre-treatment with CBG (at 2.5 and 5 µM doses) alone and in combination with CBD (at 2.5 and 5 µM doses) was able to reduce neuroinflammation induced by a culture medium of LPS-stimulated macrophages. In particular, the pre-treatment with CBD at a 5 µM dose decreased TNF-α levels and increased IL10 and IL-37 expression. CBG-CBD association at a 5 µM dose also reduced NF-kB nuclear factor activation with low degradation of the inhibitor of kappaB alpha (IkBα). CBG and CBD co-administered at a 5 µM dose decreased iNOS expression and increased Nrf2 levels. Furthermore, the pre-treatment with the association of two non-psychoactive cannabinoids downregulated Bax protein expression and upregulated Bcl-2 expression. Our data show the anti-inflammatory, anti-oxidant, and anti-apoptotic effects PPARγ-mediated. Conclusions: Our results provide preliminary support on the potential therapeutic application of a CBG-CBD combination for further preclinical studies.

GPNMB ameliorates mutant TDP-43-induced motor neuron cell death.

Glycoprotein nonmetastatic melanoma protein B (GPNMB) aggregates are observed in the spinal cord of amyotrophic lateral sclerosis (ALS) patients, but the detailed localization is still unclear. Mutations of transactive response DNA binding protein 43kDa (TDP-43) are associated with neurodegenerative diseases including ALS. In this study, we evaluated the localization of GPNMB aggregates in the spinal cord of ALS patients and the effect of GPNMB against mutant TDP-43 induced motor neuron cell death. GPNMB aggregates were not localized in the glial fibrillary acidic protein (GFAP)-positive astrocyte and ionized calcium binding adaptor molecule-1 (Iba1)-positive microglia. GPNMB aggregates were localized in the microtubule-associated protein 2 (MAP-2)-positive neuron and neurofilament H non-phosphorylated (SMI-32)-positive neuron, and these were co-localized with TDP-43 aggregates in the spinal cord of ALS patients. Mock or TDP-43 (WT, M337V, and A315T) plasmids were transfected into mouse motor neuron cells (NSC34). The expression level of GPNMB was increased by transfection of mutant TDP-43 plasmids. Recombinant GPNMB ameliorated motor neuron cell death induced by transfection of mutant TDP-43 plasmids and serum-free stress. Furthermore, the expression of phosphorylated ERK1/2 and phosphorylated Akt were decreased by this stress, and these expressions were increased by recombinant GPNMB. These results indicate that GPNMB has protective effects against mutant TDP-43 stress via activating the ERK1/2 and Akt pathways, and GPNMB may be a therapeutic target for TDP-43 proteinopathy in familial and sporadic ALS. © 2016 Wiley Periodicals, Inc.

Exosome derived from murine adipose-derived stromal cells: Neuroprotective effect on in vitro model of amyotrophic lateral sclerosis.

Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) have not yet provided satisfactory results. Interest in stem cells for the treatment of neurodegenerative diseases is increasing and their beneficial action seems to be due to a paracrine effect via the release of exosomes, main mediators of cell-cell communication. Here we wished to assess, in vitro, the efficacy of a novel non-cell therapeutic approach based on the use of exosomes derived from murine adipose-derived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, and used as in vitro models of disease. In particular, we set out to investigate the effect of exosomes on NSC-34 naïve cells and NSC-34 cells overexpressing human SOD1(G93A) or SOD1(G37R) or SOD1(A4V) mutants, exposed to oxidative stress. The data presented here indicate for the first time that exosomes (0.2 µg/ml) are able to protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability. These data highlight a promising role of exosomes derived from stem cells for potential therapeutic applications in motoneuron disease.

Effects of cerebrolysin on motor-neuron-like NSC-34 cells.

Although the peripheral nervous system is capable of regeneration, this capability is limited. As a potential means of augmenting nerve regeneration, the effects of cerebrolysin (CL)--a proteolytic peptide fraction--were tested in vitro on the motor-neuron-like NSC-34 cell line and organotypic spinal cord cultures. Therefore, NSC-34 cells were subjected to mechanical stress by changing media and metabolic stress by oxygen glucose deprivation. Afterwards, cell survival/proliferation using MTT and BrdU-labeling (FACS) and neurite sprouting using ImageJ analysis were evaluated. Calpain-1, Src and α-spectrin protein expression were analyzed by Western blot. In organotypic cultures, the effect of CL on motor neuron survival and neurite sprouting was tested by immunohistochemistry. CL had a temporary anti-proliferative but initially neuroprotective effect on OGD-stressed NSC-34 cells. High-dosed or repeatedly applied CL was deleterious for cell survival. CL amplified neurite reconstruction to limited extent, affected calpain-1 protein expression and influenced calpain-mediated spectrin cleavage as a function of Src expression. In organotypic spinal cord slice cultures, CL was not able to support motor neuron survival/neurite sprouting. Moreover, it hampered astroglia and microglia activities. The data suggest that CL may have only isolated positive effects on injured spinal motor neurons. High-dosed or accumulated CL seemed to have adverse effects in treatment of spinal cord injury. Further experiments are required to optimize the conditions for a safe clinical administration of CL in spinal cord injuries.

Down-regulation of apurinic/apyrimidinic endonuclease 1 (APE1) in spinal motor neurones under oxidative stress.

AIM: Apurinic/apyrimidinic endonuclease 1 (APE1) is an intermediate enzyme in base excision repair which is important for removing damaged nucleotides under normal and pathological conditions. Accumulation of damaged bases causes genome instability and jeopardizes cell survival. Our study is to examine APE1 regulation under oxidative stress in spinal motor neurones which are vulnerable to oxidative insult. METHODS: We challenged the motor neurone-like cell line NSC-34 with hydrogen peroxide and delineated APE1 function by applying various inhibitors. We also examined the expression of APE1 in spinal motor neurones after spinal root avulsion in adult rats. RESULTS: We showed that hydrogen peroxide induced APE1 down-regulation and cell death in a differentiated motor neurone-like cell line. Inhibiting the two functional domains of APE1, namely, DNA repair and redox domains potentiated hydrogen peroxide induced cell death. We further showed that p53 phosphorylation early after hydrogen peroxide treatment might contribute to the down-regulation of APE1. Our in vivo results similarly showed that APE1 was down-regulated after root avulsion injury in spinal motor neurones. Delay of motor neurone death suggested that APE1 might not cause immediate cell death but render motor neurones vulnerable to further oxidative insults. CONCLUSION: We conclude that spinal motor neurones down-regulate APE1 upon oxidative stress. This property renders motor neurones susceptible to continuous challenge of oxidative stress in pathological conditions.

Differentiated NSC-34 cells as an in vitro cell model for VX.

The US military has placed major emphasis on developing therapeutics against nerve agents (NA). Current efforts are hindered by the lack of effective in vitro cellular models to aid in the preliminary screening of potential candidate drugs/antidotes. The development of an in vitro cellular model to aid in discovering new NA therapeutics would be highly beneficial. In this regard, we have examined the response of a differentiated hybrid neuronal cell line, NSC-34, to the NA VX. VX-induced apoptosis of differentiated NSC-34 cells was measured by monitoring the changes in caspase-3 and caspase-9 activity post-exposure. Differentiated NSC-34 cells showed an increase in caspase-3 activity in a manner dependent on both time (17-23 h post-exposure) and dose (10-100 nM). The maximal increase in caspase-3 activity was found to be at 20-h post-exposure. Caspase-9 activity was also measured in response to VX and was found to be elevated at all concentrations (10-100 nM) tested. VX-induced cell death was also observed by utilizing annexin V/propidium iodide flow cytometry. Finally, VX-induced caspase-3 or -9 activities were reduced with the addition of pralidoxime (2-PAM), one of the current therapeutics used against NA toxicity, and dizocilpine (MK-801). Overall the data presented here show that differentiated NSC-34 cells are sensitive to VX-induced cell death and could be a viable in vitro cell model for screening NA candidate therapeutics.

Cannabinol (CBN) Influences the Ion Channels and Synaptic-Related Genes in NSC-34 Cell Line: A Transcriptomic Study.

Neurological disorders such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis, and schizophrenia are associated with altered neuronal excitability, resulting from dysfunctions in the molecular architecture and physiological regulation of ion channels and synaptic transmission. Ion channels and synapses are regarded as suitable therapeutic targets in modern pharmacology. Cannabinoids have received great attention as an original therapeutic approach for their effects on human health due to their ability to modulate the neurotransmitter release through interaction with the endocannabinoid system. In our study, we explored the effect of cannabinol (CBN) through next-generation sequencing analysis of NSC-34 cell physiology. Our findings revealed that CBN strongly influences the ontologies related to ion channels and synapse activity at all doses tested. Specifically, the genes coding for calcium and potassium voltage-gated channel subunits, and the glutamatergic and GABAergic receptors (Cacna1b, Cacna1h, Cacng8, Kcnc3, Kcnd1, Kcnd2, Kcnj4, Grik5, Grik1, Slc17a7, Gabra5), were up-regulated. Conversely, the genes involved into serotoninergic and cholinergic pathways (Htr3a, Htr3b, Htr1b, Chrna3, Chrnb2, Chrnb4), were down-regulated. These findings highlight the influence of CBN in the expression of genes involved into ion influx and synaptic transmission.

Cannabinol Regulates the Expression of Cell Cycle-Associated Genes in Motor Neuron-like NSC-34: A Transcriptomic Analysis.

Cannabinoids are reported to have neuroprotective properties and play a role in neurogenesis and neuroplasticity in in vitro and in vivo models. Cannabinol (CBN) is a minor cannabinoid produced by the degradation of Δ9-tetrahydrocannabinol in Cannabis sativa L. and exhibits anti-oxidant, analgesic, anti-bacterial, and anti-inflammatory effects. In this study, we explored the biological effects of 20 µM CBN (6.20 µg/mL) on differentiated NSC-34 cells by MTT assay and next-generation sequencing analysis on the transcriptome. KEGG and Gene Ontology enrichment analyses have been performed to evaluate potential CBN-associated processes. Our results highlighted the absence of any cytotoxic effect of CBN. The comparative transcriptomic analysis pointed out the downregulation of Cdkn2a, Cdkn2c and Cdkn2d genes, which are known to suppress the cell cycle. Ccne2, Cdk2, Cdk7, Anapc11, Anapc10, Cdc23, Cdc16, Anapc4, Cdc27, Stag1, Smc3, Smc1a, Nipbl, Pds5a, Pds5b, and Wapl genes, renowned for their role as cell cycle progression activators, were instead upregulated. Our work suggests that CBN regulates the expression of many genes related to the cell cycle, which are required for axonal maturation, migration, and synaptic plasticity, while not affecting the expression of genes involved in cell death or tumorigenesis.

BV2 Microglial Cell Activation/Polarization Is Influenced by Extracellular Vesicles Released from Mutated SOD1 NSC-34 Motoneuron-like Cells.

Microglia-mediated neuroinflammation is a key player in the pathogenesis of amyotrophic lateral sclerosis (ALS) as it can contribute to the progressive degeneration of motor neurons (MNs). Here, we investigated the role of mSOD1 NSC-34 MN-like cell-derived extracellular vesicles (EVs) in inducing the activation of BV2 microglial cells. NSC-34-released EVs were isolated by culture medium differential ultracentrifugation to obtain two fractions, one containing small EVs (diameter < 200 nm) and the other containing large EVs (diameter > 200 nm). BV2 cells were incubated with the two EV fractions for 12, 24, and 48 h to evaluate 1) the state of microglial inflammation through RT-PCR of IL-1ß, IL-6, IL-4, and IL-10 and 2) the expression of proteins involved in inflammasome activation (IL-ß and caspase 1), cell death (caspase 3), and glial cell recruitment (CXCR1), and presence of the TGFß cytokine receptor (TGFß-R2). The obtained results suggest a mSOD1 type-dependent polarization of BV2 cells towards an early neurotoxic phenotype and a late neuroprotective status, with an appearance of mixed M1 and M2 microglia subpopulations. A significant role in driving microglial cell activation is played by the TGFß/CX3CR1 axis. Therefore, targeting the dysregulated microglial response and modulating neuroinflammation could hold promise as a therapeutic strategy for ALS.

Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFß firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1ß-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.