Integrative miRNA-mRNA profiling of human epidermis: unique signature of SCN9A painful neuropathy.

Personalized management of neuropathic pain is an unmet clinical need due to heterogeneity of the underlying aetiologies, incompletely understood pathophysiological mechanisms and limited efficacy of existing treatments. Recent studies on microRNA in pain preclinical models have begun to yield insights into pain-related mechanisms, identifying nociception-related species differences and pinpointing potential drug candidates. With the aim of bridging the translational gap towards the clinic, we generated a human pain-related integrative miRNA and mRNA molecular profile of the epidermis, the tissue hosting small nerve fibres, in a deeply phenotyped cohort of patients with sodium channel-related painful neuropathy not responding to currently available therapies. We identified four miRNAs strongly discriminating patients from healthy individuals, confirming their effect on differentially expressed gene targets driving peripheral sensory transduction, transmission, modulation and post-transcriptional modifications, with strong effects on gene targets including NEDD4. We identified a complex epidermal miRNA-mRNA network based on tissue-specific experimental data suggesting a cross-talk between epidermal cells and axons in neuropathy pain. Using immunofluorescence assay and confocal microscopy, we observed that Nav1.7 signal intensity in keratinocytes strongly inversely correlated with NEDD4 expression that was downregulated by miR-30 family, suggesting post-transcriptional fine tuning of pain-related protein expression. Our targeted molecular profiling advances the understanding of specific neuropathic pain fine signatures and may accelerate process towards personalized medicine in patients with neuropathic pain.

MyomiRs and their multifaceted regulatory roles in muscle homeostasis and amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of both upper and lower motor neurons (MNs). The main clinical features of ALS are motor function impairment, progressive muscle weakness, muscle atrophy and, ultimately, paralysis. Intrinsic skeletal muscle deterioration plays a crucial role in the disease and contributes to ALS progression. Currently, there are no effective treatments for ALS, highlighting the need to obtain a deeper understanding of the molecular events underlying degeneration of both MNs and muscle tissue, with the aim of developing successful therapies. Muscle tissue is enriched in a group of microRNAs called myomiRs, which are effective regulators of muscle homeostasis, plasticity and myogenesis in both physiological and pathological conditions. After providing an overview of ALS pathophysiology, with a focus on the role of skeletal muscle, we review the current literature on myomiR network dysregulation as a contributing factor to myogenic perturbations and muscle atrophy in ALS. We argue that, in view of their critical regulatory function at the interface between MNs and skeletal muscle fiber, myomiRs are worthy of further investigation as potential molecular targets of therapeutic strategies to improve ALS symptoms and counteract disease progression.

MiR-146a in ALS: Contribution to Early Peripheral Nerve Degeneration and Relevance as Disease Biomarker.

Amyotrophic lateral sclerosis (ALS) is characterized by the progressive, irreversible loss of upper and lower motor neurons (UMNs, LMNs). MN axonal dysfunctions are emerging as relevant pathogenic events since the early ALS stages. However, the exact molecular mechanisms leading to MN axon degeneration in ALS still need to be clarified. MicroRNA (miRNA) dysregulation plays a critical role in the pathogenesis of neuromuscular diseases. These molecules represent promising biomarkers for these conditions since their expression in body fluids consistently reflects distinct pathophysiological states. Mir-146a has been reported to modulate the expression of the NFL gene, encoding the light chain of the neurofilament (NFL) protein, a recognized biomarker for ALS. Here, we analyzed miR-146a and Nfl expression in the sciatic nerve of G93A-SOD1 ALS mice during disease progression. The miRNA was also analyzed in the serum of affected mice and human patients, the last stratified relying on the predominant UMN or LMN clinical signs. We revealed a significant miR-146a increase and Nfl expression decrease in G93A-SOD1 peripheral nerve. In the serum of both ALS mice and human patients, the miRNA levels were reduced, discriminating UMN-predominant patients from the LMN ones. Our findings suggest a miR-146a contribution to peripheral axon impairment and its potential role as a diagnostic and prognostic biomarker for ALS.

Silk fibroin microgels as a platform for cell microencapsulation.

Cell microencapsulation has been utilized for years as a means of cell shielding from the external environment while facilitating the transport of gases, general metabolites, and secretory bioactive molecules at once. In this light, hydrogels may support the structural integrity and functionality of encapsulated biologics whereas ensuring cell viability and function and releasing potential therapeutic factors once in situ. In this work, we describe a straightforward strategy to fabricate silk fibroin (SF) microgels (µgels) and encapsulate cells into them. SF µgels (size ≈ 200 µm) were obtained through ultrasonication-induced gelation of SF in a water-oil emulsion phase. A thorough physicochemical (SEM analysis, and FT-IR) and mechanical (microindentation tests) characterization of SF µgels were carried out to assess their nanostructure, porosity, and stiffness. SF µgels were used to encapsulate and culture L929 and primary myoblasts. Interestingly, SF µgels showed a selective release of relatively small proteins (e.g., VEGF, molecular weight, MW = 40 kDa) by the encapsulated primary myoblasts, while bigger (macro)molecules (MW = 160 kDa) were hampered to diffusing through the µgels. This article provided the groundwork to expand the use of SF hydrogels into a versatile platform for encapsulating relevant cells able to release paracrine factors potentially regulating tissue and/or organ functions, thus promoting their regeneration.

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.

Dysregulation of Muscle-Specific MicroRNAs as Common Pathogenic Feature Associated with Muscle Atrophy in ALS, SMA and SBMA: Evidence from Animal Models and Human Patients.

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.

ALS mouse model SOD1G93A displays early pathology of sensory small fibers associated to accumulation of a neurotoxic splice variant of peripherin.

Growing evidence suggests that amyotrophic lateral sclerosis (ALS) is a multisystem neurodegenerative disease that primarily affects motor neurons and, though less evidently, other neuronal systems. About 75% of sporadic and familial ALS patients show a subclinical degeneration of small-diameter fibers, as measured by loss of intraepidermal nerve fibers (IENFs), but the underlying biological causes are unknown. Small-diameter fibers are derived from small-diameter sensory neurons, located in dorsal root ganglia (DRG), whose biochemical hallmark is the expression of type III intermediate filament peripherin. We tested here the hypothesis that small-diameter DRG neurons of ALS mouse model SOD1(G93A)suffer from axonal stress and investigated the underlying molecular mechanism. We found that SOD1(G93A)mice display small fiber pathology, as measured by IENF loss, which precedes the onset of the disease. In vitro small-diameter DRG neurons of SOD1(G93A)mice show axonal stress features and accumulation of a peripherin splice variant, named peripherin56, which causes axonal stress through disassembling light and medium neurofilament subunits (NFL and NFM, respectively). Our findings first demonstrate that small-diameter DRG neurons of the ALS mouse model SOD1(G93A)display axonal stress in vitro and in vivo, thus sustaining the hypothesis that the effects of ALS disease spread beyond motor neurons. These results suggest a molecular mechanism for the small fiber pathology found in ALS patients. Finally, our data agree with previous findings, suggesting a key role of peripherin in the ALS pathogenesis, thus highlighting that DRG neurons mirror some dysfunctions found in motor neurons.

FM19G11-loaded nanoparticles modulate energetic status and production of reactive oxygen species in myoblasts from ALS mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Considerable evidence indicates that early skeletal muscle atrophy plays a crucial role in the disease pathogenesis, leading to an altered muscle-motor neuron crosstalk that, in turn, may contribute to motor neuron degeneration. Currently, there is no effective treatment for ALS, highlighting the need to dig deeper into the pathological mechanisms for developing innovative therapeutic strategies. FM19G11 is a novel drug able to modulate the global cellular metabolism, but its effects on ALS skeletal muscle atrophy and mitochondrial metabolism have never been evaluated, yet. This study investigated whether FM19G11-loaded nanoparticles (NPs) may affect the bioenergetic status in myoblasts isolated from G93A-SOD1 mice at different disease stages. We found that FM19G1-loaded NP treatment was able to increase transcriptional levels of Akt1, Akt3, Mef2a, Mef2c and Ucp2, which are key genes associated with cell proliferation (Akt1, Akt3), muscle differentiation (Mef2c), and mitochondrial activity (Ucp2), in G93A-SOD1 myoblasts. These cells also showed a significant reduction of mitochondrial area and networks, in addition to decreased ROS production after treatment with FM19G11-loaded NPs, suggesting a ROS clearance upon the amelioration of mitochondrial dynamics. Our overall findings demonstrate a significant impact of FM19G11-loaded NPs on muscle cell function and bioenergetic status in G93A-SOD1 myoblasts, thus promising to open new avenues towards possible adoption of FM19G11-based nanotherapies to slow muscle degeneration in the frame of ALS and muscle disorders.

Archival skin biopsy specimens as a tool for miRNA-based diagnosis: Technical and post-analytical considerations.

Archived specimens, taken by standardized procedures in clinical practice, represent a valuable resource in translational medicine. Their use in retrospective molecular-based studies could provide disease and therapy predictors. Microfluidic array is a user-friendly and cost-effective method allowing profiling of hundreds of microRNAs (miRNAs) from a low amount of RNA. However, even though tissue miRNAs may include potentially robust biomarkers, non-uniformed post-analytical pipelines could hinder translation into clinics. In this study, epidermal RNA from archival skin biopsy specimens was isolated from patients with peripheral neuropathy and healthy individuals. Unbiased miRNA profiling was performed using RT-qPCR-based microfluidic array. We demonstrated that RNA obtained from archival tissue is appropriate for miRNA profiling, providing evidence that different practices in threshold selection could significantly influence the final results. We showed the utility of software-based quality control for amplification curves. We revealed that selection of the most stable reference and the calculation of geometric mean are suitable when utilizing microfluidic arrays without known references. By applying appropriate post-analytical settings, we obtained miRNA profile of human epidermis associated with biological processes and a list of suitable references. Our results, which outline technical and post-analytical considerations, support the broad use of archived specimens for miRNA analysis to unravel disease-specific molecular signatures.

Muscle and Muscle-like Autoantigen Expression in Myasthenia Gravis Thymus: Possible Molecular Hint for Autosensitization.

The thymus is widely recognized as an immunological niche where autoimmunity against the acetylcholine receptor (AChR) develops in myasthenia gravis (MG) patients, who mostly present thymic hyperplasia and thymoma. Thymoma-associated MG is frequently characterized by autoantibodies to the muscular ryanodine receptor 1 (RYR1) and titin (TTN), along with anti-AChR antibodies. By real-time PCR, we analyzed muscle-CHRNA1, RYR1, and TTN-and muscle-like-NEFM, RYR3 and HSP60-autoantigen gene expression in MG thymuses with hyperplasia and thymoma, normal thymuses and non-MG thymomas, to check for molecular changes potentially leading to an altered antigen presentation and autoreactivity. We found that CHRNA1 (AChR-α subunit) and AIRE (autoimmune regulator) genes were expressed at lower levels in hyperplastic and thymoma MG compared to the control thymuses, and that the RYR1 and TTN levels were decreased in MG versus the non-MG thymomas. Genes encoding autoantigens that share epitopes with AChR-α (NEFM and HSP60), RYR1 (neuronal RYR3), and TTN (NEFM) were up-regulated in thymomas versus hyperplastic and control thymuses, with distinct molecular patterns across the thymoma histotypes that could be relevant for autoimmunity development. Our findings support the idea that altered muscle autoantigen expression, related with hyperplastic and neoplastic changes, may favor autosensitization in the MG thymus, and that molecular mimicry involving tumor-related muscle-like proteins may be a mechanism that makes thymoma prone to developing MG.

Identification of a cytokine profile in serum and cerebrospinal fluid of pediatric and adult spinal muscular atrophy patients and its modulation upon nusinersen treatment.

Background and objectives: Multisystem involvement in spinal muscular atrophy (SMA) is gaining prominence since different therapeutic options are emerging, making the way for new SMA phenotypes and consequent challenges in clinical care. Defective immune organs have been found in preclinical models of SMA, suggesting an involvement of the immune system in the disease. However, the immune state in SMA patients has not been investigated so far. Here, we aimed to evaluate the innate and adaptive immunity pattern in SMA type 1 to type 3 patients, before and after nusinersen treatment. Methods: Twenty one pediatric SMA type 1, 2, and 3 patients and 12 adult SMA type 2 and 3 patients were included in this single-center retrospective study. A Bio-Plex Pro-Human Cytokine 13-plex Immunoassay was used to measure cytokines in serum and cerebrospinal fluid (CSF) of the study cohort before and after 6 months of therapy with nusinersen. Results: We detected a significant increase in IL-1ß, IL-4, IL-6, IL-10, IFN-γ, IL-17A, IL-22, IL-23, IL-31, and IL-33, in serum of pediatric and adult SMA patients at baseline, compared to pediatric reference ranges and to adult healthy controls. Pediatric patients showed also a significant increase in TNF-α and IL-17F levels at baseline. IL-4, IFN-γ, Il-22, IL-23, and IL-33 decreased in serum of pediatric SMA patients after 6 months of therapy when compared to baseline. A significant decrease in IL-4, IL-6, INF-γ, and IL-17A was detected in serum of adult SMA patients after treatment. CSF of both pediatric and adult SMA patients displayed detectable levels of all cytokines with no significant differences after 6 months of treatment with nusinersen. Notably, a higher baseline expression of IL-23 in serum correlated with a worse motor function outcome after treatment in pediatric patients. Moreover, after 6 months of treatment, patients presenting a higher IL-10 concentration in serum showed a better Hammersmith Functional Motor Scale Expanded (HFMSE) score. Discussion: Pediatric and adult SMA patients show an inflammatory signature in serum that is reduced upon SMN2 modulating treatment, and the presence of inflammatory mediators in CSF. Our findings enhance SMA knowledge with potential clinical and therapeutic implications.

LncRNAs Associated with Neuronal Development and Oncogenesis Are Deregulated in SOD1-G93A Murine Model of Amyotrophic Lateral Sclerosis.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease caused in 10% of cases by inherited mutations considered "familial". An ever-increasing amount of evidence is showing a fundamental role for RNA metabolism in ALS pathogenesis, and long non-coding RNAs (lncRNAs) appear to play a role in ALS development. Here, we aim to investigate the expression of a panel of lncRNAs (linc-Enc1, linc-Brn1a, linc-Brn1b, linc-p21, Hottip, Tug1, Eldrr, and Fendrr) which could be implicated in early phases of ALS. Via Real-Time PCR, we assessed their expression in a murine familial model of ALS (SOD1-G93A mouse) in brain and spinal cord areas of SOD1-G93A mice in comparison with that of B6.SJL control mice, in asymptomatic (week 8) and late-stage disease (week 18). We highlighted a specific area and pathogenetic-stage deregulation in each lncRNA, with linc-p21 being deregulated in all analyzed tissues. Moreover, we analyzed the expression of their human homologues in SH-SY5Y-SOD1-WT and SH-SY5Y-SOD1-G93A, observing a profound alteration in their expression. Interestingly, the lncRNAs expression in our ALS models often resulted opposite to that observed for the lncRNAs in cancer. These evidences suggest that lncRNAs could be novel disease-modifying agents, biomarkers, or pathways affected by ALS neurodegeneration.

miR-146a in Myasthenia Gravis Thymus Bridges Innate Immunity With Autoimmunity and Is Linked to Therapeutic Effects of Corticosteroids.

Toll-like receptor (TLR)-mediated innate immune responses are critically involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder affecting neuromuscular junction mainly mediated by antiacetylcholine receptor antibodies. Considerable evidence indicate that uncontrolled TLR activation and chronic inflammation significantly contribute to hyperplastic changes and germinal center (GC) formation in the MG thymus, ultimately leading to autoantibody production and autoimmunity. miR-146a is a key modulator of innate immunity, whose dysregulation has been associated with autoimmune diseases. It acts as inhibitor of TLR pathways, mainly by targeting the nuclear factor kappa B (NF-κB) signaling transducers, interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6); miR-146a is also able to target c-REL, inducible T-cell costimulator (ICOS), and Fas cell surface death receptor (FAS), known to regulate B-cell function and GC response. Herein, we investigated the miR-146a contribution to the intrathymic MG pathogenesis. By real-time PCR, we found that miR-146a expression was significantly downregulated in hyperplastic MG compared to control thymuses; contrariwise, IRAK1, TRAF6, c-REL, and ICOS messenger RNA (mRNA) levels were upregulated and negatively correlated with miR-146a levels. Microdissection experiments revealed that miR-146a deficiency in hyperplastic MG thymuses was not due to GCs, but restricted to the GC-surrounding medulla, characterized by IRAK1 overexpression. We also showed higher c-REL and ICOS mRNA levels, and lower FAS mRNA levels, in GCs than in the remaining medulla, according to the contribution of these molecules in GC formation. By double immunofluorescence, an increased proportion of IRAK1-expressing dendritic cells and macrophages was found in hyperplastic MG compared to control thymuses, along with GC immunoreactivity for c-REL. Interestingly, in corticosteroid-treated MG patients intrathymic miR-146a and mRNA target levels were comparable to those of controls, suggesting that immunosuppressive therapy may restore the microRNA (miRNA) levels. Indeed, an effect of prednisone on miR-146a expression was demonstrated in vitro on peripheral blood cells. Serum miR-146a levels were lower in MG patients compared to controls, indicating dysregulation of the circulating miRNA. Our overall findings strongly suggest that defective miR-146a expression could contribute to persistent TLR activation, lack of inflammation resolution, and hyperplastic changes in MG thymuses, thus linking TLR-mediated innate immunity to B-cell-mediated autoimmunity. Furthermore, they unraveled a new mechanism of action of corticosteroids in inducing control of autoimmunity in MG via miR-146a.

CXCL13/CXCR5 signalling is pivotal to preserve motor neurons in amyotrophic lateral sclerosis.

BACKGROUND: CXCL13 is a B and T lymphocyte chemokine that mediates neuroinflammation through its receptor CXCR5. This chemokine is highly expressed by motoneurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) SOD1G93A (mSOD1) mice during the disease, particularly in fast-progressing mice. Accordingly, in this study, we investigated the role of this chemokine in ALS. METHODS: We used in vitro and in vivo experimental paradigms derived from ALS mice and patients to investigate the expression level and distribution of CXCL13/CXCR5 axis and its role in MN death and disease progression. Moreover, we compared the levels of CXCL13 in the CSF and serum of ALS patients and controls. FINDINGS: CXCL13 and CXCR5 are overexpressed in the spinal MNs and peripheral axons in mSOD1 mice. CXCL13 inhibition in the CNS of ALS mice resulted in the exacerbation of motor impairment (n = 4/group;Mean_Diff.=27.81) and decrease survival (n = 14_Treated:19.2 ± 1.05wks, n = 17_Controls:20.2 ± 0.6wks; 95% CI: 0.4687-1.929). This was corroborated by evidence from primary spinal cultures where the inhibition or activation of CXCL13 exacerbated or prevented the MN loss. Besides, we found that CXCL13/CXCR5 axis is overexpressed in the spinal cord MNs of ALS patients, and CXCL13 levels in the CSF discriminate ALS (n = 30) from Multiple Sclerosis (n = 16) patients with a sensitivity of 97.56%. INTERPRETATION: We hypothesise that MNs activate CXCL13 signalling to attenuate CNS inflammation and prevent the neuromuscular denervation. The low levels of CXCL13 in the CSF of ALS patients might reflect the MN dysfunction, suggesting this chemokine as a potential clinical adjunct to discriminate ALS from other neurological diseases. FUNDING: Vaccinex, Inc.; Regione Lombardia (TRANS-ALS).

Circulating MyomiRs as Potential Biomarkers to Monitor Response to Nusinersen in Pediatric SMA Patients.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder caused by mutations in survival motor neuron (SMN) 1 gene, resulting in a truncated SMN protein responsible for degeneration of brain stem and spinal motor neurons. The paralogous SMN2 gene partially compensates full-length SMN protein production, mitigating the phenotype. Antisense oligonucleotide nusinersen (Spinraza®) enhances SMN2 gene expression. SMN is involved in RNA metabolism and biogenesis of microRNA (miRNA), key gene expression modulators, whose dysregulation contributes to neuromuscular diseases. They are stable in body fluids and may reflect distinct pathophysiological states, thus acting as promising biomarkers. Muscle-specific miRNAs (myomiRs) as biomarkers for clinical use in SMA have not been investigated yet. Here, we analyzed the expression of miR-133a, -133b, -206 and -1, in serum of 21 infantile SMA patients at baseline and after 6 months of nusinersen treatment, and correlated molecular data with response to therapy evaluated by the Hammersmith Functional Motor Scale Expanded (HFMSE). Our results demonstrate that myomiR serological levels decrease over disease course upon nusinersen treatment. Notably, miR-133a reduction predicted patients' response to therapy. Our findings identify myomiRs as potential biomarkers to monitor disease progression and therapeutic response in SMA patients.

FM19G11-Loaded Gold Nanoparticles Enhance the Proliferation and Self-Renewal of Ependymal Stem Progenitor Cells Derived from ALS Mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. In ALS mice, neurodegeneration is associated with the proliferative restorative attempts of ependymal stem progenitor cells (epSPCs) that normally lie in a quiescent in the spinal cord. Thus, modulation of the proliferation of epSPCs may represent a potential strategy to counteract neurodegeneration. Recent studies demonstrated that FM19G11, a hypoxia-inducible factor modulator, induces epSPC self-renewal and proliferation. The aim of the study was to investigate whether FM19G11-loaded gold nanoparticles (NPs) can affect self-renewal and proliferation processes in epSPCs isolated from G93A-SOD1 mice at disease onset. We discovered elevated levels of SOX2, OCT4, AKT1, and AKT3, key genes associated with pluripotency, self-renewal, and proliferation, in G93A-SOD1 epSPCs at the transcriptional and protein levels after treatment with FM19G11-loaded NPs. We also observed an increase in the levels of the mitochondrial uncoupling protein (UCP) gene in treated cells. FM19G11-loaded NPs treatment also affected the expression of the cell cycle-related microRNA (miR)-19a, along with its target gene PTEN, in G93A-SOD1 epSPCs. Overall our findings establish the significant impact of FM19G11-loaded NPs on the cellular pathways involved in self-renewal and proliferation in G93A-SOD1 epSPCs, thus providing an impetus to the design of novel tailored approaches to delay ALS disease progression.

Hyperexcitability in Cultured Cortical Neuron Networks from the G93A-SOD1 Amyotrophic Lateral Sclerosis Model Mouse and its Molecular Correlates.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the corticospinal tract and leading to motor neuron death. According to a recent study, magnetic resonance imaging-visible changes suggestive of neurodegeneration seem absent in the motor cortex of G93A-SOD1 ALS mice. However, it has not yet been ascertained whether the cortical neural activity is intact, or alterations are present, perhaps even from an early stage. Here, cortical neurons from this model were isolated at post-natal day 1 and cultured on multielectrode arrays. Their activity was studied with a comprehensive pool of neurophysiological analyses probing excitability, criticality and network architecture, alongside immunocytochemistry and molecular investigations. Significant hyperexcitability was visible through increased network firing rate and bursting, whereas topological changes in the synchronization patterns were apparently absent. The number of dendritic spines was increased, accompanied by elevated transcriptional levels of the DLG4 gene, NMDA receptor 1 and the early pro-apoptotic APAF1 gene. The extracellular Na+, Ca2+, K+ and Cl- concentrations were elevated, pointing to perturbations in the culture micro-environment. Our findings highlight remarkable early changes in ALS cortical neuron activity and physiology. These changes suggest that the causative factors of hyperexcitability and associated toxicity could become established much earlier than the appearance of disease symptoms, with implications for the discovery of new hypothetical therapeutic targets.

Epstein-Barr virus in tumor-infiltrating B cells of myasthenia gravis thymoma: an innocent bystander or an autoimmunity mediator?

The thymus plays a key role in myasthenia gravis (MG), a B cell-mediated autoimmune disorder affecting neuromuscular junction. Most MG patients have thymic abnormalities, including hyperplasia and thymoma, a neoplasm of thymic epithelial cells. Epstein-Barr virus (EBV) is associated with autoimmune diseases and tumors. Recently, we showed EBV persistence and reactivation in hyperplastic MG thymuses, suggesting that EBV might contribute to intra-thymic B cell dysregulation in MG patients. Here, we investigated EBV involvement in thymoma-associated MG, by searching for EBV markers in MG (n=26) and non-MG (n=14) thymomas. EBV DNA and EBV-encoded small nuclear RNA (EBER) 1 transcript were detected in 14/26 (53.8%) and 22/26 (84.6%) MG thymomas, and only in 3 of 14 (21.4%) non-MG thymomas. Latent EBNA2 and late gp350/220 lytic transcripts were undetectable in all, but one, thymomas, and early lytic BZLF1 transcript was absent in all samples, suggesting that early infection events and EBV reactivation were very rare in thymomas. EBER1 and 2-positive cells were detected in MG, but not in non-MG, thymomas, as well as cells expressing EBV latency proteins (EBNA1, LMP1, LMP2A), that were mainly of B cell phenotype, indicating EBV association with MG rather than with thymoma. Toll-like receptor (TLR) 3 transcriptional levels were higher in MG than non-MG thymomas and positively correlated with EBER1 levels, suggesting a role for EBERs in TLR3 activation. Our findings show that EBV is commonly present in thymoma-infiltrating B cells of myasthenic patients, indicating a contribution of EBV to B cell-mediated autoreactivity in MG associated with thymic tumor.

A longitudinal DTI and histological study of the spinal cord reveals early pathological alterations in G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective motor neuron degeneration in the motor cortex, brainstem and spinal cord. It is generally accepted that ALS is caused by death of motor neurons, however the exact temporal cascade of degenerative processes is not yet completely known. To identify the early pathological changes in spinal cord of G93A-SOD1 ALS mice we performed a comprehensive longitudinal analysis employing diffusion-tensor magnetic resonance imaging alongside histology and electron microscopy, in parallel with peripheral nerve histology. We showed the gradient of degeneration appearance in spinal cord white and gray matter, starting earliest in the ventral white matter, due to a cascade of pathological events including axon dysfunction and mitochondrial changes. Notably, we found that even the main sensory regions are affected by the neurodegenerative process at symptomatic disease phase. Overall our results attest the applicability of DTI in determining disease progression in ALS mice. These findings suggest that DTI could be potentially adapted in humans to aid the assessment of ALS progression and eventually the evaluation of treatment efficacy.