Interleukin-9 protects from microglia- and TNF-mediated synaptotoxicity in experimental multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a progressive neurodegenerative disease of the central nervous system characterized by inflammation-driven synaptic abnormalities. Interleukin-9 (IL-9) is emerging as a pleiotropic cytokine involved in MS pathophysiology. METHODS: Through biochemical, immunohistochemical, and electrophysiological experiments, we investigated the effects of both peripheral and central administration of IL-9 on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. RESULTS: We demonstrated that both systemic and local administration of IL-9 significantly improved clinical disability, reduced neuroinflammation, and mitigated synaptic damage in EAE. The results unveil an unrecognized central effect of IL-9 against microglia- and TNF-mediated neuronal excitotoxicity. Two main mechanisms emerged: first, IL-9 modulated microglial inflammatory activity by enhancing the expression of the triggering receptor expressed on myeloid cells-2 (TREM2) and reducing TNF release. Second, IL-9 suppressed neuronal TNF signaling, thereby blocking its synaptotoxic effects. CONCLUSIONS: The data presented in this work highlight IL-9 as a critical neuroprotective molecule capable of interfering with inflammatory synaptopathy in EAE. These findings open new avenues for treatments targeting the neurodegenerative damage associated with MS, as well as other inflammatory and neurodegenerative disorders of the central nervous system.

Pharmacological blockade of 2-AG degradation ameliorates clinical, neuroinflammatory and synaptic alterations in experimental autoimmune encephalomyelitis.

The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS. In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation. In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.

Synaptic and transcriptomic features of cortical and amygdala pyramidal neurons predict inefficient fear extinction.

Fear-related disorders arise from inefficient fear extinction and have immeasurable social and economic costs. Here, we characterize mouse phenotypes that spontaneously show fear-independent behavioral traits predicting adaptive or maladaptive fear extinction. We find that, already before fear conditioning, specific morphological, electrophysiological, and transcriptomic patterns of cortical and amygdala pyramidal neurons predispose to fear-related disorders. Finally, by using an optogenetic approach, we show the possibility to rescue inefficient fear extinction by activating infralimbic pyramidal neurons and to impair fear extinction by activating prelimbic pyramidal neurons.

MiR-142-3p is a Critical Modulator of TNF-mediated Neuronal Toxicity in Multiple Sclerosis.

BACKGROUND: TNF-dependent synaptotoxicity contributes to the neuronal damage occurring in patients with Multiple Sclerosis (pwMS) and its mouse model Experimental Autoimmune Encephalomyelitis (EAE). Here, we investigated miR-142-3p, a synaptotoxic microRNA induced by inflammation in EAE and MS, as a potential downstream effector of TNF signalling. METHODS: Electrophysiological recordings, supported by molecular, biochemical and histochemical analyses, were performed to explore TNF-synaptotoxicity in the striatum of EAE and healthy mice. MiR-142 heterozygous (miR-142 HE) mice and/or LNA-anti miR-142-3p strategy were used to verify the TNF-miR-142-3p axis hypothesis. The cerebrospinal fluid (CSF) of 151 pwMS was analysed to evaluate possible correlation between TNF and miR-142-3p levels and their impact on clinical parameters (e.g. progression index (PI), age-related clinical severity (gARMSS)) and MRI measurements at diagnosis (T0). RESULTS: High levels of TNF and miR-142-3p were detected in both EAE striatum and MS-CSF. The TNF-dependent glutamatergic alterations were prevented in the inflamed striatum of EAE miR-142 HE mice. Accordingly, TNF was ineffective in healthy striatal slices incubated with LNA-anti miR- 142-3p. However, both preclinical and clinical data did not validate the TNF-miR-142-3p axis hypothesis, suggesting a permissive neuronal role of miR-142-3p on TNF-signalling. Clinical data showed a negative impact of each molecule on disease course and/or brain lesions and unveiled that their high levels exert a detrimental synergistic effect on disease activity, PI and white matter lesion volume. CONCLUSION: We propose miR-142-3p as a critical modulator of TNF-mediated neuronal toxicity and suggest a detrimental synergistic action of these molecules on MS pathology.

Preventive exercise attenuates IL-2-driven mood disorders in multiple sclerosis.

BACKGROUND: Elevated levels of specific proinflammatory molecules in the cerebrospinal fluid (CSF) have been associated with disability progression, enhanced neurodegeneration and higher incidence of mood disorders in people with multiple sclerosis (MS). Studies in animal models of MS suggest that preventive exercise may play an immunomodulatory activity, with beneficial effects on both motor deficits and behavioral alterations. Here we explored the impact of lifestyle physical activity on clinical presentation and associated central inflammation in a large group of newly diagnosed patients with MS. Furthermore, we addressed the causal link between exercise-mediated immunomodulation and mood symptoms in the animal setting. METHODS: A cross-sectional study was conducted on 235 relapsing-remitting MS patients at the time of the diagnosis. Patients were divided into 3 groups ("sedentary", "lifestyle physical activity" and "exercise") according to the level of physical activity in the six months preceding the evaluation. Patients underwent clinical, neuropsychological and psychiatric evaluation, magnetic resonance imaging and lumbar puncture for diagnostic purposes. The CSF levels of proinflammatory and anti-inflammatory cytokines were analyzed and compared with a group of 80 individuals with non-inflammatory and non-degenerative diseases. Behavioral and electrophysiological studies were carried out in control mice receiving intracerebral injection of IL-2 or vehicle. Behavior was also assessed in mice with experimental autoimmune encephalomyelitis (EAE), animal model of MS, reared in standard (sedentary group) or running wheel-equipped (exercise group) cages. RESULTS: In exercising MS patients, depression and anxiety were reduced compared to sedentary patients. The CSF levels of the interleukin-2 and 6 (IL-2, IL-6) were increased in MS patients compared with control individuals. In MS subjects exercise was associated with normalized CSF levels of IL-2. In EAE mice exercise started before disease onset reduced both behavioral alterations and striatal IL-2 expression. Notably, a causal role of IL-2 in mood disorders was shown. IL-2 administration in control healthy mice induced anxious- and depressive-like behaviors and impaired type-1 cannabinoid (CB1) receptor-mediated neurotransmission at GABAergic synapses, mimicking EAE-induced synaptic dysfunction. CONCLUSIONS: Our results indicate an immunomodulatory effect of exercise in MS patients, associated with reduced CSF expression of IL-2, which might result in reduced mood disorders. These data suggest that exercise in the early stages may act as a disease-modifying therapy in MS although further longitudinal studies are needed to clarify this issue.

Transcriptomic and Network Analyses Reveal Immune Modulation by Endocannabinoids in Approach/Avoidance Traits.

Approach and avoidance (A/A) tendencies are stable behavioral traits in responding to rewarding and fearful stimuli. They represent the superordinate division of emotion, and individual differences in such traits are associated with disease susceptibility. The neural circuitry underlying A/A traits is retained to be the cortico-limbic pathway including the amygdala, the central hub for the emotional processing. Furthermore, A/A-specific individual differences are associated with the activity of the endocannabinoid system (ECS) and especially of CB1 receptors whose density and functionality in amygdala differ according to A/A traits. ECS markedly interacts with the immune system (IS). However, how the interplay between ECS and IS is associated with A/A individual differences is still ill-defined. To fill this gap, here we analyzed the interaction between the gene expression of ECS and immune system (IS) in relation to individual differences. To unveil the deep architecture of ECS-IS interaction, we performed cell-specific transcriptomics analysis. Differential gene expression profiling, functional enrichment, and protein-protein interaction network analyses were performed in amygdala pyramidal neurons of mice showing different A/A behavioral tendencies. Several altered pro-inflammatory pathways were identified as associated with individual differences in A/A traits, indicating the chronic activation of the adaptive immune response sustained by the interplay between endocannabinoids and the IS. Furthermore, results showed that the interaction between the two systems modulates synaptic plasticity and neuronal metabolism in individual difference-specific manner. Deepening our knowledge about ECS/IS interaction may provide useful targets for treatment and prevention of psychopathology associated with A/A traits.

MiR-142-3p regulates synaptopathy-driven disease progression in multiple sclerosis.

AIM: We recently proposed miR-142-3p as a molecular player in inflammatory synaptopathy, a new pathogenic hallmark of multiple sclerosis (MS) and of its mouse model experimental autoimmune encephalomyelitis (EAE), that leads to neuronal loss independently of demyelination. MiR-142-3p seems to be unique among potential biomarker candidates in MS, since it is an inflammatory miRNA playing a dual role in the immune and central nervous systems. Here, we aimed to verify the impact of miR-142-3p circulating in the cerebrospinal fluid (CSF) of MS patients on clinical parameters, neuronal excitability and its potential interaction with disease modifying therapies (DMTs). METHODS AND RESULTS: In a cohort of 151 MS patients, we found positive correlations between CSF miR-142-3p levels and clinical progression, IL-1ß signalling as well as synaptic excitability measured by transcranial magnetic stimulation. Furthermore, therapy response of patients with 'low miR-142-3p' to dimethyl fumarate (DMF), an established disease-modifying treatment (DMT), was superior to that of patients with 'high miR-142-3p' levels. Accordingly, the EAE clinical course of heterozygous miR-142 mice was ameliorated by peripheral DMF treatment with a greater impact relative to their wild type littermates. In addition, a central protective effect of this drug was observed following intracerebroventricular and ex vivo acute treatments of EAE wild type mice, showing a rescue of miR-142-3p-dependent glutamatergic alterations. By means of electrophysiology, molecular and biochemical analysis, we suggest miR-142-3p as a molecular target of DMF. CONCLUSION: MiR-142-3p is a novel and potential negative prognostic CSF marker of MS and a promising tool for identifying personalised therapies.

Exercise protects from hippocampal inflammation and neurodegeneration in experimental autoimmune encephalomyelitis.

Exercise is increasingly recommended as a supportive therapy for people with Multiple Sclerosis (pwMS). While clinical research has still not disclosed the real benefits of exercise on MS disease, animal studies suggest a substantial beneficial effect on motor disability and pathological hallmarks such as central and peripheral dysregulated immune response. The hippocampus, a core area for memory formation and learning, is a brain region involved in MS pathophysiology. Human and rodent studies suggest that the hippocampus is highly sensitive to the effects of exercise, the impact of which on MS hippocampal damage is still elusive. Here we addressed the effects of chronic voluntary exercise on hippocampal function and damage in experimental autoimmune encephalomyelitis (EAE), animal model of MS. Mice were housed in standard or wheel-equipped cages starting from the day of immunization and throughout the disease course. Although running activity was reduced during the symptomatic phase, exercise significantly ameliorated motor disability. Exercise improved cognition that was assessed through the novel object recognition test and the nest building in presymptomatic and acute stages of the disease, respectively. In the acute phase exercise was shown to prevent EAE-induced synaptic plasticity abnormalities in the CA1 area, by promoting the survival of parvalbumin-positive (PV+) interneurons and by attenuating inflammation. Indeed, exercise significantly reduced microgliosis in the CA1 area, the expression of tumour necrosis factor (TNF) in microglia and, to a lesser extent, the hippocampal level of interleukin 1 beta (IL-1ß), previously shown to contribute to aberrant synaptic plasticity in the EAE hippocampus. Notably, exercise exerted a precocious and long-lasting mitigating effect on microgliosis that preceded its neuroprotective action, likely underlying the improved cognitive function observed in both presymptomatic and acute phase EAE mice. Overall, these data provide evidence that regular exercise improves cognitive function and synaptic and neuronal pathology that typically affect EAE/MS brains.

Cerebrospinal fluid from frontotemporal dementia patients is toxic to neurons.

Frontotemporal Lobar Degeneration (FTD) is a neurodegenerative disease characterized by a progressive deterioration of cognitive functions. Currently, no effective treatment exists. We have studied cytotoxicity and neuronal functionality in cortical and spinal cord cultures upon exposure to cerebrospinal fluid (CSF) from 39 FTD patients. FTD-CSF alters the miniature excitatory postsynaptic currents in the cortical cultures and it is toxic to spinal cord cultures, particularly to GABAergic+ and calbindin-D28k + neurons.

The microRNA let-7b-5p Is Negatively Associated with Inflammation and Disease Severity in Multiple Sclerosis.

The identification of microRNAs in biological fluids for diagnosis and prognosis is receiving great attention in the field of multiple sclerosis (MS) research but it is still in its infancy. In the present study, we observed in a large sample of MS patients that let-7b-5p levels in the cerebrospinal fluid (CSF) were highly correlated with a number of microRNAs implicated in MS, as well as with a variety of inflammation-related protein factors, showing specific expression patterns coherent with let-7b-5p-mediated regulation. Additionally, we found that the CSF let-7b-5p levels were significantly reduced in patients with the progressive MS compared to patients with relapsing-remitting MS and were negatively correlated with characteristic hallmark processes of the two phases of the disease. Indeed, in the non-progressive phase, let-7b-5p inversely associated with both central and peripheral inflammation; whereas, in progressive MS, the CSF levels of let-7b-5p negatively correlated with clinical disability at disease onset and after a follow-up period. Overall, our results uncovered, by the means of a multidisciplinary approach and multiple statistical analyses, a new possible pleiotropic action of let-7b-5p in MS, with potential utility as a biomarker of MS course.

Optogenetic Stimulation of Prelimbic Pyramidal Neurons Maintains Fear Memories and Modulates Amygdala Pyramidal Neuron Transcriptome.

Fear extinction requires coordinated neural activity within the amygdala and medial prefrontal cortex (mPFC). Any behavior has a transcriptomic signature that is modified by environmental experiences, and specific genes are involved in functional plasticity and synaptic wiring during fear extinction. Here, we investigated the effects of optogenetic manipulations of prelimbic (PrL) pyramidal neurons and amygdala gene expression to analyze the specific transcriptional pathways associated to adaptive and maladaptive fear extinction. To this aim, transgenic mice were (or not) fear-conditioned and during the extinction phase they received optogenetic (or sham) stimulations over photo-activable PrL pyramidal neurons. At the end of behavioral testing, electrophysiological (neural cellular excitability and Excitatory Post-Synaptic Currents) and morphological (spinogenesis) correlates were evaluated in the PrL pyramidal neurons. Furthermore, transcriptomic cell-specific RNA-analyses (differential gene expression profiling and functional enrichment analyses) were performed in amygdala pyramidal neurons. Our results show that the optogenetic activation of PrL pyramidal neurons in fear-conditioned mice induces fear extinction deficits, reflected in an increase of cellular excitability, excitatory neurotransmission, and spinogenesis of PrL pyramidal neurons, and associated to strong modifications of the transcriptome of amygdala pyramidal neurons. Understanding the electrophysiological, morphological, and transcriptomic architecture of fear extinction may facilitate the comprehension of fear-related disorders.

Emerging Role of Extracellular Vesicles in the Pathophysiology of Multiple Sclerosis.

Extracellular vesicles (EVs) represent a new reality for many physiological and pathological functions as an alternative mode of intercellular communication. This is due to their capacity to interact with distant recipient cells, usually involving delivery of the EVs contents into the target cells. Intensive investigation has targeted the role of EVs in different pathological conditions, including multiple sclerosis (MS). MS is a chronic inflammatory and neurodegenerative disease of the nervous system, one of the main causes of neurological disability in young adults. The fine interplay between the immune and nervous systems is profoundly altered in this disease, and EVs seems to have a relevant impact on MS pathogenesis. Here, we provide an overview of both clinical and preclinical studies showing that EVs released from blood-brain barrier (BBB) endothelial cells, platelets, leukocytes, myeloid cells, astrocytes, and oligodendrocytes are involved in the pathogenesis of MS and of its rodent model experimental autoimmune encephalomyelitis (EAE). Most of the information points to an impact of EVs on BBB damage, on spreading pro-inflammatory signals, and altering neuronal functions, but EVs reparative function of brain damage deserves attention. Finally, we will describe recent advances about EVs as potential therapeutic targets and tools for therapeutic intervention in MS.

Re-Examining the Role of TNF in MS Pathogenesis and Therapy.

Multiple sclerosis (MS) is a common neurological disorder of putative autoimmune origin. Clinical and experimental studies delineate abnormal expression of specific cytokines over the course of the disease. One major cytokine that has been shown to play a pivotal role in MS is tumor necrosis factor (TNF). TNF is a pleiotropic cytokine regulating many physiological and pathological functions of both the immune system and the central nervous system (CNS). Convincing evidence from studies in human and experimental MS have demonstrated the involvement of TNF in various pathological hallmarks of MS, including immune dysregulation, demyelination, synaptopathy and neuroinflammation. However, due to the complexity of TNF signaling, which includes two-ligands (soluble and transmembrane TNF) and two receptors, namely TNF receptor type-1 (TNFR1) and type-2 (TNFR2), and due to its cell- and context-differential expression, targeting the TNF system in MS is an ongoing challenge. This review summarizes the evidence on the pathophysiological role of TNF in MS and in different MS animal models, with a special focus on pharmacological treatment aimed at controlling the dysregulated TNF signaling in this neurological disorder.

Inflammation-Associated Synaptic Alterations as Shared Threads in Depression and Multiple Sclerosis.

In the past years, several theories have been advanced to explain the pathogenesis of Major Depressive Disorder (MDD), a neuropsychiatric disease that causes disability in general population. Several theories have been proposed to define the MDD pathophysiology such as the classic "monoamine-theory" or the "glutamate hypothesis." All these theories have been recently integrated by evidence highlighting inflammation as a pivotal player in developing depressive symptoms. Proinflammatory cytokines have been indeed claimed to contribute to stress-induced mood disturbances and to major depression, indicating a widespread role of classical mediators of inflammation in emotional control. Moreover, during systemic inflammatory diseases, peripherally released cytokines circulate in the blood, reach the brain and cause anxiety, anhedonia, social withdrawal, fatigue, and sleep disturbances. Accordingly, chronic inflammatory disorders, such as the inflammatory autoimmune disease multiple sclerosis (MS), have been associated to higher risk of MDD, in comparison with overall population. Importantly, in both MS patients and in its experimental mouse model, Experimental Autoimmune Encephalomyelitis (EAE), the notion that depressive symptoms are reactive epiphenomenon to the MS pathology has been recently challenged by the evidence of their early manifestation, even before the onset of the disease. Furthermore, in association to such mood disturbance, inflammatory-dependent synaptic dysfunctions in several areas of MS/EAE brain have been observed independently of brain lesions and demyelination. This evidence suggests that a fine interplay between the immune and nervous systems can have a huge impact on several neurological functions, including depressive symptoms, in different pathological conditions. The aim of the present review is to shed light on common traits between MDD and MS, by looking at inflammatory-dependent synaptic alterations associated with depression in both diseases.

Central Modulation of Selective Sphingosine-1-Phosphate Receptor 1 Ameliorates Experimental Multiple Sclerosis.

Future treatments of multiple sclerosis (MS), a chronic autoimmune neurodegenerative disease of the central nervous system (CNS), aim for simultaneous early targeting of peripheral immune function and neuroinflammation. Sphingosine-1-phosphate (S1P) receptor modulators are among the most promising drugs with both "immunological" and "non-immunological" actions. Selective S1P receptor modulators have been recently approved for MS and shown clinical efficacy in its mouse model, the experimental autoimmune encephalomyelitis (EAE). Here, we investigated the anti-inflammatory/neuroprotective effects of ozanimod (RPC1063), a S1P1/5 modulator recently approved in the United States for the treatment of MS, by performing ex vivo studies in EAE brain. Electrophysiological experiments, supported by molecular and immunofluorescence analysis, revealed that ozanimod was able to dampen the EAE glutamatergic synaptic alterations, through attenuation of local inflammatory response driven by activated microglia and infiltrating T cells, the main CNS-cellular players of EAE synaptopathy. Electrophysiological studies with selective S1P1 (AUY954) and S1P5 (A971432) agonists suggested that S1P1 modulation is the main driver of the anti-excitotoxic activity mediated by ozanimod. Accordingly, in vivo intra-cerebroventricular treatment of EAE mice with AUY954 ameliorated clinical disability. Altogether these results strengthened the relevance of S1P1 agonists as immunomodulatory and neuroprotective drugs for MS therapy.

hNGF Peptides Elicit the NGF-TrkA Signalling Pathway in Cholinergic Neurons and Retain Full Neurotrophic Activity in the DRG Assay.

In the last decade, Nerve Growth Factor (NGF)-based clinical approaches have lacked specific and efficient Tyrosine Kinase A (TrkA) agonists for brain delivery. Nowadays, the characterization of novel small peptidomimetic is taking centre stage in preclinical studies, in order to overcome the main size-related limitation in brain delivery of NGF holoprotein for Central Nervous System (CNS) pathologies. Here we investigated the NGF mimetic properties of the human NGF 1-14 sequence (hNGF1-14) and its derivatives, by resorting to primary cholinergic and dorsal root ganglia (DRG) neurons. Briefly, we observed that: 1) hNGF1-14 peptides engage the NGF pathway through TrkA phosphorylation at tyrosine 490 (Y490), and activation of ShcC/PI3K and Plc-γ/MAPK signalling, promoting AKT-dependent survival and CREB-driven neuronal activity, as seen by levels of the immediate early gene c-Fos, of the cholinergic marker Choline Acetyltransferase (ChAT), and of Brain Derived Neurotrophic Factor (BDNF); 2) their NGF mimetic activity is lost upon selective TrkA inhibition by means of GW441756; 3) hNGF1-14 peptides are able to sustain DRG survival and differentiation in absence of NGF. Furthermore, the acetylated derivative Ac-hNGF1-14 demonstrated an optimal NGF mimetic activity in both neuronal paradigms and an electrophysiological profile similar to NGF in cholinergic neurons. Cumulatively, the findings here reported pinpoint the hNGF1-14 peptide, and in particular its acetylated derivative, as novel, specific and low molecular weight TrkA specific agonists in both CNS and PNS primary neurons.

'Prototypical' proinflammatory cytokine (IL-1) in multiple sclerosis: role in pathogenesis and therapeutic targeting.

Introduction: It has been recognized for about 20 years that interleukin (IL)-1 signaling is implicated in Multiple Sclerosis (MS), a disabling, chronic, inflammatory and neurodegenerative disease of the central nervous system (CNS). Only recently, multifaceted roles of IL-1 emerged in MS pathophysiology as a result of both clinical and preclinical studies. Notably, drugs that directly target the IL-1 system have not been tested so far in MS.Areas covered: Recent studies in animal models, together with the development of ex vivo chimeric MS models, have disclosed a critical role for IL-1 not only at the peripheral level but also within the CNS. In the present review, we highlight the IL-1-dependent neuropathological aspects of MS, by providing an overview of the cells of the immune and CNS systems that respond to IL-1 signaling, and by emphasizing the subsequent effects on the CNS, from demyelinating processes, to synaptopathy, and excitotoxicity.Expert opinion: Drugs that act on the IL-1 system show a therapeutic potential in several autoinflammatory diseases and preclinical studies have highlighted the effects of these compounds in MS. We will discuss why anti-IL-1 therapies in MS have been neglected to date.

Corrigendum: Inflammation-Associated Synaptic Alterations as Shared Threads in Depression and Multiple Sclerosis.

[This corrects the article DOI: 10.3389/fncel.2020.00169.].

Modified age-dependent expression of NaV1.6 in an ALS model correlates with motor cortex excitability alterations.

Cortical hyperexcitability is an early and intrinsic feature of Amyotrophic Lateral Sclerosis (ALS), but the mechanisms underlying this critical neuronal dysfunction are poorly understood. Recently, we have demonstrated that layer V pyramidal neurons (PNs) in the primary motor cortex (M1) of one-month old (P30) G93A ALS mice display an early hyperexcitability status compared to Control mice. In order to investigate the time-dependent evolution of the cortical excitability in the G93A ALS model, here we have performed an electrophysiological and immunohistochemical study at three different mouse ages. M1 PNs from 14-days old (P14) G93A mice have shown no excitability alterations, while M1 PNs from 3-months old (P90) G93A mice have shown a hypoexcitability status, compared to Control mice. These age-dependent cortical excitability dysfunctions correlate with a similar time-dependent trend of the persistent sodium current (INaP) amplitude alterations, suggesting that INaP may play a crucial role in the G93A cortical excitability aberrations. Specifically, immunohistochemistry experiments have indicated that the expression level of the NaV1.6 channel, one of the voltage-gated Na+ channels mainly distributed within the central nervous system, varies in G93A primary motor cortex during disease progression, according to the excitability and INaP alterations, but not in other cortical areas. Microfluorometry experiments, combined with electrophysiological recordings, have verified that P30 G93A PNs hyperexcitability is associated to a greater accumulation of intracellular calcium ([Ca2+]i) compared to Control PNs, and that this difference is still present when G93A and Control PNs fire action potentials at the same frequency. These results suggest that [Ca2+]i de-regulation in G93A PNs may contribute to neuronal demise and that the NaV1.6 channels could be a potential therapeutic target to ameliorate ALS disease progression.

Modelling the pathogenesis of Myotonic Dystrophy type 1 cardiac phenotype through human iPSC-derived cardiomyocytes.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease, autosomal dominant, caused by a CTG repeat expansion in DMPK gene. We assessed the appropriateness of patient-specific induced pluripotent stem cell-derived cardiomyocytes (CMs) as a model to recapitulate some aspects of the pathogenetic mechanism involving cardiac manifestations in DM1 patients. Once obtained in vitro, CMs have been characterized for their morphology and their functionality. CMs DM1 show intranuclear foci and transcript markers abnormally spliced respect to WT ones, as well as several irregularities in nuclear morphology, probably caused by an unbalanced lamin A/C ratio. Electrophysiological characterization evidences an abnormal profile only in CMs DM1 such that the administration of antiarrythmic drugs to these cells highlights even more the functional defect linked to the disease. Finally, Atomic Force Measurements reveal differences in the biomechanical behaviour of CMs DM1, in terms of frequencies and synchronicity of the beats. Altogether the complex phenotype described in this work, strongly reproduces some aspects of the human DM1 cardiac phenotype. Therefore, the present study provides an in vitro model suggesting novel insights into the mechanisms leading to the development of arrhythmogenesis and dilatative cardiomyopathy to consider when approaching to DM1 patients, especially for the risk assessment of sudden cardiac death (SCD). These data could be also useful in identifying novel biomarkers effective in clinical settings and patient-tailored therapies.