Agonist of growth hormone-releasing hormone improves the disease features of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is a severe autosomal recessive neuromuscular disease affecting children and young adults, caused by mutations of the survival motor neuron 1 gene (SMN1). SMA is characterized by the degeneration of spinal alpha motor neurons (αMNs), associated with muscle paralysis and atrophy, as well as other peripheral alterations. Both growth hormone-releasing hormone (GHRH) and its potent agonistic analog, MR-409, exert protective effects on muscle atrophy, cardiomyopathies, ischemic stroke, and inflammation. In this study, we aimed to assess the protective role of MR-409 in SMNΔ7 mice, a widely used model of SMA. Daily subcutaneous treatment with MR-409 (1 or 2 mg/kg), from postnatal day 2 (P2) to euthanization (P12), increased body weight and improved motor behavior in SMA mice, particularly at the highest dose tested. In addition, MR-409 reduced atrophy and ameliorated trophism in quadriceps and gastrocnemius muscles, as determined by an increase in fiber size, as well as upregulation of myogenic genes and inhibition of proteolytic pathways. MR-409 also promoted the maturation of neuromuscular junctions, by reducing multi-innervated endplates and increasing those mono-innervated. Finally, treatment with MR-409 delayed αMN death and blunted neuroinflammation in the spinal cord of SMA mice. In conclusion, the present study demonstrates that MR-409 has protective effects in SMNΔ7 mice, suggesting that GHRH agonists are promising agents for the treatment of SMA, possibly in combination with SMN-dependent strategies.

Moxifloxacin rescues SMA phenotypes in patient-derived cells and animal model.

Spinal muscular atrophy (SMA) is a genetic disease resulting in the loss of α-motoneurons followed by muscle atrophy. It is caused by knock-out mutations in the survival of motor neuron 1 (SMN1) gene, which has an unaffected, but due to preferential exon 7 skipping, only partially functional human-specific SMN2 copy. We previously described a Drosophila-based screening of FDA-approved drugs that led us to discover moxifloxacin. We showed its positive effect on the SMN2 exon 7 splicing in SMA patient-derived skin cells and its ability to increase the SMN protein level. Here, we focus on moxifloxacin's therapeutic potential in additional SMA cellular and animal models. We demonstrate that moxifloxacin rescues the SMA-related molecular and phenotypical defects in muscle cells and motoneurons by improving the SMN2 splicing. The consequent increase of SMN levels was higher than in case of risdiplam, a potent exon 7 splicing modifier, and exceeded the threshold necessary for a survival improvement. We also demonstrate that daily subcutaneous injections of moxifloxacin in a severe SMA murine model reduces its characteristic neuroinflammation and increases the SMN levels in various tissues, leading to improved motor skills and extended lifespan. We show that moxifloxacin, originally used as an antibiotic, can be potentially repositioned for the SMA treatment.

Recent Advances in High-Content Imaging and Analysis in iPSC-Based Modelling of Neurodegenerative Diseases.

In the field of neurodegenerative pathologies, the platforms for disease modelling based on patient-derived induced pluripotent stem cells (iPSCs) represent a valuable molecular diagnostic/prognostic tool. Indeed, they paved the way for the in vitro recapitulation of the pathological mechanisms underlying neurodegeneration and for characterizing the molecular heterogeneity of disease manifestations, also enabling drug screening approaches for new therapeutic candidates. A major challenge is related to the choice and optimization of the morpho-functional study designs in human iPSC-derived neurons to deeply detail the cell phenotypes as markers of neurodegeneration. In recent years, the specific combination of high-throughput screening with subcellular resolution microscopy for cell-based high-content imaging (HCI) screening allowed in-depth analyses of cell morphology and neurite trafficking in iPSC-derived neuronal cells by using specific cutting-edge microscopes and automated computational assays. The present work aims to describe the main recent protocols and advances achieved with the HCI analysis in iPSC-based modelling of neurodegenerative diseases, highlighting technical and bioinformatics tips and tricks for further uses and research. To this end, microscopy requirements and the latest computational pipelines to analyze imaging data will be explored, while also providing an overview of the available open-source high-throughput automated platforms.

Mesenchymal Stem Cell Treatment Perspectives in Peripheral Nerve Regeneration: Systematic Review.

Traumatic peripheral nerve lesions affect hundreds of thousands of patients every year; their consequences are life-altering and often devastating and cause alterations in movement and sensitivity. Spontaneous peripheral nerve recovery is often inadequate. In this context, nowadays, cell therapy represents one of the most innovative approaches in the field of nerve repair therapies. The purpose of this systematic review is to discuss the features of different types of mesenchymal stem cells (MSCs) relevant for peripheral nerve regeneration after nerve injury. The published literature was reviewed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A combination of the keywords "nerve regeneration", "stem cells", "peripheral nerve injury", "rat", and "human" were used. Additionally, a "MeSH" research was performed in PubMed using the terms "stem cells" and "nerve regeneration". The characteristics of the most widely used MSCs, their paracrine potential, targeted stimulation, and differentiation potentials into Schwann-like and neuronal-like cells are described in this paper. Considering their ability to support and stimulate axonal growth, their remarkable paracrine activity, their presumed differentiation potential, their extremely low immunogenicity, and their high survival rate after transplantation, ADSCs appear to be the most suitable and promising MSCs for the recovery of peripheral nerve lesion. Clinical considerations are finally reported.

How to Build and to Protect the Neuromuscular Junction: The Role of the Glial Cell Line-Derived Neurotrophic Factor.

The neuromuscular junction (NMJ) is at the crossroad between the nervous system (NS) and the muscle. Following neurotransmitter release from the motor neurons (MNs), muscle contraction occurs and movement is generated. Besides eliciting muscle contraction, the NMJ represents a site of chemical bidirectional interplay between nerve and muscle with the active participation of Schwann cells. Indeed, signals originating from the muscle play an important role in synapse formation, stabilization, maintenance and function, both in development and adulthood. We focus here on the contribution of the Glial cell line-Derived Neurotrophic Factor (GDNF) to these processes and to its potential role in the protection of the NMJ during neurodegeneration. Historically related to the maintenance and survival of dopaminergic neurons of the substantia nigra, GDNF also plays a fundamental role in the peripheral NS (PNS). At this level, it promotes muscle trophism and it participates to the functionality of synapses. Moreover, compared to the other neurotrophic factors, GDNF shows unique peculiarities, which make its contribution essential in neurodegenerative disorders. While describing the known structural and functional changes occurring at the NMJ during neurodegeneration, we highlight the role of GDNF in the NMJ-muscle cross-talk and we review its therapeutic potential in counteracting the degenerative process occurring in the PNS in progressive and severe diseases such as Alzheimer's disease (AD), Amyotrophic Lateral Sclerosis (ALS) and Spinal Muscular Atrophy (SMA). We also describe functional 3D neuromuscular co-culture systems that have been recently developed as a model for studying both NMJ formation in vitro and its involvement in neuromuscular disorders.

Mitochondrial Dysfunctions: A Red Thread across Neurodegenerative Diseases.

Mitochondria play a central role in a plethora of processes related to the maintenance of cellular homeostasis and genomic integrity. They contribute to preserving the optimal functioning of cells and protecting them from potential DNA damage which could result in mutations and disease. However, perturbations of the system due to senescence or environmental factors induce alterations of the physiological balance and lead to the impairment of mitochondrial functions. After the description of the crucial roles of mitochondria for cell survival and activity, the core of this review focuses on the "mitochondrial switch" which occurs at the onset of neuronal degeneration. We dissect the pathways related to mitochondrial dysfunctions which are shared among the most frequent or disabling neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, Amyotrophic Lateral Sclerosis, and Spinal Muscular Atrophy. Can mitochondrial dysfunctions (affecting their morphology and activities) represent the early event eliciting the shift towards pathological neurobiological processes? Can mitochondria represent a common target against neurodegeneration? We also review here the drugs that target mitochondria in neurodegenerative diseases.

Motor neuron degeneration, severe myopathy and TDP-43 increase in a transgenic pig model of SOD1-linked familiar ALS.

Amyotrophic Lateral Sclerosis (ALS) is a neural disorder gradually leading to paralysis of the whole body. Alterations in superoxide dismutase SOD1 gene have been linked with several variants of familial ALS. Here, we investigated a transgenic (Tg) cloned swine model expressing the human pathological hSOD1G93A allele. As in patients, these Tg pigs transmitted the disease to the progeny with an autosomal dominant trait and showed ALS onset from about 27 months of age. Post mortem analysis revealed motor neuron (MN) degeneration, gliosis and hSOD1 protein aggregates in brainstem and spinal cord. Severe skeletal muscle pathology including necrosis and inflammation was observed at the end stage, as well. Remarkably, as in human patients, these Tg pigs showed a quite long presymptomatic phase in which gradually increasing amounts of TDP-43 were detected in peripheral blood mononuclear cells. Thus, this transgenic swine model opens the unique opportunity to investigate ALS biomarkers even before disease onset other than testing novel drugs and possible medical devices.

Mesenchymal Stem Cells for Spinal Cord Injury: Current Options, Limitations, and Future of Cell Therapy

Spinal cord injury (SCI) constitutes an inestimable public health issue. The most crucial phase in the pathophysiological process of SCI concerns the well-known secondary injury, which is the uncontrolled and destructive cascade occurring later with aberrant molecular signaling, inflammation, vascular changes, and secondary cellular dysfunctions. The use of mesenchymal stem cells (MSCs) represents one of the most important and promising tested strategies. Their appeal, among the other sources and types of stem cells, increased because of their ease of isolation/preservation and their properties. Nevertheless, encouraging promise from preclinical studies was followed by weak and conflicting results in clinical trials. In this review, the therapeutic role of MSCs is discussed, together with their properties, application, limitations, and future perspectives.

Cognitive deficits and brain myo-Inositol are early biomarkers of epileptogenesis in a rat model of epilepsy.

One major unmet clinical need in epilepsy is the identification of therapies to prevent or arrest epilepsy development in patients exposed to a potential epileptogenic insult. The development of such treatments has been hampered by the lack of non-invasive biomarkers that could be used to identify the patients at-risk, thereby allowing to design affordable clinical studies. Our goal was to test the predictive value of cognitive deficits and brain astrocyte activation for the development of epilepsy following a potential epileptogenic injury. We used a model of epilepsy induced by pilocarpine-evoked status epilepticus (SE) in 21-day old rats where 60-70% of animals develop spontaneous seizures after around 70days, although SE is similar in all rats. Learning was evaluated in the Morris water-maze at days 15 and 65 post-SE, each time followed by proton magnetic resonance spectroscopy for measuring hippocampal myo-Inositol levels, a marker of astrocyte activation. Rats were video-EEG monitored for two weeks at seven months post-SE to detect spontaneous seizures, then brain histology was done. Behavioral and imaging data were retrospectively analysed in epileptic rats and compared with non-epileptic and control animals. Rats displayed spatial learning deficits within three weeks from SE. However, only epilepsy-prone rats showed accelerated forgetting and reduced learning rate compared to both rats not developing epilepsy and controls. These deficits were associated with reduced hippocampal neurogenesis. myo-Inositol levels increased transiently in the hippocampus of SE-rats not developing epilepsy while this increase persisted until spontaneous seizures onset in epilepsy-prone rats, being associated with a local increase in S100ß-positive astrocytes. Neuronal cell loss was similar in all SE-rats. Our data show that behavioral deficits, together with a non-invasive marker of astrocyte activation, predict which rats develop epilepsy after an acute injury. These measures have potential clinical relevance for identifying individuals at-risk for developing epilepsy following exposure to epileptogenic insults, and consequently, for designing adequately powered antiepileptogenesis trials.

Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs), after intraparenchymal, intrathecal and endovenous administration, have been previously tested for cell therapy in amyotrophic lateral sclerosis in the SOD1 (superoxide dismutase 1) mouse. However, every administration route has specific pros and cons. METHODS: We administrated human MSCs (hMSCs) in the cisterna lumbaris, which is easily accessible and could be used in outpatient surgery, in the SOD1 G93A mouse, at the earliest onset of symptoms. Control animals received saline injections. Motor behavior was checked starting from 2 months of age until the mice were killed. Animals were killed 2 weeks after transplantation; lumbar motoneurons were stereologically counted, astrocytes and microglia were analyzed and quantified after immunohistochemistry and cytokine expression was assayed by means of real-time polymerase chain reaction. RESULTS: We provide evidence that this route of administration can exert strongly positive effects. Motoneuron death and motor decay were delayed, astrogliosis was reduced and microglial activation was modulated. In addition, hMSC transplantation prevented the downregulation of the anti-inflammatory interleukin-10, as well as that of vascular endothelial growth factor observed in saline-treated transgenic mice compared with wild type, and resulted in a dramatic increase in the expression of the anti-inflammatory interleukin-13. CONCLUSIONS: Our results suggest that hMSCs, when intracisternally administered, can exert their paracrine potential, influencing the inflammatory response of the host.

Synergistically Acting on Myostatin and Agrin Pathways Increases Neuromuscular Junction Stability and Endurance in Old Mice.

Sarcopenia is the primary cause of impaired motor performance in the elderly. The current prevailing approach to counteract such condition is increasing the muscle mass through inhibition of the myostatin system: however, this strategy only moderately improves muscular strength, not being able to sustain the innervation of the hypertrophic muscle per se, leading to a progressive worsening of motor performances. Thus, we proposed the administration of ActR-Fc-nLG3, a protein that combines the soluble activin receptor, a strong myostatin inhibitor, with the C-terminal agrin nLG3 domain. This compound has the potential of reinforcing neuro-muscular stability to the hypertrophic muscle. We previously demonstrated an enhancement of motor endurance and ACh receptor aggregation in young mice after ActR-Fc-nLG3 administration. Now we extended these observations by demonstrating that also in aged (2 years-old) mice, long-term administration of ActR-Fc-nLG3 increases in a sustained way both motor endurance and muscle strength, compared with ActR-Fc, a myostatin inhibitor, alone. Histological data demonstrate that the administration of this biological improves neuromuscular stability and fiber innervation maintenance, preventing muscle fiber atrophy and inducing only moderate hypertrophy. Moreover, at the postsynaptic site we observe an increased folding in the soleplate, a likely anatomical substrate for improved neurotransmission efficiency in the NMJ, that may lead to enhanced motor endurance. We suggest that ActR-Fc-nLG3 may become a valid option for treating sarcopenia and possibly other disorders of striatal muscles.

Administration of adipose-derived stem cells extracellular vesicles in a murine model of spinal muscular atrophy: effects of a new potential therapeutic strategy.

BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. METHODS: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. RESULTS: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. CONCLUSIONS: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.

Biochemical characterization on muscle tissue of a novel biallelic ACO2 mutation in an infant with progressive encephalopathy.

The ACO2 gene encodes the mitochondrial protein aconitate hydratase, which is responsible for catalyzing the interconversion of citrate into isocitrate in the tricarboxylic acid (TCA) cycle. Mitochondrial aconitase is expressed ubiquitously, and deficiencies in TCA-cycle enzymes have been reported to cause various neurodegenerative diseases due to disruption of cellular energy metabolism and development of oxidative stress. We investigated a severe early infantile-onset neurometabolic syndrome due to a homozygous novel variant in exon 13 of the ACO2 gene. The in vitro pathogenicity of this variant of unknown significance was demonstrated by the loss of both protein expression and its enzymatic activity on muscle tissue sample taken from the patient. The patient presented with progressive encephalopathy soon after birth, characterized by hypotonia, progressive severe muscle atrophy, and respiratory failure. Serial brain magnetic resonance imaging showed progressive abnormalities compatible with a metabolic disorder, possibly mitochondrial. Muscle biopsy disclosed moderate myopathic alterations and features consistent with a mitochondriopathy albeit nonspecific. The course was characterized by progressive worsening of the clinical and neurological picture, and the patient died at 5 months of age. This study provides the first report on the validation in muscle from human subjects regarding in vitro analysis for mitochondrial aconitase activity. To our knowledge, no prior reports have demonstrated a correlation of phenotypic and diagnostic characteristics with in vitro muscle enzymatic activity of mitochondrial aconitase in humans. In conclusion, this case further expands the genetic spectrum of ACO2 variants and defines a complex case of severe neonatal neurometabolic disorder.

hESC-derived striatal progenitors grafted into a Huntington's disease rat model support long-term functional motor recovery by differentiating, self-organizing and connecting into the lesioned striatum.

BACKGROUND: Huntington's disease (HD) is a motor and cognitive neurodegenerative disorder due to prominent loss of striatal medium spiny neurons (MSNs). Cell replacement using human embryonic stem cells (hESCs) derivatives may offer new therapeutic opportunities to replace degenerated neurons and repair damaged circuits. METHODS: With the aim to develop effective cell replacement for HD, we assessed the long-term therapeutic value of hESC-derived striatal progenitors by grafting the cells into the striatum of a preclinical model of HD [i.e., adult immunodeficient rats in which the striatum was lesioned by monolateral injection of quinolinic acid (QA)]. We examined the survival, maturation, self-organization and integration of the graft as well as its impact on lesion-dependent motor alterations up to 6 months post-graft. Moreover, we tested whether exposing a cohort of QA-lesioned animals to environmental enrichment (EE) could improve graft integration and function. RESULTS: Human striatal progenitors survived up to 6 months after transplantation and showed morphological and neurochemical features typical of human MSNs. Donor-derived interneurons were also detected. Grafts wired in both local and long-range striatal circuits, formed domains suggestive of distinct ganglionic eminence territories and displayed emerging striosome features. Moreover, over time grafts improved complex motor performances affected by QA. EE selectively increased cell differentiation into MSN phenotype and promoted host-to-graft connectivity. However, when combined to the graft, the EE paradigm used in this study was insufficient to produce an additive effect on task execution. CONCLUSIONS: The data support the long-term therapeutic potential of ESC-derived human striatal progenitor grafts for the replacement of degenerated striatal neurons in HD and suggest that EE can effectively accelerate the maturation and promote the integration of human striatal cells.

Bicalutamide and Trehalose Ameliorate Spinal and Bulbar Muscular Atrophy Pathology in Mice.

Spinal and bulbar muscular atrophy (SBMA) is characterized by motor neuron (MN) degeneration that leads to slowly progressive muscle weakness. It is considered a neuromuscular disease since muscle has a primary role in disease onset and progression. SBMA is caused by a CAG triplet repeat expansion in the androgen receptor (AR) gene. The translated poly-glutamine (polyQ) tract confers a toxic gain of function to the mutant AR altering its folding, causing its aggregation into intracellular inclusions, and impairing the autophagic flux. In an in vitro SBMA neuronal model, we previously showed that the antiandrogen bicalutamide and trehalose, a natural disaccharide stimulating autophagy, block ARpolyQ activation, reduce its nuclear translocation and toxicity and facilitate the autophagic degradation of cytoplasmic AR aggregates. Here, in a knock-in SBMA mouse model (KI AR113Q), we show that bicalutamide and trehalose ameliorated SBMA pathology. Bicalutamide reversed the formation of the AR insoluble forms in KI AR113Q muscle, preventing autophagic flux blockage. We demonstrated that apoptosis is activated in KI AR113Q muscle, and that both compounds prevented its activation. We detected a decrease of mtDNA and an increase of OXPHOS enzymes, already at early symptomatic stages; these alterations were reverted by trehalose. Overall, bicalutamide and/or trehalose led to a partial recovery of muscle morphology and function, and improved SBMA mouse motor behavior, inducing an extension of their survival. Thus, bicalutamide and trehalose, by counteracting ARpolyQ toxicity in skeletal muscle, are valuable candidates for future clinical trials in SBMA patients.

Activation of the Hepcidin-Ferroportin1 pathway in the brain and astrocytic-neuronal crosstalk to counteract iron dyshomeostasis during aging.

During physiological aging, iron accumulates in the brain with a preferential distribution in regions that are more vulnerable to age-dependent neurodegeneration such as the cerebral cortex and hippocampus. In the brain of aged wild-type mice, alteration of the Brain Blood Barrier integrity, together with a marked inflammatory and oxidative state lead to increased permeability and deregulation of brain-iron homeostasis. In this context, we found that iron accumulation drives Hepcidin upregulation in the brain and the inhibition of the iron exporter Ferroportin1. We also observed the transcription and the increase of NCOA4 levels in the aged brain together with the increase of light-chain enriched ferritin heteropolymers, more efficient as iron chelators. Interestingly, in cerebral cortex and hippocampus, Ferroportin1 is mainly expressed by astrocytes, while the iron storage protein ferritin light-chain by neurons. This differential distribution suggests that astrocytes mediate iron shuttling in the nervous tissue and that neurons are unable to metabolize it. Our findings highlight for the first time that Hepcidin/Ferroportin1 axis and NCOA4 are directly involved in iron metabolism in mice brain during physiological aging as a response to a higher brain iron influx.

Genes and miRNAs as Hurdles and Promoters of Corticospinal Tract Regeneration in Spinal Cord Injury.

Spinal cord injury (SCI) is a devastating lesion to the spinal cord, which determines the interruption of ascending/descending axonal tracts, the loss of supraspinal control of sensory-motor functions below the injured site, and severe autonomic dysfunctions, dramatically impacting the quality of life of the patients. After the acute inflammatory phase, the progressive formation of the astrocytic glial scar characterizes the acute-chronic phase: such scar represents one of the main obstacles to the axonal regeneration that, as known, is very limited in the central nervous system (CNS). Unfortunately, a cure for SCI is still lacking: the current clinical approaches are mainly based on early vertebral column stabilization, anti-inflammatory drug administration, and rehabilitation programs. However, new experimental therapeutic strategies are under investigation, one of which is to stimulate axonal regrowth and bypass the glial scar. One major issue in axonal regrowth consists of the different genetic programs, which characterize axonal development and maturation. Here, we will review the main hurdles that in adulthood limit axonal regeneration after SCI, describing the key genes, transcription factors, and miRNAs involved in these processes (seen their reciprocal influencing action), with particular attention to corticospinal motor neurons located in the sensory-motor cortex and subjected to axotomy in case of SCI. We will highlight the functional complexity of the neural regeneration programs. We will also discuss if specific axon growth programs, that undergo a physiological downregulation during CNS development, could be reactivated after a spinal cord trauma to sustain regrowth, representing a new potential therapeutic approach.

Organotypic spinal cord cultures: An in vitro 3D model to preliminary screen treatments for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe neuromuscular disease affecting children, due to mutation/deletion of survival motor neuron 1 (SMN1) gene. The lack of functional protein SMN determines motor neuron (MN) degeneration and skeletal muscle atrophy, leading to premature death due to respiratory failure. Nowadays, the Food and Drug Administration approved the administration of three drugs, aiming at increasing the SMN production: although assuring noteworthy results, all these therapies show some non-negligible limitations, making essential the identification of alternative/synergistic therapeutic strategies. To offer a valuable in vitro experimental model for easily performing preliminary screenings of alternative promising treatments, we optimized an organotypic spinal cord culture (derived from murine spinal cord slices), which well recapitulates the pathogenetic features of SMA. Then, to validate the model, we tested the effects of human Mesenchymal Stem Cells (hMSCs) or murine C2C12 cells (a mouse skeletal myoblast cell line) conditioned media: 1/3 of conditioned medium (obtained from either hMSCs or C2C12 cells) was added to the conventional medium of the organotypic culture and maintained for 7 days. Then the slices were fixed and immunoreacted to evaluate the MN survival. In particular we observed that the C2C12 and hMSCs conditioned media positively influenced the MN soma size and the axonal length respectively, without modulating the glial activation. These data suggest that trophic factors released by MSCs or muscular cells can exert beneficial effects, by acting on different targets, and confirm the reliability of the model. Overall, we propose the organotypic spinal cord culture as an excellent tool to preliminarily screen molecules and drugs before moving to in vivo models, in this way partly reducing the use of animals and the costs.

miR-7b-3p Exerts a Dual Role After Spinal Cord Injury, by Supporting Plasticity and Neuroprotection at Cortical Level.

Spinal cord injury (SCI) affects 6 million people worldwide with no available treatment. Despite research advances, the inherent poor regeneration potential of the central nervous system remains a major hurdle. Small RNAs (sRNAs) 19-33 nucleotides in length are a set of non-coding RNA molecules that regulate gene expression and have emerged as key players in regulating cellular events occurring after SCI. Here we profiled a class of sRNA known as microRNAs (miRNAs) following SCI in the cortex where the cell bodies of corticospinal motor neurons are located. We identified miR-7b-3p as a candidate target given its significant upregulation after SCI in vivo and we screened by miRWalk PTM the genes predicted to be targets of miR-7b-3p (among which we identified Wipf2, a gene regulating neurite extension). Moreover, 16 genes, involved in neural regeneration and potential miR-7b-3p targets, were found to be downregulated in the cortex following SCI. We also analysed miR-7b-3p function during cortical neuron development in vitro: we observed that the overexpression of miR-7b-3p was important (1) to maintain neurons in a more immature and, likely, plastic neuronal developmental phase and (2) to contrast the apoptotic pathway; however, in normal conditions it did not affect the Wipf2 expression. On the contrary, the overexpression of miR-7b-3p upon in vitro oxidative stress condition (mimicking the SCI environment) significantly reduced the expression level of Wipf2, as observed in vivo, confirming it as a direct miR-7b-3p target. Overall, these data suggest a dual role of miR-7b-3p: (i) the induction of a more plastic neuronal condition/phase, possibly at the expense of the axon growth, (ii) the neuroprotective role exerted through the inhibition of the apoptotic cascade. Increasing the miR-7b-3p levels in case of SCI could reactivate in adult neurons silenced developmental programmes, supporting at the same time the survival of the axotomised neurons.

Imaging of Inflammation in Spinal Cord Injury: Novel Insights on the Usage of PFC-Based Contrast Agents.

Labeling of macrophages with perfluorocarbon (PFC)-based compounds allows the visualization of inflammatory processes by 19F-magnetic resonance imaging (19F-MRI), due to the absence of endogenous background. Even if PFC-labeling of monocytes/macrophages has been largely investigated and used, information is lacking about the impact of these agents over the polarization towards one of their cell subsets and on the best way to image them. In the present work, a PFC-based nanoemulsion was developed to monitor the course of inflammation in a model of spinal cord injury (SCI), a pathology in which the understanding of immunological events is of utmost importance to select the optimal therapeutic strategies. The effects of PFC over macrophage polarization were studied in vitro, on cultured macrophages, and in vivo, in a mouse SCI model, by testing and comparing various cell tracking protocols, including single and multiple administrations, the use of MRI or Point Resolved Spectroscopy (PRESS), and application of pre-saturation of Kupffer cells. The blood half-life of nanoemulsion was also investigated by 19F Magnetic Resonance Spectroscopy (MRS). In vitro and in vivo results indicate the occurrence of a switch towards the M2 (anti-inflammatory) phenotype, suggesting a possible theranostic function of these nanoparticles. The comparative work presented here allows the reader to select the most appropriate protocol according to the research objectives (quantitative data acquisition, visual monitoring of macrophage recruitment, theranostic purpose, rapid MRI acquisition, etc.). Finally, the method developed here to determine the blood half-life of the PFC nanoemulsion can be extended to other fluorinated compounds.