Peripheral neuropathic symptoms have been reported in Gaucher disease (GD), a rare lysosomal storage disorder caused by mutations in β-glucocerebrosidase gene (GBA1), albeit poorly investigated only in clinical settings. To shed light on the involvement of peripheral myelination by Schwann cells (SCs) in GD, we generated a conditional knockout mouse line in which β-glucocerebrosidase is depleted in myelinating glia (Gba1f/f::cre). Adult Gba1f/f::cre peripheral nerves presented hypomyelination of large caliber axons and higher frequency of myelin infoldings, accompanied by evidence of repair-SC program activation, as indicated by the expression of p75ntr and c-Jun. The Gba1f/f::cre mice displayed reduced motor performance, associated with altered neuromuscular junction morphology and neuromuscular transmission. Given the well-established role of β-glucocerebrosidase in lysosomal function and autophagy, we also investigated whether its deficiency in SCs could affect nerve injury response. Despite the ability of conditional knockout mice to reach a full recovery, the initial steps of myelinophagy, a process required to eliminate myelin debris, thus prompting axon regeneration, were impaired in β-glucocerebrosidase deficient SCs in vivo. Consistently, when in vitro nerve degeneration was induced in presence of the β-glucocerebrosidase inhibitor conduritol-B-epoxide (CBE), a block in the autophagic flux was observed. Our data show that decreased degradation efficiency and/or accumulation of bioactive lipids in SCs lacking β-glucocerebrosidase sustain the activation of a repair-SC program, leading to myelin and axonal defects. These results indicate a novel role for GBA1 in guaranteeing SC lysosomal function as relevant for peripheral nerve homeostasis.

>The gut microbiota: The human body is colonized by a diverse and complex microbial community – including bacteria, viruses, archaea, and unicellular eukaryotes – that plays a central role in human wellbeing. Indeed, microbiota is crucial for several functions, including host metabolism, physiology, maintenance of the intestinal epithelial integrity, nutrition, and immune function, earning it the designation of a “vital organ”(Guinane and Cotter, 2013).

The glucocorticoid (GC) resistance onset in pediatric T-cell acute lymphoblastic leukemia (T-ALL) patients remains one of the biggest challenges in current cancer treatment. The mechanisms driving this resistance are still not fully understood, making it difficult to predict patient outcomes and to develop effective therapies. Our study uncovered critical insights into the biological processes underlying GC resistance, offering potential breakthroughs for future treatments. Building on our previous research on lymphocyte cell-specific protein-tyrosine kinase (LCK) hyperactivation in GC-resistant T-ALL patients, we have now delved deeper into the LCK downstream nuclear factor of activated T cells (NFAT) transcription factor family’s contribution to GC resistance. We discovered that, even at the time of diagnosis, GC resistant T-ALL patients exhibit an intrinsic low glucocorticoid receptor (GR) activity coupled with high NFATc1 and NFATc2 activity. This dysregulation creates a roadblock to effective GC therapy. Indeed, in the absence of either NFATc1 or NFATc2, the normal transcriptional activity of GR is restored, re-sensitizing leukemia cells to dexamethasone treatment both in vitro and in vivo. This suggests that NFATc1 and NFATc2 are central to driving GC resistance, as they directly regulate crucial pathways like cholesterol biosynthesis and WNT/β-catenin signaling. The identification of NFAT transcription factors as key players in leukemia therapy resistance offers a promising target for future therapeutic strategies, potentially transforming the way we approach treatment for these challenging conditions or autoimmune disorders where glucocorticoids are a cornerstone of treatment.

IntroductionRecent evidence supports the hypothesis of an association between gut microbiota and the pathogenesis of retinal and eye diseases, suggesting the existence of a gut-eye axis. However, no data are available on the possible effect of the gut microbiota on the optic nerve fiber maturation and myelin development.MethodsWe investigated the impact of gut microbiota on the optic nerves collected from neonatal and young adult germ-free (GF), gnotobiotic (stably colonized with 12 bacteria strains, OMM12) and control (colonized with a complex gut microbiota, CGM) mice, by performing stereological and morphoquantitative analyses with transmission electron microscopy and gene expression analysis by quantitative real-time PCR.ResultsYoung adult GF and OMM12 optic nerve axons are smaller and hypermyelinated compared to CGM ones, while no such differences were detected in neonatal optic nerves. The transcription factors Olig1, Olig2, and Sox10 (oligodendrocyte myelination positive regulators) are downregulated in CGM and OMM12 young adult mice compared to the respective neonates. Such developmental downregulation was not observed in GF optic nerves, suggesting that the absence of the gut microbiota prolongs the stimulation of optic nerve fiber myelination, possibly through mechanisms that are yet to be identified.DiscussionAltogether, these data underscore the gut microbiota pivotal role in driving optic nerve myelination, contributing to our knowledge about both the gut-eye axis and the gut-brain axis, and opening new horizons for further investigations that will explore the role of the microbiota also in pathologies, injuries and regeneration associated with the optic nerve.

Skeletal muscle development, homeostasis, and function rely on complex interactions among multiple cell types and the extracellular matrix (ECM). Developing in vitro models that recapitulate both intrinsic cellular and extrinsic ECM elements of innervated skeletal muscle is crucial for advancing basic biology and disease modeling studies. Here, we combine tissue engineering approaches with human induced pluripotent stem cell (hiPSC) technology to create tissue-engineered neuromuscular organoids (t-NMOs). Using decellularized muscles as scaffolds, hiPSCs differentiate to form organoids that establish a continuum with the provided biomaterial. After 30 days, t-NMOs exhibit compartmentalized neural and muscular components that establish functional interactions, allowing muscle contraction. We demonstrate the model's potential by creating Duchenne Muscular Dystrophy patient-specific t-NMOs, that recapitulate the reduced skeletal muscle contraction and altered calcium dynamics typical of the disease. Altogether, our study presents a tissue-engineered organoid that model the human neuromuscular system (dys)function, highlighting the potential of applying the ECM in organoid engineering.

β-glucocerebrosidase in health and disease:Mutations in the β-glucocerebrosidase(GBA)gene do cause the rare lysosomal storage disorder Gaucher's disease(GD)with an estimated global prevalence of 1∶200,000(Imbalzano et al.,2024).

Background Mutations in the β-glucocerebrosidase ( GBA1 ) gene do cause the lysosomal storage Gaucher disease (GD) and are among the most frequent genetic risk factors for Parkinson’s disease (PD). So far, studies on both neuronopathic GD and PD primarily focused on neuronal manifestations, besides the evaluation of microglial and astrocyte implication. White matter alterations were described in the central nervous system of paediatric type 1 GD patients and were suggested to sustain or even play a role in the PD process, although the contribution of oligodendrocytes has been so far scarcely investigated. Methods We exploited a system to study the induction of central myelination in vitro, consisting of Oli-neu cells treated with dibutyryl-cAMP, in order to evaluate the expression levels and function of β-glucocerebrosidase during oligodendrocyte differentiation. Conduritol-B-epoxide, a β-glucocerebrosidase irreversible inhibitor was used to dissect the impact of β-glucocerebrosidase inactivation in the process of myelination, lysosomal degradation and α-synuclein accumulation in vitro. Moreover, to study the role of β-glucocerebrosidase in the white matter in vivo, we developed a novel mouse transgenic line in which β-glucocerebrosidase function is abolished in myelinating glia, by crossing the Cnp1-cre mouse line with a line bearing loxP sequences flanking Gba1 exons 9–11, encoding for β-glucocerebrosidase catalytic domain. Immunofluorescence, western blot and lipidomic analyses were performed in brain samples from wild-type and knockout animals in order to assess the impact of genetic inactivation of β-glucocerebrosidase on myelination and on the onset of early neurodegenerative hallmarks, together with differentiation analysis in primary oligodendrocyte cultures. Results Here we show that β-glucocerebrosidase inactivation in oligodendrocytes induces lysosomal dysfunction and inhibits myelination in vitro. Moreover, oligodendrocyte-specific β-glucocerebrosidase loss-of-function was sufficient to induce in vivo demyelination and early neurodegenerative hallmarks, including axonal degeneration, α-synuclein accumulation and astrogliosis, together with brain lipid dyshomeostasis and functional impairment. Conclusions Our study sheds light on the contribution of oligodendrocytes in GBA1-related diseases and supports the need for better characterizing oligodendrocytes as actors playing a role in neurodegenerative diseases, also pointing at them as potential novel targets to set a brake to disease progression.

Adult muscle stem cells (MuSCs) are critical for muscle homeostasis and regeneration, and their behavior relies on a finely regulated niche made of specific extracellular matrix (ECM) components and soluble factors. Among ECM proteins, collagen VI (Col6) influences the mechanical properties of the niche and, in turn, MuSC self -renewal capabilities. Here, we investigated whether Col6 can exert a direct function as a biochemical signal for regulating the stemness and differentiation of murine MuSCs and myoblasts. Native Col6, but not its pepsin -resistant fragment, counteracts the early differentiation of myogenic cells by reducing the expression of differentiation marker genes and preserving stemness features, with inhibition of the canonical Wnt pathway. Our data indicate that extracellular Col6 acts as a soluble ligand in delaying early myogenic differentiation by regulating intracellular signals involved in adult myogenesis.

Dear Editor, Collagen VI (ColVI)-related myopathies are a distinct group of progressive muscle disorders, which include Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), for which no therapy is yet available. In the last two decades, much effort was spent on the elucidation of the pathogenic mechanisms underlying ColVI-related myopathies, also by taking advantage of the ColVI null (Col6a1−/−) mouse model,1 in order to identify druggable targets for prospective therapies.2-4 The results obtained within this work provide a proof-of-concept for the repurposing of the Food and Drug Administration (FDA)-approved salbutamol in the context of ColVI-related myopathies, as they demonstrate that systemic salbutamol administration can recover the major structural and functional neuromuscular junction (NMJ) defects occurring in murine muscles downstream ColVI deficiency, and previously described also in patients.5 We selected salbutamol for three main reasons: (i) it was previously reported to ameliorate NMJ structure and electrophysiological cues in animal models of human NMJ-related disorders; (ii) it is an FDA-approved drug already used with success as an off-label medication in several diseases displaying not only primary but also secondary NMJ defects, including Pompe disease, spinal muscular atrophy and Duchenne, Becker and facioscapulohumeral muscular dystrophies; (iii) it was proven to be safe in the majority of the open clinical trials and therefore eligible for future rapid translational applications in the clinical settings.6 Five-month-old wild type (WT) and Col6a1−/− male mice were treated with either vehicle or salbutamol at two different doses, 4 and 8 mg/kg/die, subcutaneously administered via Alzet osmotic minipumps for 28 days (Figure S1 and Supporting Information). First, we investigated the impact of salbutamol on NMJ structural defects and found that the abnormally increased NMJ fragmentation displayed by vehicle-treated Col6a1−/− diaphragms, compared to WT animals (Figure 1A–C), was recovered by treatment with salbutamol at the higher dose (Figure 1D–F). Furthermore, morphometric analysis of NMJs showed that both low- and high-dose salbutamol induced a significant increase in postsynaptic terminal areas and compactness in Col6a1−/− mice (Figures 1G–L and S2A), coherently with reports of salbutamol beneficial effects in expanding NMJ size in animal models of congenital myasthenic syndromes (CMS).7, 8 The analyses performed on treated WT mice confirmed the general trend of salbutamol in increasing NMJ size (Figure S2B–H). To evaluate whether salbutamol also ameliorated neuromuscular transmission, we first monitored the four-limb hanging test performance. Besides confirming a marked difference between WT and Col6a1−/− mice (Figure 1M), the test highlighted an improved performance of Col6a1−/− mice treated with high-dose salbutamol (Figure 1N,O). Moreover, ex vivo electrophysiological recordings performed in diaphragm muscles revealed that high-dose salbutamol treatment recovered the NMJ functional parameters found to be altered in Col6a1−/− mice, when compared to WT animals, including a lower amplitude of nerve-evoked endplate potentials and of miniature endplate potentials (mEPP), a reduced quantal content and input resistance, as well as decreased endplate currents, miniature endplate currents and mEPP frequency (Figure 1P–V). Notably, assessment of neuromuscular transmission by repetitive stimulation of the phrenic nerve for 25 pulses at 5 Hz further highlighted that the higher decrement registered upon vehicle treatment in Col6a1−/− mice, when compared to WT animals, was also rescued by salbutamol (Figure 1W), clearly indicating that high-dose salbutamol treatment not only ameliorates NMJ fragmentation but also elicits NMJ functional improvement in Col6a1−/− mice. Since β2-agonists are well-known anabolic agents,9 we hypothesised that Col6a1−/− muscles could benefit per se from salbutamol treatment. Thus, we evaluated salbutamol impact on muscle mass, attesting a significant increase of body weight and of the absolute and normalised tibialis anterior (TA) and gastrocnemius muscle weight both in Col6a1−/− and WT mice (Figures 2A–C and S3A–F). Consistently, morphometric analysis of TA cross-sections revealed a significant increase in average cross-sectional area and minimum Feret's diameter elicited by high-dose salbutamol treatment in Col6a1−/− mice (Figure 2E,F), paralleled by a shift of myofibre size distribution towards larger myofibre classes (Figure 2G–L) and by a reduction in myofibre density (Figure 2M). A similar shift in fibre size was detectable in salbutamol-treated WT muscles (Figure S3G–O), whereas myofibre density was not significantly affected (Figure S3P). Interestingly, while salbutamol treatment in either Col6a1−/− and WT mice did not influence the occurrence of centronucleated fibres (Figures 2N and S3Q), a higher proportion of regenerating fibres, together with increased Myh3 transcript levels, was detectable in muscles of both genotypes upon high-dose treatment (Figures 2O–Q and S3R–T). Such results validated the concept that salbutamol not only promotes muscle remodelling in WT animals but also counteracts muscle mass loss and promotes muscle regeneration in Col6a1−/− mice. In keeping with literature, showing that β2-agonists can induce slow-oxidative to fast-glycolytic myofibre switch in rodents,9 despite the absence of any overt change in succinate dehydrogenase staining in WT and Col6a1−/− muscles (Figures 3A,B and S4A,B), an upregulation of Myh4 gene (Figures 3C,D and S4C,D) and an increased percentage of type IIB fibres (Figure 3E,F) were detected upon salbutamol treatment in Col6a1−/− mice, compared to the corresponding vehicle-treated mice. Similar changes were not detectable in salbutamol-treated WT muscles (Figure S4E,F). We then evaluated salbutamol's ability to increase muscle strength, by performing in vivo tetanic force measurements on gastrocnemius muscles of WT and Col6a1−/− mice, following either salbutamol or vehicle administration. The impaired muscle strength of Col6a1−/−, compared to WT mice, both in terms of absolute and normalised force (Figure 4A,B), resulted ameliorated by salbutamol since it induced a significant gain in the absolute force, mostly evident at intermediate stimulation frequencies (Figure 4C), which, notably, remained significant even when normalised to muscle weight upon high-dose treatment (Figure 4D). In WT mice, treatments determined a significant gain only in absolute force at intermediate frequencies of stimulation (Figure S5A,B). Of note, the curves corresponding to the normalised force of vehicle-treated WT mice displayed a complete overlap with those of salbutamol-treated Col6a1−/− mice at intermediate stimulation frequencies (Figure S5C,D), demonstrating that salbutamol induces a substantial amelioration of in vivo muscle strength. Finally, β2-adrenergic receptor protein levels were significantly reduced in both TA and diaphragm muscles of Col6a1−/− mice after the 4-week salbutamol treatment at both doses (Figure 4E,F,H,I), consistently with receptor desensitisation9 and with a potential undesired mitigation of the biological response to the treatment. Considering that salbutamol is a sympathomimetic drug and supposing it might reconstitute a defective sympathetic innervation of NMJs of Col6a1−/− mice, we monitored tyrosine hydroxylase (TH) as a marker for sympathetic neurons. TH protein levels appeared only mildly affected in TA muscles (Figure 4E,G), while in diaphragms, a decrease in TH levels was detectable in high-dose salbutamol-treated Col6a1−/− mice, when compared to vehicle-treated ones (Figure 4H,J). In situ quantification of NMJs presenting sympathomimetic innervation did not highlight any further difference between vehicle-treated WT and vehicle- or high-dose salbutamol-treated Col6a1−/− mice (Figure 4K,L), and norepinephrine concentration in serum was not affected as well by the different treatments (Figure S6). Therefore, we cannot infer that salbutamol efficacy relies on an overtly impaired sympathetic innervation in Col6a1−/− mice. In conclusion, our results show that 1-month systemic treatment with salbutamol stabilises NMJ structures while ameliorating the myopathic phenotype of Col6a1−/− mice. By limiting muscle wasting and improving neuromuscular transmission in mice, salbutamol administration may provide clinical benefits for patients. Of note, salbutamol doses adopted here are comparable to those proven to be successful in counteracting CMS phenotype in murine models.8 On the other hand, a direct conversion to human application, where the highest effective dosages used in clinics for CMS adult patients range between 6 and 12 mg per die,10 is not realistic. In addition, subcutaneous infusion for salbutamol administration is less frequently opted in patients, where the per os route is preferred in the context of CMS and other dystrophies.6 Nonetheless, being salbutamol already approved by the FDA, optimised salbutamol-based therapeutic strategies might be more rapidly translated into clinical trials, an aspect of high relevance in the clinical setting for BM/UCMD patients. Matilde Cescon designed the study, acquired funding and supervised analysis; Sonia Calabrò and Matilde Cescon performed in vivo treatments; Sonia Calabrò performed in vivo and ex-vivo analysis; Leonardo Nogara performed in vivo muscle force measurements; Yongzhi Jian and Manuel Valentin performed ex-vivo electrophysiological analysis; Dario Bizzotto and Paola Braghetta managed mouse colonies; Loris Russo and Lisa Gambarotto contributed to the analysis; Bert Blaauw and Said Hashemolhosseini supervised in vivo and ex-vivo electrophysiological analysis, respectively; Paolo Bonaldo provided Col6a1−/− mice and acquired funding; Sonia Calabrò and Matilde Cescon wrote the original draft; all the other authors contributed to revision of the manuscript. We acknowledge the light microscopy facility of the Biology Department of the University of Padova. This work was funded by AFM Telethon (Trampoline Grant Number #22360 to Matilde Cescon), University of Padova (PRID-DMM to Matilde Cescon); Telethon Foundation (Grant GGP19229 to Paolo Bonaldo), and the Italian Ministry of Education, University and Research (Grant P2022Y2A3L funded in the framework of NRRP, Mission 4.2, Investment 1.1 "progetti di ricerca di Rilevante Interesse Nazionale - PRIN", funded by the European Union - Next Generation EU, CUP C53D23007520001, to Matilde Cescon, Grant P20227YB93 to Matilde Cescon; Grant 201742SBXA to Paolo Bonaldo), German Research Council (DFG) Grants (HA3309/3-1, HA3309/6-1 and HA3309/7-1 to Said Hashemolhosseini). Moreover, as part of the activities of the National Center for Gene Therapy and Drugs based on RNA Technology, funded in the framework of the National Recovery and Resilience Plan (NRRP), Mission 4 "Education and Research", Component 2 "From Research to Business", Investment 1.4 "Strengthening research structures for supporting the creation of National Centres, national R&D leaders on some Key Enabling Technologies", this work was funded by the Europena Union - Next Generation EU, Project CN00000041, CUP B93D21010860004, Spoke n. 5 "Inflammatory and infectious diseases". The authors declare no conflicts of interest. Animal procedures were approved by the Animal Ethics Committee of the University of Padova and authorised by the Italian Ministry of Health (Project No. 98/2020-PR). All data needed to evaluate the conclusions in the paper are present in the paper and the Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

The core component of the class III phosphatidylinositol 3-kinase complex, Beclin 1, takes part in different protein networks, thus switching its role from inducing autophagy to regulating autophagosomal maturation and endosomal trafficking. While assessed in neurons, astrocytes, and microglia, its role is far less investigated in myelinating glia, including Schwann cells (SCs), responsible for peripheral nerve myelination. Remarkably, the dysregulation in endosomal trafficking is emerging as a pathophysiological mechanism underlying peripheral neuropathies, such as demyelinating Charcot-Marie-Tooth (CMT) diseases. By knocking out Beclin 1 in SCs here a novel mouse model (Becn1 cKO) is generated, developing a severe and progressive neuropathy, accompanied by involuntary tremors, body weight loss, and premature death. Ultrastructural analysis revealed abated myelination and SCs displaying enlarged cytoplasm with progressive accumulation of intracellular vesicles. Transcriptomic and histological analysis from sciatic nerves of 10-day and 2-month-old mice revealed pro-mitotic gene deregulation and increased SCs proliferation at both stages with axonal loss and increased immune infiltration in adults, well reflecting the progressive motor and sensory functional impairment that characterizes Becn1 cKO mice, compared to controls. The study establishes a further step in understanding key mechanisms in SC development and points to Beclin 1 and its regulated pathways as targets for demyelinating CMT forms.

Collagen VI (Col-VI) is an extracellular matrix protein primarily known for its bridging role in connective tissues that has been suggested to play a neuroprotective role. In the present study we report increased mRNA and protein expression of Col-VI in the hippocampus and cortex at a late stage of epileptogenesis in a post-status epilepticus (SE) model of epilepsy and in brain tissue from patients with epilepsy. We further present a novel finding that exposure of mouse hippocampal slices to Col-VI augments paired-pulse facilitation in Schaffer collateral-CA1 excitatory synapses indicating decreased release probability of glutamate. In line with this finding, lack of Col-VI expression in the knock-out mice show paired-pulse depression in these synapses, suggesting increased release probability of glutamate. In addition, we observed dynamic changes in Col-VI blood plasma levels in rats after Kainate-induced SE, and increased levels of Col-VI mRNA and protein in autopsy or postmortem brain of humans suffering from epilepsy. Thus, our data indicate that elevated levels of ColVI following seizures leads to attenuated glutamatergic transmission, ultimately resulting in less overall network excitability. Presumably, increased Col-VI may act as part of endogenous compensatory mechanism against enhanced excitability during epileptogenic processes in the hippocampus, and could be further investigated as a potential functional biomarker of epileptogenesis, and/or a novel target for therapeutic intervention.

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 (Egr2), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel ‘Gut Microbiota-Peripheral Nervous System-axis.’

Background: The overall survival of pediatric T-cell Acute Lymphoblastic Leukemia (T-ALL) is significantly improved (˜75-80%) with the current therapeutic approaches. Nevertheless, for those patients that do not respond to conventional treatment and experience relapse, the prognosis is extremely poor. In this context, it is well defined that resistance to glucocorticoids (GCs), pillar drugs in the treatment protocol, can predispose pediatric T-ALL patients to a poor outcome. A pivotal role of LCK kinase has been elucidated in supporting GC resistance in T-ALL cells in our previous study and recently confirmed by other colleagues. However, to date, the biological processes modulated by LCK in this context are not yet defined. Aim: the identification of new mechanisms underlying GC resistance can lead to alternative therapeutic approaches to prevent or overcome GC resistance and ameliorate the outcome of this subgroup of patients. Methods and Results: we uncovered the involvement of NFATc1 and NFATc2 transcription factors, that act downstream LCK kinase, in guiding GC resistance in T-ALL cells. Of note, a high NFATc1 and NFATc2 transcriptional activity characterizes pediatric GC resistant T-ALL patients at diagnosis, that in turns show a low Glucocorticoid Receptor (GR) activity. In agreement, NFATc1 or NFATc2 specific gene silencing in 3 T-ALL GC resistant cell line models and primary cells increases dexamethasone response, by restoring GR canonical transcriptional activity through the increaseexpression of BIM GR targetgene (p value <0.05). Conversely, NFATc1 or NFATc2 overexpression in a murine T-ALL GC sensitive cell line confers resistance to dexamethasone treatment. Interestingly, by Gene Expression Profile (GEP) and Nuclear Magnetic Resonance (NMR) analysis, we observed that NFATc1 gene silencing in GC resistant cells significantly downregulates intracellular cholesterol abundance. Additionally, by Chromatin Immunoprecipitation (ChIP) we revealed that NFATc1 can directly control the transcription of HMGCS1, EBP and DHCR7, key enzymes of cholesterol biosynthesis process (n≥3, p value <0.05 for all the three genes). In agreement, exogenous cholesterol addition to NFATc1 knock-down cell lines rebuild dexamethasone resistance. Conversely, by GEP and flow cytometry analysis we observed that NFATc2 gene silencing in T-ALL GC resistant cells leads to a downregulation of the Wnt/β-catenin signaling pathway and to an increased T-cell differentiation. Furthermore, by ChIP analysis we revealed that in T-ALL GC resistant cells NFATc2 can directly affect the transcription of LRP6, a key Wnt/β-catenin signaling component (n=3, p value <0.05). In agreement, the Wnt/β-catenin signaling activation, by Wnt3a stimulation, restores dexamethasone resistance in NFATc2 knock-down T-ALL cells. Finally, we observed that the inhibition of cholesterol biosynthesis by simvastatin or of Wnt/β-catenin by PRI-724 increases GC sensitivity in T-ALL GC resistant cells by the High-Throughput drug synergism Screening (HTS) and the Highest Single Agent (HSA) approach. Conclusions: Overall, we revealed for the first time the involvement of NFATc1 and NFATc2 transcription factors in supporting GC resistance in T-ALL cells by the modulation of cholesterol biosynthesis and Wnt/β-catenin signaling, both processes well-described in sustaining chemotherapy resistance, paving the rationale to alternative therapeutic options for T-ALL GC resistant pediatric patients.

COL6 (collagen type VI)-related myopathies (COL6-RM) are a distinct group of inherited muscle disorders caused by mutations of COL6 genes and characterized by early-onset muscle weakness, for which no cure is available yet. Key pathophysiological features of COL6-deficient muscles involve impaired macroautophagy/autophagy, mitochondrial dysfunction, neuromuscular junction fragmentation and myofiber apoptosis. Targeting autophagy by dietary means elicited beneficial effects in both col6a1 null (col6a1(-/-)) mice and COL6-RM patients. We previously demonstrated that one-month per os administration of the nutraceutical spermidine reactivates autophagy and ameliorates myofiber defects in col6a1(-/-) mice but does not elicit functional improvement. Here we show that a 100-day-long spermidine regimen is able to rescue muscle strength in col6a1(-/-) mice, with also a beneficial impact on mitochondria and neuromuscular junction integrity, without any noticeable side effects. Altogether, these data provide a rationale for the application of spermidine in prospective clinical trials for COL6-RM.

Human neuromuscular organoids (NMOs) derived from induced pluripotent stem cells (hiPSCs) hold a great potential to study (dys)functional human skeletal muscle (SkM) in vitro. The three-dimensional (3D) self-assembly of NMOs leads to the generation of spheroids, whose 3D organization cannot be controlled. Indeed, proper development, maturation and function of the innervated SkM require a well-defined multiscale 3D organization of the cells in a tissue-specific extracellular matrix (ECM) context. We hypothesized that extracellular structural imprinting along with hiPSC small-molecule-based differentiation could provide self-assembly guidance driving NMO morphogenesis and promoting the maturation and function of the human neuronal-coupled SkM in vitro models. We found that SkM ECM, provided as decellularized skeletal muscle, is able to reproducibly guide the morphogenesis of differentiating hiPSC toward multiscale structured tissue-like NMOs (t-NMOs). T-NMOs show contractile activity and possess functional neuromuscular junctions (NMJs), with mature neuromuscular system upon 30 days of hiPSC differentiation. We found that t-NMO could mimic altered muscle contraction upon administration of neurotoxins that act at NMJ level. Finally, we used hiPSCs derived from patients affected by Duchenne Muscular Dystrophy (DMD) to produce DMD t-NMOs that, upon neuronal stimulation, were able to mimic the altered SkM contractility and calcium dynamics typical of the disease. Altogether, our data confirm the ability of t-NMO platform to model in vitro human neuromuscular system (patho)physiology.

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.

Microenvironmental factors are known fundamental regulators of the phenotype and aggressiveness of glioblastoma (GBM), the most lethal brain tumor, characterized by fast progression and marked resistance to treatments. In this context, the extracellular matrix (ECM) is known to heavily influence the behavior of cancer cells from several origins, contributing to stem cell niches, influencing tumor invasiveness and response to chemotherapy, mediating survival signaling cascades, and modulating inflammatory cell recruitment. Here, we show that collagen VI (COL6), an ECM protein widely expressed in both normal and pathological tissues, has a distinctive distribution within the GBM mass, strongly correlated with the most aggressive and phenotypically immature cells. Our data demonstrate that COL6 sustains the stem-like properties of GBM cells and supports the maintenance of an aggressive transcriptional program promoting cancer cell proliferation and survival. In particular, we identified a specific subset of COL6-transcriptionally co-regulated genes, required for the response of cells to replicative stress and DNA damage, supporting the concept that COL6 is an essential stimulus for the activation of GBM cell response and resistance to chemotherapy, through the ATM/ATR axis. Altogether, these findings indicate that COL6 plays a pivotal role in GBM tumor biology, exerting a pleiotropic action across different GBM hallmarks, including phenotypic identity and gene transcription, as well as response to treatments, thus providing valuable information for the understanding of the complex microenvironmental cues underlying GBM malignancy.