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Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (Aβ), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the Aβ accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-β Forty-Two Inducer named Aftin-5. Secreted, soluble Aβ fragment concentrations were analyzed in MB-conditioned media. An increase in the Aβ42 fragment secretion was observed as was an increased Aβ42/Aβ40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent Aβ accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological Aβ concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both Aβ oligomers and their target, the cellular prion protein (PrPC), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.

The neuromuscular junction (NMJ), a synapse between the motor neuron terminal and a skeletal muscle fiber, is crucial throughout life in maintaining the reliable neurotransmission required for functional motricity. Disruption of this system leads to neuromuscular disorders, such as autoimmune myasthenia gravis (MG), the most common form of NMJ disease. MG is caused by autoantibodies directed mostly against the acetylcholine receptor (AChR) or the muscle-specific kinase MuSK. Several studies report immunoreactivity to the Frizzled-like cysteine-rich Wnt-binding domain of MuSK (CRD) in patients, although the pathogenicity of the antibodies involved remains unknown. We showed here that the immunoreactivity to MuSK CRD induced by the passive transfer of anti-MuSKCRD antibodies in mice led to typical MG symptoms, characterized by a loss of body weight and a locomotor deficit. The functional and morphological integrity of the NMJ was compromised with a progressive decay of neurotransmission and disruption of the structure of presynaptic and postsynaptic compartments. We found that anti-MuSKCRD antibodies completely abolished Agrin-mediated AChR clustering by decreasing the Lrp4-MuSK interaction. These results demonstrate the role of the MuSK CRD in MG pathogenesis and improve our understanding of the underlying pathophysiological mechanisms.

Background Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of survival of motor neuron (SMN) protein inducing progressive muscle weakness and atrophy due to motor neurons degeneration. Despite benefits of SMN restoration therapies in patients, motor defects are still persistent. We investigated the potential of BIO101, a new drug candidate promoting muscle growth by activating the protective arm of the renin‐angiotensin system through the MAS receptor, as monotherapy or in combination with the SMN‐based therapy ASO‐10‐27 (Nusinersen). Methods BIO101 was administrated daily on severe or mild Taiwanese SMA mouse models or diluted in culture medium of SMA patient‐derived myoblasts. The BIO101 effects were evaluated on severe SMA mouse model in vivo (growth, survival and motor function), ex vivo (motor neuron, neuromuscular junction maturation, skeletal muscle phenotype) and on muscle SMN expression, while motor function effects were evaluated on mild SMA mouse model. The in vitro effects on proliferation, differentiation, metabolism and SMN expression of SMA patient‐derived myoblasts were analysed. Effects of the combination of BIO101 with ASO‐10‐27 were evaluated on severe SMA mouse model, in vivo and on tissular intracellular AKT signalling and SMN expression. Results In severe SMA mice, BIO101 alone protected lateral motor neurons (+20%, p < 0.05), limited muscular atrophy (+30%, p < 0.01), accelerated maturation of muscular fibres (+70% for fast‐twitch muscles) and neuromuscular junctions (+50% of perforated clustering, p < 0.05) with more prominent effects on fast‐twitch muscles. Those adaptations led to an improvement of muscular function, significant at 7, 9 and 10 days post‐natal (+2‐fold for crossed squares and time of suspension, p < 0.01), which was also observed in mild SMA mice at 8 and 9 months of age (p < 0.01). Interestingly, BIO101 treatment also improved SMA patient‐derived myoblast differentiation (+20% myotube diameter and nuclei/myotube, p < 0.05) and anaerobic performances (ECAR, + 10%; p ≤ 0.05) without any impact on the proliferative state and aerobic capacities through MAS receptor activation. All BIO101 effects were independent of SMN protein expression. When combined with the ASO‐10‐27, BIO101 enhanced even more muscle resistance to fatigue (> 3‐fold over 27 days for time of suspension, p < 0.05) when compared with severe SMA mice treated with ASO‐10‐27 alone, without effects on survival through the activation of AKT intracellular pathway and independently of SMN protein expression. Conclusions We showed that BIO101 constitutes an efficient SMN‐independent therapy to improve muscle performance in SMA, which could open new therapeutic avenues for patients in combination with SMN‐based therapies, or as monotherapy for less severe forms.

Dysregulated RNA metabolism caused by SMN deficiency leads to motor neuron disease spinal muscular atrophy (SMA). Current therapies improve patient outcomes but achieve no definite cure, prompting renewed efforts to better understand disease mechanisms. The calcium channel blocker flunarizine improves motor function in Smn-deficient mice and can help uncover neuroprotective pathways. Murine motor neuron-like NSC34 cells were used to study the molecular cell-autonomous mechanism. Following RNA and protein extraction, RT-qPCR and immunodetection experiments were performed. The relationship between flunarizine mRNA targets and RNA-binding protein GEMIN5 was explored by RNA-immunoprecipitation. Flunarizine increases demethylase Kdm6b transcripts across cell cultures and mouse models. It causes, in NSC34 cells, a temporal expression of GEMIN5 and KDM6B. GEMIN5 binds to flunarizine-modulated mRNAs, including Kdm6b transcripts. Gemin5 depletion reduces Kdm6b mRNA and protein levels and hampers responses to flunarizine, including neurite extension in NSC34 cells. Moreover, flunarizine increases the axonal extension of motor neurons derived from SMA patient-induced pluripotent stem cells. Finally, immunofluorescence studies of spinal cord motor neurons in Smn-deficient mice reveal that flunarizine modulates the expression of KDM6B and its target, the motor neuron-specific transcription factor HB9, driving motor neuron maturation. Our study reveals GEMIN5 regulates Kdm6b expression with implications for motor neuron diseases and therapy.

ABSTRACT Background Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of survival of motor neuron (SMN) protein inducing progressive muscle weakness and atrophy due to motor neurons degeneration. Despite benefits of SMN restoration therapies in patients, motor defects are still persistent. We investigated the potential of BIO101, a new drug candidate promoting muscle growth by activating the protective arm of the renin‐angiotensin system through the MAS receptor, as monotherapy or in combination with the SMN‐based therapy ASO‐10‐27 (Nusinersen). Methods BIO101 was administrated daily on severe or mild Taiwanese SMA mouse models or diluted in culture medium of SMA patient‐derived myoblasts. The BIO101 effects were evaluated on severe SMA mouse model in vivo (growth, survival and motor function), ex vivo (motor neuron, neuromuscular junction maturation, skeletal muscle phenotype) and on muscle SMN expression, while motor function effects were evaluated on mild SMA mouse model. The in vitro effects on proliferation, differentiation, metabolism and SMN expression of SMA patient‐derived myoblasts were analysed. Effects of the combination of BIO101 with ASO‐10‐27 were evaluated on severe SMA mouse model, in vivo and on tissular intracellular AKT signalling and SMN expression. Results In severe SMA mice, BIO101 alone protected lateral motor neurons (+20%, p < 0.05), limited muscular atrophy (+30%, p < 0.01), accelerated maturation of muscular fibres (+70% for fast‐twitch muscles) and neuromuscular junctions (+50% of perforated clustering, p < 0.05) with more prominent effects on fast‐twitch muscles. Those adaptations led to an improvement of muscular function, significant at 7, 9 and 10 days post‐natal (+2‐fold for crossed squares and time of suspension, p < 0.01), which was also observed in mild SMA mice at 8 and 9 months of age ( p < 0.01). Interestingly, BIO101 treatment also improved SMA patient‐derived myoblast differentiation (+20% myotube diameter and nuclei/myotube, p < 0.05) and anaerobic performances (ECAR, + 10%; p ≤ 0.05) without any impact on the proliferative state and aerobic capacities through MAS receptor activation. All BIO101 effects were independent of SMN protein expression. When combined with the ASO‐10‐27, BIO101 enhanced even more muscle resistance to fatigue (> 3‐fold over 27 days for time of suspension, p < 0.05) when compared with severe SMA mice treated with ASO‐10‐27 alone, without effects on survival through the activation of AKT intracellular pathway and independently of SMN protein expression. Conclusions We showed that BIO101 constitutes an efficient SMN‐independent therapy to improve muscle performance in SMA, which could open new therapeutic avenues for patients in combination with SMN‐based therapies, or as monotherapy for less severe forms.

Human mini-brains (MB) are cerebral organoids that recapitulate in part the complexity of the human brain in a unique three-dimensional in vitro model, yielding discrete brain regions reminiscent of the cerebral cortex. Specific proteins linked to neurodegenerative disorders are physiologically expressed in MBs, such as APP-derived amyloids (A[beta]), whose physiological and pathological roles and interactions with other proteins are not well established in humans. Here, we demonstrate that neuroectodermal organoids can be used to study the A[beta] accumulation implicated in Alzheimer's disease (AD). To enhance the process of protein secretion and accumulation, we adopted a chemical strategy of induction to modulate post-translational pathways of APP using an Amyloid-[beta] Forty-Two Inducer named Aftin-5. Secreted, soluble A[beta] fragment concentrations were analyzed in MB-conditioned media. An increase in the A[beta].sub.42 fragment secretion was observed as was an increased A[beta].sub.42 /A[beta].sub.40 ratio after drug treatment, which is consistent with the pathological-like phenotypes described in vivo in transgenic animal models and in vitro in induced pluripotent stem cell-derived neural cultures obtained from AD patients. Notably in this context we observe time-dependent A[beta] accumulation, which differs from protein accumulation occurring after treatment. We show that mini-brains obtained from a non-AD control cell line are responsive to chemical compound induction, producing a shift of physiological A[beta] concentrations, suggesting that this model can be used to identify environmental agents that may initiate the cascade of events ultimately leading to sporadic AD. Increases in both A[beta] oligomers and their target, the cellular prion protein (PrP.sup.C ), support the possibility of using MBs to further understand the pathophysiological role that underlies their interaction in a human model. Finally, the potential application of MBs for modeling age-associated phenotypes and the study of neurological disorders is confirmed.

Abstract The development of the neuromuscular junction (NMJ) requires dynamic trans-synaptic coordination orchestrated by secreted factors, including the morphogens of the Wnt family. Yet, how the signal of these synaptic cues is transduced, and particularly during the regulation of acetylcholine receptor (AChR) accumulation in the postsynaptic membrane remains unclear. We explored the function of Van Gogh-Like protein 2 (Vangl2), a core component of Wnt planar cell polarity signaling. We showed that the conditional genetic ablation of Vangl2 in muscle reproduces the NMJ differentiation defects in mice with constitutive Vangl2 deletion. These alterations persisted into adulthood with NMJs disassembly leading to an impairment of neurotransmission and motor function deficits. Mechanistically, we found that Vangl2 and the muscle-specific kinase MuSK acted in the same genetic pathway and that Vangl2 binds MuSK, thus controlling its signaling activity. Our results identify Vangl2 as a key player of the core complex of molecules shaping neuromuscular synapses and shed light on the molecular mechanisms underlying NMJ assembly. Competing Interest Statement The authors have declared no competing interest.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with an unpredictable course. An observational study was set up using the French hospital discharge database to describe the reasons, outcomes and costs of hospitalisations related to this disease. Patients newly hospitalised for idiopathic pulmonary fibrosis (ICD-10 code: J84.1) in 2008 were identified and followed for 5 years. As J84.1 includes other fibrotic pulmonary diseases, an algorithm excluding age<50 years and presence of a differential diagnosis in the following year was defined. Overall, 6,476 patients were identified; of whom 30% were admitted through the emergency unit and 12% died during their first hospitalisation. Most of patients were hospitalised at least once for one or several acute events (n = 5,635; 87.0% of patients), of whom 36.5% of patients with an acute respiratory worsening (in-hospital mortality of 17.0% and median cost of €3,224; interquartile range (IQR €889-6,092)), 43.7% of patients with a respiratory infection (in-hospital mortality of 29.5% and median cost of €5,432 (IQR, €3,620-9,115)) and 51.7% of patients with a cardiac event (in-hospital mortality of 35.7% and median cost of €4,584 (IQR, €2,803-6,399)); 30.2% of these events occurred during the first hospitalisation. Finally, the 3-year in-hospital mortality crude rate was 36.8%. This study is the first providing extensive data on hospitalisations in patients with pulmonary fibrosis, mostly idiopathic, in France, demonstrating high burden and hospital cost.