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Spinal Muscular Atrophy (SMA) is a severe genetic neuromuscular disorder that occurs in childhood and is caused by misexpression of the survival motor neuron (SMN) protein. SMN reduction induces spinal cord motoneuron (MN) degeneration, which leads to progressive muscular atrophy and weakness. The link between SMN deficiency and the molecular mechanisms altered in SMA cells remains unclear. Autophagy, deregulation of intracellular survival pathways and ERK hyperphosphorylation may contribute to SMN-reduced MNs collapse, offering a useful strategy to develop new therapies to prevent neurodegeneration in SMA. Using SMA MN in vitro models, the effect of pharmacological inhibition of PI3K/Akt and ERK MAPK pathways on SMN and autophagy markers modulation was studied by western blot analysis and RT-qPCR. Experiments involved primary cultures of mouse SMA spinal cord MNs and differentiated SMA human MNs derived from induced pluripotent stem cells (iPSCs). Inhibition of the PI3K/Akt and the ERK MAPK pathways reduced SMN protein and mRNA levels. Importantly, mTOR phosphorylation, p62, and LC3-II autophagy markers protein level were decreased after ERK MAPK pharmacological inhibition. Furthermore, the intracellular calcium chelator BAPTA prevented ERK hyperphosphorylation in SMA cells. Our results propose a link between intracellular calcium, signaling pathways, and autophagy in SMA MNs, suggesting that ERK hyperphosphorylation may contribute to autophagy deregulation in SMN-reduced MNs. This work was supported by grants from Instituto de Salud Carlos III, PI20/00098 y cofinanciado por la Unión Europea; Fundació La Marató TV3 (202005-30); Ministerio de Ciencia e Innovación /FEDER (grant number: PID2021-122785OB-I00), and CERCA Program/Generalitat de Catalunya. AG is a Serra Hunter Fellow from Generalitat de Catalunya, AS and MB hold Biomedical Research Talent grant from Diputació de Lleida-IRBLleida, FCN and MPM hold a fellow from the Generalitat de Catalunya, AGAUR (Programa INVESTIGO 2022 finançat per la Unió Europea, Next Generation EU and Ajuts per a la contractació de personal investigador predoctoral en formació, finançat per Fons Social Europeu, FSE Inverteix en el teu futur).

This work was supported by Abbott and a grant from the Spanish Ministerio de Ciencia, Innovacion y Universidades cofinanced by Fondo Europeo de Desarrollo Regional (RTI2018-099278-B-I00 to JC and JE) ., Besides skeletal muscle wasting, sarcopenia entails morphological and molecular changes in distinct components of the neuromuscular system, including spinal cord motoneurons (MNs) and neuromuscular junctions (NMJs); moreover, noticeable microgliosis has also been observed around aged MNs. Here we examined the impact of two flavonoid-enriched diets containing either green tea extract (GTE) catechins or cocoa flavanols on age-associated regressive changes in the neuromuscular system of C57BL/6J mice. Compared to control mice, GTE- and cocoa-supplementation significantly improved the survival rate of mice, reduced the proportion of fibers with lipofuscin aggregates and central nuclei, and increased the density of satellite cells in skeletal muscles. Additionally, both supplements significantly augmented the number of innervated NMJs and their degree of maturity compared to controls. GTE, but not cocoa, prominently increased the density of VAChT and VGluT2 afferent synapses on MNs, which were lost in control aged spinal cords; conversely, cocoa, but not GTE, significantly augmented the proportion of VGluT1 afferent synapses on aged MNs. Moreover, GTE, but not cocoa, reduced aging-associated microgliosis and increased the proportion of neuroprotective microglial phenotypes. Our data indicate that certain plant flavonoids may be beneficial in the nutritional management of age-related deterioration of the neuromuscular system., Abbott Laboratories, Spanish Ministerio de Ciencia, Innovacion y Universidades, European Commission RTI2018-099278-B-I00

Spinal muscular atrophy (SMA) is caused by a homozygous deletion or mutation in the survival motor neuron 1 (SMN1) gene that leads to reduced levels of SMN protein resulting in degeneration of motor neurons (MNs). The best known functions of SMN is the biogenesis of spliceosomal snRNPs. Linked to this function, Cajal bodies (CBs) are involved in the assembly of spliceosomal (snRNPs) and nucleolar (snoRNPs) ribonucleoproteins required for pre-mRNA and pre-rRNA processing. Recent studies support that the interaction between CBs and nucleoli, which are especially prominent in neurons, is essential for the nucleolar rRNA homeostasis. We use the SMN∆7 murine model of type I SMA to investigate the cellular basis of the dysfunction of RNA metabolism in MNs. SMN deficiency in postnatal MNs produces a depletion of functional CBs and relocalization of coilin, which is a scaffold protein of CBs, in snRNP-free perinucleolar caps or within the nucleolus. Disruption of CBs is the earliest nuclear sign of MN degeneration. We demonstrate that depletion of CBs, with loss of CB-nucleolus interactions, induces a progressive nucleolar dysfunction in ribosome biogenesis. It includes reorganization and loss of nucleolar transcription units, segregation of dense fibrillar and granular components, retention of SUMO-conjugated proteins in intranucleolar bodies and a reactive, compensatory, up-regulation of mature 18S rRNA and genes encoding key nucleolar proteins, such as upstream binding factor, fibrillarin, nucleolin and nucleophosmin. We propose that CB depletion and nucleolar alterations are essential components of the dysfunction of RNA metabolism in SMA. This work was supported by the following grants: “Dirección General de Investigación” of Spain (BFU2014-54754-P), “Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED; CB06/05/0037)” from Spain, and “Ministerio de Economía y Competitividad” of Spain cofinanced by FEDER (SAF2015-70801-R). Dr. Tapia is a recipient of a grant from SMA Europe cofinanced by FundAME (Spain) and "Instituto de Investigación Valdecilla" (IDIVAL; Next-Val) from Santander, Spain.

Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the pre-mRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal nuclear accumulation in euchromatin domains of poly(A) RNA granules (PARGs) enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were accompanied by changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. PARG-containing MNs were observed at presymptomatic SMA stage, increasing their number during the symptomatic stage. Moreover, the massive accumulations of poly(A) RNA granules in MNs was accompanied by the cytoplasmic depletion of polyadenylated mRNAs for their translation. We suggest that the SMN-dependent abnormal accumulation of polyadenylated mRNAs and Sam68 in PARGs reflects a severe dysfunction of both mRNA processing and translation, which could contribute to SMA pathogenesis. This work was supported by grants from: “Dirección General de Investigación” of Spain (BFU2014-54754-P and SAF2015-70801-R, cofinanced by FEDER) and “Instituto de Investigación Marqués de Valdecilla-IDIVAL (NVAL17/22). Dr. Tapia is the recipient of a grant from SMA Europe and FundAME (Spain).

The present study uses the embryonic chick to examine in vivo the mechanisms and regulation of Schwann cell programmed cell death (PCD) in spinal and cranial peripheral nerves. Schwann cells are highly dependent on the presence of axons for survival because the in ovo administration of NMDA, which excitotoxically eliminates motoneurons and their axons by necrosis, results in a significant increase in apoptotic Schwann cell death. Additionally, pharmacological and surgical manipulation of axon numbers also affects the relative amounts of Schwann cell PCD. Schwann cells undergoing both normal and induced PCD display an apoptotic-like cell death, using a caspase-dependent pathway. Furthermore, axon elimination results in upregulation of the p75 and platelet-derived growth factor receptors in mature Schwann cells within the degenerating ventral root. During early development, Schwann cells are also dependent on axon-derived mitogens; the loss of axons results in a decrease in Schwann cell proliferation. Axon removal during late embryonic stages, however, elicits an increase in proliferation, as is expected from these more differentiated Schwann cells. In rodents, Schwann cell survival is regulated by glial growth factor (GGF), a member of the neuregulin family of growth factors. GGF administration to chick embryos selectively rescued Schwann cells during both normal PCD and after the loss of axons, whereas other trophic factors tested had no effect on Schwann cell survival. In conclusion, avian Schwann cells exhibit many similarities to mammalian Schwann cells in terms of their dependence on axon-derived signals during early and later stages of development.

We have previously shown that chronic treatment of chick embryos [from embryonic day 5 (E5) to E9] with NMDA rescues spinal cord motoneurons (MNs) from programmed cell death. In this situation, MNs exhibit a reduced vulnerability to acute excitotoxic lesions and downregulate NMDA and AMPA-kainate receptors. Here, we report that this treatment results in long-lasting sublethal structural changes in MNs. In Nissl-stained sections from the spinal cord of NMDA-treated embryos, MNs display an area adjacent to an eccentrically positioned nucleus in which basophilia is excluded. Ultrastructurally, MNs accumulate tubulovesicular structures surrounded by Golgi stacks. Thiamine pyrophosphatase but not acid phosphatase was detected inside the tubulovesicular structures, which are resistant to disruption by brefeldin A or monensin. Immunocytochemistry reveals changes in the content and distribution of calcitonin gene-related peptide, the KDEL receptor, the early endosomal marker EEA1, and the recycling endosome marker Rab11, indicating that a dysfunction in membrane trafficking and protein sorting occurs in these MNs. FM1-43, a marker of the endocytic pathway, strongly accumulates in MNs from isolated spinal cords after chronic NMDA treatment. Changes in the distribution of cystatin C and presenilin-1 and an accumulation of amyloid precursor protein and beta-amyloid product were also observed in NMDA-treated MNs. None of these alterations involve an interruption of MN-target (muscle) connections, as detected by the retrograde tracing of MNs with cholera toxin B subunit. These results demonstrate that chronic NMDA treatment induces severe changes in the motoneuronal endomembrane system that may be related to some neuropathological alterations described in human MN disease.

Unilateral limb-bud removal (LBR) before the outgrowth of sensory or motor neurons to the leg of chick embryos was used to examine the role of limb (target)-derived signals in the development and survival of lumbar motoneurons and sensory neurons in the dorsal root ganglia (DRG). After LBR, motor and sensory neurons underwent normal initial histological differentiation, and cell growth in both populations was unaffected. Before their death, target-deprived motoneurons also expressed a cell-specific marker, the homeodomain protein islet-1. Proliferation of sensory and motor precursor cells was also unaffected by LBR, and the migration of neural crest cells to the DRG and of motoneurons into the ventral horn occurred normally. During the normal period of programmed cell death (PCD), increased numbers of both sensory and motor neurons degenerated after LBR. However, whereas motoneuron loss increased by 40–50% (90% total), only ∼25% more sensory neurons degenerated after LBR. A significant number of the surviving sensory neurons projected to aberrant targets in the tail after LBR, and many of these were lost after ablation of both the limb and tail. Treatment with neurotrophic factors (or muscle extract) rescued sensory and motor neurons from cell death after LBR without affecting precursor proliferation of either population. Activity blockade with curare failed to rescue motoneurons after LBR, and combined treatment with curare plus muscle extract was no more effective than muscle extract alone. Treatment with the antioxidantN-acetylcysteine rescued motoneurons from normal cell death but not after LBR. Two specific inhibitors of the interleukin β1 converting enzyme (ICE) family of cysteine proteases also failed to prevent motoneuron death after LBR. Taken together these data provide definitive evidence that the loss of spinal neurons after LBR cannot be attributed to altered proliferation, migration, or differentiation. Rather, in the absence of limb-derived trophic signals, the affected neurons fail to survive and undergo PCD. Although normal cell death and cell death after target deprivation share many features in common, the intracellular pathways of cell death in the two may be distinct.

In the present work, we show that chick embryo Schwann cells die by apoptosis both during normal development and after axonal degeneration induced by neurotoxin treatment. Schwann cell apoptosis during development takes place during a period roughly coincidental with normally occurring motoneuron death. Administration of NMDA to chick embryos on embryonic day 7 induces extensive excitotoxic motoneuronal damage in the spinal cord without any apparent effects on neurons in the dorsal root ganglia (DRG). The death of Schwann cells inventralnerve roots after NMDA treatment causes degenerative changes that display ultrastructural features of apoptosis and exhibitin situdetectable DNA fragmentation. By contrast, NMDA treatment does not increase the death of Schwann cells indorsalnerve roots.In situdetection of DNA fragmentation in combination with the avian Schwann cell marker 1E8 antibody demonstrates that dying cells in ventral nerve roots are in the Schwann cell lineage. Administration of cycloheximide does not prevent the toxic effects of NMDA on motoneurons, but dramatically reduces the number of pyknotic Schwann cells and DNA fragmentation profiles in the ventral nerve roots.In ovoadministration of various tissue extracts (muscle, brain, and spinal cord) from the chick embryo or of the motoneuron conditioned medium fails to prevent Schwann cell apoptosis in NMDA-treated embryos. Intramuscular administration of the snake toxin β-bungarotoxin produces a massive death of both lateral motor column motoneurons and DRG neurons, resulting in a substantial increase in the number of pyknotic Schwann cells in both ventral and dorsal nerve roots. It is concluded that during development, axonal-derived trophic signals are involved in the regulation of Schwann cell survival in peripheral nerves.

Spinal muscular atrophy (SMA) is a devastating autosomal recessive neuromuscular disease characterized by degeneration of spinal cord alpha motor neurons (αMNs). SMA is caused by the homozygous deletion or mutation of the survival motor neuron 1 (SMN1) gene, resulting in reduced expression of SMN protein, which leads to αMN degeneration and muscle atrophy. The majority of transcripts of a second gene (SMN2) generate an alternative spliced isoform that lacks exon 7 and produces a truncated nonfunctional form of SMN. A major function of SMN is the biogenesis of spliceosomal snRNPs, which are essential components of the pre-mRNA splicing machinery, the spliceosome. In recent years, new potential therapies have been developed to increase SMN levels, including treatment with antisense oligonucleotides (ASOs). The ASO-nusinersen (Spinraza) promotes the inclusion of exon 7 in SMN2 transcripts and notably enhances the production of full-length SMN in mouse models of SMA. In this work, we used the intracerebroventricular injection of nusinersen in the SMN∆7 mouse model of SMA to evaluate the effects of this ASO on the behavior of Cajal bodies (CBs), nuclear structures involved in spliceosomal snRNP biogenesis, and the cellular distribution of polyadenylated mRNAs in αMNs. The administration of nusinersen at postnatal day (P) 1 normalized SMN expression in the spinal cord but not in skeletal muscle, rescued the growth curve and improved motor behavior at P12 (late symptomatic stage). Importantly, this ASO recovered the number of canonical CBs in MNs, significantly reduced the abnormal accumulation of polyadenylated RNAs in nuclear granules, and normalized the expression of the pre-mRNAs encoding chondrolectin and choline acetyltransferase, two key factors for αMN homeostasis. We propose that the splicing modulatory function of nusinersen in SMA αMN is mediated by the rescue of CB biogenesis, resulting in enhanced polyadenylated pre-mRNA transcription and splicing and nuclear export of mature mRNAs for translation. Our results support that the selective restoration of SMN expression in the spinal cord has a beneficial impact not only on αMNs but also on skeletal myofibers. However, the rescue of SMN expression in muscle appears to be necessary for the complete recovery of motor function. The authors wish to thank Raquel García-Ceballos for technical assistance and Alfonso Nieva for video editing. We are also grateful to Prof. A. I. Lamond (University of Dundee, UK) for anti-coilin antibody (204.10) and Prof. Larry Gerace (The Scripps Research Institute, La Jolla, USA) for anti-LaminA/C antibody. The authors are also indebted to Prof. Josep E. Esquerda for critical reading of the manuscript and helpful suggestions. This work was supported by the following grants: Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED, CB06/05/0037) Spain; Instituto de Investigación Sanitaria Valdecilla (IDIVAL, Next-Val 17/22), Santander, Spain; and Spanish Ministerio de Ciencia, Innovación y Universidades cofinanced by Fondo Europeo de Desarrollo Regional (FEDER) (RTI2018-099278-B-I00).