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Apoptosi; Motoneurones; Atròfia muscular espinal Apoptosis; Motoneuronas; Atrofia muscular espinal Apoptosis; Motoneurons; Spinal muscular atrophy Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function. This work was supported by grants from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias, Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” (PI17/00231 and PI20/00098) to RMS and AG; CERCA Program/Generalitat de Catalunya; and Spanish Agency of Research (Agencia Estatal de Investigacion-PID2019-107286RB-I00) and CIBERNED to JXC. AS holds a fellowship from Universitat de Lleida and SdF holds a fellowship from “Ajuts de Promoció de la Recerca en Salut” (IRBLleida-Diputació de Lleida). We thank Elaine Lilly, PhD, for English language revision of the paper.

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons., The adaptor protein p11 and K+ channel TASK1 have overlapping distributions in the CNS. Here, the authors demonstrate that the transcription factor Sp1 regulates p11 levels, which in turn affects intrinsic membrane properties and can contribute to degeneration of motor neurons in disease and injury models.

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy. This work was financially supported by grants from the Instituto de Salud Carlos III, Fondo de Inversiones Sanitarias, Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” (PI14/00060 and PI17/00231).

During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease. This study was supported by Fundación Tatiana Pérez de Guzmán el Bueno, the Spanish Ministry of Science and Innovation/FEDER (BFU2013-43763-P and BFU2016-78934-P), Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI14/00060), Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) ‘‘Una manera de hacer Europa’’ and Generalitat de Catalunya (SGR740).

Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach. This work was supported by grants from Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI14/00060), Unión Europea, Fondo europeo de Desarrollo Regional (FEDER) "Una manera de hacer Europa", and Generalitat de Catalunya (SGR740). AP holds a fellowship from Comissionat d’Universitats i Recerca, Departament d’Innovació, Universitats i Empresa de la Generalitat de Catalunya i Fons Social Europeu, SA and SdF hold a fellowship from Universitat de Lleida. We thank Elaine Lilly, Ph.D., for the English language revision of the manuscript.

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well. The project was supported by grants of the Associazione Girotondo Onlus (Trivero, Italy), the Smarathon Association (Milan, Italy), and by a grant of the Italian Ministry of Health (Project RF-2009-1475235). AP was the recipient of a fellowship (3 years) from the ‘’Master and Back” project 2009 by the Regione Autonoma della Sardegna (Cagliari, Italy).

Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.

Ground level and vertical measurements (coupled with modelling) of ozone (O3), other gaseous pollutants (NO, NO2, CO, SO2) and aerosols were carried out in the plains (Vic Plain) and valleys of the northern region of the Barcelona Metropolitan Area (BMA) in July 2015; an area typically recording the highest O3 episodes in Spain. Our results suggest that these very high O3 episodes were originated by three main contributions: (i) the surface fumigation from high O3 reservoir layers located at 1500–3000 m a.g.l., and originated during the previous day(s) injections of polluted air masses at high altitude; (ii) local/regional photochemical production and transport (at lower heights) from the BMA and the surrounding coastal settlements, into the inland valleys; and (iii) external (to the study area) contributions of both O3 and precursors. These processes gave rise to maximal O3 levels in the inland plains and valleys northwards from the BMA when compared to the higher mountain sites. Thus, a maximum O3 concentration was observed within the lower tropospheric layer, characterised by an upward increase of O3 and black carbon (BC) up to around 100–200 m a.g.l. (reaching up to 300 µg/m3 of O3 as a 10-s average), followed by a decrease of both pollutants at higher altitudes, where BC and O3 concentrations alternate in layers with parallel variations, probably as a consequence of the atmospheric transport from the BMA and the return flows (to the sea) of strata injected at certain heights the previous day(s). At the highest altitudes reached in this study (900–1000 m a.g.l.) during the campaign, BC and O3 were often anti-correlated or unrelated, possibly due to a prevailing regional/hemispheric contribution of O3 at those altitudes. In the central hours of the days a homogeneous O3 distribution was evidenced for the lowest 1 km of the atmosphere, although probably important variations could be expected at higher levels, where the high O3 return strata are injected according to the modelling results and free sounding data. Relatively low concentrations of ultrafine particles (UFP) were recorded in the 100–200 m a.g.l. atmospheric layer where concentrations of O3 were high; and nucleation episodes were only detected into the boundary layer. Two types of O3 episodes were identified: Type A) with major exceedances of the O3 information threshold (180 µg/m3 on an hourly basis) caused by a clear daily concatenation of local/regional production with accumulation (at upper levels), fumigation and direct transport from the BMA (closed circulation); and Type B) with regional O3 production without major recirculation (neither fumigation) of the polluted BMA/regional air masses (open circulation), and relatively lower O3 levels. The interpretation of OX (O3 + NO2) experimental data from strategically selected monitoring sites on the coast and inland, together with the photochemical modelling results have allowed to study the O3 phenomenology associated with the onset and development of severe episodes in the region of Catalonia in NE Spain. To implement potential O3 control and abatement strategies two major key tasks are proposed: (i) meteorological forecasting, from June to August, to predict recirculation episodes so that NOX and VOCs abatement measures can be applied before these episodes start; (ii) sensitivity analysis with high resolution modelling to evaluate the effectiveness of these potential abatement measures of precursors for O3 reduction.

In vivoandin vitromotoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKβ kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKβ phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKβ, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-xLoverexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.

FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5–15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor κB (NF-κB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75NTR, in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-κB-mediated control of neurite growth in developing neurons. This work was funded by Ministerio de Sanidad y Consumo (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) Grant CB06/05/1104), Ministerio de Educación y Ciencia Grant SAF2007-60287, Generalitat de Catalunya (Suport als Grups de Recerca Consolidats) (J.X.C.), Ministerio de Ciencia e Innovación Grants BFU2008-01328 (V.J.Y.) and BFU2008-3980 (E.S.), and Fundació Caixa Catalunya Obra Social (E.S.). C.S. was supported by postgraduate fellowships from Ministerio de Educación y Ciencia and Fondo de Investigación Sanitaria. V.I.-G. holds a postgraduate fellowship from the Generalitat de Catalunya. R.S.M. and V.J.Y. are under the Juan de la Cierva and Ramon y Cajal programs, respectively, from the Ministerio de Educación y Ciencia (Spain), cofinanced by the European Social Fund. We thank D. Trono (Geneva, Switzerland) for providing the lentiviral plasmids and Dr. Joaquin Lopez-Soriano and Patricia Ortega for excellent technical support.

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.

To determine which intracellular pathways mediate the survival effects of ciliary neurotrophic factor and cardiotrophin-1 cytokines on motoneurons, we studied the activation of the Jak/STAT, the PI 3-kinase/Akt, and the ERK pathways. At shorter time points, cytokines induced the activation of STAT3 and ERK, but not PI 3-kinase. Jak3 inhibitor suppressed cytokine- and muscle extract-induced survival. In contrast, PD 98059, a MEK inhibitor, was not able to prevent cytokine-induced survival, demonstrating that ERK is not involved. Surprisingly, the PI 3-kinase inhibitor LY 294002 prevented the survival-promoting effects of cytokines. When assays of PI 3-kinase activity were performed at later stages following cytokine treatment a significant increase was observed compared to control cultures. This delayed increase of activity could be completely prevented by treatment with protein synthesis or Jak3 inhibitors. Collectively, these results demonstrate that cytokines induce motoneuron survival through a PI 3-kinase activation requiring de novo protein synthesis dependent on Jak pathway.

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells.

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promotein vivoandin vitrosurvival of different neuronal populations, including spinal cord motoneurons. These factors signal via multicomponent receptors that consist of the Ret receptor tyrosine kinase plus a member of the GDNF family receptor α (GRFα) family of glycosylphosphatidylinositol-linked coreceptors. Activation of the receptor induces Ret phosphorylation that leads the survival-promoting effects. Ret phosphorylation causes the activation of several intracellular pathways, but the biological effects caused by the activation of each of these pathways are still unknown. In the present work, we describe the ability of the GDNF family members to promote chicken motoneuron survival in culture. We show the presence of Ret and GFRα-1, GFRα-2, and GFRα-4 in chicken motoneurons usingin situhybridization and reverse transcription-PCR techniques. By Western blot analysis and kinase assays, we demonstrate the ability of these factors to induce the phosphatidylinositol 3 kinase (PI 3-kinase) and the extracellular regulated kinase (ERK)–mitogen-activated protein (MAP) kinase pathways activation. To characterize the involvement of these pathways in the survival effect, we used the PI 3-kinase inhibitor LY 294002 and the MAP kinase and ERK kinase (MEK) inhibitor PD 98059. We demonstrate that LY 294002, but not PD 98059, prevents GDNF-, neurturin-, and persephin-induced motoneuron survival, suggesting that PI 3-kinase intracellular pathway is responsible in mediating the neurotrophic effect.

In the present work, we find that the elevation of extracellular K+concentration promotes the survival of chick spinal cord motoneuronsin vitrodeprived of any neurotrophic support. This treatment induces chronic depolarization of the neuronal plasma membrane, which activates L-type voltage-dependent Ca2+channels, resulting in Ca2+influx and elevation of the cytosolic free Ca2+concentration. Pharmacological reduction of intracellular free Ca2+or withdrawal of extracellular Ca2+reversed the effects of depolarization on survival. The intracellular Ca2+response to membrane depolarization developed as an initial peak followed by a sustained increase in intracellular Ca2+concentration. The depolarizing treatment caused tyrosine phosphorylation of mitogen-activated protein kinase (MAPK) without involving tyrosine kinase receptor activation. The calmodulin antagonist W13 inhibited the survival-promoting effect induced by membrane depolarization but not the tyrosine phosphorylation of MAPK. Moreover, depolarization did not induce phosphatidylinositol-3 kinase (PI-3K) phosphorylation in our cells, and the PI-3K inhibitor wortmannin did not suppress the survival-promoting effect of K+treatment. These results suggest that calmodulin is involved in calcium-mediated survival of motoneurons through the activation of PI-3K- and MAPK-independent pathways.

Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons. This work was supported by grants from Fundación Genoma España, GENAME to JL, LT and RMS, and from Instituto de Salud Carlos III-Fondo de Investigaciones Sanitarias (PI11/01047) to RMS. AG holds a postdoctoral contract from Genoma España. VC-M and AC-R have been supported by a predoctoral fellowship from “Govern de les Illes Balears, Conselleria d’Educació, Cultura i Universitats” under a program of joint financing with the European Social Fund.

Intracellular pathways related to cell survival regulate neuronal physiology during development and neurodegenerative disorders. One of the pathways that have recently emerged with an important role in these processes is nuclear factor- κB (NF-κB). The activity of this pathway leads to the nuclear translocation of the NF-κB transcription factors and the regulation of anti-apoptotic gene expression. Different stimuli can activate the pathway through different intracellular cascades (canonical, non-canonical, and atypical), contributing to the translocation of specific dimers of the NF-κB transcription factors, and each of these dimers can regulate the transcription of different genes. Recent studies have shown that the activation of this pathway regulates opposite responses such as cell survival or neuronal degeneration. These apparent contradictory effects depend on conditions such as the pathway stimuli, the origin of the cells, or the cellular context. In the present review, the authors summarize these findings and discuss their significance with respect to survival or death in the nervous system. This work was supported by grants to RMS from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI080267/PI1101047), and Consolider-Ingenio 2010 (CSD2007-00020, Ministerio de Ciencia e Innovación), and by the Generalitat de Catalunya (Suport Grups de Recerca Emergents) 2009SGR740. SMT holds a fellowship from Consolider-Ingenio 2010 (CSD2007-00020).

In the present work we study the functional role of muscle cell extracellular matrix components in axonal guidance during synaptic regeneration. We focused on components recognized by the N-acetylglucosamine-specific lectin called wheat germ agglutinin (WGA) that has been shown to bind to the muscle cell extracellular matrix. We have used a cryoculture bioassay which is based on the ability of chick ciliary ganglion neurons to grow on rat skeletal muscle cryostat sections [Covault, J., et al., J. Neurosci., 105 (1987) 2479-2488.]. In control cultures neurites extended upon the muscle sections closely associated to the muscle cell surface. Masking WGA lectin receptors on the muscle cell surface perturbed the behavior of neurites. On WGA-treated sections, most of the neurites extended indiscriminately on intercellular and intracellular regions. These results indicate that N-acetylglucosamine-bearing molecules on muscle cell surfaces may play functional roles in the guidance of neurites through the extracellular matrix.

The purpose of the work reported here is to identify molecular components of the synaptic basal lamina of skeletal muscle fibres which allow recognition of original synaptic sites by regenerating motor axons. We focused on s-laminin and components recognized by the lectin Dolichos biflorus agglutinin previously shown to be specifically located at the synaptic basal lamina. We used a cryoculture bioassay in which chick ciliary ganglion neurons grow on rat skeletal muscle cryostat sections. In control cultures, neurites extended over the muscle sections in close association with the muscle cell surface. It was observed that most of the neurites that extended towards the endplate zone and reached an area of 40 microns around the neuromuscular junction ceased to grow when they contacted the synaptic site. Masking either lectin receptors or some s-laminin molecule epitopes prior to the culture of neurons alters the behaviour of growing neurites. On sections treated either with Dolichos biflorus agglutinin or anti s-laminin monoclonal antibodies (D5 and C4) most of the neurites did not stop their growth at the synaptic regions. Moreover, treating muscle sections with Dolichos biflorus agglutinin removed the gradient of substratum affinity around the endplate. These results indicate that the s-laminin and Dolichos biflorus agglutinin receptors present on muscle cell surfaces may play a functional role in the interaction of growing neurites with original synaptic sites in the process of neuromuscular regeneration.

Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord caused by homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Cell migration is critical at many stages of nervous system development; to investigate the role of SMN in cell migration, U87MG astroglioma cells were transduced with shSMN lentivectors and about 60% reduction in SMN expression was achieved. In a monolayer wound-healing assay, U87MG SMN-depleted cells exhibit reduced cell migration. In these cells, RhoA was activated and phosphorylated levels of myosin regulatory light chain (MLC), a substrate of the Rho kinase (ROCK), were found increased. The decrease in cell motility was related to activation of RhoA/Rho kinase (ROCK) signaling pathway as treatment with the ROCK inhibitor Y-27632 abrogated both the motility defects and MLC phosphorylation in SMN-depleted cells. As cell migration is regulated by continuous remodeling of the actin cytoskeleton, the actin distribution was studied in SMN-depleted cells. A shift from filamentous to monomeric (globular) actin, involving the disappearance of stress fibers, was observed. In addition, profilin I, an actin-sequestering protein showed an increased expression in SMN-depleted cells. SMN is known to physically interact with profilin, reducing its actin-sequestering activity. The present results suggest that in SMN-depleted cells, the increase in profilin I expression and the reduction in SMN inhibitory action on profilin could lead to reduced filamentous actin polymerization, thus decreasing cell motility. We propose that the alterations reported here in migratory activity in SMN-depleted cells, related to abnormal activation of RhoA/ROCK pathway and increased profilin I expression could have a role in developing nervous system by impairing normal neuron and glial cell migration and thus contributing to disease pathogenesis in SMA.

Tropomyosin-related kinase A (TrkA) receptor mediates the effects exerted by nerve growth factor on several subpopulations of neuronal cells. Ligand binding to TrkA induces receptor autophosphorylation on several tyrosine residues and the activation of signaling cascades. In this study, we describe a new site relevant for TrkA regulation, the tyrosine 701 (Y701), which is important for receptor trafficking and activation. Y701 replacement by aspartate or phenylalanine reduces receptor internalization rate and decreases the colocalization and association of TrkA with clathrin heavy chain, demonstrating that Y701 constitutes a YxxPhi (YRKF701-704) trafficking motif relevant for the regulation of receptor endocytosis. In accordance with this hypothesis, the colocalization of Y701 mutant receptors with a lysosomal marker is also reduced giving support to the involvement of the YRKF701-704 motif in the lysosomal targeting of TrkA receptors. Contrary to what was expected, substitution of Y701 for an Asp in order to mimic phosphorylation, impairs TrkA ability to mediate nerve growth factor-induced differentiation, although the mutant receptor retains its in vitro kinase activity. This is the first evidence that a Tyr residue can simultaneously regulate TrkA receptor trafficking and activity.

Neurotrophic factors promote motoneuron (MN) survival through increased intracellular calcium (Ca(2+)) and regulation of the phosphatidylinositol (PI) 3-kinase/protein kinase B (PKB) pathway by calmodulin (CaM). Activation of the PI 3-kinase/PKB pathway is one of the well established mechanisms involved in MN survival. The Ca(2+)/CaM complex interacts with and modulates the functionality of a large number of proteins, including serine/threonine protein kinases such as Ca(2+)/CaM-dependent protein kinases (CaMKs). Using a primary culture of embryonic chicken spinal cord MNs, we investigated the role of CaMKIV in mediating this process. We cloned chicken CaMKIV and demonstrated its expression in purified MNs by means of reverse transcription-PCR, Western blot, and immunofluorescence. Using RNA interference, we show that endogenous CaMKIV mediates cell survival induced by neurotrophic factors or membrane depolarization. The survival effect is independent of CaMKIV kinase activity; however, CaMKIV functionality depends on the presence of Ca(2+)/CaM. Finally, CaMKIV associates to the p85 subunit of PI 3-kinase in a Ca(2+)-dependent manner, suggesting a role in regulating PI 3-kinase/PKB activation.

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.

Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.

Chick embryo spinal cord motoneurons develop a trophic response to some neurotrophins when they are maintained in culture in the presence of muscle extract. Thus, after 2 days in culture, brain-derived neurotrophic factor (BDNF) promotes motoneuron survival. In the present study we have analyzed the intracellular pathways that may be involved in the BDNF-induced motoneuron survival. We have observed that BDNF activated the extracellular- regulated kinase (ERK) mitogen-activated protein (MAP) kinase and the phosphatidylinositol (PI) 3-kinase pathways. To examine the contribution of these pathways to the survival effect triggered by BDNF, we used PD 98059, a specific inhibitor of MAP kinase kinase, and LY 294002, a selective inhibitor of PI 3-kinase. PD 98059, at doses that significantly reduced the phosphorylation of ERKs, did not show any prominent effect on neuronal survival. However, LY 294002 at doses that inhibited the phosphorylation of Akt, a downstream element of the PI 3-kinase, completely abolished the motoneuron survival effects of BDNF. Moreover, cell death triggered by LY 294002 treatment exhibited features similar to those observed after muscle extract deprivation. Our results suggest that the PI 3-kinase pathway plays an important role in the survival effect triggered by BDNF on motoneurons, whereas activation of the ERK MAP kinase pathway is not relevant., This work was supported by grants from CICYT PN-SAF (97-0094) (J.X.C.), DGICYT (PB94-1104) (D.M.-Z.), Biotech (BIO4-CT96-0433) (J.X.C.), Generalitat de Catalunya, and Fundación Francisca de Roviralta. X.D. and J.E. are predoctoral fellows of the Ministerio de Educación y Ciencia and Generalitat de Catalunya, respectively.

During embryonic development, most neuronal populations undergo a process usually referred to as naturally occurring neuronal death. For motoneurons (MTNs) of the lumbar spinal cord of chick embryos, this process takes place in a well defined period of time, between embryonic days 6 and 10 (E6–E10). Neurotrophins (NTs) are the best characterized family of neurotrophic factors and exert their effects through activation of their specific Trk receptors.In vitroandin vivostudies have demonstrated that rodent motoneurons survive in response to BDNF, NT3, and NT4/5. In contrast, the trophic dependencies of chicken motoneurons have been difficult to elucidate, and various apparently conflicting reports have been published. In the present study, we describe how freshly isolated motoneurons from E5.5 chick embryos did not respond to any neurotrophinin vitro. Yet, because motoneurons were maintained alive in culture in the presence of muscle extract, they developed a delayed specific survival response to BDNF, NT3, and NT4/5 that is clearly dose-dependent, reaching saturation at doses of 100 pg/ml. This trophic response correlated with increasing expression of the corresponding functional receptors TrkB and TrkC. Moreover, TrkB receptor is able to become autophosphorylated and to activate classical intracellular signaling pathways such as the extracellular signal-regulated protein kinase when it is stimulated with its cognate ligand BDNF. Therefore, our results reconcile the reported differences betweenin vivoandin vitrostudies on the ability of chicken MTNs to respond to some members of the neurotrophin family of trophic factors.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs. This work was supported by grants from Instituto de Salud Carlos III, Fondo de Inversiones Sanitarias, Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) “Una manera de hacer Europa” (PI17/00231 and PI17/00134). CERCA Program/Generalitat de Catalunya. SdF holds a fellowship from Universitat de Lleida and “Ajuts de Promoció de la Recerca en Salut 2018” (IRBLLEIDA-Diputació de Lleida). AS holds a fellowship from Universitat de Lleida. AG holds a postdoctoral fellowship from Universitat de Lleida. We thank Dr. Manuel Portero from Metabolic Physiopathology Group, Universitat de Lleida-IRBLLEIDA for human iPSC differentiation protocol and assistance. We thank Elaine Lilly, PhD, for English language revision of the paper.

Spinal muscular atrophy (SMA) is a genetic disorder characterized by degeneration of spinal cord motoneurons (MNs), resulting in muscular atrophy and weakness. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene and decreased SMN protein. SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoproteins and pre-mRNA splicing requirements. SMN reduction causes neurite degeneration and cell death without classical apoptotic features, but the direct events leading to SMN degeneration in SMA are still unknown. Autophagy is a conserved lysosomal protein degradation pathway whose precise roles in neurodegenerative diseases remain largely unknown. In particular, it is unclear whether autophagosome accumulation is protective or destructive, but the accumulation of autophagosomes in the neuritic beadings observed in several neurite degeneration models suggests a close relationship between the autophagic process and neurite collapse. In the present work, we describe an increase in the levels of the autophagy markers including autophagosomes, Beclin1 and light chain (LC)3-II proteins in cultured mouse spinal cord MNs from two SMA cellular models, suggesting an upregulation of the autophagy process in Smn (murine survival motor neuron protein)-reduced MNs. Overexpression of Bcl-xL counteracts LC3-II increase, contributing to the hypothesis that the protective role of Bcl-xL observed in some SMA models may be mediated by its role in autophagy inhibition. Our in vitro experimental data indicate an upregulation in the autophagy process and autophagosome accumulation in the pathogenesis of SMA, thus providing a valuable clue in understanding the mechanisms of axonal degeneration and a possible therapeutic target in the treatment of SMA. This work was supported by grants from GENAME (Defining targets for Therapeutics in SMA) to RMS; from Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI11-01047), Generalitat de Catalunya (SGR740) and Consolider-Ingenio 2010 (CSD2007-00020) to RMS. AG holds a post-doctoral contract from Genoma Espan˜a; SA holds a fellowship from Universitat de Lleida and AP holds a fellowship from Comissionat de Universitats i Recerca, Departament d’Innovacio´, Universitats i Empresa de la Generalitat de Catalunya and Fons Social Europeu.

Spinal Muscular Atrophy (SMA), a neurodegenerative disorder primarily affecting motoneurons (MNs), is caused by the loss of the Survival Motor Neuron 1 (SMN1) gene and reduced levels of full-length survival motor neuron (SMN) protein. The exact cellular/molecular mechanisms involved in SMN-induced MN degeneration are under study. Autophagy is a degradation pathway whose precise roles in neurodegeneration remain largely unknown, but abnormal autophagy has a central role in some neurodegenerative diseases, including MN disorders. The analysis of the autophagy response in SMA and its role in the development of the disease could be essential to understand the disease. In the present work, we describe an increase of autophagosomes and LC3-II protein in spinal cord MNs of severe SMA mouse model. A time-course experiment demonstrated increased LC3-II levels from embryonic to postnatal stage, suggesting a deregulation of the autophagy process as the disease progressed. Using an in vitro model of MN culture, we analyzed the effect of autophagy modulators on Smn (murine survival motor neuron) protein level. Results suggest that the inhibitors of the autophagy flux cause reduction of Smn protein, whereas autophagy inducers increase the level of Smn protein in MNs. In order to evaluate other proteolytic systems involved to SMN degradation, we also studied the effect of the inhibition of the calcium-dependent protease, calpain, on Smn protein level. Our results demonstrate that calpain reduction increases Smn and LC3-II level in cultured MNs. Collectively, these results provide new insight into the role of autophagy and its modulation in SMN protein regulation.