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Neurocalcin delta (NCALD) is a brain-enriched neuronal calcium sensor and its reduction acts protective against spinal muscular atrophy (SMA). However, the physiological function of NCALD and implications of NCALD reduction are still elusive. Here, we analyzed the ubiquitous Ncald knockout in homozygous (Ncald(KO/KO)) and heterozygous (Ncald(KO/WT)) mice to unravel the physiological role of NCALD in the brain and to study whether 50% NCALD reduction is a safe option for SMA therapy. We found that Ncald(KO/KO) but not Ncald(KO/WT) mice exhibit significant changes in the hippocampal morphology, likely due to impaired generation and migration of newborn neurons in the dentate gyrus (DG). To understand the mechanism behind, we studied the NCALD interactome and identified mitogen-activated protein kinase kinase kinase 10 (MAP3K10) as a novel NCALD interacting partner. MAP3K10 is an upstream activating kinase of c-Jun N-terminal kinase (JNK), which regulates adult neurogenesis. Strikingly, the JNK activation was significantly upregulated in the Ncald(KO/KO) brains. Contrary, neither adult neurogenesis nor JNK activation were altered by heterozygous Ncald deletion. Taken together, our study identifies a novel link between NCALD and adult neurogenesis in the hippocampus, possibly via a MAP3K10-JNK pathway and emphasizes the safety of using NCALD reduction as a therapeutic option for SMA.

Autosomal recessive spinal muscular atrophy (SMA), the leading genetic cause of infant lethality, is caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. SMA disease severity inversely correlates with the number of SMN2 copies, which in contrast to SMN1, mainly produce aberrantly spliced transcripts. Recently, the first SMA therapy based on antisense oligonucleotides correcting SMN2 splicing, namely SPINRAZA (TM), has been approved. Nevertheless, in type I SMA-affected individuals-representing 60% of SMA patients-the elevated SMN level may still be insufficient to restore motor neuron function lifelong. Plastin 3 (PLS3) and neurocalcin delta (NCALD) are two SMN-independent protective modifiers identified in humans and proved to be effective across various SMA animal models. Both PLS3 overexpression and NCALD downregulation protect against SMA by restoring impaired endocytosis; however, the exact mechanism of this protection is largely unknown. Here, we identified calcineurin-like EF-hand protein 1 (CHP1) as a novel PLS3 interacting protein using a yeast-two-hybrid screen. Co-immunoprecipitation and pull-down assays confirmed a direct interaction between CHP1 and PLS3. Although CHP1 is ubiquitously present, it is particularly abundant in the central nervous system and at SMA-relevant sites including motor neuron growth cones and neuromuscular junctions. Strikingly, we found elevated CHP1 levels in SMA mice. Congruently, CHP1 downregulation restored impaired axonal growth in Smn-depleted NSC34 motor neuron-like cells, SMA zebrafish and primary murine SMA motor neurons. Most importantly, subcutaneous injection of low-dose SMN antisense oligonucleotide in pre-symptomatic mice doubled the survival rate of severely-affected SMA mice, while additional CHP1 reduction by genetic modification prolonged survival further by 1.6-fold. Moreover, CHP1 reduction further ameliorated SMA disease hallmarks including electrophysiological defects, smaller neuromuscular

La Atrofia Muscular Espinal (AME) es una enfermedad autosómica recesiva caracterizada por debilidad muscular y deterioro de la transmisión sináptica y pérdida de motoneuronas inferiores. La proteína SMN (Survival Motor Neuron) está codificada por dos genes: SMN1, que produce la proteína completa (SMN-FL), y SMN2, que mayoritariamente produce una forma truncada de SMN (SMN¿7) y una pequeña cantidad de proteína completa. En ausencia de SMN1, la severidad de la enfermedad depende de la cantidad de proteína completa producida por SMN2, que depende, a su vez, del número de copias de este gen en cada individuo. SMN se expresa ubicuamente en todos los tejidos, localizándose tanto en el citoplasma como en el núcleo celular. Su función mejor caracterizada es su participación en el ensamblaje de las ribonucleoproteínas pequeñas nucleares (snRNPs), que forman parte del espliceosoma. Sin embargo, los bajos niveles de SMN afectan principalmente a las motoneuronas que inervan músculos proximales, por lo que el papel específico de SMN en las motoneuronas no se conoce. Recientemente, se ha postulado sobre la existencia de genes modificadores de SMN, tales como el gen que codifica la plastina 3 (PLS3). Esta proteína se une a monómeros de actina y promueve la formación de filamentos de actina. Dado que la actina participa en funciones tan diversas e importantes como la axonogénesis o la transmisión sináptica, la PLS3 debe desempeñar un papel importante en la organización de este componente del citoesqueleto. El objetivo del presente trabajo ha sido, por un lado, investigar las alteraciones estructurales en los terminales motores y la capacidad de los terminales motores de modular la liberación de neurotransmisor, y por otro, la capacidad de la Plastina-3 (PLS3) de rescatar la patología sináptica. Para ello, se ha realizado un estudio morfológico y funcional en el modelo murino de AME SMA¿7. Se ha explorado la madurez del terminal postsináptico, la organización de las vesículas sinápt

La Atrofia Muscular Espinal (AME) es una enfermedad autosómica recesiva caracterizada por debilidad muscular y deterioro de la transmisión sináptica y pérdida de motoneuronas inferiores. La proteína SMN (Survival Motor Neuron) está codificada por dos genes: SMN1, que produce la proteína completa (SMN-FL), y SMN2, que mayoritariamente produce una forma truncada de SMN (SMN¿7) y una pequeña cantidad de proteína completa. En ausencia de SMN1, la severidad de la enfermedad depende de la cantidad de proteína completa producida por SMN2, que depende, a su vez, del número de copias de este gen en cada individuo. SMN se expresa ubicuamente en todos los tejidos, localizándose tanto en el citoplasma como en el núcleo celular. Su función mejor caracterizada es su participación en el ensamblaje de las ribonucleoproteínas pequeñas nucleares (snRNPs), que forman parte del espliceosoma. Sin embargo, los bajos niveles de SMN afectan principalmente a las motoneuronas que inervan músculos proximales, por lo que el papel específico de SMN en las motoneuronas no se conoce. Recientemente, se ha postulado sobre la existencia de genes modificadores de SMN, tales como el gen que codifica la plastina 3 (PLS3). Esta proteína se une a monómeros de actina y promueve la formación de filamentos de actina. Dado que la actina participa en funciones tan diversas e importantes como la axonogénesis o la transmisión sináptica, la PLS3 debe desempeñar un papel importante en la organización de este componente del citoesqueleto. El objetivo del presente trabajo ha sido, por un lado, investigar las alteraciones estructurales en los terminales motores y la capacidad de los terminales motores de modular la liberación de neurotransmisor, y por otro, la capacidad de la Plastina-3 (PLS3) de rescatar la patología sináptica. Para ello, se ha realizado un estudio morfológico y funcional en el modelo murino de AME SMA¿7. Se ha explorado la madurez del terminal postsináptico, la organización de las vesículas sinápt

Low levels of the Survival Motor Neuron (SMN) protein produce Spinal Muscular Atrophy (SMA), a severe monogenetic disease in infants characterized by muscle weakness and impaired synaptic transmission. We report here severe structural and functional alterations in the organization of the organelles and the cytoskeleton of motor nerve terminals in a mouse model of SMA. The decrease in SMN levels resulted in the clustering of synaptic vesicles (SVs) and Active Zones (AZs), reduction in the size of the readily releasable pool (RRP), and the recycling pool (RP) of synaptic vesicles, a decrease in active mitochondria and limiting of neurofilament and microtubule maturation. We propose that SMN is essential for the normal postnatal maturation of motor nerve terminals and that SMN deficiency disrupts the presynaptic organization leading to neurodegeneration.

Proximal spinal muscular atrophy, the most frequent genetic cause of childhood lethality, is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5, which codes for the survival motor neuron (SMN) protein. SMN plays a role in the assembly of small nuclear ribonucleoproteins and, additionally, in synaptic function. SMN deficiency produces defects in motor neuron b-actin mRNA axonal transport, neurofilament dynamics, neurotransmitter release, and synapse maturation. The underlying molecular mechanisms and, in particular, the role of the cytoskeleton on the pathogenesis of this disease are starting to be revealed.

Low levels of survival motor neuron (SMN) protein result in spinal muscular atrophy (SMA), a severe genetic disease characterized by motor impairment and premature lethality. Although SMN is a ubiquitous protein, motor neurons are much more vulnerable to low levels of SMN than other cells. To gain insight into the pathogenesis of SMA, we have compared synaptic function of motor terminals in wild-type and severe SMA mice at different ages and in two proximal muscles. Our results show that mutant muscle fibers fire normal action potentials and that multi-innervated terminals are functional. By studying the characteristics of the three main components of synaptic transmission in nerve terminals (spontaneous, evoked, and asynchronous release), we found that the kinetics of the postsynaptic potentials are slowed and evoked neurotransmitter release is decreased by 55%. In addition, asynchronous release is increased 300%, indicating an anomalous augmentation of intraterminal bulk Ca 2 during repetitive stimulation. Together, these results show that the reduction of SMN affects synaptic maturation, evoked release, and regulation of intraterminal Ca 2 levels.