Ana Garcera

A new model to study spinal muscular atrophy: Neurite degeneration and cell death is counteracted by BCL-X L Overexpression in motoneurons

Spinal muscular atrophy (SMA) is a motoneuron disorder characterized by deletions or specific mutations in the Survival Motor Neuron gene (SMN). SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoprotein (snRNP) and pre-mRNA splicing requirements. However, in motoneuron axons SMN deficiency results in inappropriate levels of certain transcripts in the distal axon, suggesting that the specific susceptibility of motoneurons to SMN deficiency is related to a specialized function in these cells. Although mouse models of SMA have been generated and are useful for in vivo and in vitro studies, the limited number of isolated MNs that could be obtained from them makes it difficult to perform biochemical, genetic and pharmacological approaches. We describe here an in vitro model of isolated embryonic mouse motoneurons in which the cellular levels of endogenous SMN are reduced. These cells show neurite degeneration and cell death after several days of SMN knockdown. We found that the over-expression of the anti-apoptotic protein Bcl-x(L) into motoneurons rescues these cells from the phenotypic changes observed. This result demonstrates that Bcl-x(L) signaling could be a possible pharmacological target of SMA therapeutics.

Survival motor neuron protein reduction deregulates autophagy in spinal cord motoneurons in vitro

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.

SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA.

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.

The Canonical Nuclear Factor-κB Pathway Regulates Cell Survival in a Developmental Model of Spinal Cord Motoneurons

In vivo and in vitro motoneuron 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-xL overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Intracellular calcium (Ca2+) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca2+ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K+) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca2+ increase. Depolarization‐induced toxicity and increased intracellular Ca2+ were blocked by treatment with antagonists to some of the voltage‐gated Ca2+ channels (VGCCs), indicating that Ca2+ influx through these channels contributed to the toxic effect of depolarization. Ca2+ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre‐treatment of MNs with calpeptin (a cell‐permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K+ concentration, which induces cell death by causing both intracellular Ca2+ increase and calpain activation.

Autophagy modulators regulate survival motor neuron protein stability in motoneurons

Abstract 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.

Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy

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.

Notch signaling pathway is activated in motoneurons of spinal muscular atrophy

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.

Smn-Deficiency Increases the Intrinsic Excitability of Motoneurons

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.

Regulation of Survival Motor Neuron Protein by the Nuclear Factor-Kappa B Pathway in Mouse Spinal Cord Motoneurons

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.