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.

Tumor necrosis factor alpha and interferon gamma cooperatively induce oxidative stress and motoneuron death in rat spinal cord embryonic explants.

The accumulation of reactive microglia in the degenerating areas of amyotrophic lateral sclerosis (ALS) tissue is a key cellular event creating a chronic inflammatory environment that results in motoneuron death. We have developed a new culture system that consists in rat spinal cord embryonic explants in which motoneurons migrate outside the explant, growing as a monolayer in the presence of glial cells. The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) have been proposed to be involved in ALS-linked microglial activation. In our explants, the combined exposure to these cytokines resulted in an increased expression of the pro-oxidative enzymes inducible nitric oxide synthase (iNOS), the catalytic subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, gp91(phox) and cyclooxygenase-2 (COX-2), as compared to each cytokine alone. This effect was related to their cooperation in the activation of the transcription factor nuclear factor kappa B (NF-kappaB). TNF-alpha and IFN-gamma also cooperated to promote protein oxidation and nitration, thus increasing the percentage of motoneurons immunoreactive for nitrotyrosine. Apoptotic motoneuron death, measured through annexin V-Cy3 and active caspase-3 immunoreactivities, was also found cooperatively induced by TNF-alpha and IFN-gamma. Interestingly, these cytokines did not affect the viability of purified spinal cord motoneurons in the absence of glial cells. It is proposed that the proinflammatory cytokines TNF-alpha and IFN-gamma have cooperative/complementary roles in inflammation-induced motoneuron death.

Pyruvate protects cerebellar granular cells from 6-hydroxydopamine-induced cytotoxicity by activating the Akt signaling pathway and increasing glutathione peroxidase expression.

Parkinson disease (PD) is the second-most common age-related neurodegenerative disease and is characterized by the selective destruction of dopaminergic neurons. Increasing evidence indicates that oxidative stress plays a crucial role in the pathogenesis of idiopathic PD. Anti-oxidant agents including catalase, manganese porphyrin and pyruvate confer cytoprotection to different cell cultures when challenged with 6-hydroxydopamine (6-OHDA). Herein we used rat cerebellar granular cell cultures to ascertain the plausible cellular pathways involved in pyruvate-induced cytoprotection against 0.1 mM 6-OHDA. Pyruvate provided cytoprotection in a concentration-dependent manner (2-10 mM). Consistent with its well-established anti-oxidant capacity, pyruvate (10 mM) prevented 6-OHDA-induced lipid peroxidation by blocking the rise in intracellular peroxides and maintaining the intracellular reduced glutathione (GSH) levels. Further experiments revealed that pyruvate increased Akt, but not extracellular signal-regulated kinase phosphorylation. Moreover, phosphatidylinositol 3-kinase (PI3K) inhibitors attenuated pyruvate-induced cytoprotection indicating that PI3K-mediated Akt activation is necessary for pyruvate to induce cytoprotection. On the other hand, pyruvate also up-regulated glutathione peroxidase mRNA levels, but not those of the anti-oxidant enzymes superoxide dismutase-1 and -2, catalase or the anti-apoptotic oncogenes Bcl-2 or Bcl-xL. In summary, our results strongly suggest that pyruvate, besides the anti-oxidant properties related to its structure, exerts cytoprotective actions by activating different anti-apoptotic routes that include gene regulation and Akt pathway activation.

Cytokines promote motoneuron survival through the Janus kinase-dependent activation of the phosphatidylinositol 3-kinase pathway.

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.

Neuronal survival induced by neurotrophins requires calmodulin.

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.

Nerve growth factor activation of the extracellular signal-regulated kinase pathway is modulated by Ca(2+) and calmodulin.

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.

Receptors of the glial cell line-derived neurotrophic factor family of neurotrophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons.

The members of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors (GDNF, neurturin, persephin, and artemin) are able to promote in vivo and in vitro survival 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 alpha (GRFalpha) 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 GFRalpha-1, GFRalpha-2, and GFRalpha-4 in chicken motoneurons using in situ hybridization 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.

Activation of phosphatidylinositol 3-kinase, but not extracellular-regulated kinases, is necessary to mediate brain-derived neurotrophic factor-induced motoneuron survival.

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 down-stream 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.

Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord.

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 vitro and in vivo studies 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 neurotrophin in 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 between in vivo and in vitro studies on the ability of chicken MTNs to respond to some members of the neurotrophin family of trophic factors.

Calmodulin is involved in membrane depolarization-mediated survival of motoneurons by phosphatidylinositol-3 kinase- and MAPK-independent pathways.

In the present work, we find that the elevation of extracellular K+ concentration promotes the survival of chick spinal cord motoneurons in vitro deprived 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.

The carbohydrate N-acetylglucosamine is involved in the guidance of neurites from chick ciliary ganglion neurons through the extracellular matrix of rat skeletal muscle fiber.

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.

S-laminin and N-acetylgalactosamine located at the synaptic basal lamina of skeletal muscle are involved in synaptic recognition by growing neurites.

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.