Disrupted in schizophrenia 1 (DISC1) is a constituent of the mammalian mitochondrial contact site and cristae organizing system (MICOS) complex, and is essential for oxidative phosphorylation.

Disrupted in Schizophrenia-1 (DISC1) has been associated with a broad spectrum of mental disorders. DISC1 is a multi-compartmentalized protein found in the cytoplasm, centrosome, nuclei and mostly enriched in mitochondria. In order to shed light on DISC1 mitochondrial function, we have studied its topology within the organelle. We show in here that in mammals DISC1 resides in the 'Mitochondrial contact site and Cristae Organizing system' (MICOS) complex, involved in cristae organization. DISC1 knockdown in SH-SY5Y cells causes MICOS disassembly and fragmentation of the mitochondrial morphology network. Moreover, DISC1 depleted cells have decreased mitochondrial DNA (mtDNA) content and steady state levels of oxidative phosphorylation (OXPHOS) subunits. As a consequence, OXPHOS complexes and supercomplexes are partially disassembled in DISC1 knockdown cells, which suffer severe bioenergetic defects, evidenced by impaired oxygen consumption, adenosine triphosphate synthesis and mitochondrial membrane potential. Transfection of recombinant full-length human DISC1 restores MICOS complex assembly and rescues OXPHOS function, meanwhile overexpression of the DISC1 truncated form Δ597-854, known to be pathogenic, fails to rescue the bioenergetic impairment caused by DISC1 knockdown. These results should contribute to reveal DISC1 physiological function and potential pathogenic role in severe mental illnesses.

Tumor necrosis factor alpha: a link between neuroinflammation and excitotoxicity.

Tumor necrosis factor alpha (TNF- α) is a proinflammatory cytokine that exerts both homeostatic and pathophysiological roles in the central nervous system. In pathological conditions, microglia release large amounts of TNF-α; this de novo production of TNF-α is an important component of the so-called neuroinflammatory response that is associated with several neurological disorders. In addition, TNF-α can potentiate glutamate-mediated cytotoxicity by two complementary mechanisms: indirectly, by inhibiting glutamate transport on astrocytes, and directly, by rapidly triggering the surface expression of Ca(+2) permeable-AMPA receptors and NMDA receptors, while decreasing inhibitory GABAA receptors on neurons. Thus, the net effect of TNF-α is to alter the balance of excitation and inhibition resulting in a higher synaptic excitatory/inhibitory ratio. This review summarizes the current knowledge of the cellular and molecular mechanisms by which TNF-α links the neuroinflammatory and excitotoxic processes that occur in several neurodegenerative diseases, but with a special emphasis on amyotrophic lateral sclerosis (ALS). As microglial activation and upregulation of TNF-α expression is a common feature of several CNS diseases, as well as chronic opioid exposure and neuropathic pain, modulating TNF-α signaling may represent a valuable target for intervention.

Role of PATJ in stroke prognosis by modulating endothelial to mesenchymal transition through the Hippo/Notch/PI3K axis.

Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient's blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4-5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, ß-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.

Neuroprotective effects of estradiol on motoneurons in a model of rat spinal cord embryonic explants.

Amyotrophic lateral sclerosis (ALS) is an adult-onset degenerative disorder characterized by motoneuron death. Clinical and experimental studies in animal models of ALS have found gender differences in the incidence and onset of disease, suggesting that female hormones may play a beneficial role. Cumulative evidence indicates that 17ß-estradiol (17ßE2) has a neuroprotective role in the central nervous system. We have previously developed a new culture system by using rat spinal cord embryonic explants in which motoneurons have the singularity of migrating outside the spinal cord, growing as a monolayer in the presence of glial cells. In this study, we have validated this new culture system as a useful model for studying neuroprotection by estrogens on spinal cord motoneurons. We show for the first time that spinal cord motoneurons express classical estrogen receptors and that 17ßE2 activates, specifically in these cells, the Akt anti-apoptotic signaling pathway and two of their downstream effectors: GSK-3ß and Bcl-2. To further validate our system, we demonstrated neuroprotective effects of 17ßE2 on spinal cord motoneurons when exposed to the proinflammatory cytokines TNF-α and IFN-γ. These effects of 17ßE2 were fully reverted in the presence of the estrogen receptor antagonist ICI 182,780. Our new culture model and the results presented here may provide the basis for further studies on the effects of estrogens, and selective estrogen receptor modulators, on spinal cord motoneurons in the context of ALS or other motoneuron diseases.

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.

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.

TNF-α potentiates glutamate-induced spinal cord motoneuron death via NF-κB.

Besides glutamate excitotoxicity, the neuroinflammatory response is emerging as a relevant contributor to motoneuron loss in amyotrophic lateral sclerosis (ALS). In this regard, high levels of circulating proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) have been shown both in human patients and in animal models of ALS. The aim of this work was to study the effects of TNF-α on glutamate-induced excitotoxicity in spinal cord motoneurons. In rat spinal cord organotypic cultures chronic glutamate excitotoxicity, induced by the glutamate-uptake inhibitor threohydroxyaspartate (THA), resulted in motoneuron loss that was associated with a neuroinflammatory response. In the presence of TNF-α, THA-induced excitotoxic motoneuron death was potentiated. Co-exposure to TNF-α and THA also resulted in down-regulation of the astroglial glutamate transporter 1 (GLT-1) and in increased extracellular glutamate levels, which were prevented by nuclear factor-kappaB (NF-κB) inhibition. Furthermore, TNF-α and THA also cooperated in the induction of oxidative stress in a mechanism involving the NF-κB signalling pathway as well. The inhibition of this pathway abrogated the exacerbation of glutamate-mediated motoneuron death induced by TNF-α. These data link two important pathogenic mechanisms, excitotoxicity and neuroinflammation, suggested to play a role in ALS and, to our knowledge, this is the first time that TNF-α-induced NF-κB activation has been reported to potentiate glutamate excitotoxicity on motononeurons.

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.

Cell stress induces TDP-43 pathological changes associated with ERK1/2 dysfunction: implications in ALS.

TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis and other neurodegenerative diseases. Here we demonstrate, using neuronal and spinal cord organotypic culture models, that chronic excitotoxicity, oxidative stress, proteasome dysfunction and endoplasmic reticulum stress mechanistically induce mislocalization, phosphorylation and aggregation of TDP-43. This is compatible with a lack of function of this protein in the nucleus, specially in motor neurons. The relationship between cell stress and pathological changes of TDP-43 also includes a dysfunction in the survival pathway mediated by mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK1/2). Thus, under stress conditions, neurons and other spinal cord cells showed cytosolic aggregates containing ERK1/2. Moreover, aggregates of abnormal phosphorylated ERK1/2 were also found in the spinal cord in amyotrophic lateral sclerosis (ALS), specifically in motor neurons with abnormal immunoreactive aggregates of phosphorylated TDP-43. These results demonstrate that cellular stressors are key factors in neurodegeneration associated with TDP-43 and disclose the identity of ERK1/2 as novel players in the pathogenesis of ALS.

Neurochemical and Cognitive Beneficial Effects of Moderate Physical Activity and Catechin in Aged Rats.

A healthy aging process is a requirement for good life quality. A relationship between physical activity, the consumption of antioxidants and brain health has been stablished via the activation of pathways that reduce the harmful effects of oxidative stress, by inducing enzymes such as SIRT1, which is a protector of brain function. We analyzed the cognitive and neurochemical effects of applying physical exercise in elderly rats, alone or in combination with the antioxidant catechin. Several tests of spatial and episodic memory and motor coordination were evaluated. In addition, brain monoaminergic neurotransmitters and SIRT1 protein levels were assessed in the brains of the same rats. The results show that physical activity by itself improved age-related memory and learning deficits, correlating with the restoration of brain monoaminergic neurotransmitters and SIRT1 protein levels in the hippocampus. The administration of the antioxidant catechin along with the exercise program enhanced further the monoaminergic pathways, but not the other parameters studied. These results agree with previous reports revealing a neuroprotective effect of physical activity, probably based on its ability to improve the redox status of the brain, demonstrating that exercise at an advanced age, combined with the consumption of antioxidants, could produce favorable effects in terms of brain health.

The conserved ASTN2/BRINP1 locus at 9q33.1-33.2 is associated with major psychiatric disorders in a large pedigree from Southern Spain.

We investigated the genetic causes of major mental disorders (MMDs) including schizophrenia, bipolar disorder I, major depressive disorder and attention deficit hyperactive disorder, in a large family pedigree from Alpujarras, South of Spain, a region with high prevalence of psychotic disorders. We applied a systematic genomic approach based on karyotyping (n = 4), genotyping by genome-wide SNP array (n = 34) and whole-genome sequencing (n = 12). We performed genome-wide linkage analysis, family-based association analysis and polygenic risk score estimates. Significant linkage was obtained at chromosome 9 (9q33.1-33.2, LOD score = 4.11), a suggestive region that contains five candidate genes ASTN2, BRINP1, C5, TLR4 and TRIM32, previously associated with MMDs. Comprehensive analysis associated the MMD phenotype with genes of the immune system with dual brain functions. Moreover, the psychotic phenotype was enriched for genes involved in synapsis. These results should be considered once studying the genetics of psychiatric disorders in other families, especially the ones from the same region, since founder effects may be related to the high prevalence.

Familial Psychosis Associated With a Missense Mutation at MACF1 Gene Combined With the Rare Duplications DUP3p26.3 and DUP16q23.3, Affecting the CNTN6 and CDH13 Genes.

Psychosis is a highly heritable and heterogeneous psychiatric condition. Its genetic architecture is thought to be the result of the joint effect of common and rare variants. Families with high prevalence are an interesting approach to shed light on the rare variant's contribution without the need of collecting large cohorts. To unravel the genomic architecture of a family enriched for psychosis, with four affected individuals, we applied a system genomic approach based on karyotyping, genotyping by whole-exome sequencing to search for rare single nucleotide variants (SNVs) and SNP array to search for copy-number variants (CNVs). We identified a rare non-synonymous variant, g.39914279 C > G, in the MACF1 gene, segregating with psychosis. Rare variants in the MACF1 gene have been previously detected in SCZ patients. Besides, two rare CNVs, DUP3p26.3 and DUP16q23.3, were also identified in the family affecting relevant genes (CNTN6 and CDH13, respectively). We hypothesize that the co-segregation of these duplications with the rare variant g.39914279 C > G of MACF1 gene precipitated with schizophrenia and schizoaffective disorder.

Cognitive and Neurochemical Changes Following Polyphenol-Enriched Diet in Rats.

Dietary recommendations are frequently developed based on nutrient deficiency or prevention of disease, but less attention has been paid to the dietary guidelines to promote brain health. Active and healthy aging is a prerequisite for improving quality of life as people age, and evidence is establishing a relationship between diet and brain health. This work studied the effect of a diet based on foods rich in antioxidants, especially polyphenols, in rats, three days a week for 20 months starting at 14 months. Behavioral analysis testing working memory, spatial and episodic memory, as well as brain monoaminergic neurotransmitters involved in these processes but also in general brain health were analyzed. In addition, hippocampal SIRT1 protein which has an important role in regulating normal brain function was evaluated. The results show that long-term intake of polyphenol-enriched diet improves memory and learning, correlating with restoration of brain monoaminergic neurotransmitters and hippocampal SIRT1 levels in aged rats. These results agree with reports revealing a neuroprotective effect of different polyphenolic compounds on age-related brain decline, based on its antioxidant and anti-inflammatory properties; and demonstrate that consumption of antioxidant-rich foods, a few days a week, gives good long-term results in terms of brain health.

Vascular endothelial growth factor protects spinal cord motoneurons against glutamate-induced excitotoxicity via phosphatidylinositol 3-kinase.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motoneurons. Recently, vascular endothelial growth factor (VEGF) has been identified as a neurotrophic factor and has been implicated in the mechanisms of pathogenesis of ALS and other neurological diseases. The potential neuroprotective effects of VEGF in a rat spinal cord organotypic culture were studied in a model of chronic glutamate excitotoxicity in which glutamate transporters are inhibited by threohydroxyaspartate (THA). Particularly, we focused on the effects of VEGF in the survival and vulnerability to excitotoxicity of spinal cord motoneurons. VEGF receptor-2 was present on spinal cord neurons, including motoneurons. Chronic (3 weeks) treatment with THA induced a significant loss of motoneurons that was inhibited by co-exposure to VEGF (50 ng/mL). VEGF activated the phosphatidylinositol 3-kinase/Akt (PI3-K/Akt) signal transduction pathway in the spinal cord cultures, and the effect on motoneuron survival was fully reversed by the specific PI3-K inhibitor, LY294002. VEGF also prevented the down-regulation of Bcl-2 and survivin, two proteins implicated in anti-apoptotic and/or anti-excitotoxic effects, after THA exposure. Together, these findings indicate that VEGF has neuroprotective effects in rat spinal cord against chronic glutamate excitotoxicity by activating the PI3-K/Akt signal transduction pathway and also reinforce the hypothesis of the potential therapeutic effects of VEGF in the prevention of motoneuron degeneration in human ALS.

Complementary roles of tumor necrosis factor alpha and interferon gamma in inducible microglial nitric oxide generation.

Proinflammatory cytokines and pathogen components activate microglia to release several substances such as nitric oxide (NO) produced after the induction of type II nitric oxide synthase (iNOS). The present study was designed to elucidate the interaction between the proinflammatory cytokines interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) on iNOS expression and NO production in microglial cells. In primary mouse microglial cells exposure to IFN-gamma (5 and 10 ng/ml; 48 h) or TNF-alpha (20 ng/ml; 48 h) alone were unable to induce iNOS expression; however, when cells were exposed to both cytokines together, the expression of this enzyme and the NO production in culture media were found significantly increased. In the BV-2 microglial cell line, IFN-gamma and TNF-alpha were shown to cooperate in nuclear factor kappa B (NF-kappa B) activation, an essential transcription factor for iNOS gene transcription. Importantly, IFN-gamma induced NF-kappa B binding to DNA was totally dependent on the endogenous TNF-alpha released via MEK/ERK signalling pathway. Thus, exposure of BV-2 cells to IFN-gamma in the presence of the selective MEK inhibitor U0126 or a neutralizing anti-TNF-alpha antibody significantly reduced IFN-gamma dependent NF-kappa B activation and iNOs expression. In addition, by activating the Jak/STAT pathway IFN-gamma potentiated TNF-alpha induced NF-kappa B binding to DNA and activated additional transcription factors (i.e. IRF-1) known to be essential for iNOs gene expression. The present findings demonstrate that the proinflammatory cytokines IFN-gamma and TNF-alpha have complementary roles on iNOS expression in microglial cells and this might be relevant to understand the molecular mechanisms of microglial activation associated with the pathogenesis of several neuroinflammatory disorders in which increased levels of IFN-gamma and TNF-alpha have been reported.

IFN-gamma prevents TNF-alpha-induced apoptosis in C2C12 myotubes through down-regulation of TNF-R2 and increased NF-kappaB activity.

Wasting of skeletal muscle (cachexia) is associated with a variety of chronic or inflammatory disorders and has long been recognized as a poor prognostic sign. It is currently accepted that the cytokine tumor necrosis factor alpha (TNF-alpha; cachectin) plays a key role in the development of this condition. TNF-alpha-induced apoptotic cell death represents a potential mechanism by which muscle wasting can occur. Evidence has accumulated that the cytokine interferon gamma (IFN-gamma) may act as a modulator of TNF-alpha signalling. Thus, the present study was designed to elucidate if TNF-alpha can directly induce apoptosis in differentiated myotubes, to assess the potential anti-apoptotic properties of IFN-gamma and to get insight into the signalling pathways implicated in the modulatory effects of IFN-gamma. Myoblasts of the murine cell line C2C12 were allowed to differentiate in a low serum containing media and myogenesis assessed by muscle specific protein expression. Non-proliferating, polynucleated, fully differentiated myotubes were obtained after seven days in differentiation media. Exposure of C2C12 myotubes to TNF-alpha for 48 h induced apoptosis characterized by enhanced caspase-3 activity, which resulted in poly(ADP-ribose) polymerase (PARP) cleavage and increased histone-associated-DNA fragmentation. These effects were fully reverted in the presence of IFN-gamma. This cytokine induced down-regulation of the subtype 2 of TNF-alpha receptors (TNF-R2), enhanced TNF-alpha-induced NF-kappaB translocation to the nucleus and binding to DNA and increased the immunoreactivity of the protein c-IAP1, a member of the inhibitor of apoptosis (IAP) gene family whose synthesis is stimulated by NF-kappaB at the transcriptional level. Together, these results demonstrate that TNF-alpha directly induces apoptosis in differentiated myotubes and suggest that the cytokine IFN-gamma, might represent a new immunoadjuvant therapeutic tool for managing cachexia.

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Therapeutic developments in the treatment of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a progressive neurodegenerative disease characterised by the selective death of motor neurones. The mechanisms and processes responsible for the selective loss of motor neurones are still unknown, however several hypotheses have been put forward, including oxidative damage and/or toxicity from intracellular aggregates due to mutant superoxide dismutase-1 activity, axonal strangulation from cytoskeletal abnormalities, loss of trophic factor support and glutamate-mediated excitotoxicity. These theories are based on a better understanding of the genetics of amyotrophic lateral sclerosis and on biochemical and pathological analysis of post-mortem tissue. They have led to the development of appropriate animal and cell culture models, allowing the sequence of events in motor neuronal degeneration to be unravelled and potential therapeutic agents to be screened. Unfortunately, the majority of therapeutics found to be efficacious in the animal and cell culture models have failed in human trials. Riluzole is still the only proven therapy in humans, shown to extend survival of amyotrophic lateral sclerosis patients by approximately 3 months, but it has no effect on muscle strength. Other potential therapeutic approaches are being identified, including inhibition of caspase-mediated cell death, maintenance of mitochondrial integrity and energy production, regulation of glutamate homeostasis, reduction of inflammation and control of neurofilament synthesis. Hopefully, in the near future some new agents will be found that can alter the course of this devastating and fatal disease.

Mechanisms involved in spinal cord central synapse loss in a mouse model of spinal muscular atrophy.

Motoneuron (MN) cell death is the histopathologic hallmark of spinal muscular atrophy (SMA), although MN loss seems to be a late event. Conversely, disruption of afferent synapses on MNs has been shown to occur early in SMA. Using a mouse model of severe SMA (SMNΔ7), we examined the mechanisms involved in impairment of central synapses. We found that MNs underwent progressive degeneration in the course of SMA, with MN loss still occurring at late stages. Loss of afferent inputs to SMA MNs was detected at embryonic stages, long before MN death. Reactive microgliosis and astrogliosis were present in the spinal cord of diseased animals after the onset of MN loss. Ultrastructural observations indicate that dendrites and microglia phagocytose adjacent degenerating presynaptic terminals. Neuronal nitric oxide synthase was upregulated in SMNΔ7 MNs, and there was an increase in phosphorylated myosin light chain expression in synaptic afferents on MNs; these observations implicate nitric oxide in MN deafferentation and suggest that the RhoA/ROCK pathway is activated. Together, our observations suggest that the earliest change occurring in SMNΔ7 mice is the loss of excitatory glutamatergic synaptic inputs to MNs; reduced excitability may enhance their vulnerability to degeneration and death.

Cellular and molecular mechanisms involved in the neuroprotective effects of VEGF on motoneurons.

Vascular endothelial growth factor (VEGF), originally described as a factor with a regulatory role in vascular growth and development, it is also known for its direct effects on neuronal cells. The discovery in the past decade that transgenic mice expressing reduced levels of VEGF developed late-onset motoneuron pathology, reminiscent of amyotrophic lateral sclerosis (ALS), opened a new field of research on this disease. VEGF has been shown to protect motoneurons from excitotoxic death, which is a relevant mechanism involved in motoneuron degeneration in ALS. Thus, VEGF delays motoneuron degeneration and increases survival in animal models of ALS. VEGF exerts its anti-excitotoxic effects on motoneurons through molecular mechanisms involving the VEGF receptor-2 resulting in the activation of the PI3-K/Akt signaling pathway, upregulation of GluR2 subunit of AMPA receptors, inhibition of p38MAPK, and induction of the anti-apoptotic molecule Bcl-2. In addition, VEGF acts on astrocytes to reduce astroglial activation and to induce the release of growth factors. The potential use of VEGF as a therapeutic tool in ALS is counteracted by its vascular effects and by its short effective time frame. More studies are needed to assess the optimal isoform, route of administration, and time frame for using VEGF in the treatment of ALS.