Jose-Luis Gonzalez de Aguilar

Sodium Valproate Exerts Neuroprotective Effects In Vivo through CREB-Binding Protein-Dependent Mechanisms But Does Not Improve Survival in an Amyotrophic Lateral Sclerosis Mouse Model

Amyotrophic lateral sclerosis (ALS) is characterized by motoneuron (MN) degeneration, generalized weakness, and muscle atrophy. The premature death of MNs is thought to be a determinant in the onset of this disease. In a transgenic mouse model of ALS expressing the G86R mutant superoxide dismutase 1 (mSOD1), we demonstrated previously that CREB (cAMP response element-binding protein)-binding protein (CBP) and histone acetylation levels were specifically decreased in nuclei of degenerating MNs. We show here that oxidative stress and mSOD1 overexpression can both impinge on CBP levels by transcriptional repression, in an MN-derived cell line. Histone deacetylase inhibitor (HDACi) treatment was able to reset proper acetylation levels and displayed an efficient neuroprotective capacity against oxidative stress in vitro. Interestingly, HDACi also upregulated CBP transcriptional expression in MNs. Moreover, when injected to G86R mice in vivo, the HDACi sodium valproate (VPA) maintained normal acetylation levels in the spinal cord, efficiently restored CBP levels in MNs, and significantly prevented MN death in these animals. However, despite neuroprotection, mean survival of treated animals was not significantly improved (<5%), and they died presenting the classical ALS symptoms. VPA was not able to prevent disruption of neuromuscular junctions, although it slightly delayed the onset of motor decline and retarded muscular atrophy to some extent. Together, these data show that neuroprotection can improve disease onset, but clearly provide evidence that one can uncouple MN survival from whole-animal survival and point to the neuromuscular junction perturbation as a primary event of ALS onset.

Nogo Provides a Molecular Marker for Diagnosis of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by the selective degeneration of upper and lower motor neurons. The lack of a molecular diagnostic marker is of increasing concern in view of the therapeutic strategies in development. Using an unbiased subtractive suppressive hybridization screen we have identified a clone encoding the neurite outgrowth inhibitor Nogo and shown that its isoforms display a characteristic altered expression in ALS. This was first confirmed by analyzing Nogo isoform expression in a transgenic ALS model at early asymptomatic stages where we found increased levels of Nogo-A and decreased Nogo-C and importantly, not following experimentally induced denervation. Furthermore, we confirmed these changes in both post-mortem and biopsy samples from diagnosed ALS patients but not control patients. Thus, the alteration in Nogo expression pattern, common to sporadic and familial ALS, represents a potential diagnosis tool and points strongly to Nogo having a central role in disease.

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

Background Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS. Methodology/Principal Findings We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model. Conclusions/Significance These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases.

Nogo expression in muscle correlates with amyotrophic lateral sclerosis severity

Nogo, a protein inhibiting axonal regeneration, exhibits a characteristic isoform‐specific pattern of expression in skeletal muscle of transgenic mice and patients with amyotrophic lateral sclerosis. Here, the increased levels of Nogo‐A or Nogo‐B in muscle biopsies of 15 amyotrophic lateral sclerosis patients significantly correlated with the severity of clinical disability and with the degree of muscle fiber atrophy. Nogo‐A immunoreactivity was observed selectively in atrophic slow‐twitch type I fibers. These results suggest that Nogo expression in muscle is a marker of amyotrophic lateral sclerosis severity. Ann Neurol 2005;57:553–556

Increased peripheral lipid clearance in an animal model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common adult motor neuron disease, causing motor neuron degeneration, muscle atrophy, paralysis, and death. Despite this degenerative process, a stable hypermetabolic state has been observed in a large subset of patients. Mice expressing a mutant form of Cu/Zn-superoxide dismutase (mSOD1 mice) constitute an animal model of ALS that, like patients, exhibits unexpectedly increased energy expenditure. Counterbalancing for this increase with a high-fat diet extends lifespan and prevents motor neuron loss. Here, we investigated whether lipid metabolism is defective in this animal model. Hepatic lipid metabolism was roughly normal, whereas gastrointestinal absorption of lipids as well as peripheral clearance of triglyceride-rich lipoproteins were markedly increased, leading to decreased postprandial lipidemia. This defect was corrected by the high-fat regimen that typically induces neuroprotection in these animals. Together, our findings show that energy metabolism in mSOD1 mice shifts toward an increase in the peripheral use of lipids. This metabolic shift probably accounts for the protective effect of dietary lipids in this model.

Loss of prion protein in a transgenic model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disorder caused in a proportion of cases by missense mutations in the gene encoding Cu/Zn superoxide dismutase (Cu/Zn-SOD) which result in unknown, lethal enzymatic activity. Based on a differential screening approach, we show here that the gene encoding the cellular prion protein (PrP(C)) was specifically repressed in a transgenic model of ALS overexpressing the mutant G86R Cu/Zn-SOD. Analysis by Northern blot, semiquantitative RT-PCR, and Western blot revealed that PrP(C) down-regulation, which appeared early in the asymptomatic phase of the pathology, occurred preferentially in those tissues primarily affected by the disease (spinal cord, sciatic nerve, and gastrocnemius muscle). This down-regulation was not accompanied by refolding of the aberrant PrP(Sc) isoform, the agent which causes transmissible spongiform encephalopathies. Furthermore, modification of PrP(C) expression was specifically linked to the presence of the G86R mutant since no changes were observed in transgenic mice overexpressing wild-type Cu/Zn-SOD. PrP(C) has been shown to play a role in the protection against oxidative stress, and we therefore propose that its down-regulation may contribute at least in part to ALS pathogenesis.

Cyclohexenonic Long-Chain Fatty Alcohols as Neuronal Growth Stimulators

Neurotrophic factors play an important role in the development and maintenance of neurons, thus providing a suitable therapeutic approach for the treatment of neurodegenerative diseases. However, their clinical use has revealed problematic because of a number of technical and biological disadvantages. Among the different strategies proposed to overcome such difficulties, the search for non-peptide substances with neurotrophic potential is giving promising results. Here we will expose major findings in this field, drawing special attention to cyclohexenonic long-chain fatty alcohols, a novel family of compounds that promote neuronal survival and neurite outgrowth.

The Role of Skeletal Muscle in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal adult‐onset disease primarily characterized by upper and lower motor neuron degeneration, muscle wasting and paralysis. It is increasingly accepted that the pathological process leading to ALS is the result of multiple disease mechanisms that operate within motor neurons and other cell types both inside and outside the central nervous system. The implication of skeletal muscle has been the subject of a number of studies conducted on patients and related animal models. In this review, we describe the features of ALS muscle pathology and discuss on the contribution of muscle to the pathological process. We also give an overview of the therapeutic strategies proposed to alleviate muscle pathology or to deliver curative agents to motor neurons. ALS muscle mainly suffers from oxidative stress, mitochondrial dysfunction and bioenergetic disturbances. However, the way by which the disease affects different types of myofibers depends on their contractile and metabolic features. Although the implication of muscle in nourishing the degenerative process is still debated, there is compelling evidence suggesting that it may play a critical role. Detailed understanding of the muscle pathology in ALS could, therefore, lead to the identification of new therapeutic targets.

Degeneration of serotonergic neurons in amyotrophic lateral sclerosis: a link to spasticity

Spasticity is a common and disabling symptom observed in patients with central nervous system diseases, including amyotrophic lateral sclerosis, a disease affecting both upper and lower motor neurons. In amyotrophic lateral sclerosis, spasticity is traditionally thought to be the result of degeneration of the upper motor neurons in the cerebral cortex, although degeneration of other neuronal types, in particular serotonergic neurons, might also represent a cause of spasticity. We performed a pathology study in seven patients with amyotrophic lateral sclerosis and six control subjects and observed that central serotonergic neurons suffer from a degenerative process with prominent neuritic degeneration, and sometimes loss of cell bodies in patients with amyotrophic lateral sclerosis. Moreover, distal serotonergic projections to spinal cord motor neurons and hippocampus systematically degenerated in patients with amyotrophic lateral sclerosis. In SOD1 (G86R) mice, a transgenic model of amyotrophic lateral sclerosis, serotonin levels were decreased in brainstem and spinal cord before onset of motor symptoms. Furthermore, there was noticeable atrophy of serotonin neuronal cell bodies along with neuritic degeneration at disease onset. We hypothesized that degeneration of serotonergic neurons could underlie spasticity in amyotrophic lateral sclerosis and investigated this hypothesis in vivo using tail muscle spastic-like contractions in response to mechanical stimulation as a measure of spasticity. In SOD1 (G86R) mice, tail muscle spastic-like contractions were observed at end-stage. Importantly, they were abolished by 5-hydroxytryptamine-2b/c receptors inverse agonists. In line with this, 5-hydroxytryptamine-2b receptor expression was strongly increased at disease onset. In all, we show that serotonergic neurons degenerate during amyotrophic lateral sclerosis, and that this might underlie spasticity in mice. Further research is needed to determine whether inverse agonists of 5-hydroxytryptamine-2b/c receptors could be of interest in treating spasticity in patients with amyotrophic lateral sclerosis.

Oxidative stress in skeletal muscle stimulates early expression of Rad in a mouse model of amyotrophic lateral sclerosis.

Motor neuron degeneration and progressive muscle atrophy characterize amyotrophic lateral sclerosis (ALS) in humans and related mutant superoxide dismutase-1 (SOD1) transgenic mice. Our previous microarray studies on ALS muscle revealed strong up-regulation of Ras-related associated with diabetes (Rad), an inhibitor of voltage-gated calcium channels. The mechanisms controlling Rad expression in disease are unknown. We analyzed Rad expression in skeletal muscle from ALS patients and animal models and investigated whether it is regulated by oxidative stress. In mutant SOD1 mice, Rad up-regulation preceded motor symptoms and markedly increased as disease progressed. Increased Rad expression was also obtained in surgically denervated muscle. No clinical signs of denervation were seen in asymptomatic mice, however. We therefore suspected that muscular mutant SOD1 toxicity causes precocious Rad up-regulation. We confirmed the accumulation of reactive oxygen species (ROS) at asymptomatic stages, coincident with the rise in Rad expression. By subjecting muscle to ischemia-reperfusion, we observed ROS accumulation and Rad overexpression. The cell-permeative antioxidant Tempol inhibited the stimulatory effect of ischemia-reperfusion. Tempol also reduced Rad up-regulation after experimental denervation. Our study provides strong evidence for the implication of oxidative stress in modulating Rad expression, in association with the initiation and progression of ALS muscle atrophy.