S. Petri

Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice.

Mitochondrial respiratory chain dysfunction, impaired intracellular Ca2+ homeostasis and activation of the mitochondrial apoptotic pathway are pathological hallmarks in animal and cellular models of familial amyotrophic lateral sclerosis associated with Cu/Zn‐superoxide dismutase mutations. Although intracellular Ca2+ homeostasis is thought to be intimately associated with mitochondrial functions, the temporal and causal correlation between mitochondrial Ca2+ uptake dysfunction and motor neuron death in familial amyotrophic lateral sclerosis remains to be established. We investigated mitochondrial Ca2+ handling in isolated brain, spinal cord and liver of mutant Cu/Zn‐superoxide dismutase transgenic mice at different disease stages. In G93A mutant transgenic mice, we found a significant decrease in mitochondrial Ca2+ loading capacity in brain and spinal cord, as compared with age‐matched controls, very early on in the course of the disease, long before the onset of motor weakness and massive neuronal death. Ca2+ loading capacity was not significantly changed in liver G93A mitochondria. We also confirmed Ca2+ capacity impairment in spinal cord mitochondria from a different line of mice expressing G85R mutant Cu/Zn‐superoxide dismutase. In excitable cells, such as motor neurons, mitochondria play an important role in handling rapid cytosolic Ca2+ transients. Thus, mitochondrial dysfunction and Ca2+‐mediated excitotoxicity are likely to be interconnected mechanisms that contribute to neuronal degeneration in familial amyotrophic lateral sclerosis.

Celastrol blocks neuronal cell death and extends life in transgenic mouse model of amyotrophic lateral sclerosis.

There is substantial evidence that both inflammation and oxidative damage contribute to the pathogenesis of motor neuron degeneration in the G93A SOD1 transgenic mouse model of amyotrophic lateral sclerosis (ALS). Celastrol is a natural product from Southern China, which exerts potent anti-inflammatory and antioxidative effects. It also acts potently to increase expression of heat shock proteins including HSP70. We administered it in the diet to G93A SOD1 mice starting at 30 days of age. Celastrol treatment significantly improved weight loss, motor performance and delayed the onset of ALS. Survival of celastrol-treated G93A mice increased by 9.4% and 13% for 2 mg/kg/day and 8 mg/kg/day doses, respectively. Cell counts of lumbar spinal cord neurons confirmed a protective effect, i.e. 30% increase in neuronal number in the lumbar spinal cords of celastrol-treated animals. Celastrol treatment reduced TNF-α, iNOS, CD40, and GFAP immunoreactivity in the lumbar spinal cord sections of celastrol-treated G93A mice compared to untreated G93A mice. TNF-α immunoreactivity co-localized with SMI-32 (neuronal marker) and GFAP (astrocyte marker). HSP70 immunoreactivity was increased in lumbar spinal cord neurons of celastrol-treated G93A mice. Celastrol has been widely used in treating inflammatory diseases in man, and is well tolerated; therefore, it may be a promising therapeutic candidate for the treatment of human ALS.

Additive neuroprotective effects of a histone deacetylase inhibitor and a catalytic antioxidant in a transgenic mouse model of amyotrophic lateral sclerosis

ALS is a devastating neurodegenerative disorder for which no effective treatment exists. Multiple molecular mechanisms are involved in the pathogenesis. We tested the catalytic antioxidant AEOL 10150, the histone deacetylase inhibitor phenylbutyrate (PBA), and the combination of PBA and AEOL 10150 in the G93A transgenic mouse model, administered from disease onset. AEOL 10150 alone improved motor function and extended survival by 11%, PBA alone significantly improved motor function and extended survival by 13%. PBA and AEOL 10150 together increased survival by 19%. Increased histone acetylation was confirmed by Western blot. Quantitative real-time RT-PCR analysis revealed upregulation of compounds capable of protecting cells against oxidative stress and apoptosis. Markers of oxidative damage were reduced in the lumbar spinal cord as compared to vehicle administration. These results suggest that agents inhibiting apoptosis and blocking oxidative stress show efficacy in treating mutant-SOD1-associated ALS and that a combination of agents targeting different disease mechanisms may exert additive therapeutic effects.

Integrative role of cPLA2 with COX‐2 and the effect of non‐steriodal anti‐inflammatory drugs in a transgenic mouse model of amyotrophic lateral sclerosis

Cyclooxygenase‐2 (COX‐2) is a key molecule in the inflammatory pathway in amyotrophic lateral sclerosis (ALS). Cytosolic phospholipase A (cPLA2) is an important enzyme providing substrate for cyclooxygenases. We therefore examined cPLA2 expression in human ALS and mutant Cu/Zn superoxide dismutase (SOD1) transgenic mice and its relation to COX‐2. Immunohistochemistry and real‐time RT‐PCR revealed elevated cPLA2 protein and its mRNA levels in the lumbar spinal cord of mutant SOD1 mice. COX‐2 immunoreactivity was increased in lumbar spinal cord sections from both familial ALS (FALS) and sporadic ALS (SALS) as compared to controls, and cPLA2 immunoreactivity was increased in a patient with FALS. Oral administration of the non‐selective cyclooxygenase (COX) inhibitor, sulindac, extended the survival (by 10%) of G93A SOD1 mice as compared to littermate controls. Sulindac, as well as the selective COX‐2 inhibitors, rofecoxib and celecoxib reduced cPLA2 immunoreactivity in the lumbar spinal cord of G93A transgenic mice. Sulindac treatment preserved motor neurons, and reduced microglial activation and astrocytosis, in the spinal cord of G93A SOD1 transgenic mice. These results suggest that cPLA2 plays an important role in supplying arachidonic acid to the COX‐2 driven inflammatory pathway in ALS associated with SOD1 mutations.

The lipophilic metal chelators DP-109 and DP-460 are neuroprotective in a transgenic mouse model of amyotrophic lateral sclerosis.

One of the hypotheses for the development of familial amyotrophic lateral sclerosis (ALS) is that mutations in the superoxide dismutase 1 enzyme lead to aberrant properties of the copper within the active site of the enzyme which then causes increased oxidative damage. The lipophilic metal chelators DP‐109 and DP‐460 which chelate calcium, copper, and zinc were tested in the G93A‐transgenic ALS mouse model. Both compounds significantly extended survival, DP‐109 (5 mg/kg/day) by 10%, DP‐460 (10 mg/kg/day) by 9%. While the effect on survival was relatively small, chelator treatment also improved motor performance, dramatically reduced cell loss in the lumbar spinal cord and decreased reactive astrocytosis and microgliosis. Markers of oxidative damage, tumor necrosis factor (TNF)‐αand α‐synuclein were reduced in the lumbar spinal cord of G93A mice treated with DP‐109 or DP‐460 as compared with vehicle‐treated animals. Furthermore, the treatment induced protein expression of the transcription factor hypoxia inducible factor‐1α and mRNA levels of vascular endothelial growth factor as a corresponding target gene. In line with previous studies using metal chelators in the G93A animal model, our results suggest that these compounds have neuroprotective capacities in ALS.

Loss of Fas ligand-function improves survival in G93A-transgenic ALS mice.

ALS is a devastating neurodegenerative disorder for which no effective treatment exists. The precise molecular mechanisms underlying the selective degeneration of motor neurons are still unknown. A motor neuron specific apoptotic pathway involving Fas and NO has been discovered. Motor neurons from ALS-mice have an increased sensitivity to Fas-induced cell death via this pathway. In this study we therefore crossed G93A-SOD1 overexpressing ALS mice with Fas ligand (FasL) mutant (gld) mice to investigate whether the reduced Fas signaling could have beneficial effects on motor neuron death. G93A-SOD1 mutant mice with a homozygous FasL mutant showed a modest but statistically significant extension of survival, and reduced loss of motor neurons. These results indicate that motor neuron apoptosis triggered by Fas is relevant in ALS pathogenesis.

The cellular mRNA expression of GABA and glutamate receptors in spinal motor neurons of SOD1 mice

ALS is a fatal neurodegenerative disorder characterized by a selective loss of upper motor neurons in the motor cortex and lower motor neurons in the brain stem and spinal cord. About 10% of ALS cases are familial, in 10-20% of these, mutations in the gene coding for superoxide dismutase 1 (SOD1) can be detected. Overexpression of mutated SOD1 in mice created animal models which clinically resemble ALS. Abnormalities in glutamatergic and GABAergic neurotransmission presumably contribute to the selective motor neuron damage in ALS. By in situ hybridization histochemistry (ISH), we investigated the spinal mRNA expression of the GABAA and AMPA type glutamate receptor subunits at different disease stages on spinal cord sections of mutant SOD1 mice and control animals overexpressing wild-type SOD1 aged 40, 80, 120 days and at disease end-stage, i.e. around 140 days) (n=5, respectively). We detected a slight but statistically significant decrease of the AMPA receptor subunits GluR3 and GluR4 only in end stage disease animals.

Cell-permeable peptide antioxidants as a novel therapeutic approach in a mouse model of amyotrophic lateral sclerosis

Reactive oxygen species (ROS) play a major role in the pathogenesis of neurodegenerative diseases. They are important contributors to necrotic and apoptotic cell death. A major proportion of cellular ROS is generated at the inner mitochondrial membrane by the respiratory chain. In the present study, we investigated a novel peptide antioxidant (SS-31) targeted to the inner mitochondrial membrane for its therapeutic effects both in vitro and in vivo in the G93A mouse model of amyotrophic lateral sclerosis (ALS). SS-31 protected against cell death induced by hydrogen peroxide in vitro in neuronal cells stably transfected with either wild-type or mutant Cu/Zn superoxide dismutase (SOD1). Daily intraperitoneal injections of SS-31 (5 mg/kg), starting at 30 days of age, led to a significant improvement in survival and motor performance. In comparison with vehicle-treated G93A mice, SS-31-treated mice showed a decreased cell loss and a decrease in immunostaining for markers of oxidative stress in the lumbar spinal cord. This further enhances the concept that pharmacological modification of oxidative stress is a therapeutic option for the treatment of ALS.