Mahmoud Kiaei

Mutated Human SOD1 Causes Dysfunction of Oxidative Phosphorylation in Mitochondria of Transgenic Mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.

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.

Peroxisome proliferator-activated receptor-gamma agonist extends survival in transgenic mouse model of amyotrophic lateral sclerosis.

Accumulating evidence suggests that inflammation plays a major role in the pathogenesis of motoneuron death in amyotrophic lateral sclerosis (ALS) both in humans and transgenic mouse models. Peroxisome proliferator-activated receptors (PPARs) are involved in the inflammatory process. Agonists of PPAR-alpha, -gamma, and -delta show anti-inflammatory effects both in vitro and in vivo. We investigated the therapeutic effect of pioglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist, in the G93A SOD1 transgenic mouse model of ALS. Orally administered pioglitazone improved motor performance, delayed weight loss, attenuated motor neuron loss, and extended survival of G93A mice as compared to the untreated control littermate group. Pioglitazone treatment extended survival by 13%, and it reduced gliosis as assessed by immunohistochemical staining for CD-40 and GFAP. Pioglitazone also reduced iNOS, NFkappa-B, and 3-nitrotyrosine immunoreactivity in the spinal cords of G93A transgenic mice. These results suggest that pioglitazone may have therapeutic potential for human ALS.

Impaired PGC-1α function in muscle in Huntington's disease

We investigated the role of PPAR gamma coactivator 1alpha (PGC-1alpha) in muscle dysfunction in Huntingtons disease (HD). We observed reduced PGC-1alpha and target genes expression in muscle of HD transgenic mice. We produced chronic energy deprivation in HD mice by administering the catabolic stressor beta-guanidinopropionic acid (GPA), a creatine analogue that reduces ATP levels, activates AMP-activated protein kinase (AMPK), which in turn activates PGC-1alpha. Treatment with GPA resulted in increased expression of AMPK, PGC-1alpha target genes, genes for oxidative phosphorylation, electron transport chain and mitochondrial biogenesis, increased oxidative muscle fibers, numbers of mitochondria and motor performance in wild-type, but not in HD mice. In muscle biopsies from HD patients, there was decreased PGC-1alpha, PGC-1beta and oxidative fibers. Oxygen consumption, PGC-1alpha, NRF1 and response to GPA were significantly reduced in myoblasts from HD patients. Knockdown of mutant huntingtin resulted in increased PGC-1alpha expression in HD myoblast. Lastly, adenoviral-mediated delivery of PGC-1alpha resulted increased expression of PGC-1alpha and markers for oxidative muscle fibers and reversal of blunted response for GPA in HD mice. These findings show that impaired function of PGC-1alpha plays a critical role in muscle dysfunction in HD, and that treatment with agents to enhance PGC-1alpha function could exert therapeutic benefits. Furthermore, muscle may provide a readily accessible tissue in which to monitor therapeutic interventions.

Additive neuroprotective effects of creatine and cyclooxygenase 2 inhibitors in a transgenic mouse model of amyotrophic lateral sclerosis

There is substantial evidence implicating both inflammation and mitochondrial dysfunction in amyotrophic lateral sclerosis (ALS) pathogenesis. We investigated the therapeutic effects of cyclooxygenase 2 (COX‐2) inhibitors both alone and in combination with creatine in the G93A transgenic mouse model of ALS. Oral administration of either celecoxib or rofecoxib significantly improved motor performance, attenuated weight loss and extended survival. The administration of COX‐2 inhibitors significantly reduced prostaglandin E2 levels at 110 days of age. The combination of creatine with COX‐2 inhibitors produced additive neuroprotective effects and extended survival by approximately 30%. The COX‐2 inhibitors significantly protected against depletion of anterior horn motor neurons and creatine with COX‐2 inhibitors showed greater protection than COX‐2 inhibitors alone. These results suggest that combinations of therapies targeting different disease mechanisms may be a useful strategy in the treatment of ALS.

Thalidomide and Lenalidomide Extend Survival in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

Accumulating evidence suggests that inflammation plays a major role in the pathogenesis of motor neuron death in amyotrophic lateral sclerosis (ALS). Important mediators of inflammation such as the cytokine tumor necrosis factor-α (TNF-α) and its superfamily member fibroblast-associated cell-surface ligand (FasL) have been implicated in apoptosis. We found increased TNF-α and FasL immunoreactivity in lumbar spinal cord sections of ALS patients and G93A transgenic mice. Both increased TNF-α and FasL immunostaining in the lumbar spinal cord of the G93A SOD1 transgenic mice occurred at 40–60 d, well before the onset of symptoms and loss of motor neurons. We tested the neuroprotective effect of thalidomide and its analog lenalidomide, pharmacological agents that inhibit the expression of TNF-α and other cytokines by destabilizing their mRNA. Treatment with either thalidomide or lenalidomide attenuated weight loss, enhanced motor performance, decreased motor neuron cell death, and significantly increased the life span in G93A transgenic mice. Treated G93A mice showed a reduction in TNF-α and FasL immunoreactivity as well as their mRNA in the lumbar spinal cord. Both compounds also reduced interleukin (IL)-12p40, IL-1α, and IL-1β and increased IL-RA and TGF-β1 mRNA. Therefore, both thalidomide and lenalidomide bear promise as therapeutic interventions for the treatment of ALS.

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.

Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F2-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinsons disease and Huntingtons disease.

Matrix metalloproteinase-9 regulates TNF-α and FasL expression in neuronal, glial cells and its absence extends life in a transgenic mouse model of amyotrophic lateral sclerosis

Abstract Whether increased levels of matrix metalloproteinases (MMPs) correspond to a role in the pathogenesis of amyotrophic lateral sclerosis (ALS) needs to be determined and it is actively being pursued. Here we present evidence suggesting that MMP-9 contributes to the motor neuron cell death in ALS. We examined the role of MMP-9 in a mouse model of familial ALS and found that lack of MMP-9 increased survival (31%) in G93A SOD1 mice. Also, MMP-9 deficiency in G93A mice significantly attenuated neuronal loss, and reduced neuronal TNF-α and FasL immunoreactivities in the lumbar spinal cord. These findings suggest that MMP-9 is an important player in the pathogenesis of ALS. Our data suggest that the mechanism for MMP-9 neurotoxicity in ALS may be by upregulating neuronal TNF-α and FasL expression and activation. This study provides new mechanism and suggests that MMP inhibitors may offer a new therapeutic strategy for ALS.