SOD1 Rats Research Articles

Novel Role for Vascular Endothelial Growth Factor (VEGF) Receptor-1 and Its Ligand VEGF-B in Motor Neuron Degeneration

Although vascular endothelial growth factor-B (VEGF-B) is a homolog of the angiogenic factor VEGF, it has only minimal angiogenic activity, raising the question of whether this factor has other (more relevant) biological properties. Intrigued by the possibility that VEGF family members affect neuronal cells, we explored whether VEGF-B might have a role in the nervous system. Here, we document that the 60 kDa VEGF-B isoform, VEGF-B(186), is a neuroprotective factor. VEGF-B(186) protected cultured primary motor neurons against degeneration. Mice lacking VEGF-B also developed a more severe form of motor neuron degeneration when intercrossed with mutant SOD1 mice. The in vitro and in vivo effects of VEGF-B(186) were dependent on the tyrosine kinase activities of its receptor, Flt1, in motor neurons. When delivered intracerebroventricularly, VEGF-B(186) prolonged the survival of mutant SOD1 rats. Compared with a similar dose of VEGF, VEGF-B(186) was safer and did not cause vessel growth or blood-brain barrier leakiness. The neuroprotective activity of VEGF-B, in combination with its negligible angiogenic/permeability activity, offers attractive opportunities for the treatment of neurodegenerative diseases.

Vascular endothelial growth factor counteracts the loss of phospho‐Akt preceding motor neurone degeneration in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that results in the selective loss of motor neurones. In the present study, the involvement of the antiapoptotic protein, Akt (protein kinase B), was studied. We found that motor neurones of both sporadic and familial ALS patients lack phospho-Akt, and that motor neurones of mutant SOD1 mice lose activated Akt early in the disease, before the onset of clinical symptoms. In vitro, overexpression of constitutively active Akt protects against mutant SOD1-dependent cell death. In vivo, levels of phospho-Akt in the spinal cord increase after intracerebroventricular administration of vascular endothelial growth factor to mutant SOD1 rats, a treatment we previously described to significantly protect motor neurones. From these results, we conclude that the loss of phospho-Akt could be involved in motor neurone death in ALS, and that therapies upregulating phospho-Akt thus might be of clinical relevance.

Pyrrolidine Dithiocarbamate Inhibits Induction of Immunoproteasome and Decreases Survival in a Rat Model of Amyotrophic Lateral Sclerosis

Pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear transcription factor kappa-B (NF-kappaB) and an antioxidant, has beneficial effects in animal models of various diseases, including arthritis, brain ischemia, spinal cord injury, Alzheimer's disease, and Duchenne muscular dystrophy. Because inflammation and oxidative damage are also hallmarks of amyotrophic lateral sclerosis (ALS), we studied the effect of oral PDTC treatment on G93A-superoxide dismutase 1 (SOD1) transgenic (TG) rat model of human ALS and observed that PDTC treatment significantly decreases the survival. PDTC treatment evoked the end stage of the disease at 121 +/- 21 days, whereas untreated TG animals reached the end stage at 141 +/- 13 days (p < 0.01). The DNA binding activity of NF-kappaB was not altered in G93A-SOD1 TG rats by PDTC treatment. The copper concentration in the spinal cord was increased after PDTC treatment both in G93A-SOD1 TG and wild-type rats, suggesting that increased copper may enhance the neurotoxicity of mutant SOD1. The amount of ubiquitinated proteins were significantly higher and proteasomal activity was decreased in the spinal cords of PDTC-treated TG rats compared with other groups, suggesting that PDTC treatment decreases proteasome function. Immunoblotting and immunocytochemistry showed that the level of immunoproteasome but not constitutive proteasome was increased in glia of G93A-SOD1 TG rats along with disease development. PDTC treatment completely blocked the induction of immunoproteasome expression without affecting constitutive proteasome. These results suggest that PDTC acts as an immunoproteasome inhibitor in mutant SOD1 rats and that immunoproteasome may help the nervous system to cope with deleterious effects of SOD1-G93A mutation.

Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease leading to motor neuron cell death, but recent studies suggest that non-neuronal cells may contribute to the pathological mechanisms involved. Myostatin is a negative regulator of muscle growth whose function can be inhibited using neutralizing antibodies. In this study, we used transgenic mouse and rat models of ALS to test whether treatment with anti-myostatin antibody slows muscle atrophy, motor neuron loss, or disease onset and progression. Significant increases in muscle mass and strength were observed in myostatin-antibody-treated SOD1(G93A) mice and rats prior to disease onset and during early-stage disease. By late stage disease, only diaphragm muscle remained significantly different in treated animals in comparison to untreated controls. Myostatin inhibition did not delay disease onset nor extend survival in either the SOD1(G93A) mouse or rat. Together, these results indicate that inhibition of myostatin does not protect against the onset and progression of motor neuron degenerative disease. However, the preservation of skeletal muscle during early-stage disease and improved diaphragm morphology and function maintained through late stage disease suggest that anti-myostatin therapy may promote some improved muscle function in ALS.

Degeneration of respiratory motor neurons in the SOD1 G93A transgenic rat model of ALS

The transgenic mutant superoxide dismutase (SOD1) mice and rats have been important tools in attempting to understand motor neuron pathology and degeneration but the mechanism behind death in this model has not been studied. We studied the electrophysiologic and pathologic properties of the cervical motor neurons and phrenic nerves in mutant SOD1 rats and demonstrated motor neuron loss, progressive reduction of phrenic nerve compound muscle action potential amplitudes, phrenic nerve fiber loss, and diaphragm atrophy suggesting respiratory insufficiency as a significant contributing factor leading to SOD1 rat death. Unlike previous observations suggesting that a dying-back process may be occurring in the mouse model of the disease, we did not observe differences between proximal and distal axon loss in phrenic nerves of SOD1 rats. This may reflect a unique feature of respiratory motor neuron biology or may be related to the relatively rapid course of decline in the rat model when compared with the mouse SOD1 model. Significant motor neuron loss was also noted in the lumbosacral spinal cord with relative sparing of motor neurons in the cranial nuclei. Taken together, these data suggest that respiratory motor neuron loss results in significant electrophysiologic changes and diaphragmatic atrophy. These changes may play a significant role resulting in death of these animals.

Inflammatory mediators and growth factors in the spinal cord of G93A SOD1 rats

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. One mechanism involved in ALS pathology is neuroinflammation. Neuroinflammation is mediated by soluble pro-inflammatory molecules such as cytokines, prostaglandins and nitric oxide. Studies on transgenic mice demonstrated the expression of pro-inflammatory mediators in early stages of murine ALS. Recently a transgenic rat model became available. Since species differences in regard to cytokine expression have been reported in other disease models we set out to validate the neuroinflammatory hypothesis in the ALS-transgenic rat. We investigated the expression of inflammatory mediators and growth factors in the spinal cord by semi-quantitative RT-PCR. We found that several pro-inflammatory mediators are up-regulated at asymptomatic and end-stages, whereas VEGF, a neuroprotective factor was down-regulated.

Synaptosomal glutamate transport studies in a transgenic rat model of amyotrophic lateral sclerosis

Transgenic rats over‐expressing the G93A Co/Zn superoxide dismutase (SOD1) mutation have recently been generated (Howland et al. PNAS99, 1604–1609) and found to replicate an amyotrophic lateral sclerosis (ALS) – like motor neuron disease previously described in transgenic mice harboring the same mutation. Impairments in synaptosomal glutamate transport capacity have been documented both in the transgenic mouse model and with tissue obtained from human ALS patients. We have measured synaptosomal glutamate transport in the G93A SOD1 rat to further characterize these animals and to provide a comparison with the data described above. We observed a 36% decrease in the maximum velocity of glutamate uptake in synaptosomes prepared from spinal cord of G93A SOD1 rats compared with age matched control animals (Vmax values for glutamate transport were 152 and 98 pmol/min/mg, respectively, in control and transgenic animals) while the affinity for glutamate (Km 3–5 μm) was unchanged. In contrast, no differences in Vmax values were observed when synaptosomes were prepared from cortex, hippocampus, striatum, brainstem or cerebellum. Pharmacological sensitivity to the selective GLT‐1 transport inhibitor dihydrokainate (IC50 74 μm) demonstrates that glutamate uptake in spinal cord synaptosomes is predominantly mediated by this subtype. Incubation of spinal cord synaptosomes with riluzole (100 μm) resulted in a 20–30% increase in glutamate uptake in the control animals while in the G93A SOD1 rats this effect was less pronounced. Our results indicate both a reduction in spinal cord glutamate transport capacity in the SOD1 rats and an altered functional response to riluzole.