Male-specific differences in proliferation, neurogenesis, and sensitivity to oxidative stress in neural progenitor cells derived from a rat model of ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1) gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs). E14 pups were bred using SOD1(G93A) transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1(G93A) transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1(G93A) over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1(G93A) over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1(G93A) transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1(G93A) over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression

Expression of cell cycle marker proteins was reduced in male SOD1^G93A rNPCs.

<p>(<b>A</b>) Western blot analysis was used to measure the protein expression of proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase (cdc2), two known cell proliferation markers. (<b>B</b>) Semi-quantitative densitometric analysis of PCNA protein expression. (<b>C and D</b>) Semi-quantitative densitometric analysis using Western blotting revealed that SOD1^G93A transgene altered the phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2) in male rNPCs. Three independent rNPC lines in each group were used for densitometric analysis. The data were expressed as means ± SEM.</p

The number of mature neurons was decreased in differentiated cells with SOD1^G93A transgene.

<p>(<b>A</b>) After 7 days of differentiation, TuJ1 (green) and GFAP (red)-positive cells were found in male wild type [SOD(−)] or transgenic [SOD(+)] cells. The number of TuJ1-postive neurons was significantly decreased by SOD1^G93A over-expression in male cells derived from the spinal cord (<b>B</b>) and cortex (<b>C</b>). There was no difference in the number of GFAP-positive astrocytes differentiated from the spinal cord (<b>D</b>) and cortex (<b>E</b>) rNPCs. *: P<0.05 vs. other groups.</p

Synergistic Effects of GDNF and VEGF on Lifespan and Disease Progression in a Familial ALS Rat Model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons in the brain and spinal cord. We have recently shown that human mesenchymal stem cells (hMSCs) modified to release glial cell line-derived neurotrophic factor (GDNF) decrease disease progression in a rat model of ALS when delivered to skeletal muscle. In the current study, we determined whether or not this effect could be enhanced by delivering GDNF in concert with other trophic factors. hMSC engineered to secrete GDNF (hMSC-GDNF), vascular endothelial growth factor (hMSC-VEGF), insulin-like growth factor-I (hMSC-IGF-I), or brain-derived neurotrophic factor (hMSC-BDNF), were prepared and transplanted bilaterally into three muscle groups. hMSC-GDNF and hMSC-VEGF prolonged survival and slowed the loss of motor function, but hMSC-IGF-I and hMSC-BDNF did not have any effect. We then tested the efficacy of a combined ex vivo delivery of GDNF and VEGF in extending survival and protecting neuromuscular junctions (NMJs) and motor neurons. Interestingly, the combined delivery of these neurotrophic factors showed a strong synergistic effect. These studies further support ex vivo gene therapy approaches for ALS that target skeletal muscle

SOD1^G93A over-expressing rNPCs were more sensitive to oxidative stress.

<p>H₂O₂ (<b>A</b>) and SIN-1 (<b>B</b>) significantly increased the LDH released into the media from the cells derived from SOD1 transgenic males compared to the wild type male cells. *: P<0.01 and **: P<0.05 vs. wild-type male.</p

Over-expression of the mutant SOD1 gene significantly reduces proliferation of rat NPCs.

<p>(<b>A</b>) Photomicrographs represent BrdU immunoreactivity in wild type [SOD1 (−)] or transgenic [SOD1 (+)] NPC in male cells derived from the spinal cord. (<b>B and C</b>) The number of BrdU positive cells was decreased in male SOD1^G93A (+) cells derived from the spinal cord (<b>B</b>) and cortex tissues (<b>C</b>). *: P<0.05 vs. other groups.</p