scAAV9-VEGF alleviates symptoms of amyotrophic lateral sclerosis (ALS) mice through regulating aromatase.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, chronic, progressive, and fatal neurodegenerative disease that leads to progressive atrophy and weakness of the muscles throughout the body. Herein, we found that the intrathecal injection of adeno-associated virus (AAV)-delivered VEGF in SOD1-G93A transgenic mice, as well as ALS mice, could significantly delay disease onset and preserve motor functions and neurological functions, thus prolonging the survival of mice models. Moreover, we found that VEGF treatment could induce the elevated expression of aromatase, which is a key enzyme in estrogen synthesis, in neurons but not in astrocytes. On the other hand, the changes in the expression of oxidative stress-related factors HO-1 and GCLM and autophagy-related proteins p62 and LC3II upon the administration of VEGF revealed the involvement of oxidative stress and autophagy underlying the downstream of the VEGF-induced mitigation of ALS. In conclusion, this study proved the protective effects of VEGF in the onset and development of ALS and revealed the involvement of estrogen, oxidative stress and autophagy in the VEGF-induced alleviation of ALS. Our results highlighted the potential of VEGF as a promising therapeutic agent in the treatment of ALS.

Raloxifene, a promising estrogen replacement, limits TDP-25 cell death by enhancing autophagy and suppressing apoptosis.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease, and at present, therapies for ALS are limited. Estrogen is a potential therapeutic agent for ALS but has undesirable effects that might increase the risk of breast and uterine cancers or stroke. Raloxifene (Ral) has estrogenic properties but does not exhibit these adverse effects. However, the mechanism of Ral in ALS has not been studied. We thus investigated the effects of Ral in an NSC34 model of ALS that stably expresses the 25-kDa C-terminal fragment of TDP-43 (i.e., TDP-25 cells) and found that GPR30 (G protein-coupled receptor 30) and ER (estrogen receptor) α/ERß were expressed in TDP-25 cells, which show significantly different morphology compared with controls. Both E2 (17ß-estradiol) and Ral increased the expression of ERα and GPR30 and enhanced TDP-25 cell viability, and these effects were completely abolished by treatment with an ERα/ß antagonist (ICI 182,780) or GPR30 antagonist (G15). The P62, caspase-9 and Bax levels were significantly decreased in TDP-25 cells treated with Ral or E2, and the LC3-II levels were elevated in E2-treated cells but reduced in Ral-treated cells. All these changes were abolished by treatment with ICI 182,780 or G15. These data suggest that Ral, similar to E2, enhances autophagy and suppresses apoptosis to limit motor neuron death by binding to ERα/ß or GPR30 in TDP-25 cells. These results demonstrate the protective effects of Ral in an ALS cell model and suggest that Ral is a promising replacement for estrogen and a promising therapeutic strategy for ALS.

scAAV9-VEGF-165 inhibits neuroinflammatory responses and invasion of macrophages into the peripheral nervous system of ALS transgenic mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that leads to paralysis and death within 3-5 years. Although the vast majority of studies have focused on vulnerable neurons, growing evidence has shown that non-neuronal cells contribute to the pathogenesis and disease progression. Here, we showed that intrathecal injection of scAAV9-VEGF at 60 days of age significantly reduced the number of microglia and inhibited the neuroinflammatory response in the CNS. Meanwhile, we found that administration of VEGF inhibited the invasion of macrophages into the PNS, including ventral nerve roots, sciatic nerves and muscles. Overall, our study indicated the anti-inflammation effect of VEGF in the CNS and PNS of ALS mice when delivered by intrathecal injection.

Effects of Ovariectomy in an hSOD1-G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS).

BACKGROUND Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive muscular dystrophy and paralysis; most ALS patients die from respiratory failure within 3 to 5 years, and there is currently no effective treatment. Some studies have indicated sex differences in the incidence of ALS, and evidence suggests a neuroprotective role for estrogen. MATERIAL AND METHODS We used human Cu/Zn superoxide dismutase (hSOD1-G93A) transgenic mice to determine the effects of ovariotomy on the onset of disease and behavior; we also used Western blotting to measure the expression of aromatase and estrogen receptors, as well as the inflammatory cytokines and apoptosis markers, in the lumbar spinal cord to determine the mechanism of estrogen-mediated neuroprotection. RESULTS Ovariectomy advanced the onset of disease, down-regulated aromatase and estrogen receptor alpha (ER-a) expression, and inhibited expression of the anti-inflammatory factors arginase-1 and the anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) in the lumbar spinal cord of hSOD1-G93A transgenic mice. CONCLUSIONS Ovariectomy resulted in earlier disease onset and attenuated the anti-inflammatory and anti-apoptotic actions of estrogen in hSOD1-G93A transgenic mice. Therefore, estrogen may play an important role in protecting spinal cord motor neurons.

Characterization of aromatase expression in the spinal cord of an animal model of familial ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving motor neurons in the motor cortex, brainstem and spinal cord. ALS leads to progressive, aggravated muscle weakness and paralysis. Although the precise pathogenesis remains unknown, several studies have shown that estrogens exert neuroprotective effects during the course of the disease. Aromatase is the key enzyme in estrogen synthesis. In the present study, we used immunohistochemistry, immunofluorescence and western blotting to observe the characteristics of aromatase expression in the spinal cords of copper-zinc superoxide dismutase-1 (SOD1)-G93A transgenic mice. Under normal and nearly normal (pre-symptomatic stage) conditions, the motor neurons in the spinal anterior horn expressed aromatase. After disease onset, astrocytes began to express aromatase. The total level of aromatase expression decreased with disease progression. These findings may provide the basis for the pathogenesis of ALS through glial aromatization during the progression of this disease.