Abstract

Riluzole is the only FDA approved drug for Amyotrophic Lateral Sclerosis (ALS) treatment. Riluzole enhances astrocytic production of trophic factors. We hypothesize that the protective effect of riluzole in ALS is partially mediated by stimulation of trophic factors production by motor neuron associated cells. Mice were treated with riluzole in the drinking water (100μg/ml). The levels of trophic factors were evaluated in the spinal cord, muscle, sciatic nerve and brain at 3, 6, 15 and 30 days (d). 1) In the spinal cord, riluzole significantly increased the levels of GDNF, BDNF and cardiotrophin 1 (CT-1) at 3, 6 and up to 15 d. However, these levels were significantly decreased below basal levels after 30 d treatment. 2) In muscle, a similar pattern was observed, but the decrease occurred at 15 d of riluzole treatment. 3) In sciatic nerve, a 75% decrease on the levels of CT-1 occurred at 15 d of treatment, and 4) in the brain, riluzole increased the BDNF levels by 15 d, but its levels were decreased by 30 d. In contrast, both GDNF and CT-1 increased by 6 d and declined at 15 d. The direct effect of riluzole on astrocytes and Schwann cells was investigated in culture. Incubation of primary astrocytes for 24h with riluzole (1 μM) induced gene expression of CT-1 without affecting BDNF and GDNF expression. After continuous incubation of astrocyte cultures with riluzole for 6 d the expression of the mRNA for GDNF and CT-1 was below controls, while the expression of BDNF was increased by 50%. Conditioned media by astrocytes supported the survival of trophic factor deprived (TFD) motor neurons. Astrocytes and Schwann cells were cultured in the presence of riluzole for 1, 6, and 15 d. After removal of the drug the cells were allowed to conditioned media for 24 h. Riluzole pretreatment of astrocytes for up to 6 d significantly increased TFD motor neuron survival when compare to conditioned media alone. However, this increased was lost after 15 d incubation with the drug. Neutralizing antibodies to CT-1 (N-CT-1) had little effect on TFD motor neuron survival stimulated by conditioned media alone, but partially abolished the effects of riluzole pretreatment. Similar results were obtained with conditioned media from primary Schwann cells, but the N-CT-1 completely blocked the conditioned media effects on motor neuron survival independently of the pretreatment with riluzole. Our results show that an acute pretreatment of astrocytes and Schwann cells with riluzole increased the production of trophic factors, but chronic exposure elicited opposite effect. The limited effect of riluzole in ALS patients could be due to a paradoxical effect, which inhibits the production of trophic factors for motor neurons.