Deletion of Ripk3 prevents Motor Neuron death in vitro but not in vivo

Abstract

Increasing evidence suggests that necroptosis, a form of programmed cell death, contributes to neurodegeneration in several disorders, including amyotrophic lateral sclerosis (ALS). Supporting this view, both in vitro and in vivo investigations in models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons. To confirm the pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors Ripk1, Ripk3 and Mlkl in the spinal cord of mutant superoxide dismutase-1 (SOD1^G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of RIPK1 (but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1^G93A mice. In post-mortem motor cortex samples from sporadic and familial ALS patients, no changes in protein levels of RIPK1 were detected. Silencing of Ripk3 in cultured motor neurons protected them from toxicity associated with SOD1^G93A astrocytes. However, constitutive deletion of Ripk3 in Tg SOD1^G93A mice failed to provide behavioral and neuropathological improvement, demonstrating no similar benefit of Ripk3 silencing in vivo. Lastly, no genotype-specific myelin decompaction – proposed to be a proxy of necroptosis in ALS – was detected in either Tg SOD1^G93A or Optineurin knockout mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1^G93A-induced neurodegeneration and call for further preclinical investigations prior to concluding that necroptosis plays a critical pathogenic role in ALS.

Significance Statement Studies in models of amyotrophic lateral sclerosis (ALS) suggest an instrumental role for necroptosis in this currently incurable disease. Given the availability of a growing number of small molecules that inhibit key determinants of necroptosis, implication of this form of programmed cell death in ALS may open new therapeutic avenues. We found that despite changes in the expression of key necroptosis determinants, targeting RIPK3 prevents motor neuron death in vitro but not in vivo. These results challenge the suggestion that targeting RIPK3 to abrogate necroptosis will provide therapeutic benefit in ALS.