Deletion of<i>Ripk3</i>Prevents Motor Neuron Death<i>In Vitro</i>but not<i>In Vivo</i>

Abstract

Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in both <i>in vitro</i> and <i>in vivo</i> models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors <i>Ripk1</i>, <i>Ripk3</i>, and <i>Mlkl</i> in the spinal cord of mutant superoxide dismutase-1 (SOD1^G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1^G93A mice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing of <i>Ripk3</i> in cultured MNs protected them from toxicity associated with SOD1^G93A astrocytes. However, constitutive deletion of <i>Ripk3</i> in Tg SOD1^G93A mice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit of <i>Ripk3</i> silencing <i>in vivo</i>. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1^G93A or <i>Optineurin</i> knock-out 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 to determine if necroptosis plays a critical role in the pathogenesis of ALS.