Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is proposed to occur by necroptosis, an inflammatory form of regulated cell death. Prior studies implicated necroptosis in ALS based on accumulation of necroptotic markers in affected tissues of patients and mouse models, and amelioration of disease in mutant superoxide dismutase 1 (SOD1 G93A ) mice with inhibition of the upstream necroptotic mediators, receptor interacting protein kinase 1 (RIPK1), and RIPK3. To definitively address the pathogenic role of necroptosis in ALS, we genetically ablated the critical terminal executioner of necroptosis, mixed lineage kinase domain-like protein (MLKL), in SOD1 G93A mice. Disease onset, progression, and survival were not affected in SOD1 G93A mice lacking MLKL. Motor neuron degeneration and activation of neuroinflammatory cells, astrocytes, and microglia, were independent of MLKL expression in SOD1 G93A mice. While RIPK1 accumulation occurred in spinal cords of SOD1 G93A mice in late stage disease, RIPK3 and MLKL expression levels were not detected in central nervous system tissues from normal or SOD1 G93A mice at any disease stage. These findings demonstrate that necroptosis does not play an important role in motor neuron death in ALS, which may limit the potential of therapeutic targeting of necroptosis in the treatment of neurological disorders.

Background and Purpose Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1G93A mouse model. Experimental Approach The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1G93A mice as a slow‐release subcutaneous implant (5 mg·day−1). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC–MS–MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. Key Results Flutamide was metabolised to 2‐hydroxyflutamide achieving steady‐state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1G93A mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1G93A mice treated with flutamide, although motor neuron pathology was not affected. Conclusion and Implications The androgen receptor antagonist accelerated disease onset in male SOD1G93A mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.

Objective: To compare the frequency and impact on the channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-offunction KCNH2 variants as predictive biomarkers of SUDEP risk. Methods: We searched for KCNH2 variants with a minor allele frequency of <5%. Functional analysis in Xenopus laevis oocytes was performed for all KCNH2 variants identified. Results: KCNH2 variants were found in 11.1% (10/90) of SUDEP individuals compared to 6.0% (20/332) of epilepsy controls (p = 0.11). Loss-offunction KCNH2 variants, defined as causing >20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about threefold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (<1% allele frequency) associated with greater loss of function and an~11-fold enrichment in the SUDEP cohort (nominal p = 0.03). In silico tools were unable to predict the impact of a variant on function highlighting the need for electrophysiological analysis. Interpretation: These data show that loss-of-function KCNH2 variants are enriched in SUDEP patients when compared to an epilepsy population older than 50 years, suggesting that cardiac mechanisms contribute to SUDEP risk. We propose that genetic screening in combination with functional analysis can identify loss-of-function KCNH2 variants that could act as biomarkers of an individual's SUDEP risk.