Ubiquitin Proteasome System BTB-Adaptor Protein Insomniac Modifies Seizure Severity and Response to the Ketogenic Diet in a Drosophila Model of Mitochondrial Encephalomyopathy

Background/Objectives: The ubiquitin proteasome system (UPS) serves critical cellular homeostatic functions that contribute to neurological health, including neuronal metabolism, synaptic plasticity, and the molecular circadian clock. The UPS is tightly regulated to ensure the correct balance of healthy proteins and those targeted for degradation and recycling; malfunction in this quality control process can contribute to disease states such as neurodegeneration and cancer. Target specificity in this system is due in part to ubiquitin ligase adaptor proteins, such as those of the BTB superfamily of proteins, which connect substrates to the larger proteasomal complex. In Drosophila, the BTB family member insomniac (human orthologs KTCD2, 5, and 17) has been shown to regulate sleep, arousal, and synaptic plasticity, but has not been examined for a role in neuronal excitability disease phenotypes. Methods: We utilized a genetic approach to interrogate neurobehavioral dysfunction in mitochondrial encephalomyopathy (ME) and the ability of dietary therapy to modulate this dysfunction. Results: Here, we report that genetic interference with insomniac modifies seizure-like behavior in a neuronal-subtype-specific manner in the ATP61 model of ME, ameliorating seizure severity when knocked down in GABAergic or dopaminergic neurons. Furthermore, while ATP61 typically responds to ketogenic dietary therapy with a robust reduction in seizure phenotype, we report that knockdown of insomniac or its binding partner Cullin RING Ligase 3 (Cul3) in neurons ablates this effect entirely, a previously unreported role for the UPS in downstream neurophysiological effects of the KD. Conclusions: These results implicate UPS BTB adaptor proteins as therapeutic targets in seizure pathology and identify them as a novel contributor to the ketogenic suppression of neuronal hyperexcitability. These two neurological roles are likely to be proceeding via cellular and molecularly distinct pathways, including a non-CRL3-based mechanism of seizure modulation.