Authors :
Presenting Author: Bianca Brenha, BS – Case Western Reserve University
Tingwei Mu, PhD – Case Westerbn Reserve University
Rationale:
Mutations in GABRA1, which encodes the α1 subunit of GABAA receptors responsible for primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system, have been associated with various forms of epilepsy. Around 35% of cases are not adequately controlled with medication, partially because the effects of GABAA receptor mutations and other proteins on the development and function of human neurons are unknown. Published data from our lab demonstrated that surface trafficking deficiency is one major disease-causing mechanism for GABAA receptor variants. The α1 G251D mutation was also characterized by its cellular aggregation. The accumulation of misfolded proteins in insoluble aggregates is one of the most common pathological hallmarks of most human neurodegenerative diseases. The clearance of these misfolded proteins may represent a promising therapeutic strategy in these diseases. It has been suggested that autophagy prevents the development and progression of epilepsy through the regulation of the balance between inhibitory GABA and excitatory glutamate. Impaired autophagy may contribute to the onset and progression of epilepsy by altering the expression or function of ion channels such as the GABAA receptors, resulting in decreased or increased neuronal excitability. Understanding the molecular mechanisms connecting autophagy defects to epilepsy could provide novel insights into therapeutic strategies for managing this debilitating condition.
Methods:
In this study, we used HEK293T cells stably expressing GABAA receptors subunits (α1β2γ2) with the GABRA1 G251D mutation and human induced pluripotent stem cells (iPSCs)-derived neurons with CRISPR gene editing to establish a disease model. We measured the functional properties of differentiated excitatory and inhibitory neurons to understand the impact of the G251D mutation on neuronal activity. Results:
We differentiated the iPSC-derived neurons over 15 days on a multi-electrode array (MEA). The mutant neurons were observed to have significantly higher firing rate and number of bursts after day 10. The cultures were observed to express neuronal markers MAP2 and FOXG1, confirming the presence of mature neuronal cultures suitable for assessment of α1 G251D mutation. We examined p62 protein expression in neurons at day 10 as a marker to assess autophagy levels, the GABRA1 G251D mutants showed significantly increased p62 indicating a potential impairment in the autophagy process. We performed a drug screening assessing 70 autophagy activators to identify those that increase membrane trafficking in HEK293T cells stably expressing α1 G251D mutation. Of these, we observed that some of them can successfully rescue the surface trafficking deficiency. Furthermore, we explored the mechanism of action of these small molecules about how it alters the autophagy pathways associated with the GABAA receptor homeostasis network. Conclusions:
Our findings indicate the critical role of autophagy in regulating proteostasis of pathogenic GABAA receptor mutations. Moreover, our study paved the foundation to develop novel therapeutic strategies for epilepsy-associated genes.Funding: Tingwei Mu R01 funding from NIH.