P3.02. ASSESSMENT OF MITOPHAGY IN HUMAN SUBPALLIAL ORGANOIDS ENRICHED WITH GABAERGIC NEURONS IN DRAVET SYNDROME
Paulina Tyszko1,2, Marzena Zychowicz1, Valery Zayat1, Michał Liput1, Zuzanna Kuczyńska1, Erkan Metin1, Leonora Bużańska1
1 Mossakowski Medical Research Institute, Department of Stem Cell Bioengineering, Pawinskiego 5 str, Warsaw, Poland
2 Warsaw University of Life Science, Faculty of Biology and Biotechnology, Nowoursynowska 166 str, Warsaw, Poland
INTRODUCTION: Mitochondria are essential for cellular energy and homeostasis, with mitophagy (a specialized form of autophagy) playing a key role in removing damaged mitochondria. Recent studies suggest mitochondrial dysfunction may contribute to neurodevelopmental disorders. Dravet Syndrome (DRVT), a severe developmental and epileptic encephalopathy associated with SCN1A mutations, has unclear underlying mechanisms, but emerging evidence points to possible mitophagy dysregulation.
AIM(S): The aim of this study was to assess the mitophagy in a human 3D ventral forebrain organoid model enriched for GABAergic neurons derived from Dravet Syndrome patients.
METHOD(S): Two induced pluripotent stem cell (iPSC) lines derived from DRTV patients with SCN1A mutations and healthy control iPSC line were used for derivation of ventral (subpalial) forebrain organoids enriched for GABAergic neurons using dual SMAD inhibition, SHH agonist and WNT pathway inhibitor, and sequential treatment with EGF, bFGF, BDNF and NT3. Organoids were collected on days 60, 75, and 100 for immunocytochemistry and on 100 day for gene expression analysis.
RESULTS: Ventralisation and GABAergic neuron enrichment were confirmed by expression of markers including GABA, EOMES, DLX2, GAD67, and somatostatin. Mitophagy was detected by the presence of BNIP3 and LC3B-positive cells at day 100. The downregulation of mitophagy-related genes (PINK1, PARK2, BNIP3) was detected in DRVT organoids compared to control. Additionally, altered expression of mitochondrial biogenesis genes, such as NRF1, PPARGC1, TFAM was observed in DRVT organoids.
CONCLUSIONS: Our findings suggest that mitophagy and mitochondrial biogenesis are impaired in Dravet Syndrome ventral forebrain organoids, supporting the hypothesis that mitochondrial dysregulation may contribute to the pathogenesis of DRVT. These results highlight mitophagy as a potential therapeutic target for further investigation.
FINANCIAL SUPPORT: Mossakowski Medical Research Institute internal grant FBW-035 and Statutory Funds