Ligustilide inhibits Purkinje cell ferritinophagy via the ULK1/NCOA4 pathway to attenuate valproic acid-induced autistic features

Background: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder in which social impairment is the core symptom. Presently, there are no definitive medications to cure core symptoms of ASD, and most therapeutic strategies ameliorate ASD symptoms. Treatments with proven efficacy in autism are imminent. Ligustilide (LIG), an herbal monomer extracted from Angelica Sinensis and Chuanxiong, is mainly distributed in the cerebellum and widely used in treating neurological disorders. However, there are no studies on its effect on autisticlike phenotypes and its mechanism of action. Purpose: Investigate the efficacy and mechanism of LIG in treating ASD using two Valproic acid(VPA)-exposed and BTBR T + Itpr3tf/J (BTBR) mouse models of autism. Methods: VPA-exposed mice and BTBR mice were given LIG for treatment, and its effect on autistic-like phenotype was detected by behavioral experiments, which included a three-chamber social test. Subsequently, RNA-Sequence(RNA-Seq) of the cerebellum was performed to observe the biological changes to search target pathways. The autophagy and ferroptosis pathways screened were verified by WB(Western Blot) assay, and the cerebellum was stained by immunofluorescence and examined by electron microscopy. To further explore the therapeutic mechanism, ULK1 agonist BL -918 was used to block the therapeutic effect of LIG to verify its target effect. Results: Our work demonstrates that LIG administration from P12 -P14 improved autism-related behaviors and motor dysfunction in VPA-exposed mice. Similarly, BTBR mice showed the same improvement. RNA-Seq data identified ULK1 as the target of LIG in regulating ferritinophagy in the cerebellum of VPA-exposed mice, as evidenced by activated autophagy, increased ferritin degradation, iron overload, and lipid peroxidation. We found that VPA exposure-induced ferritinophagy occurred in the Purkinje cells, with enhanced NCOA4 and Lc3B expressions. Notably, the therapeutic effect of LIG disappeared when ULK1 was activated. Conclusion: LIG treatment inhibits ferritinophagy in Purkinje cells via the ULK1/NCOA4-dependent pathway. Our study reveals for the first time that LIG treatment ameliorates autism symptoms in VPA-exposed mice by reducing aberrant Purkinje ferritinophagy. At the same time, our study complements the pathogenic mechanisms of autism and introduces new possibilities for its therapeutic options.