¹National Health Research Institutes, Center for Neuropsychiatric Research, Miaoli County, Taiwan, ²Taipei City Hospital, Department of Psychiatry, Taipei City Psychiatric Center, Taipei, Taiwan, ³Taipei Medical University, Department of Psychiatry, School of Medicine, College of Medicine, Taipei, Taiwan

Background: In our previous study on Admission Delirium Tremens (ADT), a severe form of alcohol withdrawal marked by tremors and confusion, we found a significant increase in the neuronal biomarker neurofilament light chain (NfL) [1]. Although haloperidol, used for DT by antagonizing dopamine D2 and serotonin 5-HT2A receptors [2]., was once a common treatment, it did not impact the duration of DT and is no longer the primary therapy. Genetic association analyses revealed a correlation between the serotonin 5-HT2A receptor gene (HTR2A) and NfL.

Aims & Objectives: This study aims to investigate whether the expression level of HTR2A correlates with the release of NfL in cell culture media.

Method: A total of 221 alcohol-dependent patients were enrolled for genotyping verification. Plasma and culture media NfL levels were measured using the Simple Plex™ NfL Assay on the Ella™ instrument, following the manufacturer's instructions. HTR2A SNPs were selected from the imputed dataset generated by the Michigan Imputation Server, and the polymorphism rs9567746 was validated using TaqMan Real-Time PCR assays. Its association with NfL levels was then evaluated through linear regression analysis. The SK-N-SH human neuroblastoma cell line was cultured, and HTR2A expression was knocked down using HTR2A siRNA targeting exon 4 (1466–1486, NM_000621.5) in the presence or absence of TNF-α or haloperidol. HTR2A mRNA expression levels were quantified by real-time PCR. Data visualization was conducted using GraphPad Prism 5 software, with statistical significance set at P < 0.05.

Results: ADT patients exhibited significantly higher plasma NfL levels compared to non-DT patients and controls. Notably, DT patients’ HTR2A genetic polymorphisms located at intron 3 were significantly associated with NfL levels (P = 0.001). Carriers of the minor allele at rs9567746 showed significantly elevated NfL levels than the major genotype carriers. In vitro, selective knockdown of HTR2A significantly increased NfL release in SK-N-SH cell culture media, particularly when co-incubated with TNF-α (100 ng/mL). These findings suggest a mechanistic link between HTR2A expression and NfL release.

Discussion & Conclusions: In rs9567746 polymorphism of HTR2A, the ADT patient’s minor allele type carrier, present in approximately 20% of ADT patients, showed significant increase of plasma NfL levels than major genotype carriers. This minor genotype carriers could be having lower HTR2A expression, this could be demonstrated by HTR2A knock-down and increase the NfL release through pathways with or without TNF-α, highlighting the low expression of HTR2A could be a pathway toward more severe brain neuronal injury in ADT.