Tmlhe deficiency induces neurodevelopmental dysfunction and synaptic excitatory/inhibitory imbalance linked to autism-like phenotypes in zebrafish

Mutations in TMLHE, the initial enzyme for carnitine biosynthesis, are linked to autism spectrum disorder (ASD). However, the molecular mechanisms by which TMLHE defects contribute to neurodevelopmental abnormalities remain elusive. This study aimed to elucidate the role of tmlhe deficiency in vertebrate neural development and its link to ASD-like behaviors using a zebrafish model. We established a tmlhe knockdown zebrafish model using morpholino microinjection. The tmlhe gene is specifically expressed in the developing nervous system, as characterized by whole-mount in situ hybridization (WISH). Its knockdown induces severe morphological defects and significant ASD-like behaviors, including impaired social interaction and delayed environmental response. Metabolomic analysis confirmed a blockage in carnitine biosynthesis, leading to deficient long-chain acylcarnitine levels and impaired fatty acid transport. Transcriptomic profiling revealed downregulation of fatty acid transport and β-oxidation pathways and negative enrichment of peroxisome proliferator-activated receptor (PPAR) signaling. Consequently, tmlhe morphants exhibited severe neurodevelopmental defects, including blocked neuronal progenitor cell differentiation, increased apoptosis in the brain, and profound disruption of synaptic function. Transcriptional alterations in glutamatergic and GABAergic receptor-related genes suggest a potential excitatory/inhibitory (E/I) imbalance. Exogenous l-carnitine supplementation successfully rescued the morphological and behavioral deficits. Our findings demonstrate that tmlhe deficiency disrupts carnitine biosynthesis, impairing energy and glycerophospholipid metabolism, which is critical for early neurogenesis. This may lead to impaired neural development and a synaptic E/I imbalance, ultimately causing ASD-like phenotypes. This study establishes a crucial link between carnitine metabolism and ASD etiology, proposing a potential therapeutic target.