ENT1 inhibitor J4 restores cognitive function and white-matter integrity in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a hereditary disease caused by pathogenic mutations in the TSC1 or TSC2 genes, leading to overactivation of mTOR signaling and dysregulation of downstream pathways. The majority of individuals with TSC develop psychiatric and neurodevelopmental comorbidities-including intellectual disability, autism spectrum disorders, anxiety and other behavioral manifestations-collectively termed TSC-associated neuropsychiatric disorders. Over the past decade, increasing evidence from clinical and preclinical studies has highlighted a significant myelination deficit in the brains of TSC patients and animal models. Although mTOR inhibition can alleviate myelination defects and improve cognition in rodent models, clinical studies using the mTOR inhibitor, everolimus, has limited cognitive benefits in TSC individuals. Adenosine signaling has been recognized as a key player in oligodendrocyte maturation and myelin formation and may represent a promising target for therapeutic intervention. In the present study, we treated Tsc2+/- mice with J4, an equilibrative nucleoside transporter 1 inhibitor that has been demonstrated to increase adenosine level, and used diffusion MRI and relevant imaging modalities to assess its therapeutical effects on behavioral deficits and white matter defects in Tsc2+/- mice. We also analyzed the myelin ultrastructural changes using transmission electron microscopy and examined the expression oligodendrocytes- and myelination-associated proteins after the treatment. J4 treatment improved both cognitive deficits and anxiety-like behavior in Tsc2+/- mice, and ameliorated white matter abnormalities through enhancing myelin sheath integrity. We also demonstrated that J4 mitigates gray matter cytoskeletal disorganization, along with increased expression of key mature oligodendrocyte- and myelin-associated proteins. Furthermore, J4 significantly reduced the aberrant overexpression of pS6 and cFos, both of which are elevated in Tsc2+/- mice as a result of hyperactivation of mTOR and heightened neuronal activity. Altogether, these findings indicate that J4 modulates oligodendroglial lineage populations, enhances myelination, and improves neural connectivity by regulating neuronal hyperactivity. Our results suggest that J4 is a strong therapeutic candidate for addressing the neuropsychiatric manifestations of TSC.