Tuberous Sclerosis Complex (TSC) Inactivation Increases Neuronal Network Activity by Enhancing Ca²⁺ Influx via L-Type...

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs, such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurologic impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remains unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2^-/-) neurons exhibited elevated neuronal activity with highly synchronized Ca²⁺ spikes. Notably, TSC2^-/- neurons presented enhanced Ca²⁺ influx via L-type Ca²⁺ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2^-/- neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2^-/- neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.SIGNIFICANCE STATEMENT There is a close relationship between elevated mammalian target of rapamycin (mTOR) activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2^-/- neurons, we identified elevated Ca²⁺ influx via L-type Ca²⁺ channels as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2^-/- neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca²⁺ signaling via L-type Ca²⁺ channels in human TSC neurons, which could cause epilepsy in TSC.