Authors :
Presenting Author: Nolan Dvorak, PhD – Baylor College of Medicine
Jeffery Noebels, M.D., Ph.D. – Baylor College of Medicine
Rationale:
Hyperexcitability is a prominent phenotype in the early stage of Alzheimer’s Disease (AD). However, the cellular signaling mechanisms that become corrupted to produce the hyperexcitability remain underdefined. Previous research has shown that mammalian target of rapamycin (mTOR) signaling is increased in the AD brain. In addition, our group has shown that increased mTOR signaling promotes network hyperexcitability and seizures through an mTOR complex 2 (mTORC2), but not mTOR complex 1 (mTORC1), dependent mechanism. Furthermore, we showed that mTORC2 forms a complex with and alters phosphorylation of the voltage-gated Na+ channel isoform 1.2 (Nav1.2), a key ion channel that regulates the excitability of neocortical pyramidal neurons. Based upon the aforementioned, we tested the hypothesis that enhanced mTORC2-Nav1.2 signaling could contribute to hyperexcitability in early-stage AD.Methods:
Patch-clamp recordings in HEK-Nav1.2 cell line and cortical brain slices from J20 mice.
Results:
We tested the effect of elevated mTOR signaling on Nav1.2 channel function. We performed patch-clamp recordings in HEK-Nav1.2 cells that were treated with an inhibitor of PTEN, a negative regulator of mTOR signaling. These studies revealed that hyperactivation of mTOR signaling increases Nav1.2 current, which was reversed by pharmacological inhibition of mTORC1 and mTORC2, but not mTORC1 alone, suggesting that PTEN inhibition potentiates Nav1.2 channel function through a mechanism requiring mTORC2. Next, we investigated if hyperactive mTOR signaling contributes to increased Nav1.2 channel function in pyramidal neurons (PNs) in the anterior cingulate cortex (ACC), a brain region that shows the most age-related metabolic dysfunction. We performed patch-clamp recordings in brain slices from WT mice and 4–5-month-old J20 mice. We found that PNs in the ACC of 4–5-month-old J20 mice displayed increased Nav1.2 currents. Notably, these Nav1.2 gain-of-function cellular phenotypes observed in PNs of J20 mice were reversible by pharmacological inhibition of mTORC1 and mTORC2, but not by inhibition of mTORC1 alone. Given the key role of the Nav1.2 channel in regulating neuronal excitability, we next investigated if PNs in J20 mice would display altered firing behavior. Current-clamp recordings revealed that ACC PNs in the J20 mice display hyperexcitability, and that this phenotype is reversible via inhibition of mTORC1 and mTORC2, but not mTORC1 alone, supporting the prominent role of mTORC2-Nav1.2 signaling in the hyperexcitability observed in early-stage AD. Conclusions:
These findings provide the first mechanistic evidence for cellular hyperexcitability mediated by mTOR activation, and highlight regulation of the Nav1.2 channel conferred by mTORC2 in the context of AD. These findings suggest that therapeutic targeting of the mTORC2-Nav1.2 interaction in early-stages of AD could prevent hyperexcitability and thereby potentially slow neurodegeneration.
Funding:
NIH NS R01124145 (JLN); R01 NS 29709 (JLN) and The Blue Bird Circle Foundation.