Regulation of serotonin transport by geranylgeranyl transferase and Ca 2+ /calmodulin‐dependent protein kinase II

Abstract

The serotonin transporter (SERT) transports extracellular serotonin (5HT) into neuron terminals and serves as the primary mechanism by which 5HT is regulated in the brain. Recently, it has been shown that SERT activity is increased after treatment with cholesterol-lowering statin drugs in a manner independent of cholesterol but dependent on the cholesterol biosynthetic intermediate geranylgeranyl pyrophosphate (GGPP). GGPP and its corresponding enzyme geranylgeranyl transferase (GGT) lipidate proteins for targeting to the plasma membrane. Although statins are known to increase constitutive activity of the canonical targets of GGPP including Rac1, CDC42, and RhoA, the mechanism by which GGPP regulates SERT activity is unknown. To investigate the role of GGPP on neuronal SERT function, serotonin neurons were modeled in serotonergic RN46A-B14 cells with stably overexpressed SERT. Simvastatin or a GGT inhibitor that specifically blocks geranylgeranylation by GGPP were applied to cells for 24hr and 5HT transport was assayed by measuring 5HT uptake in cells after incubation with exogenous 5HT. Simvastatin and GGT inhibition increased 5HT uptake by 58% and 82% (Mean (SEM): Veh=2.09 (0.30), Sim=4.99 (0.53), GGT=11.84 (2.63), p<0.05), respectively, in a manner blocked by fluoxetine. Pharmacological inhibition of CDC42 or knockdown of the mRNA for RhoA did not block statin-increased 5HT uptake. Protein kinases p38 mitogen-activated protein kinase (P38MAPK) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) were also investigated based on their regulation downstream of GGPP. While phosphorylation of P38MAPK was significantly increased by 37% after 24hr simvastatin treatment (Mean (SEM): Veh=100 (6.7), Sim=159 (19.0), p<0.02), inhibition of P38MAPK did not block statin-increased uptake. However, inhibition of CaMKII with CaMKII inhibitor Tat-CN21 dose-dependently blocked statin-increased 5HT uptake (Mean (SEM): Veh=100 (0), Sim=263 (25.8), Tat-CN21=119.48 (12.2), p<0.001). Ongoing studies of the inhibition of GGT on 5HT uptake are being conducted using a panel of CaMKII inhibitors including Tat-CN21, myristoylated autocamtide-2-related inhibitory peptide, and small molecule inhibitors with corresponding control agents. Our results indicate that SERT is regulated through the isoprenylation pathway in a GGPP, GGT and CaMKII-dependent manner.