Expression of a Constitutively Active Human Insulin Receptor in Hippocampal Neurons Does Not Alter VGCC Currents

Abstract

Memory and cognitive decline are the product of numerous physiological changes within the aging brain. Multiple theories have focused on the oxidative, calcium, cholinergic, vascular, and inflammation hypotheses of brain aging, with recent evidence suggesting that reductions in insulin signaling may also contribute. Specifically, a reduction in insulin receptor density and mRNA levels has been implicated, however, overcoming these changes remains a challenge. While increasing insulin receptor occupation has been successful in offsetting cognitive decline, alternative molecular approaches should be considered as they could bypass the need for brain insulin delivery. Moreover, this approach may be favorable to test the impact of continued insulin receptor signaling on neuronal function. Here we used hippocampal cultures infected with lentivirus with or without IRβ, a constitutively active, truncated form of the human insulin receptor, to characterize the impact continued insulin receptor signaling on voltage-gated calcium channels. Infected cultures were harvested between DIV 13 and 17 (48 h after infection) for Western blot analysis on pAKT and AKT. These results were complemented with whole-cell patch-clamp recordings of individual pyramidal neurons starting 96 h post-infection. Results indicate that while a significant increase in neuronal pAKT/AKT ratio was seen at the time point tested, effects on voltage-gated calcium channels were not detected. These results suggest that there is a significant difference between constitutively active insulin receptors and the actions of insulin on an intact receptor, highlighting potential alternate mechanisms of neuronal insulin resistance and mode of activation.

Keywords: Calcium; Electrophysiology; Insulin resistance; Memory.

Fig. 1

Insulin signaling with and without exogenous insulin. a. Representative Western blots of mixed primary hippocampal cultures treated with saline or 10 nM Apidra^® for 5, 15, or 30 min. Each sample was run in duplicate and probed separately across gels. Blots were probed with Cell Signaling Technology anti-phospho AKT (Ser473; #4051) 1:1000 and total AKT (pan #4685) 1:1000. b. Quantification reveals signaling increases after 15 (trend #) and 30 min compared to timed saline controls (n=3) c. Representative Western blots of mixed primary hippocampal cultures infected with Ef1a or EF1a-IRβ lentiviruses. Each sample was run in duplicate and probed separately across gels. Blots were probed as in b. d. Western blot quantification reports significant pAKT/AKT between EF1a and EF1a-IRβ, suggesting constitutive activity (n=3). # indicates p<0.10; * indicates p<0.05. All data represent means +/− SEM

Fig. 2

Constitutive activity of the human truncated insulin receptor β subunit does not alter voltage sensitivity of VGCCs. a. Plasmid map of synapsin-dTomato construct. The IRβ sequence was inserted between XbaI and BamHI sites using PCR ligation for production of the synapsin-IRβ-dTomato plasmid. b. Photomicrograph of hippocampal neurons probed for IRβ expression using a fluorescent HA-tag antibody. Cells in green indicate presence of IRβ. c. Representative inward currents obtained from a holding potential of −70 mV during determination of IV relationships (−60 to + 30 mV). d. Quantification of VGCC currents across groups showed no significant difference. e. Current density (pA/pF) of peak, late, and 50 ms tail currents were not altered by production of the constitutively active form of the human brain insulin receptor. All data represent means +/− SEM

Fig. 3

A second constitutively active form of the human truncated insulin receptor β subunit does not alter voltage sensitivity of VGCCs. a. Plasmid map of synapsin-mCherry IR β subunit construct. Note replacement of the IRES sequence with the P2A site. The IRβ sequence was inserted between XbaI and BamHI sites using PCR ligation for production of the synapsin-IRβ-mCherry plasmid. b. Photomicrograph of cultured neurons exposed 48 hrs to the synapsin-IRβ-mCherry, we estimate ~70–80% infection efficacy. Uninfected cells (dark) where used as controls. Inset shows both fluorescent and non-fluorescent cells. c. Representative inward currents obtained from a holding potential of −70 mV during determination of IV relationships (−60 to + 30 mV). d. Quantification of VGCC currents across groups shows no significant difference. e. Current density (pA/pF) of peak, late, and 50 ms tail were not altered by production of this second constitutively active form of the human insulin receptor. All data represent means +/− SEM