Neurotoxin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling
Senthil Selvaraj, … , Lutz Birnbaumer, Brij B Singh
Senthil Selvaraj, … , Lutz Birnbaumer, Brij B Singh
Published March 26, 2012
Citation Information: J Clin Invest. 2012;122(4):1354-1367. https://doi.org/10.1172/JCI61332.
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Research Article

Neurotoxin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling

Abstract

Individuals with Parkinson’s disease (PD) experience a progressive decline in motor function as a result of selective loss of dopaminergic (DA) neurons in the substantia nigra. The mechanism(s) underlying the loss of DA neurons is not known. Here, we show that a neurotoxin that causes a disease that mimics PD upon administration to mice, because it induces the selective loss of DA neurons in the substantia nigra, alters Ca2+ homeostasis and induces ER stress. In a human neuroblastoma cell line, we found that endogenous store-operated Ca2+ entry (SOCE), which is critical for maintaining ER Ca2+ levels, is dependent on transient receptor potential channel 1 (TRPC1) activity. Neurotoxin treatment decreased TRPC1 expression, TRPC1 interaction with the SOCE modulator stromal interaction molecule 1 (STIM1), and Ca2+ entry into the cells. Overexpression of functional TRPC1 protected against neurotoxin-induced loss of SOCE, the associated decrease in ER Ca2+ levels, and the resultant unfolded protein response (UPR). In contrast, silencing of TRPC1 or STIM1 increased the UPR. Furthermore, Ca2+ entry via TRPC1 activated the AKT pathway, which has a known role in neuroprotection. Consistent with these in vitro data, Trpc1–/– mice had an increased UPR and a reduced number of DA neurons. Brain lysates of patients with PD also showed an increased UPR and decreased TRPC1 levels. Importantly, overexpression of TRPC1 in mice restored AKT/mTOR signaling and increased DA neuron survival following neurotoxin administration. Overall, these results suggest that TRPC1 is involved in regulating Ca2+ homeostasis and inhibiting the UPR and thus contributes to neuronal survival.

Authors

Senthil Selvaraj, Yuyang Sun, John A. Watt, Shouping Wang, Saobo Lei, Lutz Birnbaumer, Brij B Singh

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Figure 5

AKT modulation is crucial for TRPC1-mediated neuroprotection.

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AKT modulation is crucial for TRPC1-mediated neuroprotection. (A) Brain ...
(A) Brain lysates from control and PD samples were immunoblotted with the respective antibodies. (B) Cells were treated with MPP+ (500 μM, 12 hours) with or without Ad-TRPC1, and Western blots were performed. For control, membranes were reprobed with anti-AKT1. (C) Relative expression of phospho-AKT1 (Ser473) is shown. Values are mean ± SD; *P < 0.05. (D) Cells were transfected with control vector or TRPC1pm (36 hours) and treated with MPP+ (500 μM) for 12 hours, lysed, and subjected to Western blotting with the indicated antibodies. (E) SH-SY5Y cells were treated with 5 μM Tg in SES buffer for 10 minutes with or without Ca2+ (2 mM), lysed, resolved using SDS-PAGE, and immunoblotted with AKT1 and phosphorylated AKT antibodies. (F) SH-SY5Y cells were treated with 5 μM Tg or 100 μM CCh for 15 minutes with or without pretreatment with SKF-96365 (50 μM, 45 minutes), processed, and probed with phospho-AKT1 (Ser473). Diagram shows the densitometric values of phospho-AKT1 (Ser473). Values are mean ± SEM. *P < 0.05 versus untreated controls. (G) SH-SY5Y cells were transfected with AKT1 siRNA and/or with Ad-TRPC1, followed by the addition of MPP+ (12 hours) and assayed for cell survival. Values represent mean ± SD from at least 3 independent experiments. *P < 0.05 versus untreated control. (H) SH-SY5Y cells were transfected with 50 pmol AKT1 siRNA, lysed after 36 hours, resolved, and immunoblotted with anti-AKT1 and anti–β-actin.