Neurotoxin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling
Senthil Selvaraj, Yuyang Sun, John A. Watt, Shouping Wang, Saobo Lei, Lutz Birnbaumer, Brij B Singh
Senthil Selvaraj, Yuyang Sun, John A. Watt, Shouping Wang, Saobo Lei, Lutz Birnbaumer, Brij B Singh
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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 2

TRPC1 mediates SOCE in SH-SY5Y cells, and MPP+ selectively decreases TRPC1 expression/function.

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TRPC1 mediates SOCE in SH-SY5Y cells, and MPP+ selectively decreases TRP...
(A) SH-SY5Y cells were treated with MPP+ (500 μM) for 12 hours. RNA was extracted, and quantitative RT-PCR was performed. Values represent mean ± SD from at least 3 independent experiments. *P < 0.05 versus untreated control. TRPC1 was evaluated after 15 cycles, whereas other TRPCs required at least 25 cycles. (B) Cells were treated with vehicle control or MPP+ and lysed, and proteins were subjected to SDS-PAGE followed by Western blotting with the indicated antibodies. Membranes were reprobed with anti–β-actin antibody to confirm equal loading. (C) Quantification (mean ± SD) of individual proteins from 3 or more independent experiments. Relative expression of each individual proteins was normalized to β-actin. *P < 0.05 versus untreated control. (D and E) Immunoprecipitation with anti-STIM1 antibody of lysates from control or MPP+-treated cells with or without Tg. Immunoblotting was performed using anti-TRPC1, -Orai1, and -STIM1 antibodies. (E) Brain lysates from control and PD samples were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (F) Paraffin-embedded sections of postmortem human SNpc samples obtained from control and PD patients were immunostained using TRPC1 and TH antibodies. Original magnification, ×40.