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 3

TRPC1 functions as an endogenous SOCE channel, and knockdown of TRPC1 induces ER stress.

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TRPC1 functions as an endogenous SOCE channel, and knockdown of TRPC1 in...
(A) Tg-induced currents (mean ± SEM) were evaluated in control siRNA– and TRPC1 siRNA–transfected cells. The holding potential for the recordings was –80 mV, and an I-V curve (mean current ± SEM) under these conditions is shown in B. (C) Analog plots of the 340/380 ratio from an average of 40–60 cells are shown. (D) Quantification (mean ± SEM) of fluorescence ratio. *P < 0.05 versus untreated control; numbers of cells imaged are indicated. (E) SH-SY5Y cells were transfected with control siRNA or TRPC1 siRNA, or treated with 50 or 100 μM SKF-96365 for 24 hours. Cells were lysed, subjected to SDS-PAGE, and immunoblotted with the respective antibodies. (F) SH-SY5Y cells transfected with control or STIM1 siRNA were lysed and immunoblotted with respective antibodies. (G) MTT assay was performed in control, TRPC1 siRNA–transfected, SKF-96365–treated (100 μM for 24 hours), or STIM1 siRNA–transfected cells. Values represent mean ± SD from at least 3 independent experiments. *P < 0.05 versus control. (H) Tissue lysates from the SNpc region of wild-type and Trpc1–/– mice were subjected to SDS-PAGE and immunoblotted with the respective antibodies. (I and J) Endogenous currents (mean ± SEM) and relative I-V curves (mean currents ± SEM) upon Tg stimulation in DA neurons in the SNpc of Trpc1+/+ and Trpc1–/– mice. The currents shown were recorded at a holding potential of –70 mV. (K) DA neurons induced a large inward current by a hyperpolarizing pulse of 60 mV, indicating the electrical signature of DA neurons.