Neurotoxin-induced ER stress in mouse dopaminergic neurons involves downregulation of TRPC1 and inhibition of AKT/mTOR signaling (original) (raw)
Evidence that loss of ER Ca2+ induces ER stress in cultured cells and that ER stress is increased in PD and in neurotoxin-induced animal models that mimic PD. Previous studies have suggested that the unfolded protein response (UPR) could be one of the reasons for the loss of DA neurons (17); however, the mechanism that triggers the UPR is not known. Thus, we examined this mechanism by evaluating the status of UPR proteins, critical for initiating ER stress in in vivo and in vitro PD models. As shown in Figure 1, UPR markers (GRP78 and CHOP) were upregulated at both the mRNA (Figure 1B) and the protein levels (Figure 1A) in the SNpc region of postmortem brains from PD patients when compared with age-matched control samples (quantification shown in Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI61332DS1). Based on these findings, we assessed whether neurotoxin-induced experimental PD models show signs of an activated UPR. As shown in Figure 1C, GRP78 and CHOP were also increased in the SNpc of mice treated with MPTP. Moreover, addition of MPP+ to SH-SY5Y neuroblastoma cells significantly increased the expression of ER chaperones GRP78/Bip and GRP94 (Figure 1D). Importantly, increased expression of both GRP78 and GRP94 was observed after 3 hours MPP+ treatment and remained elevated for 12 hours (Figure 1D). In addition, CHOP, which is an important mediator of ER stress–induced apoptosis, was upregulated at 6 hours of MPP+ treatment (Figure 1D). Quantification of individual proteins showed a 60% increase in their expression after 12 hours of MPP+ treatment when compared with control cells (Figure 1E), indicating that MPP+ activates a persistent UPR in SH-SY5Y cells. To confirm these results, we performed luciferase assays to evaluate the activation of the ER stress response element (ERSE), which is present in the promoter region of various UPR target genes, including CHOP. As shown in Figure 1F, a time-dependent increase in ERSE activity was observed after MPP+ treatment, further suggesting that addition of MPP+ induces ER stress. Overall, the results obtained from PD patients and experimental models of PD clearly revealed that ER stress is activated in PD and could lead to neurodegeneration.
MPP+ induces ER Ca2+ depletion by attenuating SOCE, which causes ER stress. (A) Representative blots from the SNpc region of postmortem human PD (n = 5) and age-matched controls (n = 4). (B) RNA was extracted, and RT-PCR was performed. Values represent mean ± SD from 3 independent experiments (*P < 0.05). (C) Mice received 25 mg/kg MPTP as described in ref. 19, and SNpc samples were removed, processed, and immunoblotted using the respective antibodies. (D) SH-SY5Y cells were treated with 500 μM MPP+, and cell lysates were resolved and analyzed by Western blotting. Antibodies are labeled; β-actin was used as loading control. (E) Quantification (mean ± SD) from 3 or more independent experiments. The OD of GRP78 and GRP94 was normalized to β-actin. (F) Cells transfected with the ERSE promoter were lysed after MPP+ treatment, and luciferase assays were performed. Values represent mean ± SD from 3 independent experiments (*P < 0.05). (G) Analog plots of the fluorescence ratio (340/380 nm) from an average of 30–40 cells in each condition. (H) Quantification (mean ± SEM) of 340/380 ratio. *P < 0.05 versus untreated. (I) Tg-induced currents (mean ± SEM) were evaluated in control and MPP+-treated (12 hours) cells. The holding potential for current recordings was –80 mV. (J) I-V curves (mean current ± SEM) under these conditions; the average (8–10 recordings) current intensity under various conditions is shown in K. *P < 0.05 compared with control; values are shown as mean ± SEM. SKF, SKF-96365.
To determine the mechanism(s) underlying MPP+-induced ER stress, we investigated the effect of MPP+ on SOC-mediated Ca2+ entry, since SOC-mediated Ca2+ entry is essential for maintaining ER Ca2+ levels and loss of ER Ca2+ can initiate UPR. For evaluation of SOC-mediated Ca2+ entry, ER Ca2+ stores were depleted by the addition of thapsigargin (Tg, 2 μM), a sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump blocker. Importantly, in the absence of extracellular Ca2+, the increase in intracellular Ca2+ ([Ca2+]i) evoked by Tg (first peak) was significantly decreased following 3 hours of MPP+ treatment, when compared with control untreated cells (Figure 1, G and H). Subsequently, addition of external Ca2+ (1 mM), which initiates SOC-mediated Ca2+ entry, was decreased even within 1 hour of MPP+ treatment. Together these results suggest that loss of SOC-mediated Ca2+ entry could decrease ER Ca2+ levels and initiate the UPR response. To establish the identity of the SOC channel, we performed electrophysiological recordings. Addition of Tg induced an inward current that was nonselective and reversed between 0 and –5 mV (Figure 1, I–K; characterization of the currents shown in Supplemental Figure 1, B–I). The currents shown are recorded at a holding potential of –80 mV, and maximum peak currents were used for tabulation. The current-voltage (I-V) curves were made using a ramp protocol wherein current density was evaluated at various membrane potentials and plotted in the figure. Importantly, the channel properties were similar to those previously observed with TRPC1 channels and the activity was blocked by Gd3+ (26), suggesting that TRPC1 could contribute to the endogenous SOC-mediated Ca2+ entry channel in SH-SY5Y cells. Also, SKF-96365, a nonspecific TRPC channel blocker, decreased these inward currents in SH-SY5Y cells (Figure 1K and Supplemental Figure 1, H and I). Importantly, the MPP+ treatment significantly decreased SOC currents without altering the I-V relationship (Figure 1J). Similar results were also obtained in differentiated SH-SY5Y cells (retinoic acid treatment), where MPP+ treatment decreased SOC-mediated Ca2+ entry (Supplemental Figure 1J). Collectively, these results suggest that MPP+ decreases ER Ca2+ by diminishing SOC-mediated Ca2+ entry, which could lead to the activation of the UPR in these cells. Importantly, although 1-hour treatment with MPP+ or addition of MPP+ in the patch pipette decreased SOC-mediated Ca2+ entry, no cell death was observed until 12 hours of treatment with MPP+ (Supplemental Figure 1, K–M). Importantly, since ER Ca2+ was decreased after 3 hours and ER stress was induced after 6 hours of MPP+ treatment, it can be hypothesized that the loss of SOC-mediated Ca2+ entry is the early event that could lead to ER stress followed by neurotoxin-induced neuronal loss.
MPP+ decreases SOC-mediated Ca2+ entry by reducing TRPC1 expression. Given the importance of MPP+-induced ER stress caused by the loss of Ca2+ homeostasis, we next studied the expression of SOC(s) that were affected by prolonged treatment with MPP+. Although the molecular component(s) of SOCs in neurons are not known, members of TRPC and Orai that have been shown as candidates of SOC channels in many cell types (24, 27) could be present in neuronal cells. To address this issue, we performed real-time RT-PCR analysis to evaluate changes in TRPC mRNA. As shown in Figure 2A, a significant decrease in expression of TRPC1, but not other TRPCs (TRPC3, TRPC5, and TRPC6), was observed in MPP+-treated cells. TRPC4 and TRPC7 were not expressed in these cells. Western blot analysis confirmed the loss of TRPC1 after MPP+ treatment (12 hours), whereas no change in the expression of either Orai1 or STIM1 (a regulator for TRPC1 and Orai1) (24, 27) was observed (Figure 2, B and C).
TRPC1 mediates SOCE in SH-SY5Y cells, and MPP+ selectively decreases TRPC1 expression/function. (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.
Previous studies have shown that upon store depletion, STIM1 interacts with TRPC1 as well as with Orai1 and thereby initiates Ca2+ entry (24, 28). Thus, to further confirm that TRPC1 is critical for Ca2+ entry in these cells, we performed co-immunoprecipitation experiments. Importantly, Tg-mediated store depletion induced STIM1-TRPC1 interaction in SH-SY5Y cells, which was decreased in MPP+-treated cells (Figure 2D). In addition, association of STIM1 with Orai11, which is also shown to increase upon store depletion (28), was unaffected upon MPP+ treatment (Figure 2D; quantification provided in Supplemental Figure 2, A and B). Together these data suggest that TRPC1 is essential for store-operated Ca2+ entry (SOCE) in SH-SY5Y cells and that MPP+ decreases SOCE by decreasing TRPC1 expression and TRPC1-STIM1 interaction. While the above results suggest the significance of TRPC1 in an in vitro PD model, nothing is known about its function in PD patients. Thus, we further explored the potential relevance of TRPC1 in PD by evaluating TRPC1 expression in the SNpc of control and PD patients. Expression of TRPC1, but not Orai1 or STIM1, was decreased in the SNpc of PD patients as compared with age-matched control SNpc tissues (Figure 2E; quantification is shown in Supplemental Figure 2C). Moreover, TRPC1 was localized in or near the plasma membrane of the DA neurons, and expression was decreased in PD patients (Figure 2F). Similar results were also obtained in mouse primary DA cells, which also showed a significant decrease in TRPC1 expression when treated with MPP+ (Supplemental Figure 2F). Taken together, these findings suggest that PD patients have decreased TRPC1 expression in DA neurons; however, since these samples were obtained from patients at stages 3 and 4 of PD, caution should be used before interpreting these results, and more samples from patients at the early stage of disease are needed to confirm the loss of TRPC1 in PD samples.
Attenuation of SOC-mediated Ca2+ entry or deletion of TRPC1 induces ER stress. TRPC1 is ubiquitously expressed, including in the SNpc (30), and although TRPC1 allows plasma membrane Ca2+ influx in response to ER Ca2+ depletion, so far there are no reports showing that TRPC1 mediates SOCE in SH-SY5Y cells. To address this, we silenced TRPC1 using TRPC1 siRNA and assessed both ER Ca2+ release and Ca2+ influx upon store depletion. Interestingly, Tg-induced SOC currents were significantly decreased in TRPC1 siRNA–transfected cells when compared with control siRNA–transfected cells (Figure 3, A and B). RNAi-mediated knockdown of TRPC1 not only abolished Ca2+ entry activated by store depletion, but also led to a significant decrease in ER Ca2+ (Figure 3, C and D). The efficiency of siRNA-mediated TRPC1 knockdown in SH-SY5Y cells was confirmed by Western blotting (Supplemental Figure 2D). These results suggested that TRPC1 is essential for SOC-mediated Ca2+ entry and that deletion of TRPC1 affects ER/cytosolic Ca2+ homeostasis. We further studied whether blocking of TRPC1 function or silencing of TRPC1 induces ER stress in SH-SY5Y cells. Indeed, TRPC1 silencing induced a UPR, which was clearly evidenced by increased expression of GRP78, GRP94, and CHOP (Figure 3E; quantification shown in Supplemental Figure 2E). Also, silencing of TRPC1 led to increased phosphorylation of PERK and its downstream effector eIF2α. Similarly, blocking TRPC1 channel activity with SKF-96365 led to an increase in the UPR and inhibited protein translation by increasing eIF2α phosphorylation (Figure 3E; quantification shown in Supplemental Figure 3, A–G). Interestingly, silencing of TRPC3 failed to upregulate UPR (Supplemental Figure 3F). These results indicate that inhibition of SOC-mediated Ca2+ entry could be critical in inducing ER stress in SH-SY5Y cells.
TRPC1 functions as an endogenous SOCE channel, and knockdown of TRPC1 induces ER stress. (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.
To further validate this hypothesis, we repressed SOC-mediated Ca2+ entry by silencing STIM1, which again induced ER stress by increasing the expression of GRP78, GRP94, CHOP, and phospho-eIF2α (Figure 3F; quantification shown in Supplemental Figure 4, A–C). Moreover, either silencing of TRPC1 or STIM1 or blocking of TRPC channel activity decreased the survival of SH-SY5Y cells (Figure 3G). Consistent with these results, Trpc1–/– mice had increased GRP78, GRP94, CHOP, and p-eIF2α levels (Figure 3H and Supplemental Figure 4D) compared with wild-type (Trpc1+/+). To determine whether SOC channels are also present in native DA neurons, we performed electrophysiological recordings in DA neurons (SNpc) of Trpc1+/+ and Trpc1–/– mice. Interestingly, addition of Tg in the SNpc of Trpc1+/+ initiated a linear, nonselective current in DA neurons, which was similar to the currents observed in SH-SY5Y cells and was absent in Trpc1–/– mice (Figure 3, I–K, and Supplemental Figure 4E). The electrical signature present in DA neurons was used to confirm that indeed these are DA neurons (Figure 3K and Supplemental Figure 4F). Collectively, these results reveal that TRPC1 mediates SOC-mediated Ca2+ entry in DA cells/neurons and that inhibition of Ca2+ entry (by TRPC1 or STIM1 silencing or blocking of TRPC1 channel activity) prevents optimal refilling of ER Ca2+, thereby inducing ER stress.
Overexpression of TRPC1 restores SOC-mediated Ca2+ entry and attenuates ER stress. The results shown above suggest that TRPC1 could be critical for SOC-mediated Ca2+ entry and in maintaining ER Ca2+ homeostasis; however, to confirm the role of TRPC1, we next overexpressed TRPC1 and evaluated its role in ER Ca2+ homeostasis and the ER stress response. SH-SY5Y cells were infected with Ad-HA-TRPC1 at an MOI of 5, and Ad-GFP (MOI of 5) was used as control. The efficiency of TRPC1 expression (HA-TRPC1) was confirmed by Western blotting (Supplemental Figure 5A). Importantly, overexpression of TRPC1, but not TRPC3 or Orai1, increased SOC currents (without changing the I-V relationship) and led to increased cell survival (Figure 4, A and B, and Supplemental Figure 5, B and C). The transfection efficiency of myc-tagged TRPC3 and Orai1 was confirmed by Western blotting (Figure 4F and Supplemental Figure 5A). Overexpression of TRPC1 also amended ER Ca2+ and restored SOC-mediated Ca2+ entry in MPP+-treated SH-SY5Y cells when compared with control GFP-expressing cells treated with MPP+ (Figure 4, C and D). In agreement with this finding, TRPC1 overexpression decreased the elevations in GRP78, GRP94, and CHOP that were induced after MPP+ treatment, indicating that TRPC1 could prevent prolonged UPR activation (Figure 4E; quantification shown in Supplemental Figure 5, D and F). Phosphorylation of PERK, an important transducer of the UPR, and downstream signaling targets (eIF2α) was also increased after MPP+ treatment, but decreased in TRPC1-overexpressing cells (Figure 4E). Similarly, prolonged activation of the UPR, which has been shown to activate JNK and leads to cell death (31), was increased in MPP+-treated cells but restored to normal in TRPC1-overexpressing cells.
TRPC1 overexpression restores SOCE and attenuates ER stress. (A) Tg-induced currents (at –80 mV, mean ± SEM) were evaluated in control and TRPC1-overexpressing SH-SY5Y cells. (B) Average (mean ± SEM from 7–9 recordings) current intensity under various conditions. (C) SH-SY5Y cells were treated with MPP+ for 12 hours with or without TRPC1 overexpression, and analog plots (mean ± SEM) of the fluorescence ratio (340/380 nm) are shown. Fluorescence ratio was measured in the presence of 2 μM Tg with and without 1 mM Ca2+. (D) Quantification (mean ± SEM) of fluorescence ratio; *P < 0.05 versus MPP+-treated cells. (E–G) TRPC1-, Orai1-, or TRPC1pm-overexpressing SH-SY5Y cells were treated with MPP+ for 12 hours, lysed, resolved, and subjected to Western blotting with the indicated antibodies. (H) Cell survival (MTT assay) under different conditions. Values are expressed as mean ± SD. *P < 0.05 versus MPP+-treated cells.
Although Orai1 overexpression did not increased Tg-induced SOC-mediated Ca2+ entry in SH-SY5Y cells (Figure 4B), we still evaluated its role in regulating ER stress, since it has been also shown to contribute to SOC current in some cells (27–29). As shown in Figure 4F, Orai1 overexpression did not prevent the MPP+-induced ER stress response (quantification of individual proteins shown in Supplemental Figure 5, G–I). To further confirm that the TRPC1-dependent decrease in UPR was mediated by SOC-mediated Ca2+ entry through TRPC1, we overexpressed a TRPC1 pore mutant (TRPC1pm) in SH-SY5Y cells. Consistent with our previous results (32), overexpression of TRPC1pm failed to increase Tg-induced SOC currents in SH-SY5Y cells (Figure 4B). Interestingly, SH-SY5Y cells overexpressing TRPC1pm also failed to inhibit MPP+-induced UPR and did not protect against neurotoxin-induced cell death (Figure 4, G and H; quantification shown in Supplemental Figure 5, J–L). Taken together, these results suggest that MPP+ induces ER stress by downregulating the function of TRPC1 and that overexpression of functional TRPC1 is crucial for maintaining ER Ca2+ homeostasis and inhibiting MPP+-induced ER stress response, thereby preventing neurodegeneration.
Modulation of AKT is essential for TRPC1-mediated neuroprotection. To better understand the link between TRPC1 and cell survival, we searched for downstream signaling molecules that could be responsible for TRPC1-mediated protection. Given the already known relationship between AKT and neuroprotection (33), we studied whether AKT plays a role in TRPC1-mediated neuroprotection. As shown in Figure 5A, a decrease in AKT phosphorylation was observed in PD patient samples (quantification of individual blots shown in Supplemental Figure 6, A and B). Interestingly, MPP+ treatment also significantly decreased AKT1 phosphorylation without affecting total AKT1 levels in SH-SY5Y cells (Figure 5B). In addition, overexpression of full-length TRPC1, but not TRPC1pm, prevented the decrease in AKT phosphorylation seen after MPP+ treatment (Figure 5, B–D; quantification of phospho-AKT [Ser473] shown in Supplemental Figure 6C). In addition, quantification of the phospho-AKT (Ser473) indicated an approximately 50% inhibition of the AKT activity after MPP+ treatment, which was restored to approximately 75% in cells overexpressing TRPC1 and treated with MPP+ (Figure 5C). We next examined whether SOCE that is dependent on TRPC1 activates AKT phosphorylation in SH-SY5Y cells. Interestingly, SH-SY5Y cells treated with Tg (5 μM) in the absence of external Ca2+ failed to show AKT phosphorylation (Figure 5E), suggesting that Ca2+ influx through SOCs was necessary for AKT1 phosphorylation, as Ca2+ release from internal ER stores by itself was not sufficient to activate AKT1 phosphorylation (quantification shown in Supplemental Figure 6D). Moreover, stimulation of TRPC1 by Tg or carbachol (CCh) (known activators of TRPC1) significantly increased AKT1 phosphorylation (Ser473) when compared with control untreated cells (Figure 5F). Furthermore, addition of SKF-96365 prevented the activation of AKT1 induced by Tg and CCh (Figure 5F). To evaluate whether other sources of Ca2+ influx can also stimulate AKT phosphorylation, we stimulated SH-SY5Y cells with oleyl-acetyl-glycerol (OAG), which is known to activate other TRPC channels and is independent of store depletion (34). Interestingly, AKT phosphorylation was not altered upon OAG stimulation (Supplemental Figure 6E), suggesting that the effect observed in AKT phosphorylation is dependent on Ca2+ entry via the SOC channel. In addition, expression of brain-derived neurotrophic factor (BDNF) was also evaluated, since Ca2+ entry is known to induce the expression of these factors, which has been shown to increase protection of DA cells. As indicated in Supplemental Figure 6F, addition of MPP+ significantly decreased BDNF expression; however, no increase in BDNF expression was observed in cells overexpressing TRPC1, suggesting that TRPC1-mediated protection is independent of BDNF. To further evaluate the role of TRPC1 and AKT in cell survival, we performed MTT assays. MPP+-treated cells showed a significant decrease in neuronal survival, which was inhibited by TRPC1 overexpression (Figure 5G). Additionally, silencing of AKT1 completely blocked TRPC1-mediated neuroprotection against MPP+, indicating that AKT1 plays a crucial role in TRPC1-mediated neuroprotection (Figure 5, G and H). These results strongly suggest that TRPC1-mediated Ca2+ influx is vital for AKT1 activation in SH-SY5Y cells, which is critical for their survival.
AKT modulation is crucial for TRPC1-mediated neuroprotection. (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.
TRPC1 overexpression protects DA neurons in an in vivo MPTP model of PD. While the above results strongly suggest the importance of TRPC1 in cellular models of PD, nothing is known regarding the role of TRPC1 in an in vivo PD model. Thus, we overexpressed HA-TRPC1 in the SNpc region by intranigral injection of Ad-TRPC1 as shown in Figure 6A. Control mice received intranigral injection of Ad-GFP, and as indicated in Figure 6B, GFP was expressed in DA neurons of the SNpc and colocalized with tyrosine hydroxylase (TH, a marker for DA neurons), indicating that we had been successful in targeting the SNpc with our injections. Thus, we next injected Ad-HATRPC1 and confirmed by confocal microscopy the overexpression of TRPC1 (HA-TRPC1), which also colocalized with TH-positive neurons (DA neurons) of SNpc (Figure 6C). Also as expected, MPTP treatment decreased the expression of TH and TRPC1 in SNpc (Figure 6D; quantification of individual proteins shown in Supplemental Figure 7, A and B). Importantly, MPTP treatment induced ER stress in DA neurons by activating the UPR, which was inhibited in mice treated with MPTP but overexpressing TRPC1 (Figure 6D; quantification of individual proteins shown in Supplemental Figure 7, C–E). To further understand the role of TRPC1 in the protection of DA neurons, we evaluated TH staining under these conditions. MPTP induces neuronal degeneration of DA neurons, which was indicated by the decrease in TH levels in MPTP-injected mice (Figure 6E). Importantly, a significant increase in TH-positive neurons was observed in TRPC1-overexpressing mice treated with MPTP. Quantification of the data indicated approximately 80% survival of DA neurons in TRPC1-overexpressing mice following MPTP treatment (Figure 6F). To further confirm these results, we quantified TH-positive neurons in wild-type and Trpc1–/– mice, since the results shown above indicated that Trpc1–/– mice have decreased SOC-mediated Ca2+ entry and increased ER stress. A significant decrease in TH-positive neurons was observed in Trpc1–/– mice even without MPTP treatment (Figure 6G).
TRPC1 overexpression protects DA neuron in an in vivo MPTP model of PD. (A) Graphic representation of intranigral injection protocol. (B and C) Unilateral injection of GFP or HA-TRPC1 adenovirus (3 × 107 particles) into the SNpc (n = 6). Brain samples containing the SNpc region were sectioned (12 μm) and stained for TH immunofluorescence. Expression of GFP and TH in the SNpc and in ventral tegmental area (VTA) were evaluated. HA-TRPC1 colocalized with TH (C, bottom row). Scale bars in C: 20 μm. (D) HA-TRPC1 or GFP adenovirus was injected directly into the SNpc of animals (n = 6–8 per group) 7 days before MPTP treatment. After 1 week of MPTP injection, SNpc samples were removed and subjected to SDS-PAGE and immunoblotted with the respective antibodies. Control GFP virus–injected mice received an equal volume of saline. (E) Representative TH staining of the ipsilateral sides of animals injected with the indicated virus with and without MPTP. (F) Quantification of the number of TH-positive neurons from ipsilateral or contralateral sides for the indicated treatment groups. Data are mean ± SEM. *P < 0.05. (G) Brain samples containing the SNpc from wild-type and Trpc1–/– mice were sectioned (12 μm) and stained for TH. Quantification of TH-positive neurons is shown in the graph. Data are presented as mean ± SEM. *P < 0.05. Original magnification, ×40; magnified images in B, ×100.
In vivo TRPC1 overexpression activates the AKT/mTOR pathway. The above results clearly suggest that TRPC1 overexpression prevented prolonged UPR activation and attenuated the degeneration of DA neurons in an in vivo PD model. However, the signaling intermediates linking TRPC1 and DA neuron survival in PD are still unknown. We therefore examined whether in vivo overexpression of TRPC1 would activate the AKT/mTOR pathway. Importantly, MPTP treatment attenuated the activation of mTOR, a kinase that regulates neuronal survival, in SNpc (Figure 7A; quantification shown in Supplemental Figure 7F). This mTOR suppression could in turn suppress its downstream proteins that are involved in cellular signaling. Consistent with our in vitro observations (Figure 5B), as shown in Figure 7B, treatment with MPTP decreased the phosphorylation of AKT at both Ser473 and Thr378 in the SNpc, as indicated by Western blotting. These observations indicate that MPTP impaired the functions of AKT/mTOR in DA neurons and thereby induced neurodegeneration. Interestingly, TRPC1 overexpression in SNpc significantly restored the activation of mTOR and its downstream targets (Figure 7A). Consistent with this, TRPC1 overexpression in SNpc prevented the suppression of AKT1 activation by MPTP (Figure 7B; quantification shown in Supplemental Figure 7, G and H). These results suggest that TRPC1 is necessary to restore AKT/mTOR activation and in the protection of DA neurons.
TRPC1 overexpression activates the AKT/mTOR pathway in mice. (A and B) SNpc regions were removed from control animals and animals overexpressing TRPC1 that had been treated or not with MPTP, and were subjected to SDS-PAGE and immunoblotting with the respective antibodies. Data are representative of 2–3 independent experiments. (C) Model for MPP+/MPTP-induced DA loss and TRPC1-mediated neuroprotection. MPP+/MPTP decreases the expression of TRPC1 and SOC-mediated Ca2+ influx either directly or indirectly via mitochondrial dysfunction. This leads to prolonged ER Ca2+ depletion and activation of the UPR and subsequent ER stress–mediated neurodegeneration. In contrast, TRPC1 overexpression restores SOCE function and maintains ER Ca2+ homeostasis. Further, Ca2+ influx through TRPC1 activates AKT/mTOR-mediated survival mechanisms in DA cells, which leads to increased neuronal survival.