Thioredoxin-1 Inhibits Golgi Stress Induced by Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine

Abstract

Aims:

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc) and the aggregation of alpha-synuclein (α-syn) in Lewy bodies. Emerging studies find that disruption of the Golgi structure and Golgi stress are involved in PD. Thioredoxin-1 (Trx-1) is a redox regulatory protein that protects DA neurons from methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) damage. However, whether Trx-1 can protect DA neurons against MPTP-induced Golgi stress is still unknown.

Results:

We first made sure that MPTP led to the loss of DA neurons in the SNpc and motor impairment in mice, which was reversed in Trx-1 overexpression mice. Trx-1 overexpression suppressed Golgi apparatus fragmentation, α-syn aggregation, oxidative stress, and protein kinase C zeta expression increased by MPTP. Trx-1 overexpression restored the colocalization of Trx-1 and tyrosine hydroxylase with Golgi matrix protein 130 (GM130), decreased by MPTP. Moreover, Trx-1 overexpression suppressed the increased co-localization of Leucine-rich repeat kinase 2 and Ras-associated binding protein 29 with vacuolar protein sorting-associated protein 52 induced by MPTP. Trx-1 overexpression suppressed the expression changes of ADP-ribosylation factor 4 and heat shock protein 47, and their colocalization with GM130 induced by MPTP.

Innovation:

Our study reveals a novel mechanism, whereby Trx-1 inhibits Golgi stress in DA neuron induced by MPTP.

Conclusions:

These results suggest that Trx-1 may regulate the development of PD through inhibiting Golgi stress and is a potential new molecular target and therapeutic strategy for Golgi stress involved in PD. Antioxid. Redox Signal. 44, 661–675.

Keywords

Parkinson’s disease thioredoxin-1 oxidative stress Golgi stress

Introduction

Parkinson’s disease (PD) is the second most common neurodegenerative disease, seriously affecting the quality of daily life of millions of patients worldwide (Xu et al., 2024). The pathological hallmarks of PD are the loss of dopaminergic (DA) neurons in the midbrain substantia nigra pars compacta (SNpc) and the accumulation of aggregated alpha-synuclein (α-syn) in Lewy bodies (Zalon et al., 2024). Potential risk factors for PD mainly include genetic factors (Lim and Klein, 2024) and environmental factors (Aravindan et al., 2024). Genetic studies have shown that mutations in Leucine-rich repeat kinase 2 (LRRK2) are associated with an increased risk of PD (Yuan et al., 2024). PKC zeta, an atypical protein kinase C (PKC) isoenzyme, has been implicated in oxidative stress regulation (Banan et al., 2002). Ras-associated binding proteins (Rab) are a class of small GTPases involved in intracellular vesicle transport and play an important regulatory role in membrane transport in eukaryotic cells, whose destruction is a pathological hallmark of PD (Ebanks et al., 2019). A subset of Rab GTPases, including Rab29 (also known as Rab7L1), is a substrate for LRRK2 (Steger et al., 2016). The gene encoding Rab29 is located in a genetic locus known as parkinson's disease locus 16 (PARK16), which has been linked to an increased risk of PD (Simon-Sanchez et al., 2009).

Innovation

Although Golgi stress is recognized to play a critical role in the pathogenesis of PD, its exact mechanisms remain unclear. This study demonstrated that GA-related molecules, ROS, PKC zeta, CREB3/ARF4, and HSP47 were involved in MPTP-induced Golgi stress. Trx-1 could reverse the above changes, thereby ameliorating the DA neurons in PD mice. These findings indicate that targeting Trx-1 may represent a potential therapeutic strategy for the treatment of PD (Fig. 1).

In addition, there is a growing body of evidence suggesting that Golgi apparatus (GA) stress also plays a crucial role in neurodegenerative diseases, such as PD (Shirai and Yamauchi, 2024). GA is an organelle consisting of several flat cisternae including cis-, medial-, and trans-Golgi, which make up the cis-Golgi network and trans-Golgi network (TGN) (Li et al., 2019). Its primary function is to modify, sort, and transport proteins produced in the endoplasmic reticulum (ER) (Shirai and Yamauchi, 2024). Vacuolar protein sorting-associated protein 52 (VPS52) is a subunit of the Golgi-associated retrograde protein (GARP) complex (Beilina et al., 2020), which is primarily localized in the TGN, and is necessary for proper sorting of different kinds of proteins (O’Brien et al., 2023). Golgi reassembly stacking proteins (GRASPs) are another type of Golgi matrix proteins that have been shown to play a crucial role in the formation and maintenance of Golgi stacks and the biological function of the GA (Rabouille and Linstedt, 2016). Currently, several pathways have been identified to regulate the mammalian GA stress response, especially the heat shock protein 47 (HSP47) and cAMP responsive element binding protein 3 (CREB3)/Adenosine Diphosphate (ADP)-ribosylation factor 4 (ARF4) pathways (Mohan et al., 2023).

Thioredoxin-1 (Trx-1) is a multifunctional protein that is widely presented in living organisms (Holmgren, 1985). Trx-1 plays a crucial role in a variety of cellular functions, including promoting proliferation, inhibiting apoptosis, and maintenance of redox homeostasis (Powis and Montfort, 2001). Our previous research has demonstrated that Trx-1 protects DA neurons in the SNpc of mice from the toxicity of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), one of the compounds commonly used to create PD models, by modulating the ER stress and ferroptosis pathways (Bai et al., 2021; Zeng et al., 2014). Recently, we have also discovered that Trx-1 reduces α-syn accumulation in MPTP PD mice through the autophagy–lysosome pathway (Gu et al., 2024). However, it is still unclear whether Trx-1 regulates GA stress in PD.

To investigate the role of Trx-1 in the GA stress of PD, we established a mouse model of PD in the mice of overexpression of Trx-1. We detected Golgi morphology and expression of structure-related proteins in the SNpc of mice through immunofluorescence (IF) and Western blot. Finally, we examined the effects of Trx-1 on the molecules involved in GA stress. These results provide a new insight into the role of GA stress in the development of PD and suggest a potential treatment approach for PD.

FIG. 1.

Summary graphic illustration showing how overexpression of Trx-1 inhibits MPTP-induced Golgi stress in PD mice. In PD, MPTP treatment induces ROS and increases PKC zeta level, and induces the expression changes of ARF4 and HSP47, thereby leading to Golgi stress and Golgi fragmentation. Mechanistically, MPTP increases the accumulation of α-syn on the GA and promotes colocalization of LRRK2, Rab29, and VPS52. Trx-1 overexpression can counteract MPTP-induced Golgi stress by modulating these molecules. α-syn, alpha-synuclein; ARF4, ADP-ribosylation factor 4; GA, Golgi apparatus; GM130, Golgi matrix protein 130; GRASP65, Golgi reassembly stacking protein 65; HSP47, heat shock protein 47; LRRK2, leucine-rich repeat kinase 2; MPTP, methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD, Parkinson’s disease; PKC, protein kinase C; Rab29, Ras-associated binding protein 29; ROS, reactive oxygen species; Trx-1, Thioredoxin-1; VPS52, vacuolar protein sorting-associated protein 52.

Results

Overexpression of Trx-1 improved motor impairment in MPTP-induced PD mice

To assess the effect of Trx-1 on motor performance in MPTP-treated mice, a series of behavioral tests were conducted (Supplementary Fig. S1A). The results showed that MPTP caused a significant decrease in the distance between the two forepaws, indicating unstable gait in the mice. However, overexpression of Trx-1 significantly improved this gait instability (Supplementary Fig. S1B). In addition, the traction test revealed a decrease in the grasping ability of the hind paws in MPTP-treated mice, which was restored by Trx-1 overexpression (Supplementary Fig. S1C).

In PD, the reduction of dopamine released from the SNpc neurons projecting to the striatum disrupts the balance of basal ganglia motor circuits, which constitutes the primary cause of motor deficits (Giguere et al., 2018). Tyrosine hydroxylase (TH), as the rate-limiting enzyme in dopamine biosynthesis, is commonly used as a specific marker for DA neurons. As shown in Supplementary Figure S1D, MPTP reduced TH expression level, while its level was significantly increased in the Trx-1 overexpression group, indicating that Trx-1 was able to resist MPTP-induced TH reduction.

These results suggest that overexpression of Trx-1 suppressed the loss of DA neurons caused by MPTP, ultimately improving motor impairment in PD mice, as our previous studies (Gu et al., 2024).

Overexpression of Trx-1 inhibited GA fragmentation in the SNpc of MPTP-induced mice

Growing evidence suggests that GA fragmentation serves as an early hallmark of neurodegenerative diseases such as PD (Mohan et al., 2023). However, whether Trx-1 can protect against MPTP-induced GA fragmentation remains unknown. As shown in Figure 2A, Trx-1 expression in the SNpc of MPTP-treated mice was significantly reduced compared with the control group, but overexpression of Trx-1 restored its level. Moreover, electron microscopy (EM) results showed that the GA structure was damaged in the SNpc of MPTP-treated mice. As shown in Figure 2B, the flat vesicles of the GA in the control group were regular and structurally intact, while the vesicles were destruction and fragmentation in MPTP group, which were restored in Trx-1 overexpression mice. Quantitative analysis also revealed that the proportion of fragmented GA in the SNpc was significantly increased in MPTP mice compared with the control group, whereas Trx-1 overexpression effectively suppressed MPTP-induced GA fragmentation (Fig. 2C). These results demonstrated that overexpression of Trx-1 effectively protected the GA from MPTP-induced damage.

FIG. 2.

Overexpression of Trx-1 suppressed GA-fragmented damage induced by MPTP in the SNpc of mice. (A) Western blot analysis of Trx-1 expression in the SNpc in each group (n = 6). (B) Representative electron micrographs illustrate the Golgi morphology in the SNpc in each group (n = 3). Areas in black boxes were enlarged and shown in the right panel. The white arrowheads indicate the Golgi structure. G, Golgi apparatus; N, nucleus; M, mitochondria. (C) Quantitative analysis of proportion of fragmented GA in each group (n = 3). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, **p < 0.01, ***p < 0.001. SEM, standard error of the mean; SNpc, substantia nigra pars compacta.

Upregulated PKC zeta expression and oxidative stress in GA induced by MPTP were attenuated in Trx-1 overexpression mice

To further elucidate the mechanism of Trx-1 in maintaining GA structural integrity, we performed IF costaining for Golgi matrix protein 130 (GM130) and Trx-1 in the SNpc of mice. GM130 is a component of the cis-Golgi matrix and commonly used as one of the Golgi-specific markers (Nakamura et al., 1995). As shown in Figure 3A, the control group exhibited intact GA distribution around the nucleus, while the MPTP induced a significant amount of fragmentation in the GA in the SNpc. Interestingly, GA fragmentation induced by MPTP was significantly rescued in the Trx-1 Tg + MPTP mice, indicating a protective role of Trx-1 in maintaining the morphology and structure of the GA. Overexpression of Trx-1 also restored the MPTP-induced reduction in GM130 fluorescence intensity (Fig. 3B). Furthermore, there was a noticeable colocalization between Trx-1 and GM130 in the MPTP group, suggesting that Trx-1 specifically located in GA is related to GA stress (Fig. 3A, C). To determine whether the GA of DA neurons was also affected by MPTP, we performed TH and GM130 costaining of the SNpc (Fig. 3D). Compared with the control group, the MPTP group showed obvious GA fragmentation and a significant decrease in TH level. However, in the Trx-1 overexpression group, the GA structure was intact, and TH expression was increased, consistent with the results in Supplementary Figure S1D. Overexpression of Trx-1 effectively inhibited MPTP-induced reduction of TH level and GA fragmentation. In addition, colocalization of TH and GM130 was restored in the Trx-1 overexpression group (Fig. 3D–E), indicating that Trx-1 can protect the structural stability of the GA in DA neurons.

FIG. 3.

Overexpression of Trx-1 suppressed GA-fragmented damage in DA neurons induced by MPTP. (A) Immunofluorescence costaining of GM130 (green) and Trx-1 (red) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of GM130 and Trx-1. (B) Quantitative analysis of GM130 relative fluorescence intensity in each group (n = 3). (C) Quantitative analysis of colocalization of GM130 and Trx-1 in each group (n = 3). (D) Immunofluorescence costaining of GM130 (green) and TH (red) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of GM130 and TH. (E) Quantitative analysis of colocalization of GM130 and TH in each group (n = 3). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. GM130, Golgi matrix protein 130; TH, tyrosine hydroxylase.

GA has been demonstrated to be involved in oxidative stress, and excessive oxidative stress can disrupt the integrity of GA structure (Alvarez-Miranda et al., 2015). Accumulating evidence suggests that PKC zeta also plays a pivotal role in oxidative stress regulation (Banan et al., 2002). To determine whether PKC zeta is involved in oxidative stress in PD, its expression in the SNpc was measured by Western blot. Compared with the control group, MPTP induced significant upregulation of PKC zeta in the SNpc of mice. Interestingly, PKC zeta expression was higher in Trx-1 overexpression mice than control mice; however, PKC zeta expression was not higher in these Trx-1 overexpression mice treated by MPTP than that in Trx-1 overexpression mice (Fig. 4A). To quantitatively assess oxidative stress, dihydroethidium (DHE) staining was employed. As shown in Figure 4B–C, MPTP induced a significant increase in reactive oxygen species (ROS) level, which was effectively mitigated by Trx-1 overexpression in GA. Importantly, the GM130 level exhibited a negative correlation with ROS level (Fig. 4D). These results suggest that PKC zeta induction is related to oxidative stress induced by MPTP in GA.

FIG. 4.

Overexpression of Trx-1 upregulated PKC zeta expression and attenuated oxidative stress in MPTP-induced PD mice. (A) Western blot analysis of PKC zeta expression in the SNpc in each group (n = 6). (B) Immunofluorescence costaining of GM130 (green) and ROS (red, measured by DHE staining) in the SNpc in each group (n = 3). (C) Quantitative analysis of ROS relative fluorescence intensity in each group (n = 3). (D) Correlation between ROS level and GM130 expression per cell analyzed by two-tailed Pearson’s test (n = 12). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, **p < 0.01, ***p < 0.001.

Trx-1 restored the expression of GRASP65 decreased by MPTP in GA

GM130 and GRASP65 are two Golgi matrix proteins which are essential for maintaining its normal structure and function (Barr et al., 1998; Hu et al., 2015). We further investigated GRASP65 expression in the GA by costaining of GM130 and GRASP65. In the control group, there was a high level of colocalization between GM130 and GRASP65. However, in the MPTP group, there was a significant decrease in colocalization between the two proteins. Similar to the control group, in the Trx-1 overexpression group, a large amount of colocalization was observed (Fig. 5A–B).

FIG. 5.

Trx-1 restored GRASP65 expression in the GA. (A) Immunofluorescence costaining of GM130 (green) and GRASP65 (red) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of GM130 and GRASP65. (B) Quantitative analysis of colocalization of GM130 and GRASP65 in each group (n = 3). (C) Western blot analysis of GRASP65 expression in the SNpc in each group (n = 6). (D) Western blot analysis of GM130 expression in the SNpc in each group (n = 6). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Next, the expression levels of GM130 and GRASP65 in the SNpc were also detected by Western blot. Similar to the IF results, MPTP significantly decreased the expression of GRASP65 and GM130 in the SNpc compared with the control group, and overexpression of Trx-1 inhibited the MPTP-induced reduction of GM130 and GRASP65 expression (Fig. 5C–D), suggesting that Trx-1 has a protective effect against MPTP-induced GA damage.

Overexpression of Trx-1 reduced α-syn expression in GA in MPTP-induced PD mice

Our previous study revealed a significant increase in the expression level of α-syn in MPTP-treated mice. In addition, we found that overexpression of Trx-1 was able to effectively reduce the accumulation of α-syn in the SNpc caused by MPTP (Gu et al., 2024). Studies have shown that overexpression of either wild-type or mutant (A53T) α-syn increases Golgi fragmentation in the ventral midbrain of rats (Furlong et al., 2020; Koch et al., 2015). Therefore, we aimed to investigate whether α-syn is accumulated in GA. As shown in Figure 6A, MPTP-treated group showed a significant increase in α-syn expression compared with control group. However, overexpression of Trx-1 effectively reduced α-syn accumulation induced by MPTP in GA. Quantitative analysis demonstrated that Trx-1 overexpression significantly attenuated MPTP-induced colocalization of α-syn with GM130 (Fig. 6B). These results suggest that Trx-1 may prevent MPTP-induced α-syn aggregation in GA.

FIG. 6.

Overexpression of Trx-1 inhibited α-syn expression in GA in MPTP-induced PD mice. (A) Immunofluorescence costaining of GM130 (green) and α-syn (red) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of GM130 and α-syn. (B) Quantitative analysis of colocalization of GM130 and α-syn in each group (n = 3). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, ***p < 0.001.

Overexpression of Trx-1 inhibited colocalization of LRRK2, Rab29, and VPS52 in MPTP-induced PD mice

LRRK2 is located at the cytoplasm and various membranous organelles, including the GA and lysosomes. Rab29 is primarily localized in the Golgi complex and has been shown to physically interact with LRRK2 (McGrath et al., 2021; Steger et al., 2017). In addition, Rab29 can be directly phosphorylated by LRRK2 (Fujimoto et al., 2018). VPS52 is a subunit of GARP complex and primarily localized in the TGN. To demonstrate the potential effect of Trx-1 on these three proteins, we first performed IF staining. As shown in Figure 7A, colocalization of LRRK2, Rab29, and VPS52 was observed. MPTP treatment significantly increased the colocalization of LRRK2, Rab29, and VPS52 in the SNpc of mice, and this change was effectively suppressed by Trx-1 overexpression (Fig. 7B).

FIG. 7.

Overexpression of Trx-1 inhibited the colocalization of LRRK2, Rab29, and VPS52. (A) Immunofluorescence costaining of LRRK2 (red), Rab29 (white), and VPS52 (green) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of LRRK2, Rab29, and VPS52. (B) Quantitative analysis of colocalization of LRRK2, Rab29, and VPS52 in each group (n = 3). (C) Western blot analysis of LRRK2 expression in the SNpc in each group (n = 6). (D) Western blot analysis of Rab29 expression in the SNpc in each group (n = 6). (E) Western blot analysis of VPS52 expression in the SNpc in each group (n = 6). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.

Western blot results showed that the expression of these three proteins was significantly increased in the SNpc of MPTP-treated mice, and overexpression of Trx-1 could significantly inhibit this increase (Fig. 7C–E), which was consistent with IF result.

Overexpression of Trx-1 suppressed the changes of ARF4 and HSP47 expression induced by MPTP

To further explore the mechanism of Trx-1 regulating GA stress induced by MPTP, the major signaling pathways involved in GA stress, such as CREB3/ARF4 and HSP47, were examined. Our IF results showed a significant upregulation in ARF4 expression in the SNpc of MPTP-treated mice, along with an obvious increase in its colocalization with GM130. However, overexpression of Trx-1 effectively inhibited the elevation of ARF4-GM130 colocalization induced by MPTP (Fig. 8A–B). In addition, Western blot results revealed that Trx-1 also prevented the increase in ARF4 expression in the MPTP-treated group (Fig. 8C). In contrast to CREB3/ARF4, the HSP47 has been shown to play an antiapoptotic role in GA stress (Taniguchi and Yoshida, 2017). As expected, MPTP reduced the colocalization of HSP47 and GM130 in the GA (Fig. 9A–B). At the same time, the expression of HSP47 was decreased in MPTP-treated group measured by Western blot. However, overexpression of Trx-1 could effectively restore HSP47 expression (Fig. 9C). These results suggest that Trx-1 inhibition of MPTP-induced GA stress is also related to CREB3/ARF4 and HSP47 expression.

FIG. 8.

Overexpression of Trx-1 inhibited ARF4 expression in GA induced by MPTP. (A) Immunofluorescence costaining of ARF4 (red) and GM130 (green) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of ARF4 and GM130. (B) Quantitative analysis of colocalization of ARF4 and GM130 in each group (n = 3). (C) Western blot analysis of ARF4 expression in the SNpc in each group (n = 6). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, ***p < 0.001.

FIG. 9.

Overexpression of Trx-1 restored HSP47 expression in GA induced by MPTP. (A) Immunofluorescence costaining of HSP47 (red) and GM130 (green) in the SNpc in each group (n = 3). Areas in white boxes were enlarged and shown in the right panel. The white arrowheads indicate colocalization of HSP47 and GM130. (B) Quantitative analysis of colocalization of HSP47 and GM130 in each group (n = 3). (C) Western blot analysis of HSP47 expression in the SNpc in each group (n = 6). Each bar represented the mean ± SEM. Statistical significance: ns, p > 0.05, **p < 0.01, ***p < 0.001.

Discussion

Our previous studies have demonstrated that Trx-1 protects against the death of DA neurons induced by MPTP through various mechanisms, including ER stress (Zeng et al., 2014), calcium (Ca²⁺) homeostasis (Zhang et al., 2021), ferroptosis (Bai et al., 2021), and autophagy–lysosome pathways (Gu et al., 2024). In our current study, we found a new role of Trx-1 in protecting DA neurons from MPTP-induced GA stress.

First, we made sure that TH in the SNpc was decreased and a significant impairment in the motor capacity of mice was induced by MPTP, which was reversed in Trx-1 overexpression mice (Supplementary Fig. S1). Previous studies have found that GA fragmentation appeared in the nigral neurons of patients with PD (Tomas et al., 2021), so we detected the GA fragmentation in the SNpc of MPTP-treated PD mice through EM. GM130 is located on the cis-surface of the GA and used as a mark of GA (Nakamura et al., 1995). The colocalization between TH and GM130 was significantly decreased by MPTP, which was rescued in Trx-1 overexpression mice (Figs. 2–3). These findings suggest that GA stress is involved in the development of PD, and Trx-1 may play an important role in suppressing GA stress in PD mice.

A large number of evidence indicates that oxidative stress disrupts the structure and physiological functions of GA (Alborzinia et al., 2018). More and more evidence indicates that PKC family plays an important role in the regulation of oxidative stress and related to many diseases, including cancer, cardiovascular diseases (Silnitsky et al., 2023), and neurodegenerative diseases (Lorden and Newton, 2021). The PKC family contains a variety of isoenzymes, and PKC zeta is an important atypical PKC isoenzyme. Banan et al. found that overexpression of PKC zeta inhibited the upregulation of inducible nitric oxide synthase and the generation of reactive oxygen radicals induced by H₂O₂ in Caco-2 cells (Banan et al., 2002). In our study, the expression of PKC zeta was increased after MPTP treatment, and ROS was also increased in GA (Fig. 4A–C). GM130 level was inversely correlated with ROS, which may represent an oxidative stress in GA in MPTP-treated mice (Fig. 4D). Importantly, MPTP-induced PKC zeta and ROS increase was suppressed in Trx-1 overexpression mice. In the present study, PKC zeta level was increased in MPTP mice, suggesting a response increase to ROS. Moreover, PKC zeta level was higher in Trx-1 overexpression mice. Our result is consistent with Kahlos’ study in which Trx-1 is required for restoring PKC zeta level inhibited by nitric oxide in lung endothelial cells (Kahlos et al., 2003). Thus, increased PKC zeta level in Trx-1 overexpression mice resisted increase of ROS induced by MPTP (Fig. 4).

GRASP65, a cis-Golgi protein, via its C-terminal participates in the maintenance of the cis-face ribbon structure of the GA (Zhang and Seemann, 2021) and ensures normal functioning of the GA in protein transport and processing (Zhang and Wang, 2020). In our study, MPTP treatment reduced the colocalization of GM130 with GRASP65 and decreased the expression of GM130 and GRASP65, which was restored in MPTP-treated Trx-1 overexpression mice (Fig. 5). This result further suggests that GA stress is induced by MPTP, which is inhibited by Trx-1.

Studies have shown that overexpression of either wild-type or mutant (A53T) α-syn increases in Golgi fragments in the ventral midbrain of rats (Furlong et al., 2020; Koch et al., 2015). Our results of colocalization of α-syn with GM130 in the SNpc of MPTP-treated mice are consistent with previous study. Moreover, α-syn expression in GA was decreased in Trx-1 overexpression mice (Fig. 6). Since increase of α-syn in GA may cause accumulated toxins, ultimately leading to oxidative stress (Simon et al., 2020), these results suggest that Trx-1 plays an important role in inhibiting MPTP-induced α-syn expression in GA.

Mutations in LRRK2 are one of the most common genetic risk factors for PD (Sosero and Gan-Or, 2023). LRRK2 mutants disrupt GA integrity and vesicle trafficking (Mohan et al., 2023). In our study, the expression of LRRK2 was significantly increased in the SNpc of MPTP-treated mice (Fig. 7A, C). Rab29 belongs to the Rab GTPase family, which is a master regulator of membrane trafficking and intracellular signaling (Pfeffer, 2017). LRRK2 and Rab29 gene polymorphisms increase the risk of PD (MacLeod et al., 2013; Pihlstrom et al., 2015). VPS52 has been shown to be a subunit of the GARP complex, which sensitizes DA neurons to the toxicity of mutant LRRK2 (Beilina et al., 2020). Our results showed that LRRK2, Rab29, and VPS52 colocalization and their expression were increased in the SNpc of MPTP-treated mice, which was suppressed in Trx-1 overexpression mice (Fig. 7). These results suggest that LRRK2 and Rab29 are also related to GA stress induced by MPTP, and Trx-1 inhibits these alterations.

CREB3/ARF4 plays an important role in inducing apoptosis in response to GA stress. Under GA stress, CREB3 is released from the ER into the GA, inducing cell death by upregulating the expression of ARF4, which is necessary for the transport of cargo from the GA (Shirai and Yamauchi, 2024). When the expression of ARF4 was inhibited by RNA interference, apoptosis induced by GA stress inducers such as Brefeldin A was significantly reduced (Reiling et al., 2013). HSP47 is another important molecule in mammalian response to GA, which mainly prevents GA stress (Miyata et al., 2013). Our results showed that MPTP increased the expression of ARF4 and decreased the expression of HSP47 in GA in the SNpc of mice, providing a possible explanation for the GA stress induced by MPTP. Importantly, the MPTP-induced changes in ARF4 and HSP47 expression were reversed in Trx-1 overexpressed mice. These results suggest that CREB3/ARF4 and HSP47 are also involved in Trx-1 protecting against MPTP-induced GA stress (Figs. 8–9).

Taken together, our results showed that GA stress was induced by MPTP, which was suppressed by Trx-1. PKC zeta expression, oxidative stress, and α-syn expression in GA were increased by MPTP. In addition, MPTP increased distribution of LRRK2 and Rab29 in the TGN, as well as MPTP altered the expression the CREB3/ARF4 and HSP47 in GA in the SNpc of PD mice. All the above alterations are reversed by overexpression of Trx-1 (Fig. 1). Nevertheless, our findings demonstrate that Trx-1 plays an important role in regulating GA stress induced by MPTP, which will provide new strategies for the treatment of PD.

Materials and Methods

Animals

Male C57BL/6 wild-type mice (22–25 g, 8–9 weeks) were obtained from Chongqing Medical University (Chongqing, China), and C57BL/6 human TRX-1 (hTRX-1) overexpression transgenic mice were constructed by Cyagen Biosciences Inc. (Guangzhou, China). The construction of transgenic mice was based on previous articles (Takagi et al., 1999). Briefly, hTRX-1 cDNA was inserted between the β-actin promoter and the β-actin terminator. The gene fragment was cut out from the plasmid using VspI and XbaI endonuclease, purified, and then injected into the pronuclei of fertilized eggs of C57BL/6 mice to produce transgenic mice. The mice were housed in an air-conditioned room (temperature 20–25°C, humidity 40%–70%) with a 12 h light/dark cycle and could eat and drink freely. All animal experiments were approved by the Animal Ethics Committee of Kunming University of Science and Technology.

Drugs and treatment

MPTP-HCl was purchased from Sigma-Aldrich (#M0896).

The mice were randomly divided into four groups: control group, MPTP group, Trx-1 overexpression group (Trx-1 Tg), and Trx-1 Tg + MPTP group. The mice in the control and Trx-1 Tg groups were intraperitoneally injected with saline daily over a period of 7 days, while the mice in the MPTP and Trx-1 Tg + MPTP groups were given intraperitoneal injection of MPTP-HCl at a dose of 23.4 mg/kg/d for 7 days, twice daily.

Behavioral tests

After the administration period, the mice were given 2 days to acclimate before undergoing behavioral tests.

Gait analysis test

Both forepaws of the mice were dipped in ink and placed at the top of a slope where white paper had been laid out in advance. After the mice walked freely to the bottom of the slope, the white paper covered with paw prints was taken out, and the stride length and step width were measured.

Traction test

Mice were allowed to hold onto a horizontal piece of wire with the forepaws, and the position of the hind limb was observed. Mice that gripped the wire with both hind paws scored 3 points, mice that gripped the wire with one hind paw scored 2 points, and mice that did not grip the wire with either paw scored 1 point. The higher the score, the better the movement ability.

Western blot analysis

After behavioral tests, the mice were sacrificed by cervical dislocation. The SNpc tissues were rapidly dissected out and lysed in a protein lysis buffer. The protein concentration was determined with Bio-Rad protein assay reagent (Hercules, CA, USA).

Protein samples were separated by 10%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a 0.45 μm polyvinylidene fluoride membrane (Millipore Corp., Billerica, MA, USA). Then, the membranes were soaked in 10% skim milk (in a phosphate buffer containing 0.1% Tween 20, pH 7.2) and blocked at room temperature for 2 h (h). The membranes were incubated by primary antibodies against TH (1:5000; ab137869, abcam), Trx-1 (1:1000; ab273877, abcam), PKC zeta (1:1000; ab108970, abcam), LRRK2 (1: 1000; ab186334, abcam), Rab29 (1:1000; ab256526, abcam), VPS52 (1:1000; 11662–2-AP, Proteintech), GM130 (1:1000; 610822, BD Biosciences), GRASP65 (1:1000; 10747–2-AP, Proteintech), ARF4 (1: 1000; 11673–1-AP, Proteintech), HSP47 (1: 500; 10875–1-AP, Proteintech), and β-actin (1: 5000; 66009–1-Ig, Proteintech) overnight at 4°C. The next day, remove the primary antibody and incubate with Anti-Mouse IgG (H + L) Antibody (1:10,000; SeraCare, 5450–0011) or Anti-Rabbit IgG (H + L) Antibody (1:10,000; SeraCare, 5450–0010) at room temperature for 1 h. Finally, the membranes were washed and visually detected using an ECL Western blot detection kit (Millipore, Billerica, USA). Protein expression was analyzed by Image J software.

IF staining

The brain tissues were fixed in 4% paraformaldehyde (BL539A, Biosharp), then dehydrated using a sucrose density gradient, rapidly frozen, and finally cut into 20 µm slices. After being washed with phosphate buffered saline (PBS), the slices were immersed in a sodium citrate antigen retrieval solution at 95°C for 40 min, then treated with a blocking solution (0.3% TritonX-100 and 10% goat serum in 0.01 M PBS) for 1.5 h, and incubated with the primary antibodies against α-syn (1:200; ab212184, abcam), Trx-1 (1:200; ab273877, abcam), TH (1:200; ab137869, abcam), GM130 (1:200; 610822, BD Biosciences), GRASP65 (1:200; 10747–2-AP, Proteintech), LRRK2 (1:200; ab186334, abcam), Rab29 (1:200; ab256526, abcam), VPS52 (1:200; 11662–2-AP, Proteintech), ARF4 (1: 200; 11673–1-AP, Proteintech), or HSP47 (1: 200; 10875–1-AP, Proteintech) at 4°C overnight. On the following day, the primary antibodies were washed off with PBS, and then the corresponding fluorescent secondary antibodies were added. The secondary antibodies were as follows: Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ Plus 488 (1:1000; A32731TR, Invitrogen); Goat anti-Mouse IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (1:1000; A11004, Invitrogen); Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (1:1000; A11029, Invitrogen); Goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 568 (1:1000; A11036, Invitrogen); and Goat Anti-Rabbit IgG H&L Secondary Antibody, Alexa Fluor^® 647 (1:1000; ab150079, abcam). The sections were then incubated at room temperature in the dark for 1 h. Finally, the sections were sealed with an antifluorescence quencher containing DAPI (S2110, Solarbio) and observed with obtained images using ZEISS Elyra 7 with Lattice SIM. For quantitative analysis of IF colocalization, three random fields in the SNpc region per mouse were selected, and colocalized puncta were counted. The average value from these fields was calculated as the colocalization puncta for each individual mouse. Quantitative analysis was performed using GraphPad Prism 6 software. All data were expressed as mean ± standard error of the mean (SEM) and analyzed by two-way analysis of variance (ANOVA) with Bonferroni post hoc test for comparison between multiple groups.

EM

GA submicrostructure was observed by EM. The 1 µm sections were fixed in 2.5% glutaraldehyde, then postfixed in 1% osmium tetroxide, stained with 1% uranyl acetate from matching areas of experimental, and observed with an electron microscope (JEOL1200CX) at 100 kV.

DHE staining

The generation of ROS was evaluated by DHE-ROS assay kit (BB-23021, BestBio). Briefly, the DHE staining probe was diluted 1000-fold with ultrapure water to prepare the working solution. Approximately 100 μL of the working solution was applied to frozen tissue sections, followed by incubation at 37°C in the dark for 30 min. After removing the staining solution, the sections were washed three times with PBS. Finally, the sections were mounted with an antifluorescence quencher containing DAPI (S2110, Solarbio) and imaged using ZEISS Elyra 7 with Lattice SIM.

Statistical analysis

GraphPad Prism 6 software was used for statistical analysis. All data were expressed as mean ± SEM. For correlation analysis, a two-tailed Pearson’s correlation test was conducted with a 95% confidence interval. For other analysis, results were analyzed by two-way ANOVA with Bonferroni post hoc test for comparison between multiple groups. p value less than 0.05 was considered statistically significant. The level of significance is indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Authors’ Contributions

J.B.: Conceptualization, methodology, writing—review and editing, supervision, funding acquisition. J.D.: Methodology, formal analysis, visualization. X.S.: Methodology, formal analysis, writing—original draft, visualization. Y.W.: Methodology, formal analysis, visualization. Y.P.: Methodology. L.B.: Methodology. F.Y.: Methodology.

Footnotes

Acknowledgment

Special thanks are due to the instrumental/data analysis from the Advanced Imaging Platform of Institute of Primate Translational Medicine, Kunming University of Science and Technology.

Author Disclosure Statement

The authors declare no competing financial interests.

Funding Information

This work was supported by the National Natural Science Foundation of China (82371271, U2002220), the innovation team of oxidative stress and defense of Yunnan Province (202305AS350011).

Ethical Approval and Consent to Participate

All animal experimental procedures were approved by the Animal Ethics Committee of Kunming University of Science and Technology. The license was approved by the Local Committee on Animal Use and Protection of Yunnan Province (No. LA2008305).

Availability of Data and Materials

Data and materials supporting the findings of this study are available within the article.

Supplemental Material

Abbreviations

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