Nrf2 Expression Is Decreased in LRRK2 Transgenic Mouse Brain and LRRK2 Overexpressing SH-SY5Y Cells

Abstract

Mutations in leucine rich-repeat kinase 2 (LRRK2) cause autosomal-dominant, late-onset Parkinson’s disease (PD). Accumulating evidence indicates that PD-associated LRRK2 mutations induce neuronal cell death by increasing cellular reactive oxygen species levels. However, the mechanism of increased oxidative stress associated with LRRK2 kinase activity remains unclear. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that protects cells from oxidative stress by inducing the expression of antioxidant genes. In the present, it was found that decreased expression of Nrf2 and mRNA expression of its target genes in Lrrk2-transgenic mouse brain and LRRK2 overexpressing SH-SY5Y cells. Furthermore, knockdown of glycogen synthase kinase-3β (GSK-3β) recovered Nrf2 expression and mRNA expression of its target genes in LRRK2 overexpressing SH-SY5Y cells. We concluded that since Nrf2 is transcriptional factor for antioxidative responses, therefore, reduction of Nrf2 expression by LRRK2 may be part of a mechanism that LRRK2-induces vulnerability to oxidative stress in neuronal cells.

INTRODUCTION

Leucine rich-repeat kinase 2 (LRRK2) is a 260-kDa protein, containing a leucine-rich repeat, a Ras of complex protein (Roc) guanosine 5′-triphosphatase (GTPase) domain, a C-terminal of Roc region, a kinase domain, and a WD40 domain.¹⁾ Recently, various autosomal-dominant mutations in LRRK2 have been identified, associated with both familial Parkinson’s disease (PD) and a significant proportion of sporadic PD cases.¹⁾ Several in vitro studies have demonstrated that transiently transfected mutant LRRK2 causes degeneration in SH-SY5Y neuroblastoma cells and primary neurons.²⁾ These findings suggest that increased LRRK2 kinase activity, caused by pathogenetic mutations, is a likely pathogenic mechanism. However, the physiological substrate(s) for LRRK2 kinase activity that may be involved in the neurodegeneration mechanism have not yet been identified.

Recent evidence has shown that PD-associated mutations in LRRK2 increase the levels of reactive oxygen species in cells derived from damaged mitochondria, suggesting that elevated oxidative stress is a major cellular mechanism in PD pathologies associated with LRRK2 mutations.³⁾ However, the mechanism of increased oxidative stress associated with LRRK2 kinase activity remains unclear.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that protects cells from oxidative stress by inducing the expression of antioxidant genes, such as heme oxygenase-1 (HO-1) and glutathione peroxidase (GPx).⁴⁾ Thus, activation of Nrf2 can effectively reduce neuronal cell damage caused by oxidative stress.

In the present study, we investigated effect of LRRK2 on the Nrf2 expression using brain tissue of Lrrk2 transgenic mice and LRRK2 over expressing neuronal cultured cells.

MATERIALS AND METHODS

Animals

Eight-weeks-old C57BL/6J male wild-type mice (WT) and Flag-tagged mouse wild type Lrrk2 transgenic mice (Jackson laboratory, U.S.A) were used in this study. This experiment has been approved by the Animal Experiment Committee of Kitasato University School of Medical Hygiene (Approved No. Eiken-ken 18-09-5) and the Genetic Modification Experiment Safety Committee (Approved No. 4933).

Cell Culture and Transfection of Plasmid DNA or Small Interfering RNA (siRNA)

The human dopaminergic cell lines SH-SY5Y cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) nutrient mixture F-12 HAM (Sigma, CA, U.S.A.) supplemented with 10% fetal bovine serum and penicillin-streptomycin (10000 UmL), and the cells were cultured at 37 °C and 5% CO₂ in a humidified atmosphere.⁵⁾ As previously described,^5,6) FuGENE^®HD transfection reagent (Roche, Basel, Switzerland) was used for SH-SY5Y cells to plasmid DNA containing FLAG-tagged (3×) WT LRRK2 (generous gift from Dr. Mark R Cookson, Laboratory of Neurogenetics, NIA, NIH, MD, U.S.A.) was transiently transfected into the cells.

In the knockdown experiment, we used V5-tagged human wild type LRRK2 stably expressing clone (WT4D33) and its vector control (Neo) of SH-SY5Y cells. For knockdown experiment, cells were transfected 50 pmol of siRNA using X-tremeGENE siRNA transfection reagent (Roche). The siRNAs used in this study were SMARTpool GSK-3β, LRRK2 and nontargeting control siRNA (Dharmacon, Chicago, IL, U.S.A.).⁶⁾ The effect of oxidative stress on the cell viability were measured by the WST-8 assay after 6 h incubation with indicate concentration of H₂O₂.⁶⁾

Western Blotting Analysis

Mouse brain tissue and cultured cell pellets were lysed by sonication in radio immunoprecipitation assay (RIPA) buffer [(25 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS))] supplemented with protease and phosphatase inhibitors (HALT protease and phosphatase). After blotting to the polyvinylidene difluoride (PVDF) membrane, the membrane was blocked with blocking reagent for Can get signal (TOYOBO, Osaka, Japan) and incubated with primary antibody overnight at 4 °C with Can get signal solution 1 (TOYOBO). After washing by Tris-buffered saline (TBS-T) containing 0.05% Tween-20, membrane was incubated with horseradish peroxidase-conjugated secondary antibody in Can get signal solution 2 (TOYOBO) for 1 h at room temperature. Membranes were washed three times with TBS-T and bands were visualized using enhanced chemiluminescence (Pierce ECL Plus Substrate, Thermo Fisher Scientific, Waltham, MA, U.S.A.) and detected by Odyssey Fc (LI-COR, Lincoln, NE, U.S.A.). The band signals were quantitatively analyzed using Image studio software as previously described.^5–7) Antibodies against the following proteins were used: anti-LRRK2 (Abcam, Cambridge, U.K.), anti-Nrf2 (Proteintech, Rosemont, IL, U.S.A.), anti-GSK-3β and HRP-anti-β-Actin (Cell Signaling Technologies, Danvers, MA, U.S.A.).

Real-Time PCR Analysis

Total RNA was isolated from cultured cells using TRIzol RNA isolation reagent (Invitrogen, Waltham, MA, U.S.A.) according to the manufacturer’s protocol. Primescript RT Master Mix (TaKaRa Bio, Shiga, Japan) was used to synthesize cDNA from total RNA. Real-time PCR analysis was performed using Power SYBR Green Master Mix (Thermo Fisher Scientific, A25742) and the ABI 7500 Real-time PCR System (Applied Biosystems, Waltham, MA, U.S.A.). The level of each mRNA was quantified using the normalized threshold cycle (Ct) method against the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as previously described.⁷⁾ Primers sequences were described in Supporting information 1.

Statistical Analysis

All experimental data were expressed relative to control values and are presented as means ± standard deviation (S.D.). p-Values were calculated using Student’s t-test or the one-way ANOVA combined with Tukey’s post hoc test, and significance was set at * p < 0.05, ** p < 0.01.

RESULTS AND DISCUSSION

In the present study, our Western blotting analysis revealed that decreased expression of Nrf2 and increased expression of glycogen synthase kinase-3β (GSK-3β) in the brain tissue of Lrrk2 transgenic mice (Fig. 1A). Next, we determined the mRNA expression of Nrf2 target genes in brain of Lrrk2 transgenic mice. It was found that mRNA expression of HO-1, Gclm and GPX were significantly decreased in brain of Lrrk2 transgenic mice, whereases no changes of mRNA expression of Nrf2 was observed (Fig. 1B). These results suggest that Nrf2 protein expression and mRNA expression of its target genes may be negatively regulated in LRRK2 overexpressing mouse brain. Thus, increased LRRK2 expression may implicate with reduction of antioxidative responses mediated by Nrf2. To clarify this phenomenon at cellular level, the experiments were performed using LRRK2 overexpressing human neuronal cell SH-SY5Y cells. We found that decreased expression of Nrf2 was observed in LRRK2 transiently transfected cells, but expression of GSK-3β was not changed (Fig. 2A). Furthermore, mRNA expression of Nrf2 targets genes was significantly decreased in LRRK2 transfected cells (Fig. 2B). These results revealed that increased LRRK2 expression reduced Nrf2 expression and mRNA expression of its target gene in human neuronal cells. Next, we demonstrated that decreased expression of Nrf2 in LRRK2 overexpressing cells was recovered by knockdown of GSK-3β by siRNA of GSK-3β (Fig. 3).

Fig. 1. The Determination of Nrf2 Protein Expression and mRNA Expression of Nrf2 Target Genes in LRRK2-Transgenic Mouse Brain

[A] Mouse brain lysate of Lrrk2-transgenic (Tg) and non-Tg littermate (non-Tg) control were prepared and subjected to Western blotting analysis, using antibodies against LRRK2, Nrf2, GSK-3β and β-actin (loading control). The quantification of the protein bands was performed using Image studio (Licor). [B] mRNA was isolated from these mice brain and subjected to real-time PCR analysis, using primer for NRF2, HO-1, Gclm, GPX and GAPDH (internal control). Stars represent statistical comparisons by the Student’s T-test. n = 6; *: p < 0.05, **: p < 0.01.

Fig. 2. The Effects of Overexpression of LRRK2 on the Expression of Nrf2 Protein and mRNA Expression of Nrf2 Target Genes in LRRK2 Transfected SH-SY5Y Cells

[A] SH-SY5Y cells were transfected with Flag-LRRK2 and control vector. After 48 h of transfection, cell lysates were prepared and subjected to Western blot analysis, using antibodies against LRRK2, Nrf2, GSK-3β and β-actin (loading control). The quantification of the protein bands was performed as described above. [B] After 24 h of transfection, mRNA was isolated and subjected to real-time PCR analysis, using primer for NRF2, HO-1, Gclm, GPX and GAPDH (internal control). Stars represent statistical comparisons by the one-way ANOVA combined with Tukey’s post hoc test. (n = 3) for [A] and [B]; *:p < 0.05, **: p < 0.01.

Fig. 3. Determination of Nrf2 Protein Expression in LRRK2-Stably Expressing SH-SY5Y Cells with or without siRNA for GSK-3β

[A] Cell lysates of LRRK2 stably expressing SH-SY5Y clone (WT4D33) and its vector clone (Neo) were subjected to Western blot analysis, using antibodies against LRRK2, Nrf2 and β-actin (loading control). [B] WT4D33 cells transfected siRNA for GSK-3β or siRNA control were subjected to Western blot analysis, using antibodies against LRRK2, Nrf2, GSK-3β and β-actin (loading control). The quantification of the protein bands was performed using software of Image studio (Licor). [C] Real-time PCR analysis, using primer for NRF2, HO-1, Gclm, GPX and GAPDH (internal control), as described above. Stars represent statistical comparisons by the one-way ANOVA combined with Tukey’s post hoc test. (n = 3); *: p < 0.05, **: p < 0.01.

Previously, it was reported that Nrf2 expression is reduced by GSK-3β.⁸⁾ Also, our previous report demonstrated that LRRK2 directly activates the GSK-3β activity.⁶⁾ Taken together, our present results suggest that overexpression of LRRK2 may reduce the Nrf2 protein expression through the GSK-3β activation in neuronal cells. However, the fact that GSK-3β expression was upregulated in mouse brain but not in cultured neuronal cells suggests that the upregulation of GSK-3β is not directly mediated by LRRK2. Since it has been reported that GSK-3β expression is promoted by tumor necrosis factor-α (TNF-α)⁹⁾ and that LRRK2 upregulates TNF-α via activation of TLR4,¹⁰⁾ thus we speculate that overexpression of LRRK2 in mouse brain increases GSK-3β expression via the TLR4-meditaed TNF-α production. In fact, mRNA level of inflammatory cytokines including of TNF-α was increased in LRRK2 transgenic mouse brain under the experimental conditions (Supplementary material 2).

Finally, after confirming that knockdown of LRRK2 reduces Nrf2 expression (Fig. 4A), the effect of LRRK2 on oxidative cytotoxicity was examined. We found that WT4D33 cells exhibited vulnerable effect against H₂O₂ (Fig. 4B). Furthermore, viability of LRRK2-siRNA transfected cells is significantly higher than control siRNA transfected cells (Fig. 4C). These results suggest that LRRK2 has vulnerable effect against H₂O₂-induced oxidative stress.

Fig. 4. Determination of Effect of Oxidative Stress on the Viability of LRRK2-Stably Expressing SH-SY5Y Cells with or without siRNA for LRRK2

[A] LRRK2-stably expressing SH-SY5Y cells (WT4D33) were transfected with either the LRRK2-specific siRNA or the siRNA control. After 48 h of transfection, LRRK2, Nrf2 and β-actin (loading control) was quantified by Western blotting. [B] Vector control SH-SY5Y cells (Neo) and LRRK2-stably expressing SH-SY5Y cells (WT4D33) were incubated with indicated concentration of H₂O₂ for 6 h, cell viability was measured by WST-8 assay. [C] WT4D33 cells transfected siRNA for LRRK2 or siRNA control were incubated with indicated concentration of H₂O₂ for 6 h, cell viability was measured by WST-8 assay. Stars represent statistical comparisons by the one-way ANOVA combined with Tukey’s post hoc test. (n = 3); *: p < 0.05, **: p < 0.01.

The present study concludes that LRRK2 may exerts downregulation of Nrf2-mediated antioxidative effect in neuronal cell, this is possible to one of the cellular mechanisms of LRRK2-mediated vulnerable effect against oxidative stress.

Acknowledgments

This work was supported by the JSPS KAKENHI [Grant No. 19K08985]; Kitasato University Graduate School of Medical Sciences [Integrative Research Program 2019–2020].

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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