Wild-type LRRK2 but not its mutant attenuates stress-induced cell death via ERK pathway - PubMed (original) (raw)

Wild-type LRRK2 but not its mutant attenuates stress-induced cell death via ERK pathway

Anthony K F Liou et al. Neurobiol Dis. 2008 Oct.

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is a recently identified gene that, when mutated at specific locations, results in the onset of parkinsonian symptoms with clinical features indistinguishable from idiopathic Parkinson's disease. Based on structural and domain analysis, LRRK2 is predicted to function as a stress-responsive protein scaffold mediating the regulation of mitogen activating protein kinase (MAPK) pathways. Consistent with this notion, our results supported the notion that expression of wild-type LRRK2 but not Y1699C or G2019S mutants enhanced the tolerance of HEK293 and SH-SY5Y cells towards H(2)O(2)-induced oxidative stress. This increase in stress tolerance was dependent on the presence of the kinase domain of the LRRK2 gene and manifested through the activation of the ERK pathway. Collectively, our results indicated that cells expressing LRRK2 mutants suffer a loss of protection normally derived from wild-type LRRK2, making them more vulnerable to oxidative stress.

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Figures

Figure 1. Impact of wild-type LRRK2 and its mutants Y1699C and G2019S on basal cell viability and under the insult of H2O2 in HEK293 cells

(A) Cell survival profile in response to 0–300 µM of treatment of H2O2 for 18 hours in HEK293 cells; (B) Changes in percentage cell death due to necrosis as estimated by LDH assay in response to 0–300 µM of treatment of H2O2 for 18 hours in HEK293 cells; (C) Decrease in basal cell viability in HEK293 cells expressing wild-type LRRK2 and its mutants Y1699C and G2019S. Data are means ± SEM, at least 24 readings per data point, from six independent experiments. *p < 0.01, #p < 0.01, % p< 0.01 versus viability of cells transfected with pCDNA3.1. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests; (D) Attenuation of H2O2-induced cell death for cells expressing wild-type LRRK2 but not in cells expressing mutant Y1699C or G2019S. Percentage cell survival after H2O2 insult among cells transfected with pCDNA3.1, expressing wild-type LRRK2 or its mutants Y1699C and G2019S is normalized against untreated cells transfected with the same vector or expressing the same proteins. Data are means ± SEM, at least 24 readings per data point, from six independent experiments. * p < 0.01 versus viability of cells transfected with pCDNA3.1 after 150 µM of H2O2 treatment for 18 h. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests.

Figure 2. Impact of expressing wild-type LRRK2 and Y1699C mutant on basal level of phospho-ERK (pERK) and phospho-JNK (pJNK)

(A) Changes in basal level of pERK and pJNK in HEK293 cells expressing wild-type LRRK2 and Y1699C mutant as compared to those transfected with pCDNA3.1. In the top panel, an anti-GFP antibody was used to examine expression of wild-type LRRK2 and Y1699C mutant gene in HEK293 cells via immunoblotting. The corresponding change in basal level of pERK, total ERK, pJNK, and total JNK are also shown and β-actin was used as a loading control; (B) Corresponding changes in pERK, total ERK, pJNK, and total JNK in HEK293 cells expressing wild-type LRRK2 or Y1699C mutant with and without chronic treatment with 150 µM of H2O2 for 18 hours. β-Actin was used as loading control.

Figure 3. Effect of co-expression of wild-type LRRK2, Y1699C mutant with dnERK2 respectively on basal cell viability and cell death in response to H2O2 toxicity

(A) Demonstration of the efficacy of using dnERK2 to silence ERK activation. In the top panel, cells expressing pCDNA3.1 showed activation in response to H2O2 (middle lane); on the other hand, cells expressing dnERK2 gene showed suppression of ERK activation under the same stimulus (right lane). The second panel indicates expression of the dnERK2 gene. α–Tubulin was used as loading control. (B) Co-expression of dnERK2 with LRRK2 wild-type gene or Y1699C mutant gene did not significantly alter the decrease in basal cell viability due to the expression of LRRK2 genes or its mutants. (C) Co-expression of dnERK2 with wild-type LRRK2 abrogated the protection conferred by the latter gene against cell death induced by H2O2 in HEK293 cells. No significant changes in cell death induced by H2O2 toxicity were detected when dnERK2 was co-expressed with Y1699C mutant in HEK293 cells. Percentage cell death induced by H2O2 among cells transfected with pCDNA3.1, expressing LRRK2 wt or Y1699C mt gene in the absence or presence of dnERK2 are normalized against untreated cells transfected with the same vector or expressing the same proteins. Data are means ± SEM, at least 12 readings per data point, from three independent experiments. * p < 0.05 versus cell viability of cells expressing LRRK2 wt without dnERK2 after chronic treatment with H2O2 for 18 hours. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests.

Figure 4. Impact of LRRK2 wild-type and mutants on basal viability and cell viability in response to H2O2 insult in SH-SY5Y cells

(A) Change in basal viability due to expression of LRRK2 wild-type and mutants (Y1699C and G2019S). Data are means ± SEM, 3 readings per data point, from at least free independent experiments. *p <0.05 versus basal cell viability of cells expressing LRRK2 wild-type; #p <0.05 versus basal cell viability of cells expressing LRRK2 wild-type. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests. (B) Change in percentage cell survival between cells expressing LRRK2 wild-type and mutants (Y1699C or G2019S) in response to H2O2 insult. Data are means ± SEM, 3 readings per data point, from at least free independent experiments. *p <0.01 versus percentage cell viability of cells expressing LRRK2 wild-type after H2O2 insult; #p <0.01 versus basal cell viability of cells expressing LRRK2 wild-type after H2O2 insult. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests. (C) Change in percentage cell survival between cells expressing LRRK2 wild-type and mutants (Y1699C or G2019S) in the absence and presence of 10µM U0126 in response to H2O2 insult. Each of the percentage cell survival of cells expressing LRRK2 wild-type and mutants (Y1699C or G2019S) after H2O2 insult was normalized from corresponding cells expressing the same LRRK2 gene without H2O2 insult. Data are means ± SEM, 3 readings per data point, from at least free independent experiments. *p <0.01 versus percentage cell viability of cells expressing LRRK2 wild-type after H2O2 insult; #p <0.01 versus basal cell viability of cells expressing LRRK2 wild-type after H2O2 insult. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference tests.

Figure 5. Characterization of the deletion mutants

(A) Change in basal pERK in HEK293 cells expressing wild-type LRRK2, Y1699C mutant, and their respective deletion mutants. (B) Expression of deletion mutants resulted in significantly less decrease in basal cell viability as compared to their complete gene counterpart. Data are means ± SEM, at least 12 readings per data point, from three independent experiments. * p < 0.05 versus basal cell viability of cells expressing the corresponding complete gene. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests. (C) Impact of expressing LRRK2 wild-type-kinase (deletion mutant) on ERK response in HEK293 cells towards H2O2 toxicity. The changes in pERK and total ERK with and without H2O2 treatment in cells transfected with pCDNA3.1, expressing wild-type LRRK2 (LRRK2 wt), and LRRK2 wild-type-kinase (LRRK2 wt-kinase) were visualized by immunoblotting probed with antibodies recognizing pERK, total ERK and β-actin respectively. β-actin was used as loading control. (D) Impact of expressing Y1699C mutant-kinase (deletion mutant) on ERK response in HEK293 cells towards H2O2 toxicity. The changes in pERK and total ERK with and without H2O2 treatment in cells transfected with pCDNA3.1, expresssing Y1699C mutant (Y1699C mt) and Y1699C mutant-kinase (Y1699C mt-kinase) were visualized by immunoblotting probed with antibodies recognizing pERK, total ERK and β-actin respectively. β-actin was used as loading control. (E) Comparative impact on cell death induced by treatment with 150 µM of H2O2 in cells expressing pCDNA3.1, wild-type LRRK2, LRRK2 wt-kinase, Y1699C mutant, and Y1699C mt-kinase. Percentage cell death induced by H2O2 among cells transfected with pCDNA3.1, expressing wild-type LRRK2, Y1699C mutant, or their corresponding deletion mutant are normalized against untreated cells transfected with the same vector or expressing the same proteins. Data are means ± SEM, at least 12 readings per data point, from three independent experiments. * p < 0.05 versus cell viability of cells expressing LRRK2 wt after chronic treatment with H2O2 for 18 h. Statistics were derived from ANOVA and post hoc Fisher’s protected least significant difference (PLSD) tests.

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Wild-type LRRK2 but not its mutant attenuates stress-induced cell death via ERK pathway - PubMed (original) (raw)