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Loss of parkin promotes lipid rafts-dependent endocytosis through accumulating caveolin-1: implications for Parkinson's disease.

Cha SH, Choi YR, Heo CH, Kang SJ, Joe EH, Jou I, Kim HM, Park SM - Mol Neurodegener (2015)

Bottom Line: It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD.Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells.Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Ajou University School of Medicine, 164, Worldcup-ro, Yeongtong-gu, Suwon, 16499, Korea.

ABSTRACT

Background: Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor dysfunctions. While most PD is sporadic in nature, a significant subset can be linked to either autosomal dominant or recessive mutations. PARK2, encoding the E3 ubiquitin ligase, parkin, is the most frequently mutated gene in autosomal recessive early onset PD. It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD. Therefore here, we examined the relationship between lipid rafts-related proteins and parkin.

Results: We identified caveolin-1 (cav-1), which is one of the major constituents of lipid rafts at the plasma membrane, as a substrate of parkin. Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells. Moreover, the total cholesterol level and membrane fluidity was altered by parkin deficiency, causing dysregulation of lipid rafts-dependent endocytosis. Further, cell-to-cell transmission of α-synuclein was facilitated by parkin deficiency.

Conclusions: Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

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Related in: MedlinePlus

Parkin induces the degradation of cav-1 through the proteasome-dependent pathway. a Real-time RT-PCR was performed as described in ‘Methods’. WT and parkin KO MEF cells were incubated with with 10 μg/ml cyclohexamide (CHX) for the indicated times b 10 μM MG132 for 3.5 h c and 10 μM lactacystin for 3.5 h d Lysates were analyzed by SDS-PAGE and Western blotting. P values were determined using a two way ANOVA, **p < 0.01. e WT and parkin KO MEF cells cultured on poly-D-lysine coated glass were incubated with 10 μM MG132 for 3.5 h, stained with anti-cav-1 (red), and then observed by confocal microscopy. Blue indicates DAPI staining. Scale bar indicates 10 μm
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Fig2: Parkin induces the degradation of cav-1 through the proteasome-dependent pathway. a Real-time RT-PCR was performed as described in ‘Methods’. WT and parkin KO MEF cells were incubated with with 10 μg/ml cyclohexamide (CHX) for the indicated times b 10 μM MG132 for 3.5 h c and 10 μM lactacystin for 3.5 h d Lysates were analyzed by SDS-PAGE and Western blotting. P values were determined using a two way ANOVA, **p < 0.01. e WT and parkin KO MEF cells cultured on poly-D-lysine coated glass were incubated with 10 μM MG132 for 3.5 h, stained with anti-cav-1 (red), and then observed by confocal microscopy. Blue indicates DAPI staining. Scale bar indicates 10 μm

Mentions: To investigate in more detail the manner in which the loss of parkin leads to an increase in cav-1, we tested the mRNA level of cav-1 in both WT and parkin KO MEF cells. Quantitative RT-PCR shows that the mRNA level of cav-1, as well as that of cav-2, flot-1, and flot-2 were not different between the two cell lines (Fig. 2a), suggesting that parkin does not regulate cav-1 at the transcriptional level. Parkin regulates protein degradation as an E3 ubiquitin ligase [6]. In addition, cav-1 has been reported to be partially degraded by the ubiquitin proteasome system (UPS) [26, 27], speculating that parkin may regulate cav-1 degradation via the UPS. To confirm this hypothesis, WT and parkin KO MEF cells were treated with cycloheximide (CHX), an inhibitor of protein synthesis, and the expression of cav-1 was assessed using Western blotting. As shown in Fig. 2b, the degradation of cav-1 in WT MEF cells was significantly faster than in parkin KO MEF cells. In addition, while treatment with the proteasomal inhibitors, MG-132 and lactacystin, led to an accumulation of cav-1 in WT MEF cells, it did not further enhance the steady state levels of cav-1 in parkin KO MEF cells. (Fig. 2c and d). Confocal microscopic analysis confirmed this observation (Fig. 2e), suggesting that parkin regulates cav-1 degradation via the UPS. These results also prompted speculation that cav-1 may be a substrate of parkin, thus, we tested the interaction between the two proteins. As shown in Fig. 3a, both flag-parkin and cav-1-EGFP were overexpressed in parkin KO MEF cells, and immunoprecipitation shows their interaction. Antibody staining of endogenous parkin and cav-1 in MEF cells shows the colocalization of both proteins (Fig. 3b), which was also confirmed using an in situ proximity ligation assay (PLA) (Fig. 3c), suggesting that parkin interacts with cav-1. Next, in order to investigate whether parkin ubiquitinates cav-1, we transfected parkin KO MEF cells with flag-parkin, His-ubiquitin, and cav-1-EGFP, which resulted in an increase in the ubiquitination of cav-1-EGFP by flag-parkin (Fig. 3d). Furthermore, mutants of parkin that have been identified in patients with familial PD did not rescue the increase in cav-1 by loss of parkin (Fig. 3e), suggesting that parkin mediates the ubiquitination of cav-1, thereby targeting cav-1 to the proteasome for degradation.Fig. 2


Loss of parkin promotes lipid rafts-dependent endocytosis through accumulating caveolin-1: implications for Parkinson's disease.

Cha SH, Choi YR, Heo CH, Kang SJ, Joe EH, Jou I, Kim HM, Park SM - Mol Neurodegener (2015)

Parkin induces the degradation of cav-1 through the proteasome-dependent pathway. a Real-time RT-PCR was performed as described in ‘Methods’. WT and parkin KO MEF cells were incubated with with 10 μg/ml cyclohexamide (CHX) for the indicated times b 10 μM MG132 for 3.5 h c and 10 μM lactacystin for 3.5 h d Lysates were analyzed by SDS-PAGE and Western blotting. P values were determined using a two way ANOVA, **p < 0.01. e WT and parkin KO MEF cells cultured on poly-D-lysine coated glass were incubated with 10 μM MG132 for 3.5 h, stained with anti-cav-1 (red), and then observed by confocal microscopy. Blue indicates DAPI staining. Scale bar indicates 10 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: Parkin induces the degradation of cav-1 through the proteasome-dependent pathway. a Real-time RT-PCR was performed as described in ‘Methods’. WT and parkin KO MEF cells were incubated with with 10 μg/ml cyclohexamide (CHX) for the indicated times b 10 μM MG132 for 3.5 h c and 10 μM lactacystin for 3.5 h d Lysates were analyzed by SDS-PAGE and Western blotting. P values were determined using a two way ANOVA, **p < 0.01. e WT and parkin KO MEF cells cultured on poly-D-lysine coated glass were incubated with 10 μM MG132 for 3.5 h, stained with anti-cav-1 (red), and then observed by confocal microscopy. Blue indicates DAPI staining. Scale bar indicates 10 μm
Mentions: To investigate in more detail the manner in which the loss of parkin leads to an increase in cav-1, we tested the mRNA level of cav-1 in both WT and parkin KO MEF cells. Quantitative RT-PCR shows that the mRNA level of cav-1, as well as that of cav-2, flot-1, and flot-2 were not different between the two cell lines (Fig. 2a), suggesting that parkin does not regulate cav-1 at the transcriptional level. Parkin regulates protein degradation as an E3 ubiquitin ligase [6]. In addition, cav-1 has been reported to be partially degraded by the ubiquitin proteasome system (UPS) [26, 27], speculating that parkin may regulate cav-1 degradation via the UPS. To confirm this hypothesis, WT and parkin KO MEF cells were treated with cycloheximide (CHX), an inhibitor of protein synthesis, and the expression of cav-1 was assessed using Western blotting. As shown in Fig. 2b, the degradation of cav-1 in WT MEF cells was significantly faster than in parkin KO MEF cells. In addition, while treatment with the proteasomal inhibitors, MG-132 and lactacystin, led to an accumulation of cav-1 in WT MEF cells, it did not further enhance the steady state levels of cav-1 in parkin KO MEF cells. (Fig. 2c and d). Confocal microscopic analysis confirmed this observation (Fig. 2e), suggesting that parkin regulates cav-1 degradation via the UPS. These results also prompted speculation that cav-1 may be a substrate of parkin, thus, we tested the interaction between the two proteins. As shown in Fig. 3a, both flag-parkin and cav-1-EGFP were overexpressed in parkin KO MEF cells, and immunoprecipitation shows their interaction. Antibody staining of endogenous parkin and cav-1 in MEF cells shows the colocalization of both proteins (Fig. 3b), which was also confirmed using an in situ proximity ligation assay (PLA) (Fig. 3c), suggesting that parkin interacts with cav-1. Next, in order to investigate whether parkin ubiquitinates cav-1, we transfected parkin KO MEF cells with flag-parkin, His-ubiquitin, and cav-1-EGFP, which resulted in an increase in the ubiquitination of cav-1-EGFP by flag-parkin (Fig. 3d). Furthermore, mutants of parkin that have been identified in patients with familial PD did not rescue the increase in cav-1 by loss of parkin (Fig. 3e), suggesting that parkin mediates the ubiquitination of cav-1, thereby targeting cav-1 to the proteasome for degradation.Fig. 2

Bottom Line: It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD.Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells.Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Ajou University School of Medicine, 164, Worldcup-ro, Yeongtong-gu, Suwon, 16499, Korea.

ABSTRACT

Background: Parkinson's disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor dysfunctions. While most PD is sporadic in nature, a significant subset can be linked to either autosomal dominant or recessive mutations. PARK2, encoding the E3 ubiquitin ligase, parkin, is the most frequently mutated gene in autosomal recessive early onset PD. It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD. Therefore here, we examined the relationship between lipid rafts-related proteins and parkin.

Results: We identified caveolin-1 (cav-1), which is one of the major constituents of lipid rafts at the plasma membrane, as a substrate of parkin. Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells. Moreover, the total cholesterol level and membrane fluidity was altered by parkin deficiency, causing dysregulation of lipid rafts-dependent endocytosis. Further, cell-to-cell transmission of α-synuclein was facilitated by parkin deficiency.

Conclusions: Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

Show MeSH
Related in: MedlinePlus