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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

Loss of parkin leads to an increase in cav-1. a Lysates prepared from WT and parkin KO MEF cells were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. b WT and parkin KO MEF cells were stained with anti-caveolin-1 (red) and observed by confocal microscopy. The intensity of three independent experiments was quantified. The mean intensity value of each experiment was acquired by measuring the intensity of at least 100 cells. Scale bar indicates 10 μm. c WT and parkin KO MEF cells were lysed in ice-cold 1 % Triton X-100 buffer and fractionated as described in ‘Methods’. The soluble and insoluble fractions were then analyzed using Western blotting. The transferrin receptor (TfR) was used as a marker for the non-lipid raft fractions. Band intensity of three independent experiments was quantified. d Parkin KO MEF cells were transfected with a plasmid for flag-parkin, and after 48 h, lysates were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. P values were determined using a Student’s t test. ** p < 0.01
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Fig1: Loss of parkin leads to an increase in cav-1. a Lysates prepared from WT and parkin KO MEF cells were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. b WT and parkin KO MEF cells were stained with anti-caveolin-1 (red) and observed by confocal microscopy. The intensity of three independent experiments was quantified. The mean intensity value of each experiment was acquired by measuring the intensity of at least 100 cells. Scale bar indicates 10 μm. c WT and parkin KO MEF cells were lysed in ice-cold 1 % Triton X-100 buffer and fractionated as described in ‘Methods’. The soluble and insoluble fractions were then analyzed using Western blotting. The transferrin receptor (TfR) was used as a marker for the non-lipid raft fractions. Band intensity of three independent experiments was quantified. d Parkin KO MEF cells were transfected with a plasmid for flag-parkin, and after 48 h, lysates were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. P values were determined using a Student’s t test. ** p < 0.01

Mentions: To explore whether loss of parkin induces functional alterations in lipid rafts, we first compared the expression level of the well-known lipid rafts marker proteins, caveolin-1 (cav-1), cav-2, flotillin-1 (flot-1), and flot-2 using WT and parkin KO MEF cells. Interestingly, we observed that the level of cav-1 was increased in parkin KO MEF cells specifically, while the levels of other proteins were not changed (Fig. 1a). Confocal microscopic analysis also shows an increase of cav-1 protein in parkin KO MEF cells compared with WT MEF cells (Fig. 1b). To investigate the distribution of increased cav-1 in parkin KO MEF cells, lipid rafts were isolated based on their solubility in 1 % Triton X-100 on ice [24, 25]. As shown in Fig. 1c, the increase in cav-1 protein in parkin KO MEF cells was observed only in the cold Triton X-100 insoluble fraction. Moreover, the distribution of the other proteins was not changed. The expression of parkin was relatively lower in MEF cells than that in neurons. Therefore, we loaded a 4-fold higher amount of protein for Western blotting of parkin and a small proportion of parkin was detected in the cold Triton X-100 insoluble fraction. This is also in agreement with a previous study [13]. To test whether parkin regulates cav-1 expression specifically, we overexpressed flag-parkin in parkin KO MEF cells. The overexpression of parkin in parkin KO MEF cells rescued cav-1 level without any change in the levels of other proteins (Fig. 1d), suggesting that parkin regulates cav-1 specifically.Fig. 1


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)

Loss of parkin leads to an increase in cav-1. a Lysates prepared from WT and parkin KO MEF cells were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. b WT and parkin KO MEF cells were stained with anti-caveolin-1 (red) and observed by confocal microscopy. The intensity of three independent experiments was quantified. The mean intensity value of each experiment was acquired by measuring the intensity of at least 100 cells. Scale bar indicates 10 μm. c WT and parkin KO MEF cells were lysed in ice-cold 1 % Triton X-100 buffer and fractionated as described in ‘Methods’. The soluble and insoluble fractions were then analyzed using Western blotting. The transferrin receptor (TfR) was used as a marker for the non-lipid raft fractions. Band intensity of three independent experiments was quantified. d Parkin KO MEF cells were transfected with a plasmid for flag-parkin, and after 48 h, lysates were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. P values were determined using a Student’s t test. ** p < 0.01
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666086&req=5

Fig1: Loss of parkin leads to an increase in cav-1. a Lysates prepared from WT and parkin KO MEF cells were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. b WT and parkin KO MEF cells were stained with anti-caveolin-1 (red) and observed by confocal microscopy. The intensity of three independent experiments was quantified. The mean intensity value of each experiment was acquired by measuring the intensity of at least 100 cells. Scale bar indicates 10 μm. c WT and parkin KO MEF cells were lysed in ice-cold 1 % Triton X-100 buffer and fractionated as described in ‘Methods’. The soluble and insoluble fractions were then analyzed using Western blotting. The transferrin receptor (TfR) was used as a marker for the non-lipid raft fractions. Band intensity of three independent experiments was quantified. d Parkin KO MEF cells were transfected with a plasmid for flag-parkin, and after 48 h, lysates were analyzed by SDS-PAGE and Western blotting, and the band intensity of three independent experiments was quantified. P values were determined using a Student’s t test. ** p < 0.01
Mentions: To explore whether loss of parkin induces functional alterations in lipid rafts, we first compared the expression level of the well-known lipid rafts marker proteins, caveolin-1 (cav-1), cav-2, flotillin-1 (flot-1), and flot-2 using WT and parkin KO MEF cells. Interestingly, we observed that the level of cav-1 was increased in parkin KO MEF cells specifically, while the levels of other proteins were not changed (Fig. 1a). Confocal microscopic analysis also shows an increase of cav-1 protein in parkin KO MEF cells compared with WT MEF cells (Fig. 1b). To investigate the distribution of increased cav-1 in parkin KO MEF cells, lipid rafts were isolated based on their solubility in 1 % Triton X-100 on ice [24, 25]. As shown in Fig. 1c, the increase in cav-1 protein in parkin KO MEF cells was observed only in the cold Triton X-100 insoluble fraction. Moreover, the distribution of the other proteins was not changed. The expression of parkin was relatively lower in MEF cells than that in neurons. Therefore, we loaded a 4-fold higher amount of protein for Western blotting of parkin and a small proportion of parkin was detected in the cold Triton X-100 insoluble fraction. This is also in agreement with a previous study [13]. To test whether parkin regulates cav-1 expression specifically, we overexpressed flag-parkin in parkin KO MEF cells. The overexpression of parkin in parkin KO MEF cells rescued cav-1 level without any change in the levels of other proteins (Fig. 1d), suggesting that parkin regulates cav-1 specifically.Fig. 1

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