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Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein.

Lee HJ, Cho ED, Lee KW, Kim JH, Cho SG, Lee SJ - Exp. Mol. Med. (2013)

Bottom Line: Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein.In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission.These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, SMART-IABS, Konkuk University, Seoul, South Korea. hjlee@kku.ac.kr

ABSTRACT
The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of α-synuclein aggregates and Lewy bodies, often found in PD and other α-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of α-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in α-synuclein-expressing cells would increase the secretion of α-synuclein, subsequently affecting the α-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein. In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.

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Increased α-synuclein aggregation and secretion occur when autophagy is inhibited. Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) and either 3-methyladenine (3-MA; 1 or 10 mℳ) (a) or rapamycin (Rap) (b). The α-synuclein aggregation was compared between triton-soluble and triton-insoluble fractions as well as in the culture medium. Arrows indicate the monomeric size of α-synuclein, and brackets indicate the aggregated α-synuclein. β-Actin was used to demonstrate an equal loading of proteins in all blots (*P<0.05, ***P<0.001).
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fig1: Increased α-synuclein aggregation and secretion occur when autophagy is inhibited. Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) and either 3-methyladenine (3-MA; 1 or 10 mℳ) (a) or rapamycin (Rap) (b). The α-synuclein aggregation was compared between triton-soluble and triton-insoluble fractions as well as in the culture medium. Arrows indicate the monomeric size of α-synuclein, and brackets indicate the aggregated α-synuclein. β-Actin was used to demonstrate an equal loading of proteins in all blots (*P<0.05, ***P<0.001).

Mentions: Differentiated SH-SY5Y neuroblastoma cells expressing human α-synuclein were treated with 3-MA, an inhibitor of autophagy-specific type-III phosphoinositide 3 kinases and Vps34 complexes. 3-MA inhibits the production of phosphatidylinositol-3-phosphate, which is important for autophagosome formation, thus inhibiting autophagy. We administered 3-MA to cells that were in a starving condition, which promotes starvation-induced autophagy, as 3-MA has been shown to promote autophagy under a nutrient-rich state.31 The 3-MA treatment increased the levels of sodium dodecyl sulfate-stable α-synuclein aggregates in both the triton-soluble and -insoluble fractions of the cells (Figure 1a). When the culture media were analyzed, we found that 3-MA increased the secretion of α-synuclein (Figure 1a). On the other hand, an autophagy-promoting condition induced by the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin resulted in decreased secretion of α-synuclein (Figure 1b). The secretion of α-synuclein occurs progressively over time, and the extent of the secretion strongly correlated with the concentration of 3-MA given to the cells (Figure 2a). The increase in α-synuclein secretion was not due to membrane leakage; cell viability measurements based on membrane integrity showed no changes with 3-MA treatment (Figure 2b). The inhibitory effect of 3-MA on autophagy was confirmed by observing the autophagosome marker LC3 in the treated cells. Additionally, mRFP-GFP tfLC3 (Kimura et al.32) was expressed in the SH-SY5Y cells expressing α-synuclein by transduction with the recombinant adenoviral vector (adeno/tfLC3). Autophagosome-bound tfLC3 has been shown to express both green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP) signals, but it loses the GFP signal after fusion with the lysosome.32 The cells were treated with 10 mℳ 3-MA for 18 h, and the cytosolic-free tfLC3 was removed by digitonin permeabilization to allow clearer observation of the membrane-bound tfLC3. The control cells displayed GFP and mRFP punctuates from the tfLC3, which often colocalized in the cells. However, the cells treated with 3-MA had significantly reduced GFP and mRFP signals, and the GFP signals were particularly weak (Figure 2c). This result could be explained by inhibition of autophagosome formation by 3-MA, which would cause a considerable decrease in the binding of tfLC3 to autophagosomes. The residual mRFP signals likely results from the tfLC3 that was delivered to lysosomes by the autophagosomes that survived the 3-MA inhibition.


Autophagic failure promotes the exocytosis and intercellular transfer of α-synuclein.

Lee HJ, Cho ED, Lee KW, Kim JH, Cho SG, Lee SJ - Exp. Mol. Med. (2013)

Increased α-synuclein aggregation and secretion occur when autophagy is inhibited. Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) and either 3-methyladenine (3-MA; 1 or 10 mℳ) (a) or rapamycin (Rap) (b). The α-synuclein aggregation was compared between triton-soluble and triton-insoluble fractions as well as in the culture medium. Arrows indicate the monomeric size of α-synuclein, and brackets indicate the aggregated α-synuclein. β-Actin was used to demonstrate an equal loading of proteins in all blots (*P<0.05, ***P<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3674407&req=5

fig1: Increased α-synuclein aggregation and secretion occur when autophagy is inhibited. Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) and either 3-methyladenine (3-MA; 1 or 10 mℳ) (a) or rapamycin (Rap) (b). The α-synuclein aggregation was compared between triton-soluble and triton-insoluble fractions as well as in the culture medium. Arrows indicate the monomeric size of α-synuclein, and brackets indicate the aggregated α-synuclein. β-Actin was used to demonstrate an equal loading of proteins in all blots (*P<0.05, ***P<0.001).
Mentions: Differentiated SH-SY5Y neuroblastoma cells expressing human α-synuclein were treated with 3-MA, an inhibitor of autophagy-specific type-III phosphoinositide 3 kinases and Vps34 complexes. 3-MA inhibits the production of phosphatidylinositol-3-phosphate, which is important for autophagosome formation, thus inhibiting autophagy. We administered 3-MA to cells that were in a starving condition, which promotes starvation-induced autophagy, as 3-MA has been shown to promote autophagy under a nutrient-rich state.31 The 3-MA treatment increased the levels of sodium dodecyl sulfate-stable α-synuclein aggregates in both the triton-soluble and -insoluble fractions of the cells (Figure 1a). When the culture media were analyzed, we found that 3-MA increased the secretion of α-synuclein (Figure 1a). On the other hand, an autophagy-promoting condition induced by the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin resulted in decreased secretion of α-synuclein (Figure 1b). The secretion of α-synuclein occurs progressively over time, and the extent of the secretion strongly correlated with the concentration of 3-MA given to the cells (Figure 2a). The increase in α-synuclein secretion was not due to membrane leakage; cell viability measurements based on membrane integrity showed no changes with 3-MA treatment (Figure 2b). The inhibitory effect of 3-MA on autophagy was confirmed by observing the autophagosome marker LC3 in the treated cells. Additionally, mRFP-GFP tfLC3 (Kimura et al.32) was expressed in the SH-SY5Y cells expressing α-synuclein by transduction with the recombinant adenoviral vector (adeno/tfLC3). Autophagosome-bound tfLC3 has been shown to express both green fluorescent protein (GFP) and monomeric red fluorescent protein (mRFP) signals, but it loses the GFP signal after fusion with the lysosome.32 The cells were treated with 10 mℳ 3-MA for 18 h, and the cytosolic-free tfLC3 was removed by digitonin permeabilization to allow clearer observation of the membrane-bound tfLC3. The control cells displayed GFP and mRFP punctuates from the tfLC3, which often colocalized in the cells. However, the cells treated with 3-MA had significantly reduced GFP and mRFP signals, and the GFP signals were particularly weak (Figure 2c). This result could be explained by inhibition of autophagosome formation by 3-MA, which would cause a considerable decrease in the binding of tfLC3 to autophagosomes. The residual mRFP signals likely results from the tfLC3 that was delivered to lysosomes by the autophagosomes that survived the 3-MA inhibition.

Bottom Line: Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein.In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission.These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, SMART-IABS, Konkuk University, Seoul, South Korea. hjlee@kku.ac.kr

ABSTRACT
The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of α-synuclein aggregates and Lewy bodies, often found in PD and other α-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of α-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in α-synuclein-expressing cells would increase the secretion of α-synuclein, subsequently affecting the α-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of α-synuclein. In a mixed culture of α-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular α-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of α-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated α-synuclein exocytosis, thereby promoting α-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.

Show MeSH
Related in: MedlinePlus