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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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Exocytosis-dependent secretion of α-synuclein in 3-methyladenine (3-MA)-treated cells. (a) Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) or 10 mℳ 3-MA and incubated at a normal (37 °C) or low (16 °C) temperature to inhibit exocytosis for 2 h. (a) The temperature-dependent release of α-synuclein to the medium (***P<0.001). (b) The cell survival percentage at low temperature. (c) The increase in α-synuclein aggregates in the vesicle fraction of 3-MA-treated cells is shown. The arrow represents the size of α-synuclein monomers, and the brackets show the aggregated forms of α-synuclein. (d) The graph shows the fold increase of α-synuclein in the vesicle fractions (*P<0.05).
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fig4: Exocytosis-dependent secretion of α-synuclein in 3-methyladenine (3-MA)-treated cells. (a) Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) or 10 mℳ 3-MA and incubated at a normal (37 °C) or low (16 °C) temperature to inhibit exocytosis for 2 h. (a) The temperature-dependent release of α-synuclein to the medium (***P<0.001). (b) The cell survival percentage at low temperature. (c) The increase in α-synuclein aggregates in the vesicle fraction of 3-MA-treated cells is shown. The arrow represents the size of α-synuclein monomers, and the brackets show the aggregated forms of α-synuclein. (d) The graph shows the fold increase of α-synuclein in the vesicle fractions (*P<0.05).

Mentions: Previous studies have suggested that α-synuclein is released from cells via exocytosis.18 This suggestion was based on observations that α-synuclein secretion was temperature sensitive and that a small amount of intracellular α-synuclein was present in the lumen of vesicles. To verify the involvement of exocytosis in the increased α-synuclein secretion of autophagy-compromised cells, we assessed the temperature sensitivity and vesicle localization of α-synuclein in the presence of 3-MA. An ELISA measurement showed that α-synuclein secretion was significantly decreased at 16 °C compared with the secretion at 37 °C in both the control and 3-MA-treated cultures of differentiated SH-SY5Y cells (Figure 4a). When this experiment was performed, no significant difference in cell viability was observed (Figure 4b). Next, we measured changes in the vesicular localization of α-synuclein. Upon treatment with 3-MA, the levels of α-synuclein in the vesicle fractions increased significantly (Figures 4c and d). These results suggest that the increased α-synuclein secretion occurring upon autophagic suppression is mediated by vesicle-mediated exocytosis.


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)

Exocytosis-dependent secretion of α-synuclein in 3-methyladenine (3-MA)-treated cells. (a) Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) or 10 mℳ 3-MA and incubated at a normal (37 °C) or low (16 °C) temperature to inhibit exocytosis for 2 h. (a) The temperature-dependent release of α-synuclein to the medium (***P<0.001). (b) The cell survival percentage at low temperature. (c) The increase in α-synuclein aggregates in the vesicle fraction of 3-MA-treated cells is shown. The arrow represents the size of α-synuclein monomers, and the brackets show the aggregated forms of α-synuclein. (d) The graph shows the fold increase of α-synuclein in the vesicle fractions (*P<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Exocytosis-dependent secretion of α-synuclein in 3-methyladenine (3-MA)-treated cells. (a) Differentiated SH-SY5Y cells expressing α-synuclein were treated with dimethyl sulfoxide (DMSO) or 10 mℳ 3-MA and incubated at a normal (37 °C) or low (16 °C) temperature to inhibit exocytosis for 2 h. (a) The temperature-dependent release of α-synuclein to the medium (***P<0.001). (b) The cell survival percentage at low temperature. (c) The increase in α-synuclein aggregates in the vesicle fraction of 3-MA-treated cells is shown. The arrow represents the size of α-synuclein monomers, and the brackets show the aggregated forms of α-synuclein. (d) The graph shows the fold increase of α-synuclein in the vesicle fractions (*P<0.05).
Mentions: Previous studies have suggested that α-synuclein is released from cells via exocytosis.18 This suggestion was based on observations that α-synuclein secretion was temperature sensitive and that a small amount of intracellular α-synuclein was present in the lumen of vesicles. To verify the involvement of exocytosis in the increased α-synuclein secretion of autophagy-compromised cells, we assessed the temperature sensitivity and vesicle localization of α-synuclein in the presence of 3-MA. An ELISA measurement showed that α-synuclein secretion was significantly decreased at 16 °C compared with the secretion at 37 °C in both the control and 3-MA-treated cultures of differentiated SH-SY5Y cells (Figure 4a). When this experiment was performed, no significant difference in cell viability was observed (Figure 4b). Next, we measured changes in the vesicular localization of α-synuclein. Upon treatment with 3-MA, the levels of α-synuclein in the vesicle fractions increased significantly (Figures 4c and d). These results suggest that the increased α-synuclein secretion occurring upon autophagic suppression is mediated by vesicle-mediated exocytosis.

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