Could cranking up a cellular waste removal system help rid the brain of misfolded α-synuclein, the principle component of Lewy bodies found in neurodegenerative disorders such as Parkinson’s disease (PD)? Perhaps not, suggest researchers led by Virginia Lee, University of Pennsylvania, Philadelphia, in the March 26 Journal of Biological Chemistry. They report that once formed, α-synuclein aggregates are not effectively degraded by autophagy and actually impair processing of other proteins, which may accelerate cell death. “Stimulating autophagy as a way to ramp up this cellular disposal system is not going to work to clear α-synuclein aggregates,” Lee told Alzforum. “That’s not good news in terms of using that approach for treating PD.”

In macroautophagy, double-membrane vesicles called autophagosomes encircle worn out organelles or misfolded protein aggregates before fusing with lysosomes that degrade the contents. Previous research suggested that Lewy bodies co-localize with autophagy components (see Higashi et al., 2011). However, it was unclear if Lewy body degradation followed because researchers induced α-synuclein aggregation by stressing neurons, which may have perturbed autophagy. More recently, Lee and colleagues used synthetic α-synuclein fibrils to seed Lewy body-like aggregates in either human embryonic kidney cells (HEK293) engineered to overproduce α-synuclein (see Luk et al., 2009), or primary hippocampal neurons from wild-type mice (see ARF related news story). Using these models, they tested if autophagy clears the protein clumps.

First author Selcuk Tanik and colleagues checked to see which markers of autophagy associated with synuclein. According to immunofluorescence, proteins that recruit and mark autophagosomes associated with the α-synuclein aggregates. However, Lamp1, a marker of lysosomes, did not. That suggested that the protein aggregates passed through early, but not late phases of autophagy. Autophagy inhibitors or activators did not change aggregate levels, confirming that the process did not break them down. Silencing endogenous α-synuclein expression with interfering RNAs did not facilitate clearance either, suggesting that aggregates stick around once formed. What’s more, aggregates of α-synuclein slowed autophagic processing of a 72 glutamine-repeat fragment of huntingtin (Htt Gln-72), suggesting that the Lewy bodies dampen general autophagy. Together, the findings suggest α-synuclein aggregates both resist and disrupt autophagic degradation.

How do these clumps avoid being degraded? The lysosomes appeared to be functional, as they processed non-autophagic substrates as usual and kept thier normal acidic pH. The kink probably lies somewhere in the middle of the pathway, the researchers suggest. Indeed, aggregate-associated autophagosomes showed no markers of multivesicular bodies, with which they normally fuse before lysosomes. “We think that because α-synuclein has a tendency to bind lipids, it sticks to the outside of autophagosomes, which prevents their engulfment and their degradation,” said Lee, adding “That binding also stops the fusion of autophagosomes with lysosomes.” This surface interaction does not necessarily generalize to proteins of other neurodegenerative diseases, she said. For example, autophagy clears aggregates of exon 1 of the HD gene (see Ravikumar et al., 2002), which binds less strongly to lipids.

Autophagy may be capable of tackling soluble α-synuclein or small aggregates of the protein. A recent study led by Masaki Tanaka, Kyoto Prefectural University of Medicine, Japan, reported that autophagy cleared synthetic α-synuclein fibrils from HEK293 cells expressing low levels of the protein. In that case, large aggregates of the protein did not form (see Watanabe et al., 2012). “The size of aggregates may be important,” Tanaka told Alzforum in an email. “When intracellular α-synuclein aggregates are small, they are degraded by autophagy, but they may become resistant to degradation and impair autophagy when they grow larger than a certain size,” he said.

Leonidas Stefanis, University of Athens Medical School, Greece, noted that Lee's model system only deals with larger fibrils after they have formed. “This is an interesting paper, but there is a lot more to know about how these inclusions are slowly formed in normal situations and how degradation pathways affect them in the process,” said Stefanis. The findings revive the idea that insoluble fibrils, not just oligomers, could cause major damage in PD, he noted. “Clearly they show that fully formed Lewy-body like inclusions may have detrimental effect on degradation pathways,” he told Alzforum. “We always wondered if these structures could be removed from the cell, and here we have an answer.”—Gwyneth Dickey Zakaib.

Reference:
Tanik SA, Schultheiss CE, Volpicelli-Daley LA, Brunden KR, Lee VM. Lewy body-like α-synuclein aggregates resist degradation and impair macroautophagy. J Biol Chem. 2013 Mar 26. Abstract

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References

News Citations

  1. Modeling Sporadic PD in a Dish?

Paper Citations

  1. . Localization of MAP1-LC3 in vulnerable neurons and Lewy bodies in brains of patients with dementia with Lewy bodies. J Neuropathol Exp Neurol. 2011 Apr;70(4):264-80. PubMed.
  2. . Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. Proc Natl Acad Sci U S A. 2009 Nov 24;106(47):20051-6. PubMed.
  3. . Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet. 2002 May 1;11(9):1107-17. PubMed.
  4. . p62/SQSTM1-dependent autophagy of Lewy body-like α-synuclein inclusions. PLoS One. 2012;7(12):e52868. PubMed.
  5. . Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy. J Biol Chem. 2013 May 24;288(21):15194-210. PubMed.

Further Reading

Papers

  1. . Wild type alpha-synuclein is degraded by chaperone-mediated autophagy and macroautophagy in neuronal cells. J Biol Chem. 2008 Aug 29;283(35):23542-56. PubMed.
  2. . α-Synuclein impairs macroautophagy: implications for Parkinson's disease. J Cell Biol. 2010 Sep 20;190(6):1023-37. PubMed.
  3. . Dopamine-modified alpha-synuclein blocks chaperone-mediated autophagy. J Clin Invest. 2008 Feb;118(2):777-88. PubMed.
  4. . Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science. 2004 Aug 27;305(5688):1292-5. PubMed.
  5. . Misfolded proteins partition between two distinct quality control compartments. Nature. 2008 Aug 28;454(7208):1088-95. PubMed.
  6. . Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy. J Biol Chem. 2013 May 24;288(21):15194-210. PubMed.

Primary Papers

  1. . Lewy Body-like α-Synuclein Aggregates Resist Degradation and Impair Macroautophagy. J Biol Chem. 2013 May 24;288(21):15194-210. PubMed.