For proteins, ubiquitination is a death sentence. Researchers at Harvard Medical School in Boston have now identified a long-sought enzyme that delivers the deadly molecule to α-synuclein. This, of course, is the protein that accumulates in the Lewy bodies of Parkinson’s disease (PD) and Lewy body dementia. In the September 27 Proceedings of the National Academy of Sciences online, Alfred Goldberg’s group reports that Nedd4 adds ubiquitin chains to α-synuclein in vitro and in nerve cells in culture, marking it for destruction by lysosomes. “This is a potentially highly important finding,” said David Sulzer of Columbia University in New York City, who was not involved in this work. “If it stands up to further testing, it will be a critical piece of the puzzle to understand the pathogenic mechanism [of α-synucleinopathies].”

Typically, when a chain of ubiquitin molecules gets hitched to a protein—usually a misfolded or damaged one—that protein becomes doomed for destruction in the cell. But although some α-synuclein does get ubiquitinated, much of it somehow escapes degradation, perhaps as a result of a faulty mechanism (see ARF related news story on Liu et al., 2002 and ARF related news story on Liu et al., 2009 and Cullen et al., 2009). To investigate this possibility, researchers have long been trying to pin down the enzyme that tacks ubiquitin onto α-synuclein.

Nedd4 is one of many E3 ubiquitin ligases, a class of enzymes that catalyze the last of three steps required for the addition of ubiquitin to proteins. George Tofaris, a postdoc in Goldberg’s lab, now at Oxford University in the U.K., started to have a hunch that Nedd4 might be involved in α-synuclein ubiquitination when he noticed that the protein contains a C-terminal region sequence similar to those found in other Nedd4 substrates. Tofaris and another postdoc in Goldberg’s lab, Hyoung Tae Kim, decided to test a possible α-synuclein-Nedd4 connection.

When they expressed high levels of Nedd4 in neuronal cells, the added Nedd4 activity boosted α-synuclein degradation. Conversely, when Nedd4 levels were down, α-synuclein content shot up. Having established that Nedd4 ubiquitinates α-synuclein and targets it for degradation, the researchers next turned to the mechanism involved.

Ubiquitinated proteins are degraded either by lysosomes or proteasomes, depending on the type of ubiquitin chain attached to them. Nedd4 tags proteins with ubiquitin chains linked to one another through their lysine 63 residues. That configuration normally targets membrane proteins to lysosomes. To test if this is true for synuclein, Goldberg and colleagues exposed nerve cells to different inhibitors of protein degradation. Chloroquine, a chemical that blocks lysosomal function, reduced α-synuclein degradation resulting from the increased Nedd4 activity. On the other hand, bortezomib, which blocks proteasome function, did not. Thus, Nedd4 seems to target α-synuclein for destruction in the lysosome.

Ubiquitinated membrane proteins are typically engulfed by endosomes and carried to the lysosome via a pathway known as ESCRT (which stands for endosomal sorting complex required for transport). According to Goldberg, no one had implicated the ESCRT pathway in the degradation of α-synuclein, but using RNA interference to block various molecules involved in ESCRT, his group showed that Nedd4 acts via this route. Previously, researchers reported that α-synuclein is degraded in lysosomes by chaperone-mediated autophagy—which clears cytosolic proteins independently of ubiquitination (see ARF related news story on Cuervo et al., 2004). Since α-synuclein can exist as both a membrane-associated and cytosolic protein, it is probably degraded in cells via multiple pathways, said Goldberg.

“A role for the endocytic-lysosomal pathway in neurodegeneration is emerging. Goldberg’s study further highlights the importance of this pathway and offers specific mechanistic insights that are valuable for further research in this area,” wrote Dimitri Krainc at Massachusetts General Hospital in Boston in an e-mail to ARF (see complete comment below).

To approach whether Nedd4 activity could play a role in disease, Tofaris and Goldberg first turned to a widely used model system: yeast cells overexpressing human α-synuclein (see ARF related news story on Outeiro and Lindquist, 2003). They showed that expression of Nedd4’s yeast ortholog, Rsp5p, protected cells from α-synuclein-induced toxicity. Moving to study healthy humans, they were unable to detect Nedd4 in pigmented neurons of the locus coeruleus and substantia nigra, brain areas affected by PD. In contrast, they did find Nedd4 strongly expressed in these brain regions in neurons containing Lewy bodies. “We think this increased expression of Nedd4 might reflect a compensatory change that occurs to reduce the toxic protein in these cells,” said Goldberg. “It will be important to confirm this finding in a physiological model of disease.”

More experiments will be needed before Nedd4 is confirmed to be the α-synuclein ubiquitin ligase in vivo and to play a role in slowing the development of PD. As Sulzer pointed out, other enzymes, most notably parkin, had been implicated in α-synuclein degradation but did not stand the test of time. If Nedd4 does turn out to play a role in neurodegeneration, the finding might be relevant not only to PD and other Lewy body-associated pathologies, but also other diseases involving protein aggregation. “This [endosomal-lysosomal degradation] pathway may also target the amyloid precursor protein and tau,” said Goldberg.—Laura Bonetta

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Comments on News and Primary Papers

  1. Multiple cellular systems have been implicated in the degradation of α-synuclein, including proteasomes, macroautophagy, and chaperone-mediated autophagy. The results of this study also implicate the endosomal-lysosomal pathway, and the authors speculate that this pathway may specifically catalyze the degradation of a membrane-associated pool of α-synuclein, whereas autophagy degrades the aggregated forms.

    Importantly, the authors identify Nedd4 as the E3 ligase involved in α-synuclein degradation via the endosomal pathway. Although a growing number of E3 ubiquitin ligases and their targets have been identified, much less is known about the mechanisms that regulate their activity. For example, recent work by Stenmark and colleagues showed that Nedd4 expression also controls the stability of beclin 1 (Platta et al., 2011), which plays a central role in endocytic trafficking. Previous work also showed that PTEN, a negative regulator of the PI3K pathway, is a key downstream target of Nedd4 (e.g., Wang et al., 2007). The downstream effectors of PI3K signaling, among other roles, act to regulate cytoskeletal dynamics, and thus have a profound impact on cell motility and remodeling of neuronal morphology. Therefore, it would be of interest to examine the regulation of Nedd4 in relation to these various substrates, including α-synuclein.

    Furthermore, a role for the endosomal-lysosomal pathway in neurodegeneraiton is emerging, and the study by Goldberg and colleagues further highlights the importance of this pathway and offers specific mechanistic insights that are very valuable for further research in this area.

    References:

    . Nedd4-dependent lysine-11-linked polyubiquitination of the tumour suppressor Beclin 1. Biochem J. 2012 Jan 1;441(1):399-406. PubMed.

    . NEDD4-1 is a proto-oncogenic ubiquitin ligase for PTEN. Cell. 2007 Jan 12;128(1):129-39. PubMed.

    View all comments by Dimitri Krainc

References

News Citations

  1. Jeckyl and Hyde: Protein Degradation in Parkinson's Just Added New Player, Or Did It?
  2. Striking at Synuclein by Driving Degradation
  3. Lysosomes and Proteasomes Compete for PD Researchers' Attention
  4. Yeast Teases Apart Huntington’s and Parkinson’s Protein Aggregation

Paper Citations

  1. . The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility. Cell. 2002 Oct 18;111(2):209-18. PubMed.
  2. . Membrane-associated farnesylated UCH-L1 promotes alpha-synuclein neurotoxicity and is a therapeutic target for Parkinson's disease. Proc Natl Acad Sci U S A. 2009 Mar 24;106(12):4635-40. PubMed.
  3. . Cathepsin D expression level affects alpha-synuclein processing, aggregation, and toxicity in vivo. Mol Brain. 2009;2:5. PubMed.
  4. . Impaired degradation of mutant alpha-synuclein by chaperone-mediated autophagy. Science. 2004 Aug 27;305(5688):1292-5. PubMed.
  5. . Yeast cells provide insight into alpha-synuclein biology and pathobiology. Science. 2003 Dec 5;302(5651):1772-5. PubMed.

Further Reading

Papers

  1. . A lysosomal lair for a pathogenic protein pair. Sci Transl Med. 2011 Jul 13;3(91):91ps28. PubMed.

Primary Papers

  1. . Ubiquitin ligase Nedd4 promotes alpha-synuclein degradation by the endosomal-lysosomal pathway. Proc Natl Acad Sci U S A. 2011 Oct 11;108(41):17004-9. PubMed.