2 September 2010. To release neurotransmitters, nerve terminals must repeatedly break down and rebuild specialized protein complexes, and the fidelity of this process may rely on α-synuclein, a protein that misfolds in the brains of people with Parkinson disease and other dementias. That’s the conclusion of a study published August 26 in Science. Scientists report that α-synuclein promotes assembly of SNARE complexes that mediate vesicle fusion at presynaptic terminals. α-synuclein “does not act acutely in synaptic transmission,” said lead investigator Thomas Südhof, Stanford University, Palo Alto, California, in an interview with ARF. “It is what I call an insurance policy. It backs up SNARE complex recycling to ensure its survival over an animal’s lifetime.” While the study does not directly address neurodegeneration, the data describe a physiological role for α-synuclein that explains how trapping the protein in Lewy bodies, as in slowly progressive neurodegenerative diseases such as PD, might seem initially harmless but cause neurons to suffer in the long run. Moreover, in last week’s PNAS Early Edition, Mark Gluck, Rutgers University, Newark, New Jersey, and colleagues report finding impaired learning in people who have two copies of the α-synuclein gene and are predisposed to Parkinson’s but lack, as yet, PD-like symptoms. The data hint that α-synuclein mutations could have subtle effects that result from neurotransmission problems.
The Science paper “just blows me away,” said Mark Cookson, National Institute on Aging, Bethesda, Maryland, who was not involved in the study. “It’s the first clearly defined, well-delineated function for α-synuclein that makes a lot of sense.”
For all the attention heaped on the protein’s link to neurodegenerative disease, α-synuclein’s biological role has been elusive. The brain has copious amounts of it, and various reports suggest it modifies transmitter release, though at times claiming “extremely different things,” Südhof said, including reducing and stimulating neurotransmitter release (e.g., Nemani et al., 2010; Liu et al., 2004).
He and coworkers jumped into the fray with their serendipitous discovery that modest overexpression of α-synuclein rescued neurodegeneration in mice lacking the presynaptic chaperone protein CSPα (cysteine string protein-α), whereas deletion of endogenous synucleins hastened neuronal death (Chandra et al., 2005 and ARF related news story). “We’ve been interested ever since in understanding how that comes about,” Südhof said.
Given that the CSPα knockout mice had trouble assembling SNARE complexes, the researchers figured α-synuclein might compensate for its absence by helping SNARE proteins work properly. To test this idea, first author Jacqueline Burré and colleagues immunoprecipitated SNARE complexes from the brains of wild-type mice, CSPα knockout mice, and from CSPα knockouts rescued by transgenic α-synuclein. They pulled out the complexes using antibodies to the SNARE protein SNAP-25, and found α-synuclein in the mix. Furthermore, α-synuclein antibodies immunoprecipitated SNARE proteins from mouse brains or cell lines that co-expressed the proteins. However, when individual SNARE proteins were co-expressed with α-synuclein, only synaptobrevin-2 came down with the anti-synuclein antibody. Using truncation mutants, the researchers mapped this interaction to the N-terminal part of synaptobrevin-2 and a C-terminal region of α-synuclein distinct from the domain already known to bind phospholipids (Chandra et al., 2003).
The work indicates that α-synuclein binds to SNARE complexes via synaptobrevin-2. But how could this rescue CSPα knockout mice from massive neuronal death? This puzzled the scientists because transgenic α-synuclein does not seem to affect synaptic strength. Electrophysiological readouts in brain slices of wild-type and CSFα knockout mice remained constant regardless of whether or not the animals had an α-synuclein transgene.
The researchers found that α-synuclein sustains waning neurons by helping them make SNARE complexes. In co-transfection experiments, HEK293 cells with more α-synuclein also had more SNARE complex. This relationship required the C-terminal region of α-synuclein, as judged by studies with transgenes lacking this domain.
Furthermore, Burré and colleagues generated α-, β-, and γ-synuclein triple knockout mice, with phenotypes that back up the biochemical data. As they aged, these mice made SNARE complexes less robustly, and had lower levels of synaptobrevin-2, relative to young animals. Moreover, the authors found that restoring expression of α-synuclein in cultured neurons from the triple knockout mice helped re-establish SNARE complexes, and did so in a dose-dependent way.
Behaviorally, young knockouts looked normal, whereas older mice developed motor-related neurological impairments and died prematurely—though most made it past one year. These problems were “relatively late onset,” Matthew Lavoie, Brigham and Women’s Hospital, Boston, told ARF. “I think that is in line with what one would like to see in an aging model. In PD, for instance, we're talking about onset in the fifth of sixth decade of life.”
Based on the new data, α-synuclein’s SNARE-promoting function is “akin to a proofreading activity that is essential for the continued maintenance of SNARE-mediated fusion over the lifetime of an animal,” the authors wrote.
Still, the finding that α-synuclein has no effect on synaptic strength may seem hard to reconcile with its marked effects on formation of SNARE complexes. The observations become easier to swallow in light of the discovery that SNAREs have “unbelievably high functional reserve” for acute synaptic transmission measured in brain slices, Südhof said. In his lab, RNAi knockdown of SNAP-25 to 5 percent of wild-type levels did not cause any appreciable change in synaptic transmission of cultured mouse neurons. “We were surprised by that,” he said.
“Our hypothesis is that the effects of the decrease in SNARE complex assembly are chronic, cumulative, and long term, which agrees well with the fact that these mice do live,” Südhof said.
Combined with the fact that in other species, “you can clearly make neurons and get them to fire without synucleins or their homologues,” the current data suggest that α-synuclein’s role “is more modulatory,” Cookson said. Hence, a big challenge now is to find conditions that reveal the modulation—“to figure out where synuclein is important, and why,” he said.
The PNAS paper may provide some clues as to why synuclein is important from an entirely different level of investigation. Here, first author Szabolcs Kéri of Semmelweis University, Budapest, Hungary, and colleagues studied people with a rare α-synuclein duplication—all siblings of PD patients—who themselves had not yet developed PD-like motor or cognitive symptoms. Compared with age-matched volunteers whose α-synuclein was normal, the gene duplication carriers showed defects in reward and punishment learning. Since all were in the pre-motor stage of PD at the time of behavioral testing, the data indicate that “reward learning deficits can be dissociated from motor symptoms and can precede the onset of motor deficits,” the authors wrote.—Esther Landhuis.
Burré J, Sharma M, Tsetsenis T, Buchman V, Etherton M, Südhof TC. Alpha-Synuclein Promotes SNARE-Complex Assembly in Vivo and in Vitro. Science. 26 August 2010. Abstract
Kéri S, Moustafa AA, Myers CE, Benedek G, Gluck MA. Alpha-Synuclein Gene Duplication Impairs Reward Learning. PNAS Early Edition. August 2010. Abstract