07 Feb 2014

Alpha-synuclein shape-shifts in synaptic terminals, according to a study that tracked the protein’s movements in the neurons of living mice. Published February 5 in The Journal of Neuroscience, the findings showed that while free α-synuclein moves quickly within neuronal cell bodies, it slows to a crawl and aggregates near synapses. The dynamic view may ultimately help researchers understand which forms of α-synuclein associate with pathology in Parkinson’s disease (PD) and other synucleinopathies, said study leader Vivek Unni at Oregon Health and Science University in Portland. 

Mutations that boost expression or alter the structure of α-synuclein cause familial Parkinson’s. While the normal function of the protein remains a mystery, it binds to presynaptic vesicles and may play a role in their trafficking (see Rizo and Südhof, 2012). However, the protein exists as monomers, a spectrum of oligomers, and larger aggregates, and it is unclear which form occurs in synapses or may interfere with normal processes there. Researchers don’t even agree on which states of the protein are physiological (see Feb 2012 news story). Adding to the uncertainty about its multiple personalities, most previous studies on α-synuclein aggregation have been conducted in vitro, Unni said. “You can make all kinds of aggregates in a test tube or in cell culture, but whether they are relevant to the in-vivo situation is always a question,” he said. 

To observe α-synuclein dynamics in vivo, Unni and colleagues employed fluorescence recovery after photobleaching, or FRAP. This technique measures the mobility of fluorescently labeled proteins by bleaching out their signal and then monitoring the rate at which new proteins repopulate the bleached space. The technique is normally used in vitro, but in 2010 Unni adapted the technique for use in live mice while in Bradley Hyman’s lab at Massachusetts General Hospital, Charlestown (see Unni et al., 2010). 

To monitor α-synuclein movement in cortical neurons, first author Kateri Spinelli and colleagues implanted cranial windows into mice whose neurons overexpress the protein tagged with Green Fluorescent Protein (Syn-GFP). In these animals, the uptick in synuclein is on par with the two- to threefold overexpression in people who carry an extra copy of the gene and develop early onset PD. The researchers bleached the fluorescent signal from portions of cortical neurons and monitored how quickly new Syn-GFP molecules moved into the space. 

Syn-GFP repopulated neuronal cell bodies, or soma, within milliseconds, indicating that the protein there moves quickly. However, when the researchers zapped presynaptic terminals, only 23 percent of the Syn-GFP recovered as fast. Forty-five percent took some 2.6 minutes to populate the terminals, while the remaining 32 percent took nine days or longer to come back. The researchers take this to mean that three presynaptic populations exist, which represent freely diffusible, vesicle-bound, and microaggregate forms of α-synuclein, respectively, They further hypothesize that diverse sub-pools of aggregates may exist within the most immobile fraction. 

“The existence of an extremely immobile α-synuclein pool—presumably composed of α-synuclein aggregates—is interesting, and makes one wonder about the consequences of this aggregation for synaptic physiology,” Subhojit Roy at the University of California, San Diego, wrote to Alzforum. Roy was not involved in the study.

To get a handle on the biochemical properties of the Syn-GFP populations, the researchers next treated fractionated brain extracts from Syn-GFP mice with proteinase K, which destroys mostly free soluble proteins. While the treatment wiped out Syn-GFP in cytosolic extracts, more than a third of Syn-GFP from synaptic extracts survived—indicating that the fraction of the protein that was immobile in the photobleaching experiments comprised proteinase-resistant aggregates.

Could these aggregates in the nerve terminals scupper synaptic function? Imaging brain slices, the researchers found that Syn-GFP-laden terminals contained less synapsin than did terminals void of Syn-GFP. Evidence suggests that synapsin, a synaptic vesicle protein, helps regulate neurotransmitter release. The researchers then found that by nine months of age, levels of glutamate decreased markedly in Syn-GFP terminals in these mice as well. Together, the synapsin and glutamate findings suggest that Syn-GFP in nerve terminals compromises synaptic function. 

Oddly enough, this may be a slow process. To his surprise, Unni found that the amount of slow-moving Syn-GFP in terminals remained constant in the mice from the age of one to 18 months, as did the fractions of proteinase K-resistant Syn-GFP and levels of synapsin. Only the dip in glutamate levels worsened as the mice aged. While the results don’t reveal which population of α-synuclein triggered the synaptic dysfunction, Unni hypothesized that the putative aggregates are to blame. “Over a given amount of time, even if these aggregates stay stable, it could set up a destructive process,” he said.

The researchers used an “elegant fluorescent microscopy method to measure very small things that are happening in terminals, and that’s interesting,” said Dennis Selkoe of Brigham and Women’s Hospital, Boston, who was not involved in the study. However, Selkoe, whose work suggests the predominance of a native aggregation-resistant α-synuclein tetramer (see Selkoe et al., 2013),  said he believes the conversion of stable tetramers to aggregation-prone monomers represents the earliest disease-causing event. The photobleaching method lacks the sensitivity to distinguish between monomers and small multimers (including tetramers), a limitation Unni said he hopes new techniques will one day overcome.

In the meantime, Unni plans to drill deeper into the α-synuclein pools to tease out which ones are toxic. Some reports have suggested that certain aggregates may be protective, Unni said, which raises questions about drug development strategies that seek to block all aggregation. Figuring out which aggregate is the troublemaker could aid more rational drug design, he said.—Jessica Shugart

Comments

1. • Subhojit Roy
     University of California, San Diego
   • Posted: 07 Feb 2014
   • Paper: Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease.

   These studies from Vivek Unni’s lab reiterate the importance of focusing on the synaptic accumulation and aggregation of α-synuclein, rather than somatic Lewy bodies. Specifically, they strengthen the hypothesis that small increments in α-synuclein are detrimental to synapses, and advocate that a rigorous evaluation of normal and abnormal forms of synaptic α-synuclein is needed to really move the field forward.

   The in-vivo studies by Spinelli et al. are technically challenging and are a valuable contribution to the field. They highlight the immobile fraction of synaptic α-synuclein that we and others have proposed, and also give insights into the physiological state of α-synuclein in cell bodies and synapses. The existence of an extremely immobile α-synuclein pool—presumably composed of α-synuclein aggregates—is quite interesting, and makes one wonder about the consequences of this aggregation upon synaptic physiology. 

   In light of recent studies suggesting that α-synuclein may physiologically exist as helically folded tetramers, and that these conformations resist aggregation, it will be interesting to further understand the repertoire of synaptic “aggregates/oligomers/multimers/tetramers.” Are there both “physiologic” and “pathologic” α-synuclein conformations at synapses? If so, how are these “good” and “bad” pools regulated in normal and abnormal states? A few years ago I organized a mini-symposium at SfN titled “Alpha synuclein—the Good, the Bad and the Ugly.” Clearly my timing was off and it is only now that we may be starting to frame the right questions. Does Good equal helical-tetramers, Bad equal monomers, and Ugly equal other higher-order aggregates? Maybe time will tell!

   View all comments by Subhojit Roy
2. • Walter J. Schulz-Schaeffer
     UNIVERSITÄTSMEDIZIN GÖTTINGEN GEORG-AUGUST-UNIVERSITÄT
   • Posted: 07 Feb 2014
   • Paper: Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease.

   This paper by Spinelli et al. fits well in my concept that presynaptic alpha-synuclein aggregates are pathophysiologically linked to the diseases PD, PDD, and DLB. It’s interesting to know that in-vivo multiphoton imaging may be able to monitor presynaptic alpha-synuclein aggregation. This may indeed have implications for drug development.

   View all comments by Walter J. Schulz-Schaeffer

Make a Comment

To make a comment you must login or register.

References

Alzpedia Citations

1. SNCA (α-synuclein)

News Citations

1. Synuclein—Researchers Out of Sync on Structure 17 Feb 2012

Paper Citations

1. Rizo J, Südhof TC. The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?. Annu Rev Cell Dev Biol. 2012;28:279-308. PubMed.
2. Unni VK, Weissman TA, Rockenstein E, Masliah E, McLean PJ, Hyman BT. In vivo imaging of alpha-synuclein in mouse cortex demonstrates stable expression and differential subcellular compartment mobility. PLoS One. 2010;5(5):e10589. PubMed.
3. Selkoe D, Dettmer U, Luth E, Kim N, Newman A, Bartels T. Defining the native state of α-synuclein. Neurodegener Dis. 2014;13(2-3):114-7. Epub 2013 Oct 30 PubMed.

Further Reading

Papers

1. Lashuel HA, Overk CR, Oueslati A, Masliah E. The many faces of α-synuclein: from structure and toxicity to therapeutic target. Nat Rev Neurosci. 2013 Jan;14(1):38-48. PubMed.
2. Ebrahimi-Fakhari D, McLean PJ, Unni VK. Alpha-synuclein's degradation in vivo: opening a new (cranial) window on the roles of degradation pathways in Parkinson disease. Autophagy. 2012 Feb 1;8(2):281-3. PubMed.
3. Overk CR, Masliah E. Pathogenesis of synaptic degeneration in Alzheimer's disease and Lewy body disease. Biochem Pharmacol. 2014 Apr 15;88(4):508-16. Epub 2014 Jan 21 PubMed.