Sans synuclein tetramers, mice mimic Parkinson’s disease

A new model of Parkinson’s disease re-creates key features of the disorder, and helps steady one disputed theory of what causes it. Mice carrying mutations that disrupt physiological tetramers of the α-synuclein protein develop brain pathology and neurodegeneration typical of PD, according to work from the lab of Dennis Selkoe at Brigham and Women’s Hospital in Boston. Animals as young as three months had trouble walking and endured spontaneous whole-body tremors. As in people, the symptoms were worse in males and responded partially to treatment with the drug L-DOPA. The mice offer a more complete picture of PD than existing models, said Selkoe, which bodes well for their use in testing potential treatments. The work supports the hypothesis, formulated in Selkoe’s lab, that α-synuclein toxicity stems from destabilization of physiological tetramers and accumulation of aggregation-prone monomers. The work appeared October 10 in Neuron.

  • Researchers debut α-synuclein tetramer ablation model of PD.
  • Mutations that destabilize tetramers cause spontaneous tremor and gait disturbances that respond to L-DOPA.
  • Model supports tetramer hypothesis, suggests new therapeutic approach.

“We have been trying to find evidence that the tetramer/monomer ratio matters,” said Selkoe. “These mice, with their consistent phenotype of Parkinson features—the sex difference, a resting tremor that gets worse, gait abnormalities, and the L-DOPA response—support the hypothesis that shifting tetramers to monomers is adverse,” he told Alzforum.

“The study is very exciting, and the authors need to be commended for their relentless pursuit of this unconventional (and once-unpopular) idea,” wrote Subhojit Roy, University of Wisconsin, Madison.

Other researchers also praised the work, and the new model. “This is an extremely elegant study in which the authors rigorously assess the phenotype of a transgenic mouse model in which the α-synuclein tetrameric form is destabilized. The phenotype seems more striking than observed in other mouse α-synuclein models and the partial response to L-DOPA treatment also suggests that this model may mimic human disease,” wrote Dario Alessi, University of Dundee, Scotland.

Mutations in α-synuclein cause Parkinson’s disease, dementia with Lewy bodies, and other synucleinopathies, which are all characterized by the accumulation of aggregates of the protein in the brain. A cytoplasmic resident that normally regulates trafficking of presynaptic vesicles in neurons, α-synuclein exists in cells in two forms, according to Selkoe’s hypothesis: a stable, helically folded tetramer, and free monomers (Aug 2011 news; Oct 2014 news). Mutations in α-synuclein that cause familial PD destabilize the tetramer structure in cells, and the theory holds that shifting the ratio between tetramers and aggregation-prone monomers kicks off pathological protein accumulation, and ultimately, cell death (Apr 2015 conference news).

In the new work, first author Silke Nuber parlayed tetramer-destabilizing mutations into a unique mouse model of PD. Previously, the lab had identified a series of six-residue KTKEGV repeats in α-synuclein that hold tetramers together (Dettmer et al., 2015). The PD mutation, E46K, in the middle of one repeat, was sufficient to break up tetramers, but its effects were amplified, they found, by making two additional analogous mutations in adjacent repeats. While one E46K decreased the ratio of tetramers to monomers in cells by 40 percent, the E35K/E46K/E61K triple mutant, which they call 3K, reduced that ratio by more than 90 percent, and triggered more severe α-synuclein aggregation and toxicity in cells.

What would the mutation do in mice? To find out, Nuber generated transgenic mouse strains expressing human α-synuclein with the triple mutant (3K) or wild-type human α-synuclein. To avoid overexpression artifacts, Nuber selected transgenic strains with α-synuclein levels similar to those found in human brain, and compared the animals to an existing strain expressing the E46K mutant (1K) made by Elan Pharmaceuticals. In agreement with studies in cells, the mutations decreased tetramers, while monomers became more plentiful. The ratio of tetramers to monomers in 1K brain dropped by 60 percent, while in the 3K mice, the reduction was greater than 90 percent, and was detected in multiple brain regions. In 3K brain, more monomers ended up in the insoluble fraction, and some of them appeared to be truncated, similar to what has been seen in PD brain.

The reduction in tetramers was accompanied by dramatic motor changes starting at an early age. While most PD models begin to show motor impairment at eight to 12 months, by three months the 3K mice already had developed a spontaneous head and body tremor, which gradually worsened with age. This type of tremor is not seen in other PD models but resembles the resting tremor of PD patients, Selkoe said. Their coordination also suffered: The mice had trouble climbing a pole, or balancing on a rotating rod. By six months, the animals developed a stiff gait and moved around less in their cages. Symptoms appeared at a younger age and were more robust in male mice than female, which is also true of people with Parkinson’s.

More mutations, more trouble.

Ser129 phospho-synuclein accumulates over time in cortical neurons of young mice expressing WT, E46K (1K), or triple mutant (3K) human α-synuclein, with 3K mice having the fastest and greatest accumulation. [Courtesy of Nuber et al., Neuron 2018.]

These behavioral changes tracked with early signs of brain pathology. At three months, the 3K mice harbored protease-resistant, truncated, and Ser129-phosphorylated α-synuclein deposits. Some of the α-synuclein organized into vesicle-associated, lipid-rich aggregates that grew over time and might be precursors to Lewy bodies, claimed the authors. Indeed, in 16-month-old mice, Nuber did detect rare Lewy body-like inclusions. In the 1K mice, pathological changes accrued more slowly and never reached the same severity, consistent with the milder phenotype reported for these mice.

The 3K mutations also triggered loss of dopaminergic neurons, the hallmark of PD. Six-month-old mice had 27 percent fewer dopaminergic cells than wild-type α-synuclein controls, and produced39 percent less dopamine in the striatum. Boosting the neurotransmitter with a single dose of L-DOPA improved the animals’ performance: They climbed and hung onto a pole more easily, and the fluidity of their walking especially improved. The single shot of L-DOPA did not improve the tremor, or their ability to walk on the rotating rod, which suggested to the authors that these phenotypes might be due to cortical neurodegeneration.

“The model, which recapitulates more cardinal features of PD than published PD models, supports the novel mechanistic insight that interfering with physiological α-synuclein tetramers can lead to PD,” wrote Hanseok Ko, Johns Hopkins University in Baltimore. Ko sees the model as an important step toward better animal models of PD for mechanistic and therapeutic studies. He and Ted Dawson, also at Johns Hopkins, recently reported that tetramer instability contributes to α-synuclein toxicity that comes along with mutations in glucocerebrosidase 1 (GBA1), the most common genetic risk factor for idiopathic PD (Kim et al., 2018). Restoring tetramers by either overexpressing GBA1 or lowering levels of toxic lipids that accumulate in mutant cells prevented α-synuclein toxicity in their cell models.

If the tetramers are critical to α-synuclein function, then compounds that stabilize tetramers might be good treatments, Selkoe said. That’s the idea behind an FDA-approved treatment for familial amyloid polyneuropathy, which promotes tetrameric, non-amyloidogenic assembles of the transthyretin protein (Aug 2011 news). Selkoe hopes to replicate that approach for α-synuclein. He told Alzforum that his lab has identified commercially available small molecules that restore the tetramer/monomer ratio in cells bearing α-synuclein mutations, and is now testing those in the animal model.

Clinically, the E46K mutation associates not only with motor symptoms but also with dementia in Parkinson’s patients or dementia with Lewy bodies (DLB). While the authors highlight PD symptoms in the paper, Selkoe said the 3K mice likely also model DLB. Nuber explained that it was challenging to measure cognition in the mice, because many tests rely on animals walking or swimming well. They are now testing the mice in cognitive tasks, such as object recognition, that don’t rely so much on movement.

“This is impressive work but I don’t think yet the model has been phenotyped sufficiently to recommend it as the PD animal model everyone should be using,” wrote Clive Ballard, University of Exeter. “I think we need more information re. transcriptional changes and the long-term progression of pathology and behavioral changes. We also need more understanding of how this impacts on other aspects of the disease that may be important—mitochondrial dysfunction, clearance of synuclein, neuroinflammation, etc.—and the pros and cons compared to other emerging PD models such as GBA mutations.” Different models may be called for, for different types of studies, he wrote.

Selkoe told Alzforum they are planning some of those studies. They will be tracking the progression of α-synuclein pathology over time, and since several lines of evidence point to an important role of lipids in tetramer stabilization, lipidomics approaches are on the table, too. Nuber told Alzforum she is beginning to explore the reasons for the prominent sex differences.

What about other α-synuclein mutations? The lab previously found that, among the five missense mutations that cause PD, G51D, which lies outside of the repeat domain, has the strongest tetramer-abrogating effect in cells. They want to see what that mutation will do in mice, too.

On a technical note, all the studies of 3K mice used heterozygotes, carrying just one copy of the mutated gene. Because of the young onset and exaggerated tremor symptoms, male mice had trouble breeding. With some effort, Nuber got the mice to produce one litter of homozygotes, but they had a lethal motor phenotype. The mice developed whole-body tremor shortly after birth and couldn’t get to food or water on their own. By four weeks, they had severe pSer129-postive α-syn aggregates in their brains.

Instead, Nuber mated female heterozygous mutants with wild-type males, and genotyped the offspring to identify mutation carriers. For ease of future work, she generated a lower-expressing 3K homozygous line, with intermediate pSer129 pathology, and later onset of less-dramatic motor symptoms. These mice will be easier to maintain for testing treatments, for example. Selkoe said they plan to make both of the 3K strains available to researchers through Jackson Labs in Bar Harbor, Maine.—Pat McCaffrey.

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