Alpha Synuclein Membrane-Induced Aggregation: Its Role in Parkinson's Disease and a Potential Therapeutic Target

Daniel Ysselstein, Purdue University

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that causes significant hardship for patients and caregivers for many years. The classic motor symptoms observed in PD are caused by the death of dopaminergic neurons in the substantia nigra region of the brain. The surviving dopaminergic neurons contain cytoplasmic inclusions call Lewy bodies, which predominantly contain large amyloid-like fibrils of the protein alpha-synuclein (aSyn). The production of these amyloid fibrils can be easily replicated in a test tube using purified protein. It is widely believed that the aggregation of aSyn is a key event in the development PD. Detailed insights into the process by which aSyn aggregates in the cell are critical for our understanding of the disease and for the development of new therapeutics to treat PD. Despite considerable efforts to develop inhibitors of aSyn fibrillization over the past 15 years, this approach has met with limited success. This has led to the hypothesis that the aggregation of aSyn, at least in early stages, occurs by a mechanism independent of aSyn fibril formation. In the cell, a considerable fraction of aSyn is bound to lipid membranes, which render it resistant to aggregation. We and others have hypothesized that disruption of the normal membrane interaction could promote aggregation of aSyn on the membrane surface. To examine this hypothesis, we characterized the membrane interaction and membrane-induced self-assembly of the familial aSyn mutants A30P and G51D and other aSyn variants. This study, one of the first in-depth analyses of the G51D mutant, revealed that A30P and G51D display an increased propensity for aggregation at the membrane surface and increased dopaminergic neurotoxicity compared to WT aSyn. In contrast, aSyn variants with a weak propensity for membrane-induced aggregation are non-toxic. Taken together, these results suggested a correlation between aSyn aggregation propensity at the membrane surface and the ability of aSyn to elicit dopaminergic neurotoxicity. By extension of this hypothesis, reducing membrane-induced aSyn aggregation should protect against aSyn-mediated neurodegeneration. To test this idea, we investigated whether an interacting protein could interfere with membrane-induced aSyn aggregation and alleviate aSyn neurotoxicity. The goal of this study was in part to strengthen our initial hypothesis that membrane-induced aggregation is involved in aSyn-mediated dopaminergic cell death, but in addition we aimed to determine whether membrane-bound aSyn could be a valid therapeutic target. The aSyn binding partner examined here was endosulfine-alpha (ENSA), previously shown to interact selectively with membrane-bound aSyn and to be down-regulated in the brains of individuals with Alzheimer’s disease and Down’s syndrome. We found that ENSA interfered with membrane-induced aSyn aggregation, vesicle permeabilization, and alleviated aSyn neurotoxicity in a primary midbrain culture model of PD. Both the inhibitory and the neuroprotective activities of ENSA were lost when we introduced a point mutation previously shown to disrupt the binding of ENSA to membrane-associated aSyn, suggesting that ENSA interactions with aSyn at the membrane surface are critical for the ability of ENSA to interfere with aSyn aggregation, permeabilization, and neurotoxicity. The inhibitory effects of ENSA on membrane-induced aSyn aggregation, vesicle permeabilization, and aSyn neurotoxicity led us to hypothesize that a small molecule could potentially have similar protective activities. To identify candidates, we performed a phage display screen for heptapeptides that interact with different membrane-bound aSyn conformers. High-frequency interacting peptides were synthesized and examined for their effects on membrane-induced aggregation and vesicle permeabilization of G51D aSyn. Moreover, peptidomimetic derivatives of the peptides with enhanced metabolic stability were tested for the ability to protect against neurotoxicity elicited by G51D and A30P in primary midbrain cultures. We found several peptides and peptidomimetcs that interfered with membrane-induced aggregation of G51D and alleviated A30P- and G51D-mediated dopaminergic cell death. Together these results point to a key role for the aggregation of aSyn on the membrane surface in the neurotoxic effects elicited by this protein. This conclusion is strengthened by our observation that the inhibition of membrane-induced aSyn aggregation by ENSA was sufficient to alleviate aSyn neurotoxicity. Additionally, we showed that small-molecule peptides and peptidomimetics could be designed to reduce aSyn aggregation and aSyn-mediated dopaminergic cell death. Our findings strongly implicate membrane-induced aSyn aggregation in aSyn neurotoxicity and suggest that small-molecule inhibitors of aSyn self-assembly at membrane surfaces could be viable therapies to slow progression of the disease.

Degree

Ph.D.

Advisors

Rochet, Purdue University.

Subject Area

Neurosciences|Cellular biology|Biochemistry

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