Effects of phosphatidylcholine membrane fluidity on the conformation and aggregation of N-terminally acetylated α-synuclein.

Membrane association of α-synuclein (α-syn), a neuronal protein associated with Parkinson's disease (PD), is involved in α-syn function and pathology. Most previous studies on α-syn-membrane interactions have not used the physiologically relevant N-terminally acetylated (N-acetyl) α-syn form nor the most naturally abundant cellular lipid, i.e. phosphatidylcholine (PC). Here, we report on how PC membrane fluidity affects the conformation and aggregation propensity of N-acetyl α-syn. It is well established that upon membrane binding, α-syn adopts an α-helical structure. Using CD spectroscopy, we show that N-acetyl α-syn transitions from α-helical to disordered at the lipid melting temperature (Tm ). We found that this fluidity sensing is a robust characteristic, unaffected by acyl chain length (Tm = 34-55 °C) and preserved in its homologs ß- and γ-syn. Interestingly, both N-acetyl α-syn membrane binding and amyloid formation trended with lipid order (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) > 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/sphingomyelin/cholesterol (2:2:1) ≥ DOPC), with gel-phase vesicles shortening aggregation kinetics and promoting fibril formation compared to fluid membranes. Furthermore, we found that acetylation enhances binding to PC micelles and small unilamellar vesicles with high curvature (r ∼16-20 nm) and that DPPC binding is reduced in the presence of cholesterol. These results confirmed that the exposure of hydrocarbon chains (i.e. packing defects) is essential for binding to zwitterionic gel membranes. Collectively, our in vitro results suggest that N-acetyl α-syn localizes to highly curved, ordered membranes inside a cell. We propose that age-related changes in membrane fluidity can promote the formation of amyloid fibrils, insoluble materials associated with PD.

Sexually dimorphic mechanisms of VGLUT-mediated protection from dopaminergic neurodegeneration.

Parkinson's disease (PD) targets some dopamine (DA) neurons more than others. Sex differences offer insights, with females more protected from DA neurodegeneration. The mammalian vesicular glutamate transporter VGLUT2 and Drosophila ortholog dVGLUT have been implicated as modulators of DA neuron resilience. However, the mechanisms by which VGLUT2/dVGLUT protects DA neurons remain unknown. We discovered DA neuron dVGLUT knockdown increased mitochondrial reactive oxygen species in a sexually dimorphic manner in response to depolarization or paraquat-induced stress, males being especially affected. DA neuron dVGLUT also reduced ATP biosynthetic burden during depolarization. RNA sequencing of VGLUT+ DA neurons in mice and flies identified candidate genes that we functionally screened to further dissect VGLUT-mediated DA neuron resilience across PD models. We discovered transcription factors modulating dVGLUT-dependent DA neuroprotection and identified dj-1ß as a regulator of sex-specific DA neuron dVGLUT expression. Overall, VGLUT protects DA neurons from PD-associated degeneration by maintaining mitochondrial health.

Vesicular glutamate transporter modulates sex differences in dopamine neuron vulnerability to age-related neurodegeneration.

Age is the greatest risk factor for Parkinson's disease (PD) which causes progressive loss of dopamine (DA) neurons, with males at greater risk than females. Intriguingly, some DA neurons are more resilient to degeneration than others. Increasing evidence suggests that vesicular glutamate transporter (VGLUT) expression in DA neurons plays a role in this selective vulnerability. We investigated the role of DA neuron VGLUT in sex- and age-related differences in DA neuron vulnerability using the genetically tractable Drosophila model. We found sex differences in age-related DA neurodegeneration and its associated locomotor behavior, where males exhibit significantly greater decreases in both DA neuron number and locomotion during aging compared with females. We discovered that dynamic changes in DA neuron VGLUT expression mediate these age- and sex-related differences, as a potential compensatory mechanism for diminished DA neurotransmission during aging. Importantly, female Drosophila possess higher levels of VGLUT expression in DA neurons compared with males, and this finding is conserved across flies, rodents, and humans. Moreover, we showed that diminishing VGLUT expression in DA neurons eliminates females' greater resilience to DA neuron loss across aging. This offers a new mechanism for sex differences in selective DA neuron vulnerability to age-related DA neurodegeneration. Finally, in mice, we showed that the ability of DA neurons to achieve optimal control over VGLUT expression is essential for DA neuron survival. These findings lay the groundwork for the manipulation of DA neuron VGLUT expression as a novel therapeutic strategy to boost DA neuron resilience to age- and PD-related neurodegeneration.

Interplay between α-synuclein amyloid formation and membrane structure.

Amyloid formation is a pathological hallmark of many neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's. While it is unknown how these disorders are initiated, in vitro and cellular experiments confirm the importance of membranes. Ubiquitous in vivo, membranes induce conformational changes in amyloidogenic proteins and in some cases, facilitate aggregation. Reciprocally, perturbations in the bilayer structure can be induced by amyloid formation. Here, we review studies in the last 10 years describing α-synuclein (α-syn) and its interactions with membranes, detailing the roles of anionic and zwitterionic lipids in aggregation, and their contribution to Parkinson's disease. We summarize the impact of α-syn - comparing monomeric, oligomeric, and fibrillar forms - on membrane structure, and the effect of membrane remodeling on amyloid formation. Finally, perspective on future studies investigating the interplay between α-syn aggregation and membranes is discussed. This article is part of a Special Issue entitled: Amyloids.

The ketogenic diet metabolite beta-hydroxybutyrate (ß-HB) reduces incidence of seizure-like activity (SLA) in a Katp- and GABAb-dependent manner in a whole-animal Drosophila melanogaster model.

The high-fat, low-carbohydrate ketogenic diet (KD) is an effective clinical treatment for epilepsy in juveniles, especially for drug-resistant seizures. The KD results in elevated production of ketone bodies (KB's), such as beta-hydroxybutyrate (ß-HB), which are thought to have anticonvulsant properties; however, their exact mechanism of action is unknown. In vitro, KB effects on reducing neuronal firing rates are mediated in part by Katp channel activity and GABAb signaling. In order to study metabolic and pharmacological effects in a whole-animal model, we used the eas "bang-sensitive" (BS) mutant strain of Drosophila, which exhibits seizure-like activity (SLA) upon mechanical stimulation. Direct application of the KB ß-HB to food reduced BS SLA. Application either of tolbutamide, a Katp blocker, or of CGP-55845, a GABAb antagonist, concomitantly with ß-HB, partially reversed these KB effects on SLA, verifying a role for Katp channels and GABAb signaling in mediating the anticonvulsant effects of KB's and validating this whole-animal model of KD effects on seizure.