Abstract
Backgroundα-Synuclein (aSyn) aggregation is thought to play a central role in neurodegenerative disorders termed synucleinopathies, including Parkinson’s disease (PD). Mouse aSyn contains a threonine residue at position 53 that mimics the human familial PD substitution A53T, yet in contrast to A53T patients, mice show no evidence of aSyn neuropathology even after aging. Here, we studied the neurotoxicity of human A53T, mouse aSyn, and various human-mouse chimeras in cellular and in vivo models, as well as their biochemical properties relevant to aSyn pathobiology.MethodsPrimary midbrain cultures transduced with aSyn-encoding adenoviruses were analyzed immunocytochemically to determine relative dopaminergic neuron viability. Brain sections prepared from rats injected intranigrally with aSyn-encoding adeno-associated viruses were analyzed immunohistochemically to determine nigral dopaminergic neuron viability and striatal dopaminergic terminal density. Recombinant aSyn variants were characterized in terms of fibrillization rates by measuring thioflavin T fluorescence, fibril morphologies via electron microscopy and atomic force microscopy, and protein-lipid interactions by monitoring membrane-induced aSyn aggregation and aSyn-mediated vesicle disruption. Statistical tests consisted of ANOVA followed by Tukey’s multiple comparisons post hoc test and the Kruskal-Wallis test followed by a Dunn’s multiple comparisons test or a two-tailed Mann-Whitney test.ResultsMouse aSyn was less neurotoxic than human aSyn A53T in cell culture and in rat midbrain, and data obtained for the chimeric variants indicated that the human-to-mouse substitutions D121G and N122S were at least partially responsible for this decrease in neurotoxicity. Human aSyn A53T and a chimeric variant with the human residues D and N at positions 121 and 122 (respectively) showed a greater propensity to undergo membrane-induced aggregation and to elicit vesicle disruption. Differences in neurotoxicity among the human, mouse, and chimeric aSyn variants correlated weakly with differences in fibrillization rate or fibril morphology.ConclusionsMouse aSyn is less neurotoxic than the human A53T variant as a result of inhibitory effects of two C-terminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization. Our findings highlight the importance of membrane-induced self-assembly in aSyn neurotoxicity and suggest that inhibiting this process by targeting the C-terminal domain could slow neurodegeneration in PD and other synucleinopathy disorders.
Highlights
Parkinson’s disease (PD) is a common progressive neurodegenerative disorder characterized clinically by motor symptoms attributed to a loss of dopaminergic neurons in the substantia nigra (SN)
Mouse amyloid-like fibrils of the presynaptic protein α-synuclein (aSyn) is less neurotoxic than the human A53T variant as a result of inhibitory effects of two Cterminal amino acid substitutions on membrane-induced aSyn aggregation and aSyn-mediated vesicle permeabilization
Membrane binding apparently plays an important role in the normal function of aSyn related to regulation of synaptic vesicle trafficking [12, 13], the protein has been shown to undergo accelerated aggregation when incubated in the presence of phospholipid vesicles at high protein:lipid ratios [14,15,16]. aSyn aggregation at the membrane surface is likely stimulated by the exposure of hydrophobic residues as the membrane-bound protein shifts from the long-helix form to the short-helix form [11, 16], and by the fact that molecular interactions needed for aSyn selfassembly likely occur with a higher probability on the two dimensional surface of the lipid bilayer than in solution [17, 18]
Summary
Parkinson’s disease (PD) is a common progressive neurodegenerative disorder characterized clinically by motor symptoms attributed to a loss of dopaminergic neurons in the substantia nigra (SN). A detailed understanding of molecular mechanisms by which aSyn forms neurotoxic aggregates is critical for developing therapies aimed at slowing neurodegeneration in the brains of patients with PD and other synucleinopathy disorders. The central region spanning residues 61–95 contains a sixth lysine-rich repeat and is highly hydrophobic. The C-terminal region spanning residues 96–140 is enriched with proline and acidic residues and is thought to regulate aSyn aggregation through auto-inhibitory long-range interactions [8, 9], with electrostatic interactions mediated by the acidic residues playing a major role in increasing the fibrillization lag time [10]. ASyn binds to anionic phospholipid vesicles by forming an amphipathic α-helix with varying lengths, including a short N-terminal helix spanning residues ~ 1–25 and a longer helix spanning residues ~ 1– 97 encompassing both the N-terminal and central hydrophobic domains [11, 12]. Evidence from our laboratory suggests that membrane-induced aSyn aggregation plays a key role in neurotoxicity [16], potentially via a mechanism involving membrane permeabilization [19,20,21,22]
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