Parkinson's Disease-associated Mutant Research Articles

Parkinson's disease-associated mutants shift equilibrium bound state of alpha-synuclein towards the second helix.

α-synuclein regulates neurotransmitter release by binding to the outer lipid membrane of synaptic vesicles. Mutations within the lipid-binding region of α-synuclein are associated with protein aggregates and the progression of Parkinson's Disease (PD). However, the role of lipid-binding in α-synuclein's propensity to aggregate is not fully understood. Here, we explore how a mathematical framework reveals details in protein-membrane interactions that are disrupted by PD-associated mutants. Upon binding, α-synuclein forms two α-helices that insert into the lipid membrane - a process that is mediated by lipid membrane defects. A tryptophan fluorescence assay reveals how PD-associated mutants alter the equilibrium distribution of fully vs. partially bound protein. In unraveling the role of α-synuclein in the progression of PD, we reveal a unifying difference in protein behavior between wild-type α-synuclein and disease-associated mutants. Altogether, our results show that PD-associated point mutations uniformly shift the equilibrium bound state towards the second helix, suggesting that protein aggregation and eventual neurotoxicity may be linked to the equilibrium between the two helices.

Parkinson's disease-associated mutant LRRK2 phosphorylates Rab7L1 and modifies trans-Golgi morphology

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the major genetic cause of autosomal-dominantly inherited Parkinson's disease. LRRK2 is implicated in the regulation of intracellular trafficking, neurite outgrowth and PD risk in connection with Rab7L1, a putative interactor of LRRK2. Recently, a subset of Rab GTPases have been reported as substrates of LRRK2. Here we examine the kinase activity of LRRK2 on Rab7L1 in situ in cells. Phos-tag analyses and metabolic labeling assays revealed that LRRK2 readily phosphorylates Golgi-localized wild-type Rab7L1 but not mutant forms that are distributed in the cytoplasm. In vitro assays demonstrated direct phosphorylation of Rab7L1 by LRRK2. Subsequent screening using Rab7L1 mutants harboring alanine-substitution for every single Ser/Thr residue revealed that Ser72 is a major phosphorylation site, which was confirmed by using a phospho-Ser72-specific antibody. Moreover, LRRK2 pathogenic Parkinson mutants altogether markedly enhanced the phosphorylation at Ser72. The modulation of Ser72 phosphorylation in Rab7L1 resulted in an alteration of the morphology and distribution of the trans-Golgi network. These data collectively support the involvement of Rab7L1 phosphorylation in the LRRK2-mediated cellular and pathogenetic mechanisms.

Parkinson's disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes.

Mitochondrial dysfunction represents a critical step during the pathogenesis of Parkinson disease (PD) and increasing evidence suggests abnormal mitochondrial dynamics and quality control as important underlying mechanisms. The VPS35 gene, encoding a key component of the retromer complex, is the third autosomal-dominant gene associated with PD. However, how VPS35 mutations may lead to neurodegeneration remains unclear. Here we demonstrate that PD-associated VPS35 mutations caused mitochondrial fragmentation and cell death in cultured neurons in vitro, in mouse substantia nigra neurons in vivo, and in human fibroblasts from PD patient bearing the D620N mutation. VPS35-induced mitochondrial deficits and neuronal dysfunction could be prevented by inhibition of mitochondrial fission. VPS35 mutation caused increased interactions with DLP1 which enhanced mitochondrial DLP1 complex turnover via mitochondria-derived vesicles-dependent trafficking to lysosomes for degradation. Importantly, oxidative stress increased the VPS35–DLP1 interaction which was also increased in the brains of sporadic PD cases. These results revealed a novel cellular mechanism for the involvement of VPS35 in mitochondrial fission, dysregulation of which is likely involved in the pathogenesis of familial, and possibly sporadic, PD.

Regulation of neuronal survival factor MEF2D by chaperone-mediated autophagy.

Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of alpha-synuclein transgenic mice and patients with Parkinson's disease. Wild-type alpha-synuclein and a Parkinson's disease-associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson's disease.

Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1

Aberrant molecular properties shared by familial Parkinson's disease-associated mutant UCH-L1 and carbonyl-modified UCH-L1

Structural Determinants of PLD2 Inhibition by α-Synuclein

The presynaptic protein α-synuclein has been implicated in both neuronal plasticity and neurodegenerative disease, but its normal function remains unclear. We described the induction of an amphipathic α-helix at the N terminus (exons 2–4) of α-synuclein upon exposure to phospholipid vesicles, and hypothesized that lipid-binding might serve as a functional switch by stabilizing α-synuclein in an active (α-helical) conformation. Others have shown that α and β-synucleins inhibit phospholipase D (PLD), an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking. Here, we report that all three naturally occurring synuclein isoforms (α, β, and γ-synuclein) are similarly effective inhibitors of PLD2 in vitro, as is the Parkinson's disease-associated mutant A30P. The PD-associated mutant A53T, however, is a more potent inhibitor of PLD2 than is wild-type α-synuclein. We analyze mutations of the α-synuclein protein to identify critical determinants of human PLD2 inhibition in vitro. Deletion of residues 56–102 (exon 4) decreases PLD2 inhibition significantly; this activity of exon 4 may require adoption of an α-helical conformation, as mutations that disrupt α-helicity also abrogate inhibition. Deletion of C-terminal residues 130–140 (exon 6) completely abolishes inhibitory activity. In addition, PLD2 inhibition is blocked by phosphorylation at serine 129 or at tyrosine residues 125 and 136, or by mutations that mimic phosphorylation at these sites. We conclude that PLD2 inhibition by α-synuclein is mediated by a lipid-stabilized α-helical structure in exon 4 and also by residues within exon 6, and that this inhibition can be modulated by phosphorylation of specific residues in exons 5 and 6.

Conditional control of human wild-type and Parkinson's disease-associated mutant alpha-synuclein in transgenic mouse brain

The presynaptic protein alpha-synuclein has been implicated in the pathophysiology of many neurodegenerative disorders, including Parkinson's Disease and dementia with Lewy bodies, collectively referred to as synucleinopathies. To advance the study of the function of alpha-synuclein in these diseases, we have used the tTA-system to generate transgenic mice, which express human wildtype or mutated [A30P] alpha-synuclein in a conditional manner. To obtain reversible expression of human alpha-synuclein in the brain of these mice, we combined the tTA-system with both the PrP promotor and CaMKIIalpha promotor. We investigated the conditional overexpression by western blot analysis and immunostaining of paraffin-embedded brains. Western blot analysis revealed that double-transgenic mice express alpha-synuclein at different levels in specific brain regions. The expression-pattern and level of human alpha-synuclein in the brain of double-transgenic mice depends on both the neuron-specific promotor and the integration site of the human alpha-synuclein construct. Histological analysis of the brain of transgenic mice showed aberrant expression of the protein in cell soma. However, Lewy body-like alpha-synuclein inclusions have not yet been identified. Administration of doxycycline down-regulates alpha-synuclein expression to basal levels in the brain of double-transgenic mice. We currently investigate if one transgenic mouse-line which highly expresses mutated [A30P] alpha-synuclein specifically in the olfactory bulb shows any loss of dopaminergic neurons and an impaired sense of smell. We also perform microarray expression analysis to gain insight in the pathomechanism underlying over-expression of human alpha-synuclein. Our conditional mouse-model may help to define the role of human alpha-synuclein in synucleinopathies and might be used to demonstrate whether neuropathological symptoms of these diseases are reversible.

Alpha-synuclein, especially the Parkinson's disease-associated mutants, forms pore-like annular and tubular protofibrils.

Two mutations in the alpha-synuclein gene (A30P and A53T) have been linked to autosomal dominant early-onset Parkinson's disease (PD). Both mutations promote the formation of transient protofibrils (prefibrillar oligomers), suggesting that protofibrils are linked to cytotoxicity. In this work, the effect of these mutations on the structure of alpha-synuclein oligomers was investigated using electron microscopy and digital image processing. The PD-linked mutations (A30P and A53T) were observed to affect both the morphology and the size distribution of alpha-synuclein protofibrils (measured by analytical ultracentrifugation and scanning transmission electron microscopy). The A30P variant was observed to promote the formation of annular, pore-like protofibrils, whereas A53T promotes formation of annular and tubular protofibrillar structures. Wild-type alpha-synuclein also formed annular protofibrils, but only after extended incubation. The formation of pore-like oligomeric structures may explain the membrane permeabilization activity of alpha-synuclein protofibrils. These structures may contribute to the pathogenesis of PD.

Residual Structure and Dynamics in Parkinson's Disease-associated Mutants of α-Synuclein

alpha-Synuclein (alpha S) is a pre-synaptic protein that has been implicated as a possible causative agent in the pathogenesis of Parkinson's disease (PD). Two autosomal dominant missense mutations in the alpha S gene are associated with early onset PD. Because alpha S is found in an aggregated fibrillar form in the Lewy body deposits characteristic of Parkinson's patients, aggregation of the protein is believed to be related to its involvement in the disease process. The wild type (WT) and early onset mutants A30P and A53T display diverse in vitro aggregation kinetics even though the gross physicochemical and morphological properties of the mutants are highly similar. We used high resolution solution NMR spectroscopy to compare the structural and dynamic properties of the A53T and A30P mutants with those of WT alpha S in the free state. We found that the A30P mutation disrupts a region of residual helical structure that exists in the WT protein, whereas the A53T mutation results in a slight enhancement of a small region around the site of mutation with a preference for extended conformations. Based on these results and on the anticipated effects of these mutations on elements of secondary structure, we proposed a model of how these two PD-linked mutations influence alpha S fibril formation that is consistent with the documented differences in the fibrillization kinetics of the two mutants.

Subcellular Localization of Wild-Type and Parkinson's Disease-Associated Mutant α-Synuclein in Human and Transgenic Mouse Brain

Mutations in the alpha-synuclein (alphaSYN) gene are associated with rare cases of familial Parkinson's disease, and alphaSYN is a major component of Lewy bodies and Lewy neurites. Here we have investigated the localization of wild-type and mutant [A30P]alphaSYN as well as betaSYN at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic colocalization of alphaSYN and betaSYN in human and mouse brain. In a sucrose gradient equilibrium centrifugation assay, a portion of betaSYN floated into lower density fractions, which also contained the synaptic vesicle marker synaptophysin. Likewise, wild-type and [A30P]alphaSYN were found in floating fractions. Subcellular fractionation of mouse brain revealed that both alphaSYN and betaSYN were present in synaptosomes. In contrast to synaptophysin, betaSYN and alphaSYN were recovered from the soluble fraction upon lysis of the synaptosomes. Synaptic colocalization of alphaSYN and betaSYN was directly visualized by confocal microscopy of double-stained human brain sections. The Parkinson's disease-associated human mutant [A30P]alphaSYN was found to colocalize with betaSYN and synaptophysin in synapses of transgenic mouse brain. However, in addition to their normal presynaptic localization, transgenic wild-type and [A30P]alphaSYN abnormally accumulated in neuronal cell bodies and neurites throughout the brain. Thus, mutant [A30P]alphaSYN does not fail to be transported to synapses, but its transgenic overexpression apparently leads to abnormal cellular accumulations.

Somal and neuritic accumulation of the Parkinson's disease-associated mutant [A30P]α-synuclein in transgenic mice

Somal and neuritic accumulation of the Parkinson's disease-associated mutant [A30P]α-synuclein in transgenic mice