Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

The Role of Alpha-Synuclein Oligomerization and Aggregation in Cellular and Animal Models of Parkinson’s Disease

α-synuclein (α-syn) is a synaptic protein in which four mutations (A53T, A30P, E46K and gene triplication) have been found to cause an autosomal dominant form of Parkinson’s disease (PD). It is also the major component of intraneuronal protein aggregates, designated as Lewy bodies (LBs), a prominent pathological hallmark of PD. How α-syn contributes to LB formation and PD is still not well-understood. It has been proposed that aggregation of α-syn contributes to the formation of LBs, which then leads to neurodegeneration in PD. However, studies have also suggested that aggregates formation is a protective mechanism against more toxic α-syn oligomers. In this study, we have generated α-syn mutants that have increased propensity to form aggregates by attaching a CL1 peptide to the C-terminal of α-syn. Data from our cellular study suggest an inverse correlation between cell viability and the amount of α-syn aggregates formed in the cells. In addition, our animal model of PD indicates that attachment of CL1 to α-syn enhanced its toxicity to dopaminergic neurons in an age-dependent manner and induced the formation of Lewy body-like α-syn aggregates in the substantia nigra. These results provide new insights into how α-syn-induced toxicity is related to its aggregation

The Mammalian Disaggregase Machinery: Hsp110 Synergizes with Hsp70 and Hsp40 to Catalyze Protein Disaggregation and Reactivation in a Cell-Free System

Bacteria, fungi, protozoa, chromista and plants all harbor homologues of Hsp104, a AAA+ ATPase that collaborates with Hsp70 and Hsp40 to promote protein disaggregation and reactivation. Curiously, however, metazoa do not possess an Hsp104 homologue. Thus, whether animal cells renature large protein aggregates has long remained unclear. Here, it is established that mammalian cytosol prepared from different sources possesses a potent, ATP-dependent protein disaggregase and reactivation activity, which can be accelerated and stimulated by Hsp104. This activity did not require the AAA+ ATPase, p97. Rather, mammalian Hsp110 (Apg-2), Hsp70 (Hsc70 or Hsp70) and Hsp40 (Hdj1) were necessary and sufficient to slowly dissolve large disordered aggregates and recover natively folded protein. This slow disaggregase activity was conserved to yeast Hsp110 (Sse1), Hsp70 (Ssa1) and Hsp40 (Sis1 or Ydj1). Hsp110 must engage substrate, engage Hsp70, promote nucleotide exchange on Hsp70, and hydrolyze ATP to promote disaggregation of disordered aggregates. Similarly, Hsp70 must engage substrate and Hsp110, and hydrolyze ATP for protein disaggregation. Hsp40 must harbor a functional J domain to promote protein disaggregation, but the J domain alone is insufficient. Optimal disaggregase activity is achieved when the Hsp40 can stimulate the ATPase activity of Hsp110 and Hsp70. Finally, Hsp110, Hsp70 and Hsp40 fail to rapidly remodel amyloid forms of the yeast prion protein, Sup35, or the Parkinson's disease protein, alpha-synuclein. However, Hsp110, Hsp70 and Hsp40 enhanced the activity of Hsp104 against these amyloid substrates. Taken together, these findings suggest that Hsp110 fulfils a subset of Hsp104 activities in mammals. Moreover, they suggest that Hsp104 can collaborate with the mammalian disaggregase machinery to rapidly remodel amyloid conformers

Post translational changes to α-synuclein control iron and dopamine trafficking : a concept for neuron vulnerability in Parkinson's disease

Parkinson's disease is a multifactorial neurodegenerative disorder, the aetiology of which remains elusive. The primary clinical feature of progressively impaired motor control is caused by a loss of midbrain substantia nigra dopamine neurons that have a high α-synuclein (α-syn) and iron content. α-Syn is a neuronal protein that is highly modified post-translationally and central to the Lewy body neuropathology of the disease. This review provides an overview of findings on the role post translational modifications to α-syn have in membrane binding and intracellular vesicle trafficking. Furthermore, we propose a concept in which acetylation and phosphorylation of α-syn modulate endocytic import of iron and vesicle transport of dopamine during normal physiology. Disregulated phosphorylation and oxidation of α-syn mediate iron and dopamine dependent oxidative stress through impaired cellular location and increase propensity for α-syn aggregation. The proposition highlights a connection between α-syn, iron and dopamine, three pathological components associated with disease progression in sporadic Parkinson's disease

Discovery and characterization of selective small molecule inhibitors of the mammalian mitochondrial division dynamin, DRP1

Balanced rates of mitochondrial division and fusion are required to maintain mitochondrial function, as well as cellular and organismal homeostasis. In mammals, the cellular machines that mediate these processes are dynamin-related GTPases; the cytosolic DRP1 mediates division, while the outer membrane MFN1/2 and inner membrane OPA1 mediate fusion. Unbalanced mitochondrial dynamics are linked to varied pathologies, including cell death and neurodegeneration, raising the possibility that small molecules that target the division and fusion machines to restore balance may have therapeutic potential. Here we describe the discovery of novel small molecules that directly and selectively inhibit assembly-stimulated GTPase activity of the division dynamin, DRP1. In addition, these small molecules restore wild type mtDNA copy number in MFN1 knockout mouse embryonic fibroblast cells, a phenotype linked to deficient mitochondrial fusion activity. Thus, these compounds are unique tools to explore the roles of mitochondrial division in cells, and to assess the potential therapeutic efficacy of rebalancing mitochondrial dynamics in pathologies associated with excessive mitochondrial division

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α-synuclein (αS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure-based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function