A single amino acid substitution differentiates Hsp70-dependent effects on alpha-synuclein degradation and toxicity

alpha-Synuclein aggregation and toxicity play a major role in Parkinson's disease and dementia with Lewy bodies. Hsp70 is a multipurpose stress response chaperone protein that mediates both refolding and degradation of misfolded proteins. We have shown that Hsp70 is able to block both alpha-synuclein toxicity and aggregation. Here we introduce a mutation into the ATPase domain of Hsp70 (K71S) and demonstrate that this abolishes Hsp70 refolding activity. Nonetheless, Hsp70K71S continues to mediate alpha-synuclein degradation and blocks aggregate formation. In contrast to wild type Hsp70, the ATPase domain mutant mediates alpha-synuclein degradation through a non-proteasome inhibitor sensitive pathway. Although Hsp70K71S can diminish levels of alpha-synuclein to an even greater extent than Hsp70, HSP70K71S does not protect against alpha-synuclein toxicity. The Hsp70K71S mutant appears to dissociate the formation of aggregates, which it blocks, and toxicity, which it does not block. These data suggest that the ability of Hsp70 to prevent toxicity is distinct from degradation of alpha-synuclein and is dependent on its ATPase domain.     

Rat alpha-synuclein interacts with Tat binding protein 1, a component of the 26S proteasomal complex

The alpha-synuclein gene, which encodes a brain presynaptic nerve terminal protein of unknown function, is linked to familial early-onset Parkinson's disease (PD). The finding that alpha-synuclein forms the major fibrillary component of Lewy bodies in brains of PD patients suggests that the two point mutations in alpha-synuclein (Ala(53)Thr, Ala(30)Pro) may promote the aggregation of alpha-synuclein into filaments. To address the role of alpha-synuclein in neurodegenerative diseases, we performed a yeast two-hybrid screen of a rat adult brain cDNA library using rat alpha-synuclein 2 (alphaSYN2). Here we report that alphaSYN2 interacts specifically with Tat binding protein 1, a subunit of the 700-kDa proteasome activator (PA700), the regulatory complex of the 26S proteasome and of the modulator complex, which enhances PA700 activation of the proteasome.     

The solubility of alpha-synuclein in multiple system atrophy differs from that of dementia with Lewy bodies and Parkinson's disease

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.     

A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins

We have used NMR spectroscopy and limited proteolysis to characterize the structural properties of the Parkinson's disease-related protein alpha-synuclein in lipid and detergent micelle environments. We show that the lipid or micelle surface-bound portion of the molecule adopts a continuously helical structure with a single break. Modeling alphaS as an ideal alpha-helix reveals a hydrophobic surface that winds around the helix axis in a right-handed fashion. This feature is typical of 11-mer repeat containing sequences that adopt right-handed coiled coil conformations. In order to bind a flat or convex lipid surface, however, an unbroken helical alphaS structure would need to adopt an unusual, slightly unwound, alpha11/3 helix conformation (three complete turns per 11 residues). The break we observe in the alphaS helix may allow the protein to avoid this unusual conformation by adopting two shorter stretches of typical alpha-helical structure. However, a quantitative analysis suggests the possibility that the alpha11/3 conformation may in fact exist in lipid-bound alphaS. We discuss how structural features of helical 11-mer repeats could play a role in the reversible lipid binding function of alpha-synuclein and generalize this argument to include the 11-mer repeat-containing apolipoproteins, which also require the ability to release readily from lipid surfaces. A search of protein sequence databases confirms that synuclein-like 11-mer repeats are present in other proteins that bind lipids reversibly and predicts such a role for a number of hypothetical proteins of unknown function.     

The lipid peroxidation metabolite 4-oxo-2-nonenal cross-links α-synuclein causing rapid formation of stable oligomers

Recently, the aldehyde 4-oxo-2-nonenal (ONE) was identified as a product of lipid peroxidation and found to be an effective protein modifier. In this in vitro study we investigated structural implications of the interaction between ONE and α-synuclein, a protein which forms intraneuronal inclusions in neurodegenerative disorders such as Parkinson’s disease and dementia with Lewy bodies. Our results demonstrate that ONE induced an almost complete conversion of monomeric α-synuclein into 40-80 nm wide and 6-8 nm high soluble β-sheet-rich oligomers with a molecular weight of ∼2000 kDa. Furthermore, the ONE-induced α-synuclein oligomers displayed a high stability and were not sensitive to treatment with sodium dodecyl sulfate, indicating that ONE stabilized the oligomers by cross-linking individual α-synuclein molecules. Despite prolonged incubation the oligomers did not continue to aggregate into a fibrillar state, thus suggesting that these α-synuclein species were not on a fibrillogenic pathway.     

Distinct cleavage patterns of normal and pathologic forms of alpha-synuclein by calpain I in vitro

Parkinson's disease (PD) is characterized by fibrillary neuronal inclusions called Lewy bodies (LBs) consisting largely of alpha-synuclein (alpha-syn), the protein mutated in some patients with familial PD. The mechanisms of alpha-syn fibrillization and LB formation are unknown, but may involve aberrant degradation or turnover. We examined the ability of calpain I to cleave alpha-syn in vitro. Calpain I cleaved wild-type alpha-syn predominantly after amino acid 57 and within the non-amyloid component (NAC) region. In contrast, calpain I cleaved fibrillized alpha-syn primarily in the region of amino acid 120 to generate fragments like those that increase susceptibility to dopamine toxicity and oxidative stress. Further, while calpain I cleaved wild-type alpha-syn after amino acid 57, this did not occur in mutant A53T alpha-syn. This paucity of proteolysis could increase the stability of A53T alpha-syn, suggesting that calpain I might protect cells from forming LBs by specific cleavages of soluble wild-type alpha-syn. However, once alpha-syn has polymerized into fibrils, calpain I may contribute to toxicity of these forms of alpha-syn by cleaving at aberrant sites within the C-terminal region. Elucidating the role of calpain I in the proteolytic processing of alpha-syn in normal and diseased brains may clarify mechanisms of neurodegenerative alpha-synucleinopathies.     

Cysteine and mercapturate conjugates of oxidized dopamine are in human striatum but only the cysteine conjugate impedes dopamine trafficking in vitro and in vivo

Recent results have suggested that some products of mercapturic acid pathway (MAP) metabolism of oxidized dopamine (DA) may contribute to mesostriatal dopaminergic neurodegeneration, and that at least one product, 5-S-cysteinyldopamine (Cys-DA), is elevated in patients with advanced Parkinson's disease (PD) who have been treated with L-DOPA. Here we investigated MAP enzymes and products in the midbrain and striatum of control individuals and patients with dementia with Lewy bodies (DLB) who had less severe dopaminergic degeneration than PD patients and who were not treated with L-DOPA. We also determined the biological activity of MAP metabolites of oxidized DA using primary rat mesencephalic cultures, rat cerebral synaptosomes, and rat striatum in vivo microdialysis. Our results showed that the human mesostriatal dopaminergic pathway generates Cys-DA but has limited enzymatic capacity for mercapturate formation, that striatal levels of MAP products of oxidized DA are not elevated in DLB patients compared with controls, and that Cys-DA interferes with trafficking of DA in vitro and in vivo. These results indicate that while Cys-DA is not increased in striatum of patients with mild dopaminergic neurodegeneration, it may interfere with uptake of DA in patients with advanced PD.     

Oxidative Damage to Proteins, Lipids, and DNA in Cortical Brain Regions from Patients with Dementia with Lewy Bodies

Abstract: Dementia with Lewy bodies (DLB) forms the second most common pathological subgroup of dementia after Alzheimer's disease. The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases in cortical brain areas from patients with DLB with levels in matched control tissues. Overall, there was a trend for protein carbonyl levels to be increased in all areas, but a significant difference was found only in the parietal and temporal lobes. No differences were observed in the levels of lipid peroxidation. Measurement of products of damage to DNA bases showed increased levels of thymine glycol, 8-hydroxyguanine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, and xanthine. Xanthine levels were increased in the DLB group in the parietal, occipital, and temporal lobes, indicating that peroxynitrite or other deaminating species may be involved. The finding of increased protein carbonyls and increased DNA base products in cortical regions from DLB patients indicates that oxidative stress may play a role in DLB.     

Challenges and complexities of alpha-synuclein toxicity: new postulates in unfolding the mystery associated with Parkinson's disease

The discovery of two missense mutations in alpha-synuclein gene and the identification of the alpha-synuclein as the major component of Lewy bodies and Lewy neurites have imparted a new direction in understanding Parkinson's disease. Now that alpha-synuclein has been implicated in several neurodegenerative disorders makes it increasingly clear that aggregation of alpha-synuclein is a hallmark feature in neurodegeneration. Although little has been learned about its normal function, alpha-synuclein appears to be associated with membrane phospholipids and may therefore participate in a number of cell signaling pathways. Here, we review the localization, structure, and function of alpha-synuclein and provide a new hypothesis on, (a) the disruption in the membrane binding ability of synuclein which may be the major culprit leading to the alpha-synuclein aggregation and (b) the complexity associated with nuclear localization of alpha-synuclein and its possible binding property to DNA. Further, we postulated the three possible mechanisms of synuclein induced neuronal degeneration in Parkinson's disease.     

Cysteine misincorporation in bacterially expressed human alpha-synuclein

Bacterially expressed human alpha-synuclein (alpha-syn) has been widely used in structural and functional studies. Here we show that approximately 20% of human alpha-syn expressed in Escherichia coli is mistranslated and that a Cys residue is incorporated at position 136 instead of a Tyr. Site-directed mutagenesis of codon 136 (TAC to TAT) resulted in the expression of alpha-syn lacking Cys. Although wild-type (Y136-TAC and Y136-TAT) and mutant (C136-TGC) alpha-syn had similar propensities to assemble into filaments, the levels of dimeric alpha-syn were increased by misincorporation. To avoid potential artefacts, we recommend use of the Y136-TAT construct for the expression of human alpha-syn.     

Dopamine-related and caspase-independent apoptosis in dopaminergic neurons induced by overexpression of human wild type or mutant alpha-synuclein

Human wild type (WT) and mutant alpha-synuclein (alpha-syn) genes were overexpressed using a Tet-on expression system in stably transfected dopaminergic MN9D cells. Their overexpression induced caspase-independent and dopamine-related apoptosis not rescued by general caspase inhibitor Z-VAD-FMK. While apoptosis due to overexpression of WT alpha-syn was completely abrogated by a specific tyrosine hydroxylase (TH) inhibitor, alpha-methyl-p-tyrosine (alpha-MT), the inhibitor only partially rescued apoptosis caused by overexpression of alpha-syn mutants. In addition, overexpression of mutants enhanced the toxicity of 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxyldopamine (6-OHDA) to MN9D cells, whereas overexpression of WT protected MN9D cells against MPP+ toxicity, but not against 6-OHDA. We conclude that WT alpha-syn is beneficial to dopaminergic neurons but its overexpression in the presence of endogenous dopamine makes it a potential threat to the cells. In contrast, mutant alpha-syn not only caused the loss of WT protective function but also the gain-of-toxicity which becomes more serious in the presence of dopamine and neurotoxins.     

FK506 binding protein 12 differentially accelerates fibril formation of wild type alpha-synuclein and its clinical mutants A30P or A53T

Aggregation of alpha-synuclein (α-SYN) plays a key role in Parkinson's disease. We have previously shown that aggregation of α-SYN in vitro is accelerated by addition of FK506 binding proteins (FKBP) and that this effect can be counteracted by FK506, a specific inhibitor of these enzymes. In this paper, we investigated in detail the effect of FKBP12 on early aggregation and on fibril formation of wild-type, A53T and A30P α-SYN. FKBP12 has a much smaller effect on the fibril formation of these two clinical mutants α-SYN. Using an inactive enzyme, we were able to discriminate between catalytic and non-catalytic effects that differentially influence the two processes. A model explaining non-linear concentration dependencies is proposed.     

Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.     

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.     

Dopamine differentially induces aggregation of A53T mutant and wild type alpha-synuclein: insights into the protein chemistry of Parkinson's disease

Aggregation of α-synuclein is known to be a causal factor in the genesis of Parkinson’s disease and Dementia with Lewy bodies. Duplication and/or triplication and mutation of the α-synuclein gene are associated with sporadic and familial Parkinson’s disease. Synucleinopathies appear to primarily affect dopaminergic neurons. The present studies investigate the role of dopamine in α-synuclein aggregation through NMR. Dopamine causes aggregation of both wild type and A53T mutant α-synuclein in a temperature-dependent manner, but the mutant A53T shows a greater propensity to aggregate in the presence of dopamine only at 37 °C. A single point mutation in the α-synuclein A53T mutant gene results in a structural change in the protein and drastically increases its propensity to aggregate in the presence of dopamine. The present data indicate that mutation in the α-synuclein gene may predispose the protein to dopamine-induced aggregation, thereby contributing to disease pathogenesis.     

3,4‐Dihydroxyphenylethanol and 3,4‐dihydroxyphenylacetic acid affect the aggregation process of E46K variant of α‐synuclein at different extent: Insights into the interplay between protein dynamics and catechol effect

Parkinson''s disease (PD) is a chronic multifactorial disease, whose etiology is not completely understood. The amyloid aggregation of α‐synuclein (Syn) is considered a major cause in the development of the disease. The presence of genetic mutations can boost the aggregation of the protein and the likelihood to develop PD. These mutations can lead to early onset (A30P, E46K, and A53T) or late‐onset (H50Q) forms of PD. The disease is also linked to an increase in oxidative stress and altered levels of dopamine metabolites. The molecular interaction of these molecules with Syn has been previously studied, while their effect on the pathological mutant structure and function is not completely clarified. By using biochemical and biophysical approaches, here we have studied the interaction of the familial variant E46K with two dopamine‐derived catechols, 3,4‐dihydroxyphenylacetic acid and 3,4‐dihydroxyphenylethanol. We show that the presence of these catechols causes a decrease in the formation of amyloid fibrils in a dose‐dependent manner. Native‐ and Hydrogen/deuterium exchange‐mass spectrometry (HDX‐MS) provide evidence that this effect is strongly conformation dependent. Indeed, these molecules interact differently with the interconverting conformers of Syn and its familial variant E46K in solution, selecting the most prone‐to‐aggregation one, confining it into an off‐pathway oligomer. These findings suggest that catechols could be a molecular scaffold for the design of compounds potentially useful in the treatment of Parkinson''s disease and related conditions.     

α-Synuclein Conformational Strains as Drivers of Phenotypic Heterogeneity in Neurodegenerative Diseases

The synucleinopathies, which include Parkinson’s disease, dementia with Lewy bodies, and multiple system atrophy, are a class of human neurodegenerative disorders unified by the presence of α-synuclein aggregates in the brain. Considerable clinical and pathological heterogeneity exists within and among the individual synucleinopathies. A potential explanation for this variability is the existence of distinct conformational strains of α-synuclein aggregates that cause different disease manifestations. Like prion strains, α-synuclein strains can be delineated based on their structural architecture, with structural differences among α-synuclein aggregates leading to unique biochemical attributes and neuropathological properties in humans and animal models. Bolstered by recent high-resolution structural data from patient brain-derived material, it has now been firmly established that there are conformational differences among α-synuclein aggregates from different human synucleinopathies. Moreover, recombinant α-synuclein can be polymerized into several structurally distinct aggregates that exhibit unique pathological properties. In this review, we outline the evidence supporting the existence of α-synuclein strains and highlight how they can act as drivers of phenotypic heterogeneity in the human synucleinopathies.     

Role of Autophagy Pathway in Parkinson’s Disease and Related Genetic Neurological Disorders

The elucidation of the function of the PINK1 protein kinase and Parkin ubiquitin E3 ligase in the elimination of damaged mitochondria by autophagy (mitophagy) has provided unprecedented understanding of the mechanistic pathways underlying Parkinson’s disease (PD). We provide a comprehensive overview of the general importance of autophagy in Parkinson’s disease and related disorders of the central nervous system. This reveals a critical link between autophagy and neurodegenerative and neurodevelopmental disorders and suggests that strategies to modulate mitophagy may have greater relevance in the CNS beyond PD.