Forms Of Α-synuclein Research Articles

Proteasome dysfunction in aged human α-synuclein transgenic mice

A deficit in proteasome function in Parkinson's disease has been speculated. We characterized the ubiquitin-proteasome system in three regions of brain from transgenic and nontransgenic littermates. Mice expressing a doubly mutated form of human alpha-synuclein had significant impairments whereas mice expressing the wild-type gene had lesser changes compared to nontransgenic littermates. Significant abnormalities in line hm2 alpha-SYN-39 included declines in 20S-mediated proteolytic activity, the level of the 19S proteasome subunits Rpt1 and Rpn2, and the level of soluble total high MW ubiquitin cross-reacting proteins. Line hw alpha-SYN-5 had significant, but restricted proteasome abnormalities. The severity of impairment was proportional to the substantia nigra dopaminergic neuronal loss previously identified. There were significant correlations between the level of Rpn2 with the level of Rpt1, the activity of the 20S proteasome, and the level of soluble high MW ubiquitin cross-reacting proteins. These abnormalities in symptomatic line hm2 alpha-SYN-39 mice are consistent with abnormalities identified in tissue from patients with Parkinson's disease.

Mutant α-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson's disease

Parkinson's disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode alpha-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human alpha-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, we analysed the effect of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. We have determined that co-expression of parkin can suppress phenotypes caused by expression of mutant alpha-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, we suggest that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of alpha-synuclein.

α-Synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy

There is strong evidence for the involvement of α-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which α-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of α-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce α-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α-synuclein (A53T), a familial PD-associated mutation.

Single particle detection and characterization of synuclein co-aggregation

Protein aggregation is the key event in a number of human diseases such as Alzheimer’s and Parkinson’s disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type α-synuclein forms co-aggregates with a mutant variant found in familial Parkinson’s disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects.

Parkinson's disease alpha-synuclein mutations exhibit defective axonal transport in cultured neurons.

Alpha-synuclein is a major protein constituent of Lewy bodies and mutations in alpha-synuclein cause familial autosomal dominant Parkinson's disease. One explanation for the formation of perikaryal and neuritic aggregates of alpha-synuclein, which is a presynaptic protein, is that the mutations disrupt alpha-synuclein transport and lead to its proximal accumulation. We found that mutant forms of alpha-synuclein, either associated with Parkinson's disease (A30P or A53T) or mimicking defined serine, but not tyrosine, phosphorylation states exhibit reduced axonal transport following transfection into cultured neurons. Furthermore, transfection of A30P, but not wild-type, alpha-synuclein results in accumulation of the protein proximal to the cell body. We propose that the reduced axonal transport exhibited by the Parkinson's disease-associated alpha-synuclein mutants examined in this study might contribute to perikaryal accumulation of alpha-synuclein and hence Lewy body formation and neuritic abnormalities in diseased brain.

Zeroing in on the pathogenic form of alpha-synuclein and its mechanism of neurotoxicity in Parkinson's disease.

Parkinson's disease (PD) is linked to mutations in the protein alpha-synuclein, which can exist in vitro in several aggregation states, including a natively unfolded monomer, a beta-sheet rich oligomer, or protofibril, and a stable amyloid fibril. This work reviews the current literature that is relevant to two linked questions: which of these species is pathogenic, and what is the mechanism of neurotoxicity? The amyloid fibril, fibrillar aggregates, Lewy bodies, and the alpha-synuclein monomer, which is normally expressed at high levels, are all unlikely to be pathogenic, for reasons discussed here. We therefore favor a toxic protofibril scenario, and propose that the pathogenic species is transiently populated during the process of fibrillization. Toxicity may arise from pore-like protofibrils that cause membrane permeabilization. An approach to testing this hypothesis is discussed.

Modulation of cell death by α-synuclein is stimulus-dependent in mammalian cells

α-synuclein is a neuronally-expressed protein which is mutated in familial Parkinson's disease. Previous studies have suggested that over-expression of α-synuclein can either enhance, reduce or have no effect on the degree of cell death in response to death-inducing stimuli. We resolve this discrepancy by using a well-characterised cell system to demonstrate that wild type α-synuclein can enhance cell death in response to ischaemia/reoxygenation or staurosporine treatment whilst protecting against serum removal and dopamine-induced cell death. In contrast, the two mutant forms of α-synuclein uniformly enhance cell death. Hence, the disease-associated mutations appear to convert α-synuclein from a protein which modulates cell death differently in different circumstances to forms which have a universal damaging effect.

Rare genetic mutations shed light on the pathogenesis of Parkinson disease.

Ultimately it would be of great interest to link all the multiple genes and the sporadic causes of PD into a common pathogenic biochemical pathway — much in the same way that, in AD, the metabolism of the amyloid precursor protein has been shown to be central to the degenerative process. Derangements in protein handling seem to be central in the pathogenesis of PD. Aggregation, fibrillation, and proteasom-al dysfunction seem central to the pathogenesis of α-synuclein–induced injury. Mutations in parkin lead to dysfunction in the proteasomal-degradation pathway. It will be of interest to determine whether parkin is involved in the ubiquitination and elimination of proteins whose proteasomal degradation is impaired by derangements in α-synuclein. The major nonglycosylated form of α-synuclein is linked to parkin through their mutual interactions with synphilin-1, and the minor glycosylated α-synuclein species may be directly linked to parkin, providing further evidence for a common biochemical pathway. How the unfolded-protein stress response fits into a common pathway is not clear. Oxidative stress seems to play a prominent role in sporadic PD, and oxidative stress leads to synuclein aggregation and/or proteasomal dysfunction. Recent data suggest that there may be selective derangements in the protea-somal system in the substantia nigra of sporadic PD patients (68). Thus, protein mishandling may be central to the pathogenesis of PD. Another possible common pathogenic mechanism is the disruption of synaptic function, as α-synuclein, synphilin-1, CDCrel-1, and parkin are synaptically enriched proteins, but this awaits further clarification and investigation. How DJ-1 fits into a common pathogenic pathway is not clear, but its potential role in oxidative stress would fit with the “oxidative stress” hypothesis. The fact the DJ-1 is sumoylated, a ubiquitin-like protein modification, suggests that it might be a target of parkin, tying it into the ubiquitin/proteasomal pathway. Only the future can tell whether DJ-1 and the other yet-to-be-identified PD genes will fit into one of these pathways or whether they will reveal new pathways. Surprises are undoubtedly in store as the identification of these genes leads to a better understanding of the pathogenesis of PD.

Aggregation and neurotoxicity of alpha-synuclein and related peptides.

Fibrillar deposits of alpha-synuclein occur in several neurodegenerative diseases. Two mutant forms of alpha-synuclein have been associated with early-onset Parkinson's disease, and a fragment has been identified as the non-amyloid-beta peptide component of Alzheimer's disease amyloid (NAC). Upon aging, solutions of alpha-synuclein and NAC change conformation to beta-sheet, detectable by CD spectroscopy, and form oligomers that deposit as amyloid-like fibrils, detectable by electron microscopy. These aged peptides are also neurotoxic. Experiments on fragments of NAC have enabled the region of NAC responsible for its aggregation and toxicity to be identified. NAC(8-18) is the smallest fragment that aggregates, as indicated by the concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. Fragments NAC(8-18) and NAC(8-16) are toxic, whereas NAC(12-18), NAC(9-16) and NAC(8-15) are not. Hence residues 8-16 of NAC comprise the region crucial for toxicity. Toxicity induced by alpha-synuclein, NAC and NAC(1-18) oligomers occurs via an apoptotic mechanism, possibly initiated by oxidative damage, since these peptides liberate hydroxyl radicals in the presence of iron. Molecules with anti-aggregational and/or antioxidant properties may therefore be potential therapeutic agents.

A Molotov cocktail

Dopamine and α-synuclein are a toxic combination for neurons, say Jin Xu, Bruce Yankner (Children's Hospital, Boston, Massachusetts) and colleagues. Figure α-synuclein overexpression induces apotosis in dopaminergic neurons (red). Aggregations of α-synuclein are a hallmark of Parkinson's disease (PD), and mutations in its gene are associated with familial forms of the disease. Yankner wanted to know why the protein is so toxic. When he overexpressed either wild-type or mutant forms of α-synuclein in cultured human dopaminergic neurons (DAN cells)—the cells affected in PD—a large number of the cells underwent apoptosis. In contrast, excess α-synuclein seemed to protect the nondopaminergic cortical neurons from apoptosis. If endogenous synthesis of dopamine was blocked by the addition of a tyrosine hydroxylase inhibitor (THI), overexpression of α-synuclein no longer induced apoptosis. Thus, somehow, it is the combination of α-synuclein and dopamine that causes cell death, rather than overexpression of α-synuclein alone. Yankner thinks the key to this α-synuclein–dopamine toxicity is the production of reactive oxygen species (ROS), which can damage cellular proteins. Dopamine synthesis produces ROS. But whereas a healthy cell can neutralize these ROS, neurons overexpressing α-synuclein cannot. In fact, excess α-synuclein increases the generation of ROS in DAN cells, and blocking this production with antioxidants inhibits cell death. Thus, Yankner suggests the α-synuclein potentiates ROS production by dopamine. Large α-synuclein aggregates occur in PD neurons, but Yankner's team found that soluble α-synuclein, in the form of a 53–84-kD complex, induced apoptosis in the cultured neurons. This complex included 14–3-3, a chaperone protein that sequesters the proapoptotic proteins BAD and Forkhead and prevents their functioning. Yankner says he still isn't sure what initially triggers the overexpression of α-synuclein in PD, but suggests that it may occur initially as a protective response to oxidative stress related to environmental agents and dopamine synthesis. He speculates that, once this happens, excess α-synuclein snatches up much of the 14–3-3 protein in the cell, forcing the release of the proapoptotic factors or preventing 14–3-3′s participation in ROS repair mechanisms. ▪ Reference: Xu, J., et al. 2002. Nat. Med. 8:600–606. [PubMed]

Amino acid determinants of α-synuclein aggregation: putting together pieces of the puzzle

Parkinson’s disease is the second most common neurodegenerative disease, and results from loss of dopaminergic neurons in the substantia nigra. The aggregation and fibrillation of α-synuclein in the form of intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) have been implicated as a causative factor in this disease, as well as in several other neurodegenerative disorders, including dementia with Lewy bodies, Lewy body variant of Alzheimer’s disease, multiple system atrophy and Hallervorden–Spatz disease. Thus, the aggregated forms of α-synuclein play a crucial role in the pathogenesis of the synucleinopathies. However, the molecular mechanisms underlying α-synuclein aggregation into specific filamentous inclusions remained unknown until recently. Data on the aggregation and fibrillation properties of human α-, β- and γ-synucleins, mouse α-synuclein and familial Parkinson’s disease mutants of human α-synuclein (A30P and A53T) are analyzed in order to shed light on the amino acid determinants of synuclein aggregation.

Dopaminergic Cell Loss Induced by Human A30P α-Synuclein Gene Transfer to the Rat Substantia Nigra

Somatic cell gene transfer was used to express a mutant form of alpha-synuclein (alpha-syn) that is associated with Parkinson's disease (PD) in the rat substantia nigra (SN), a brain region that, in humans, degenerates during PD. DNA encoding the A30P mutant of human alpha-syn linked to familial PD was incorporated into an adeno-associated virus vector, which was injected into the adult rat midbrain. The cytomegalovirus/chicken beta-actin promoter was used to drive transgene expression. Over a 1-year time course, this treatment produced three significant features relevant to PD: (1) accumulation of alpha-syn in SN neuron perikarya, (2) Lewy-like dystrophic neurites in the SN and the striatum, and (3) a 53% loss of SN dopamine neurons. However, motor dysfunction was not found in either rotational or rotating rod testing. The lack of behavioral deficits, despite the significant cell loss, may reflect pathogenesis similar to that of PD, where greater than 50% losses occur before motor behavior is affected.

An alternatively spliced form of rodent α-synuclein forms intracellular inclusions in vitro: role of the carboxy-terminus in α-synuclein aggregation

In the rat, the α-synuclein gene is alternatively spliced and exists in three forms, rat synuclein 1 (rSYN1), synuclein 2 (rSYN2) and synuclein 3. rSYN2 cDNA encodes a 149 amino acid protein that is homologous to rSYN1 and human α-synuclein for the first 100 amino acids, but is divergent for the 49 amino acid carboxy-terminal region. We demonstrate here that rSYN2 forms small aggregates throughout the cytoplasm when overexpressed in human H4 cells, whereas rSYN1 expression is diffuse. Inhibition of the proteasome promotes the formation of larger, cytoplasmic rSYN2 inclusions in transfected cells. Although a survey of the available databases suggests that there is no human splice form equivalent of rSYN2, thus arguing against a direct role in Lewy body formation and Parkinson's disease, these data nonetheless suggest that modifications of the carboxy-terminal region of α-synuclein predispose it to inclusion formation.

Ultrastructure of α-synuclein-positive aggregations in U373 astrocytoma and rat primary glial cells

Abnormal α-synuclein-positive glial cytoplasmic inclusions are found in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. We have recently developed an in vitro model of α-synuclein-immunoreactive aggregations in U373 astrocytoma cells. We have additionally overexpressed wild-type and a C-terminally truncated form of α-synuclein in primary rat glial cells. Astrocytes and oligodendrocytes were found to form α-synuclein-positive aggregations in vitro perinuclearly or in the processes of the cells. The morphological studies presented here demonstrate that the aggregations we have observed in vitro are not limited by a membrane but have unclear borders. They have an amorphous dense core that is intensely α-synuclein-immunopositive and a predominantly filamentous halo around. Mainly filamentous structures at the border area between the halo and the core are α-synuclein-immunoreactive. We conclude that this in vitro model of α-synuclein-positive glial aggregations mimics the morphology of the abnormal glial inclusions described in neurodegenerative disorders and could be a suitable model for studying their role in the pathogenesis of these diseases.

Human α-Synuclein over-expression increases intracellular reactive oxygen species levels and susceptibility to dopamine

α-Synuclein is a major component of Lewy bodies found in the brains of patients with Parkinson's disease (PD). Two point mutations in α-synuclein (A53T and A30P) are identified in few families with dominantly inherited PD. Yet the mechanism by which this protein is involved in nigral cell death remains poorly understood. Mounting evidence suggests the importance of oxidative stress in the pathogenesis of PD. Here we investigated the effects of wild-type and two mutant forms of α-synuclein on intracellular reactive oxygen species (ROS) levels using clonal SH-SY5Y cells engineered to over-express these proteins. All three cell lines, and particularly mutant α-synuclein-expressing cells, had increased ROS levels relative to control LacZ-engineered cells. In addition, cell viability was significantly curtailed following the exposure of all three α-synuclein-engineered cells to dopamine, but more so with mutant α-synuclein. These results suggest that over-expression of α-synuclein, and especially its mutant forms, exaggerates the vulnerability of neurons to dopamine-induced cell death through excess intracellular ROS generation. Thus, these findings provide a link between mutations or over-expression of α-synuclein and apoptosis of dopaminergic neurons by lowering the threshold of these cells to oxidative damage.

Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins.

Detergent-stable multimers of alpha-synuclein have been found specifically in the brains of patients with Parkinson's disease and other neurodegenerative diseases. Here we show that recombinant alpha-synuclein forms multimers in vitro upon exposure to vesicles containing certain polyunsaturated fatty acid (PUFA) acyl groups, including arachidonoyl and docosahexaenoyl. This process occurs at physiological concentrations and much faster than in aqueous solution. PUFA-induced aggregation involves physical association with the vesicle surface via the large apolipoprotein-like lipid-binding domain that constitutes the majority of the protein. beta- and gamma-synucleins, as well as the Parkinson's disease-associated alpha-synuclein variants A30P and A53T, show similar tendencies to multimerize in the presence of PUFAs. Multimerization does not require the presence of any tyrosine residues in the sequence. The membrane-based interaction of the synucleins with specific long chain polyunsaturated phospholipids may be relevant to the protein family's physiological functions and may also contribute to the aggregation of alpha-synuclein observed in neurodegenerative disease.

Evaluation of the γ-synuclein gene in German Parkinson's disease patients

Mutations in the α -synuclein gene are responsible for an autosomal-dominantly inherited form of Parkinson's disease (PD) and α-synuclein was found to be the major component of Lewy bodies in PD. Because of the high homology to α-synuclein and the abundance in neuronal tissues, we investigated the γ -synuclein gene in PD. We analyzed 262 German PD patients and 179 healthy German controls via two polymorphisms in the γ -synuclein gene. No significant differences in the allelic or genotypic distributions of the investigated polymorphisms were observed between patients and controls. In addition no evidence for an increased risk of combined genotypes of polymorphisms in the γ -synuclein and the α -synuclein gene was found. Therefore, our results do not support a major role of the γ -synuclein gene in PD.

Breakthrough in Understanding the Pathogenesis of Parkinson's Disease

A new international study of autopsy brain specimens from patients with inherited forms of Parkinson's disease (PD) and from non-PD controls creates a unifying theory for the pathogenesis of PD. In a nutshell, here are the theory and the findings that support it. The Theory: A particular form of the molecule α-synuclein (αS), called αSp22, is produced in dopaminergic nigrostriatal …

Copper(II)-induced self-oligomerization of α-synuclein

alpha-Synuclein is a component of the abnormal protein depositions in senile plaques and Lewy bodies of Alzheimer's disease (AD) and Parkinson's disease respectively. The protein was suggested to provide a possible nucleation centre for plaque formation in AD via selective interaction with amyloid beta/A4 protein (Abeta). We have shown previously that alpha-synuclein has experienced self-oligomerization when Abeta25-35 was present in an orientation-specific manner in the sequence. Here we examine this biochemically specific self-oligomerization with the use of various metals. Strikingly, copper(II) was the most effective metal ion affecting alpha-synuclein to form self-oligomers in the presence of coupling reagents such as dicyclohexylcarbodi-imide or N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline. The size distribution of the oligomers indicated that monomeric alpha-synuclein was oligomerized sequentially. The copper-induced oligomerization was shown to be suppressed as the acidic C-terminus of alpha-synuclein was truncated by treatment with endoproteinase Asp-N. In contrast, the Abeta25-35-induced oligomerizations of the intact and truncated forms of alpha-synuclein were not affected. This clearly indicated that the copper-induced oligomerization was dependent on the acidic C-terminal region and that its underlying biochemical mechanism was distinct from that of the Abeta25-35-induced oligomerization. Although the physiological or pathological relevance of the oligomerization remains currently elusive, the common outcome of alpha-synuclein on treatment with copper or Abeta25-35 might be useful in understanding neurodegenerative disorders in molecular terms. In addition, abnormal copper homoeostasis could be considered as one of the risk factors for the development of disorders such as AD or Parkinson's disease.

The native form of α-synuclein is not found in the cerebrospinal fluid of patients with Parkinson's disease or normal controls

α-Synuclein has recently been shown to be a major constituent of Lewy bodies in Parkinson's disease (PD). This observation led us to investigate the possibility that its detection in the cerebrospinal fluid (CSF) could be used as a marker for Lewy bodies in the central nervous system. In this study we determined the pattern of expression of α-synuclein in patients with sporadic Parkinson's disease (PD) and normal controls, using western immunoblotting in conjunction with an antibody that recognizes the carboxyl terminal of α-synuclein protein. The native 19 kDa band normally seen in brain homogenates was not found in the CSF of either parkinsonian patients or control subjects. However, a novel band was observed, which migrated at a position in the range of 42 kDa in CSF from both patients and controls. We conclude that α-synuclein cannot be used as a biomarker for Lewy bodies during life. However, further characterization of the 42 kDa protein may be of interest.