Agrin binds alpha-synuclein and modulates alpha-synuclein fibrillation

Recent studies have begun to investigate the role of agrin in brain and suggest that agrin's function likely extends beyond that of a synaptogenic protein. Particularly, it has been shown that agrin is associated with the pathological lesions of Alzheimer's disease (AD) and may contribute to the formation of beta-amyloid (Abeta) plaques in AD. We have extended the analysis of agrin's function in neurodegenerative diseases to investigate its role in Parkinson's disease (PD). Alpha-synuclein is a critical molecular determinant in familial and sporadic PD, with the formation of alpha-synuclein fibrils being enhanced by sulfated macromolecules. In the studies reported here, we show that agrin binds to alpha-synuclein in a heparan sulfate-dependent (HS-dependent) manner, induces conformational changes in this protein characterized by beta-sheet structure, and enhances insolubility of alpha-synuclein. We also show that agrin accelerates the formation of protofibrils by alpha-synuclein and decreases the half-time of fibril formation. The association of agrin with PD lesions was also explored in PD human brain, and these studies shown that agrin colocalizes with alpha-synuclein in neuronal Lewy bodies in the substantia nigra of PD brain. These studies indicate that agrin is capable of accelerating the formation of insoluble protein fibrils in a second common neurodegenerative disease. These findings may indicate shared molecular mechanisms leading to the pathophysiology in these two neurodegenerative disorders.     

Evidence for a partially folded intermediate in alpha-synuclein fibril formation

Intracellular proteinaceous aggregates (Lewy bodies and Lewy neurites) of alpha-synuclein are hallmarks of neurodegenerative diseases such as Parkinson's disease, dementia with Lewy bodies, and multiple systemic atrophy. However, the molecular mechanisms underlying alpha-synuclein aggregation into such filamentous inclusions remain unknown. An intriguing aspect of this problem is that alpha-synuclein is a natively unfolded protein, with little or no ordered structure under physiological conditions. This raises the question of how an essentially disordered protein is transformed into highly organized fibrils. In the search for an answer to this question, we have investigated the effects of pH and temperature on the structural properties and fibrillation kinetics of human recombinant alpha-synuclein. Either a decrease in pH or an increase in temperature transformed alpha-synuclein into a partially folded conformation. The presence of this intermediate is strongly correlated with the enhanced formation of alpha-synuclein fibrils. We propose a model for the fibrillation of alpha-synuclein in which the first step is the conformational transformation of the natively unfolded protein into the aggregation-competent partially folded intermediate.     

Organization of the human synphilin-1 gene, a candidate for Parkinson's disease

We have recently identified a protein we called synphilin-1, which interacts in vivo with alpha-synuclein. Mutations in alpha-synuclein cause familial Parkinson's disease (PD). Alpha-synuclein protein is present in the pathologic lesions of familial and sporadic PD, and diffuse Lewy body disease, indicating an important pathogenic role for alpha-synuclein. Here we describe the structure of the human synphilin-1 gene (SNCAIP). The open reading frame of this gene is contained within ten exons. We have designed primers to amplify each SNCAIP exon, so these primers can now be used to screen for mutations or polymorphisms in patients with Parkinson's disease or related diseases. We found a highly polymorphic GT repeat within intron 5 of SNCAIP, suitable for linkage analysis of families with PD. We have mapped SNCAIP locus to Chromosome (Chr) 5q23.1-23.3 near markers WI-4673 and AFMB352XH5. In addition, using immunohistochemistry in human postmortem brain tissue, we found that synphilin-1 protein is present in neuropil, similar to alpha-synuclein protein. Because of its association with alpha-synuclein, synphilin-1 may be a candidate for involvement in Parkinson's disease or other related disorders. Received: 21 September 1999 / Accepted: 16 March 2000     

Membrane-induced folding of the cAMP-regulated phosphoprotein endosulfine-alpha

Endosulfine-alpha (ENSA) is a 121-residue cAMP-regulated phosphoprotein, originally identified as an endogenous regulator of ATP-sensitive potassium channels. ENSA has been implicated in the regulation of insulin secretion, and expression of ENSA is decreased in brains of both Alzheimer's disease (AD) and Down's syndrome patients. We recently described membrane-dependent interactions between ENSA and the Parkinson's disease associated protein alpha-synuclein. Here we characterize the conformational change in ENSA that occurs upon binding to membranes. Secondary chemical shift analysis demonstrates formation of four helices in the lipid-bound state that are not present in the absence of lipid. The helical structure is maintained in several different lipid mimetics (sodium dodecyl sulfate, dodecyl phosphocholine, lyso 1-palmitoyl phosphatidylglycerol, and phospholipid vesicles). Introduction of a mutation (S109E) to mimic PKA phosphorylation of ENSA leads to a perturbation of the fourth helix and disrupts the interaction with alpha-synuclein. These data establish ENSA as an intrinsically unstructured protein that adopts a stable structure upon membrane binding, properties it shares with its binding partner alpha-synuclein.     

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.     

A protein-chameleon: conformational plasticity of alpha-synuclein,a disordered protein involved in neurodegenerative disorders

Under the physiological conditions in vitro, alpha-synuclein, a conservative presynaptic protein, the aggregation and fibrillation of which is assumed to be involved into the pathogenesis of Parkinson's disease and several other neurodegenerative disorders, known as synucleinopathies, is characterized by the lack of rigid well-defined structure; i.e., it belongs to the class of intrinsically unstructured proteins. Intriguingly, alpha-synuclein is characterized by a remarkable conformational plasticity, adopting a series of different conformations depending on the environment. For example, this protein may either stay substantially unfolded, or adopt an amyloidogenic partially folded conformation, or fold into alpha-helical or beta-structural species, both monomeric and oligomeric. Furthermore, it might form several morphologically different types of aggregates, including oligomers (spheres or doughnuts), amorphous aggregates, and or amyloid-like fibrils. The peculiarities of this astonishing conformational behavior are analyzed to shed light on structural plasticity of this protein-chameleon.     

Residual structure, backbone dynamics, and interactions within the synuclein family

The human synuclein protein family includes alpha-synuclein, which has been linked to both familial and sporadic Parkinson's disease, and the highly homologous beta and gamma-synuclein. Mutations in alpha-synuclein cause autosomal dominant early onset Parkinson's, and the protein is found deposited in a fibrillar form in hereditary and idiopathic forms of the disease. No genetic link between beta and gamma-synuclein, and any neurodegenerative disease has been established, and it is generally considered that these proteins are not highly pathogenic. In addition, beta and gamma-synuclein are reported to aggregate less readily than alpha-synuclein in vitro. Indeed, beta-synuclein has been reported to protect against alpha-synuclein aggregation in vitro, as well as alpha-synuclein-mediated toxicity in vivo. Earlier, we compared the structural properties of the highly helical states adopted by all three synucleins in association with detergent micelles in an attempt to delineate the basis for functional differences between the three proteins. Here, we report a comparison of the structural and dynamic properties of the free states of all three proteins in order to shed light on differences that may help to explain their different propensities to aggregate, which in turn may underlie their differing contributions to the etiology of Parkinson's disease. We find that gamma-synuclein closely resembles alpha-synuclein in its free-state residual secondary structure, consistent with the more similar propensities of the two proteins to aggregate in vitro. beta-Synuclein, however, differs significantly from alpha-synuclein, exhibiting a lower predisposition towards helical structure in the second half of its lipid-binding domain, and a higher preference for extended structures in its C-terminal tail. Both beta and gamma-synuclein show less extensive transient long-range structure than that observed in alpha-synuclein. These results raise questions regarding the role of secondary structure propensities and transient long-range contacts in directing synuclein aggregation reactions.     

Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure.

Parkinson's disease involves the aggregation of alpha-synuclein to form fibrils, which are the major constituent of intracellular protein inclusions (Lewy bodies and Lewy neurites) in dopaminergic neurons of the substantia nigra. Occupational exposure to specific metals, especially manganese, copper, lead, iron, mercury, zinc, aluminum, appears to be a risk factor for Parkinson's disease based on epidemiological studies. Elevated levels of several of these metals have also been reported in the substantia nigra of Parkinson's disease subjects. We examined the effect of various metals on the kinetics of fibrillation of recombinant alpha-synuclein and in inducing conformational changes, as monitored by biophysical techniques. Several di- and trivalent metal ions caused significant accelerations in the rate of alpha-synuclein fibril formation. Aluminum was the most effective, along with copper(II), iron(III), cobalt(III), and manganese(II). The effectiveness correlated with increasing ion charge density. A correlation was noted between efficiency in stimulating fibrillation and inducing a conformational change, ascribed to formation of a partially folded intermediate. The potential for ligand bridging by polyvalent metal ions is proposed to be an important factor in the metal-induced conformational changes of alpha-synuclein. The results indicate that low concentrations of some metals can directly induce alpha-synuclein fibril formation.     

Recent advances on alpha-synuclein cell biology: functions and dysfunctions

Alpha-synuclein is a recently discovered protein that was first identified as the major non amyloid component of senile plaques, the cerebral lesion likely responsible for Alzheimer's disease. The role of alpha-synuclein in another brain disease namely Parkinson's disease, has been more deeply documented. It appears that alpha-synuclein fills up the intracytoplasmic inclusions called Lewy bodies that likely contribute to the etiology of Parkinson's disease. Furthermore, rare familial forms of Parkinson's disease have been shown to be linked to autosomal dominant mutations of alpha-synucleins. Is alpha-synuclein a bridge between Alzheimer's and Parkinson's diseases? Could it be seen as a common denominator for these two neurodegenerative diseases? These issues could be better addressed by further delineating the physiological function of alpha-synuclein and, as a corollary, the dysfunction taking place along with the diseases. Here, I will review the recent advances concerning the physiology of alpha-synuclein and will particularly focus on the post-traductional events leading to drastic biophysical transformations. I will describe recent works suggesting that these modifications directly modulate the normal function of alpha-synuclein, likely accounting for the dysfunction associated with Parkinson's disease and perhaps contributing to Alzheimer's pathology.     

Interactions between metals and alpha-synuclein--function or artefact?

alpha-synuclein is one of a family of proteins whose function remains unknown. This protein has become linked to a number of neurodegenerative disease although its potential causative role in these diseases remains mysterious. In diseases such as Parkinson's disease and Lewy body dementias, alpha-synuclein becomes deposited in aggregates termed Lewy bodies. Also, some inherited forms of Parkinson's diseases are linked to mutations in the gene for alpha-synuclein. Studies have mostly focussed on what causes the aggregation of the protein but, like many amyloidogenic proteins associated with a neurodegenerative disorder, this protein has now been suggested to bind copper. This finding is currently controversial. This review examines the evidence that alpha-synuclein is a copper binding protein and discusses whether this has any significance in determining the function of the protein or whether copper binding is at all necessary for aggregation.     

Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins.

The pathological hallmark of Parkinson's disease is the presence of intracellular inclusions, Lewy bodies, and Lewy neurites, in the dopaminergic neurons of the substantia nigra and several other brain regions. Filamentous alpha-synuclein is the major component of these deposits and its aggregation is believed to play an important role in Parkinson's disease and several other neurodegenerative diseases. Two homologous proteins, beta- and gamma-synucleins, are also abundant in the brain. The synucleins are natively unfolded proteins. beta-Synuclein, which lacks 11 central hydrophobic residues compared with its homologs, exhibited the properties of a random coil, whereas alpha- and gamma-synucleins were slightly more compact and structured. gamma-Synuclein, unlike its homologs, formed a soluble oligomer at relatively low concentrations, which appears to be an off-fibrillation pathway species. Here we show that, although they have similar biophysical properties to alpha-synuclein, beta- And gamma-synucleins inhibit alpha-synuclein fibril formation. Complete inhibition of alpha-synuclein fibrillation was observed at 4:1 molar excess of beta- and gamma-synucleins. No significant incorporation of beta-synuclein into the fibrils was detected. The lack of fibrils formed by beta-synuclein is most readily explained by the absence of a stretch of hydrophobic residues from the middle region of the protein. A model for the inhibition is proposed.     

p25alpha Stimulates alpha-synuclein aggregation and is co-localized with aggregated alpha-synuclein in alpha-synucleinopathies

Aggregation of the nerve cell protein alpha-synuclein is a characteristic of the common neurodegenerative alpha-synucleinopathies like Parkinson's disease and Lewy body dementia, and it plays a direct pathogenic role as demonstrated by early onset diseases caused by mis-sense mutations and multiplication of the alpha-synuclein gene. We investigated the existence of alpha-synuclein pro-aggregatory brain proteins whose dysregulation may contribute to disease progression, and we identified the brain-specific p25alpha as a candidate that preferentially binds to alpha-synuclein in its aggregated state. Functionally, purified recombinant human p25alpha strongly stimulates the aggregation of alpha-synuclein in vitro as demonstrated by thioflavin-T fluorescence and quantitative electron microscopy. p25alpha is normally only expressed in oligodendrocytes in contrast to alpha-synuclein, which is normally only expressed in neurons. This expression pattern is changed in alpha-synucleinopathies. In multiple systems atrophy, degenerating oligodendrocytes displayed accumulation of p25alpha and dystopically expressed alpha-synuclein in the glial cytoplasmic inclusions. In Parkinson's disease and Lewy body dementia, p25alpha was detectable in the neuronal Lewy body inclusions along with alpha-synuclein. The localization in alpha-synuclein-containing inclusions was verified biochemically by immunological detection in Lewy body inclusions purified from Lewy body dementia tissue and glial cytoplasmic inclusions purified from tissue from multiple systems atrophy. We suggest that p25alpha plays a pro-aggregatory role in the common neurodegenerative disorders hall-marked by alpha-synuclein aggregates.     

Temperature-induced reversible conformational change in the first 100 residues of alpha-synuclein

Natively disordered proteins are a growing class of anomalies to the structure-function paradigm. The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. We noticed a dramatic difference in dilute solution 1H-15N Heteronuclear Single Quantum Coherence (HSQC) spectra of wild-type alpha-synuclein and two disease-related mutants (A30P and A53T), with spectra collected at 35 degrees C showing fewer cross-peaks than spectra acquired at 10 degrees C. Here, we show the change to be the result of a reversible conformational exchange linked to an increase in hydrodynamic radius and secondary structure as the temperature is raised. Combined with analytical ultracentrifugation data showing alpha-synuclein to be monomeric at both temperatures, we conclude that the poor quality of the 1H-15N HSQC spectra obtained at 35 degrees C is due to conformational fluctuations that occur on the proton chemical shift time scale. Using a truncated variant of alpha-synuclein, we show the conformational exchange occurs in the first 100 amino acids of the protein. Our data illustrate a key difference between globular and natively disordered proteins. The properties of globular proteins change little with solution conditions until they denature cooperatively, but the properties of natively disordered proteins can vary dramatically with solution conditions.     

Macromolecular crowding in the Escherichia coli periplasm maintains alpha-synuclein disorder

The natively disordered protein alpha-synuclein is the primary component of Lewy bodies, the cellular hallmark of Parkinson's disease. Most studies of this protein are performed in dilute solution, but its biologically relevant role is performed in the crowded environment inside cells. We addressed the effects of macromolecular crowding on alpha-synuclein by combining NMR data acquired in living Escherichia coli with in vitro NMR data. The crowded environment in the E.coli periplasm prevents a conformational change that is detected at 35 degrees C in dilute solution. This change is associated with an increase in hydrodynamic radius and the formation of secondary structure in the N-terminal 100 amino acid residues. By preventing this temperature-induced conformational change, crowding in the E.coli periplasm stabilizes the disordered monomer. We obtain the same stabilization in vitro upon crowding alpha-synuclein with 300 g/l of bovine serum albumin, indicating that crowding alone is sufficient to stabilize the disordered, monomeric protein. Two disease-associated variants (A30P and A53T) behave in the same way in both dilute solution and in the E.coli periplasm. These data reveal the importance of approaching the effects of macromolecular crowding on a case-by-case basis. Additionally, our work shows that discrete structured protein conformations may not be achieved by alpha-synuclein inside cells, implicating the commonly overlooked aspect of macromolecular crowding as a possible factor in the etiology of Parkinson's disease.     

Sept4, a component of presynaptic scaffold and Lewy bodies, is required for the suppression of alpha-synuclein neurotoxicity

In Parkinson disease (PD), alpha-synuclein aggregates called Lewy bodies often involve and sequester Septin4 (Sept4), a polymerizing scaffold protein. However, the pathophysiological significance of this phenomenon is unclear. Here, we show the physiological association of Sept4 with alpha-synuclein, the dopamine transporter, and other presynaptic proteins in dopaminergic neurons; mice lacking Sept4 exhibit diminished dopaminergic neurotransmission due to scarcity of these presynaptic proteins. These data demonstrate an important role for septin scaffolds in the brain. In transgenic mice that express human alpha-synuclein(A53T) (a mutant protein responsible for familial PD), loss of Sept4 significantly enhances neuropathology and locomotor deterioration. In this PD model, insoluble deposits of Ser129-phosphorylated alpha-synuclein(A53T) are negatively correlated with the dosage of Sept4. In vitro, direct association with Sept4 protects alpha-synuclein against self-aggregation and Ser129 phosphorylation. Taken together, these data show that Sept4 may be involved in PD as a dual susceptibility factor, as its insufficiency can diminish dopaminergic neurotransmission and enhance alpha-synuclein neurotoxicity.     

Magnesium inhibits spontaneous and iron-induced aggregation of alpha-synuclein

Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to alpha-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in alpha-synuclein as indicators of metal-related conformational changes in alpha-synuclein, we show that iron and magnesium both interact with alpha-synuclein. alpha-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of alpha-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits alpha-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases alpha-synuclein aggregation, while magnesium at concentrations >0.75 mm inhibits the aggregation of alpha-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of alpha-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation.     

PA700, the regulatory complex of the 26S proteasome, interferes with alpha-synuclein assembly

Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.     

From genetics to pathology: tau and alpha-synuclein assemblies in neurodegenerative diseases

The most common degenerative diseases of the human brain are characterized by the presence of abnormal filamentous inclusions in affected nerve cells and glial cells. These diseases can be grouped into two classes, based on the identity of the major proteinaceous components of the filamentous assemblies. The filaments are made of either the microtubule-associated protein tau or the protein alpha-synuclein. Importantly, the discovery of mutations in the tau gene in familial forms of frontotemporal dementia and of mutations in the alpha-synuclein gene in familial forms of Parkinson's disease has established that dysfunction of tau protein and alpha-synuclein can cause neurodegeneration.     

Selective detection, quantification, and subcellular location of alpha-synuclein aggregates with a protein aggregate filtration assay

Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy are caused by alpha-synuclein aggregates. At present, there is no good biochemical method defining alpha-synuclein aggregates formed in vivo versus oligomers as a means to investigate alpha-synuclein aggregation and its mechanisms of neurodegeneration. A simple method, therefore, for the selective and sensitive detection of alpha-synuclein aggregates suited for screening purposes would be useful. Since in contrast to prions a proper detection of alpha-synuclein aggregates by Western blot analysis is difficult, we developed a protein aggregate filtration (PAF) assay. It takes advantage of the inherent insolubility of aggregated alpha-synuclein using microfiltration to separate it from soluble isoforms. For the first time, this assay even makes quantitative comparisons possible. We describe how the PAF assay can be applied to human brain tissue and animal and cell culture models, as well as used as a screening method for the subcellular location of alpha-synuclein aggregates. Since it detects the pathological isoform instead of surrogate markers, the PAF assay may have also potential in diagnosis of PD and DLB.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.