α-Synuclein induces migration of BV-2 microglial cells by up-regulation of CD44 and MT1-MMP

Although there is known to be a marked concentration of reactive microglia in the substantia nigra pars compacta (SNpc) of patients with Parkinson's disease (PD), a disorder in which α-synuclein plays a key pathogenic role, the specific roles of α-synuclein and microglia remains poorly understood. In this study, we investigated the effects of α-synuclein and the mechanisms of invasive microglial migration into the SNpc. We show that α-synuclein up-regulates the expressions of the cell adhesion molecule CD44 and the cell surface protease membrane-type 1 matrix metalloproteinase through the extracellular regulated kinases 1/2 pathway. These concurrent inductions increased the generation of soluble CD44 to liberate microglia from the surrounding extracellular matrix for migration. The effects of α-synuclein were identical in BV-2 murine microglial cells subjected to cDNA transfection and extracellular treatment. These inductions in primary microglial cultures of C57Bl/6 mice were identical to those in BV-2 cells. α-Synuclein-induced microglial migration into the SNpc was confirmed in vivo using a 6-hydroxydopamine mouse model of PD. Our data demonstrate a correlation between α-synuclein-induced phenotypic changes and microglial migration. With the recruitment of the microglial population into the SNpc during dopaminergic neurodegeneration, α-synuclein may play a role in accelerating the pathogenesis of PD.     

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling

α-Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, αS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.     

Multiple Tight Phospholipid-Binding Modes of α-Synuclein Revealed by Solution NMR Spectroscopy

'In dopaminergic neurons, α-synuclein (αS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of αS to membrane phospholipids is implicated in its functional role in synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. We describe here a solution NMR study in which αS is added to small unilamellar vesicles of a composition mimicking synaptic vesicles; the results provide evidence for multiple distinct phospholipid-binding modes of αS. Exchange between the free state and the lipid-bound αS state, and between different bound states is slow on the NMR timescale, being in the range of 1-10 s^− 1. Partitioning of the binding modes is dependent on lipid/αS stoichiometry, and tight binding with slow-exchange kinetics is observed at stoichiometries as low as 2:1. In all lipid-bound states, a segment of residues starting at the N-terminus of αS adopts an α-helical conformation, while succeeding residues retain the characteristics of a random coil. The 40 C-terminal residues remain dynamically disordered, even at high-lipid concentrations, but can also bind to lipids to an extent that appears to be determined by the fraction of cis X-Pro peptide bonds in this region. While lipid-bound αS exhibits dynamic properties that preclude its direct observation by NMR, its exchange with the NMR-visible free form allows for its indirect characterization. Rapid amide-amide nuclear Overhauser enhancement buildup points to a large α-helical conformation, and a distinct increase in fluorescence anisotropy attributed to Tyr39 indicates an ordered environment for this “dark state.” Titration of αS with increasing amounts of lipids suggests that the binding mode under high-lipid conditions remains qualitatively similar to that in the low-lipid case. The NMR data appear incompatible with the commonly assumed model where αS lies in an α-helical conformation on the membrane surface and instead suggest that considerable remodeling of the vesicles is induced by αS.     

Molecular Insights into the Interaction between α-Synuclein and Docosahexaenoic Acid

α-Synuclein (α-syn) is a 140-residue protein of unknown function, involved in several neurodegenerative disorders, such as Parkinson's disease. Recently, the possible interaction between α-syn and polyunsaturated fatty acids has attracted a strong interest. Indeed, lipids are able to trigger the multimerization of the protein in vitro and in cultured cells. Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) in cerebral gray matter and is dynamically released following phospholipid hydrolysis. Moreover, it has been found in high levels in brain areas containing α-syn inclusions in patients affected by Parkinson's disease. Debated and unsolved questions regard the nature of the molecular interaction between α-syn and DHA and the effect exerted by the protein on the aggregated state of the FA. Here, we show that α-syn is able to strongly interact with DHA and that a mutual effect on the structure of the protein and on the physical state of the lipid derives from this interaction. α-Syn acquires an α-helical conformation in a simple two-state transition. The binding of the protein to the FA leads to a reduction of the size of the spontaneously formed aggregated species of DHA as well as of the critical aggregate concentration of the lipid. Specifically, biophysical methods and electron microscopy observations indicated that the FA forms oil droplets in the presence of α-syn. Limited proteolysis experiments showed that, when the protein is bound to the FA oil droplets, it is initially cleaved in the 89-102 region, suggesting that this chain segment is sufficiently flexible or unfolded to be protease-sensitive. Subsequent proteolytic events produce fragments corresponding to the first 70-80 residues that remain structured and show high affinity for the lipid. The fact that a region of the polypeptide chain remains accessible to proteases, when interacting with the lipid, suggests that this region could be involved in other interactions, justifying the ambivalent propensity of α-syn towards folding or aggregation in the presence of FAs.     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

AAV介导的α-synuclein基因过表达致多巴胺能神经元损伤-一种制作轻度帕金森病大鼠模型的新方法

目的：研究过表达α-synuclein基因是否导致大鼠黑质纹状体选择性损伤,为帕金森病（parkinson’s disease, PD）大鼠模型的制备提供一种新的方法。方法:用腺相关病毒(adeno-associated virus, AAV)做载体,将人野生型α突触核蛋白(α-synuclein, NACP)引入大鼠脑内,观察大鼠行为学的改变,通过免疫组织化学染色观察其对黑质多巴胺能神经元细胞的影响,高效液相色谱（HPLC）检测纹状体多巴胺（DA）的含量。结果:α-synuclein基因过表达后大鼠出现自发性活动减少、爬行活动减慢、暂时性躯干震颤、竖毛等类似PD初期的症状和体征;大鼠脑黑质TH阳性神经元细胞随时间的延长出现数目减少,并且纹状体DA含量也出现减少,并且出现α-synuclein的积聚。结论:上述结果表明α-synuclein基因的过表达引起黑质多巴胺能神经元细胞的死亡,对大鼠的运动行为有一定的影响,产生类似于PD早期的症状与体征,与化学毒素（如6-OHDA, MPTP）诱导的动物模型相比,此法制作的动物模型可模拟PD缓慢发展的进程,为研究PD的病程进展及发病机制提供一个理想的动物模型。     

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.     

Heat shock protein 70 inhibits alpha-synuclein fibril formation via interactions with diverse intermediates

alpha-Synuclein (AS) is a main component of Lewy bodies in midbrain dopamine neurons pathologically characteristic of Parkinson's disease. We show that heat shock protein (Hsp) 70 inhibits AS fibril formation via preventing the formation of prefibrillar AS (PreAS), binding with PreAS to impede nuclei formation, and binding with nuclei to retard fibril elongation. Also, Hsp70 suppresses the PreAS-induced permeabilization of vesicular membrane through interactions with PreAS. The substrate-binding domain alone is sufficient for Hsp70 to inhibit AS fibril formation. The binding of Hsp70 with PreAS only requires the substrate-binding subdomain, and the binding with AS nuclei requires the C-terminal lid subdomain as well. The results may form the molecular basis for elucidating the mechanism of AS fibril formation and the crucial roles of chaperones in protecting proteins from toxic conversion in many conformational diseases.     

Antioxidant compounds have potent anti-fibrillogenic and fibril-destabilizing effects for alpha-synuclein fibrils in vitro

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Various antioxidants not only inhibit the formation of beta-amyloid fibrils (fAbeta), but also destabilize preformed fAb in vitro. Using fluorescence spectroscopy with thioflavin S and electron microscopy, here we examined the effects of the antioxidants nordihydroguaiaretic acid, curcumin, rosmarinic acid, ferulic acid, wine-related polyphenols [tannic acid, myricetin, kaempferol (+)-catechin and (-)-epicatechin], docosahexaenoic acid, eicosapentaenoic acid, rifampicin and tetracycline on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules, except for docosahexaenoic acid and eicosapentaenoic acid, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: tannic acid=nordihydroguaiaretic acid=curcumin=rosmarinic acid=myricetin>kaempferol=ferulic acid>(+)-catechin=(-)-epicatechin>rifampicin=tetracycline. These compounds with anti-fibrillogenic as well as antioxidant activities could be key molecules for the development of preventives and therapeutics for LBD and MSA as well as Alzheimer's disease.