Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations

alpha-Synuclein is a major component of Lewy bodies, a neuropathological feature of Parkinson's disease. Two alpha-synuclein mutations, Ala53Thr and Ala30Pro, are associated with early onset, familial forms of the disease. Recently, synphilin-1, a protein found to interact with alpha-synuclein by yeast two hybrid techniques, was detected in Lewy bodies. In this study we report the interaction of alpha-synuclein and synphilin-1 in human neuroglioma cells using a sensitive fluorescence resonance energy transfer technique. We demonstrate that the C-terminus of alpha-synuclein is closely associated with the C-terminus of synphilin-1. A weak interaction occurs between the N-terminus of alpha-synuclein and synphilin-1. The familial Parkinson's disease associated mutations of alpha-synuclein (Ala53Thr and Ala30Pro) also demonstrate a strong interaction between their C-terminal regions and synphilin-1. However, compared with wild-type alpha-synuclein, significantly less energy transfer occurs between the C-terminus of Ala53Thr alpha-synuclein and synphilin-1, suggesting that the Ala53Thr mutation alters the conformation of alpha-synuclein in relation to synphilin-1.     

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.     

帕金森病中铁与Alpha-突触核蛋白的相互作用

帕金森病(Parkinson's disease,PD)的特异标志物Alpha-突触核蛋白的异常聚集往往伴有铁的沉积,说明铁与Alpha-突触核蛋白聚集之间存在一定联系。铁可以通过增加Alpha-突触核蛋白的产生及抑制其降解从而促进Alpha-突触核蛋白聚集。Alpha-突触核蛋白作为一种高铁还原酶也可以影响细胞铁的代谢。本文就铁与Alpha-突触核蛋白参与PD的发病以及两者之间相互作用的机制进行综述。     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis

Alpha synuclein can be phosphorylated at serine129 (P-S129), and the presence of highly phosphorylated α-synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for α-synuclein S129 phosphorylation is constitutively active in SH-SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P-S129 α-synuclein levels. Under these conditions, similar increases in P-S129 α-synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D α-synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D α-synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P-S129 α-synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson's disease pathogenesis.     

Cytopathology, Mode of Aphid Transmission and Search for the Casual Agent of Cotton Bunchy Top Disease

The cytopathological effects of cotton bunchy top (CBT) disease and its mode of transmission by Aphis gossypii Glover (cotton aphid), were studied. CBT infection affected the leaf epidermal layer producing a loose, ruptured and rough surface morphology with many stomata closed and misshapen. Roots of CBT‐infected plants showed reduced growth, small knots and a dark brown appearance. A single aphid per plant was capable of transmitting CBT at 5%, whereas three aphids per plant transmitted CBT to 50% of the cotton seedlings and 20 aphids per plant transmitted the disease agent to 80% of the cotton seedlings. Aphis gossypii acquired CBT after a minimum acquisition access period of 5 min and transmitted the agent after a minimum inoculation access period of 1 h. Both alate and apterous aphids and nymph instars 2, 3 and 4 of A. gossypii transmitted CBT. This preliminary data suggest that A. gossypii transmits CBT in a semi‐persistent manner. Myzus persicae Sulz (green peach aphid) was unable to transmit CBT. A comprehensive attempt to isolate the CBT agent, using a range of virological techniques including double‐stranded RNA extraction, two‐dimensional gel electrophoresis for viroid, circular DNA test, nanovirus polymerase chain reaction (PCR), luteovirus PCR and enzyme‐linked immunosorbent assay, phytoplasma test, nucleoprotein purification and electron microscopy, was unsuccessful, raising the possibility that CBT may be caused by a unique new pathogen.     

Microscopic analysis and seasonality of gemma production in the freshwater red alga Hildenbrandia angolensis (Hildenbrandiales, Rhodophyta)

The development and release of the unique vegetative propagules of the freshwater encrusting alga Hildenbrandia angolensis Welwitsch ex West et West, gemmae, were studied using several different microscopic and histochemical techniques. In addition, the seasonality of gemma production was monitored bimonthly over a 12‐month period in two spring‐fed streams in Texas, USA. Gemmae differentiate within the thallus and are subsequently released from the surface of the crust. Release of the gemmae most likely occurs by digestion of surrounding cells, as suggested by the presence of starch granules and lipid globules in the region between the released gemma and the thallus. The initial separation of the gemmae from the thallus occurs from the sides of the gemma or the bottom, or possibly simultaneously. Contrary to previous studies, we have observed that gemma production occurs endogenously within the thallus of freshwater Hildenbrandia, rather than on the surface of the crust in raised structures. Histochemical tests and electron microscopic examination indicate that the cells of the gemmae contain a large amount of floridean starch. The starch granules frequently form rings surrounding the nuclei of both gemma and thallus cells; a feature infrequently reported for florideophyte red algae. Our seasonality investigations indicate that large fluctuations in gemma production occur over 1 year, but at least some gemma production continues year‐round in the streams examined.     

Modifications of the iron-neuromelanin system in Parkinson's disease

Parkinson's disease is a common neurodegenerative disorder with a mainly sporadic aetiology, although a number of monogenic familiar forms are known. Most of the motor symptoms are due to selective depletion of dopaminergic, neuromelanin-containing neurones of the substantia nigra pars compacta. Neuromelanin is the dark insoluble macromolecule that confers the black (substantia nigra) or grey (locus coeruleus) colour to monoaminergic basal ganglia. In particular, nigral neurones are pigmented because of the accumulation of by-products of oxidative metabolism of the neurotransmitter dopamine. The occurrence of dopamine (and all the enzymatic machinery required for dopamine synthesis, re-uptake and disposal) and neuromelanin, and a large amount of iron ions that interact with them, makes dopaminergic nigral neurones peculiarly susceptible to oxidative stress conditions that, in turn, may become amplified by the iron-neuromelanin system itself. In this mini-review we describe biophysical evidence for iron-neuromelanin modifications that support this hypothesis. Furthermore, we discuss the formation of the covalent linkage between alpha-synuclein and neuromelanin from the early stages of the disease.     

Expression and localization of Parkinson's disease-associated leucine-rich repeat kinase 2 in the mouse brain

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinson's disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2-deficient mice is unaltered relative to wild-type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.     

α - synuclein and Parkinson's disease: the first roadblock

α-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and α-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of α-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed α-synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. α-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that α-synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of α-synuclein-induced neurodegeneration. α-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of α-synuclein with components of neuronal membrane traffic.     

The Structure and Morphogenesis of the Ventral Ciliature in Paraurostyla hymenophora*

SYNOPSIS. The structure and morphogenesis of the ventral ciliature of Paraurostyla hymenophora (Stokes) are described. The oral primordium apparently originates in association with transverse cirrus #6, from which it migrates anteriorly simultaneous with kinetosomal proliferation. The primordium eventually forms an elongate ciliary field from which the future opisthe's fronto-ventro-transverse (FVT) and undulating membrane primordial fields arise. Concomitantly, the future proter's FVT primordial field is initiated by the disaggregation of frontal cirri #4, #5, and #6. Primordia then develop simultaneously within marginal and ventral cirral rows by a disaggregation of cirri within the respective rows, and do not give rise to new cirri until the FVT fields complete segregation into discrete cirri. Near the completion of cirral production from the FVT primordia, each ventral cirral primordium (VCP) forms the 2 rightmost transverse cirri. Segregation of new cirri within the marginal cirral primordia and VCP then occurs, eventually replacing all old cirri within their respective marginal and ventral cirral rows. At the end of cortical morphogenesis, all old ciliary organelles, with the exception of the adoral zone of membranelles, are either reorganized or replaced. These results suggest an evolutionary affinity between the ventral and marginal cirral rows and raise questions about the control of the developmental competence of individual primordia.     

Structure and development of stellate trichomes in Andradea ftoribunda Fr. Allem. (Nyctaginaceae)

LOURO, R. P., MIGUENS, F. C. & MACHADO, R. D., 1992. Structure and development of stellate trichomes in Andradea ftoribunda Fr. Allem. (Nyctaginaceae). Trichomes occur on both faces of young leaves. They are peltate-stellate on the abaxial face, and comprise a stalk and radiating cells with a rudimentary central apex. On the adaxial face the trichomes arc stellate with a large apex comprising one to three cells. In both cases the stalk is formed by three to six cells of which the most distal may contain a tannoid substance. In the adult leaf only the abaxial surface exhibits stellate trichomes, with two to three celled stalks. The central region of radial cells is depressed. On the adaxial side the hairs are shed during maturation of the leaf.     

Ultrastructure of Red-Sore Lesions on Largemouth Bass (Micropterus salmoides): Association of the Ciliate Epistylis sp. and the Bacterium Aeromonas hydrophila*

SYNOPSIS. Epizootic outbreaks of red-sore disease in several reservoirs in the southeastern United States have been reported to cause heavy mortality among several species of fish having sport and commercial value. The etiologic agent is said to be the peritrich ciliate Epistylis sp.; secondary infection by the gram-negative bacterium Aeromonas hydrophila produces hemorrhagic septicemia which results in death. However, in recent studies on the largemouth bass Micropterus salmoides, Epistylis sp. could be isolated from only 35% of 114 lesions from 114 fish, while A. hydrophila was found in 96% of the same lesions. Transmission and scanning electron microscopy of lesions associated with red-sore disease indicate that neither the stalk nor the attachment structure of Epistylis sp. have organelles capable of producing lytic enzymes. Since other investigators have shown that A. hydrophila produces strong lytic toxins, and in absence of evidence to the contrary, it is concluded that Epistylis sp. is a benign ectocommensal and that A. hydrophila is the primary etiologic agent of red-sore disease.     

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.     

Adhesion of Plasmodium gallinaceum Ookinetes to the Aedes aegypti Midgut: Sites of Parasite Attachment and Morphological Changes in the Ookinete

Plasmodium gallinaceum ookinetes adhered to Aedes aegypti midgut epithelia when purified ookinetes and isolated midguts were combined in vitro. Ookinetes preferentially bound to the microvillated luminal surface of the midgut, and they seemed to interact with three types of structures on the midgut surface. First, they adhered lo and migrated through a network-like matrix, which we have termed microvilli-associated network, that covers the surface of the microvilli. This network forms on the luminal midgut surface in response to blood or protein meals. Second, the ookinetes bound directly to the microvilli on the surface of the midgut and were occasionally found immersed in the thick microvillar layer. Third, the ookinetes associated with accumulations of vesicular structures found interspersed between the microvillated cells of the midgut. The origin of these vesicular structures is unknown, but they correlated with the surface of midgut cells invaded by ookinetes as observed by TEM. After binding to the midgut. ookinetes underwent extensive morphological changes: they frequently developed one or more annular constrictions, and their surface roughened considerably, suggesting that midgut components remain bound to the parasite surface. Our observations suggest that, in a natural infection, the ookinete interacts in a sequential manner with specific components of the midgut surface. Initial binding to the midgut surface may activate the ookinete and cause morphological changes in preparation for invasion of the midgut cells.     

Alpha-synuclein up-regulates expression of caveolin-1 and down-regulates extracellular signal-regulated kinase activity in B103 neuroblastoma cells: role in the pathogenesis of Parkinson's disease

alpha-Synuclein accumulation plays an important role in the pathogenesis of Lewy body disease (LBD) and Parkinson's disease (PD). Although the mechanisms are not yet clear, it is possible that dysregulation of the extracellular signal-regulated kinase (ERK) might play a role. As caveolins form scaffolds onto which signaling molecules such as ERK can assemble, we propose that signaling alterations associated with alpha-synuclein accumulation and neurodegeneration, might be mediated via caveolae. Therefore, the objective of the present study was to investigate the potential contribution of alterations in the caveolar system in mediating alpha-synuclein effects on the ERK signaling pathway. For this, synuclein-transfected B103 neuroblastoma cells were used as a model system. In this cell line, caveolin-1 expression was up-regulated, whereas, ERK was down-regulated. ERK was weakly but consistently co-immunoprecipitated with alpha-synuclein but caveolin-1 did not co-immunoprecipitate with alpha-synuclein. Moreover, treatment of alpha-synuclein- overexpressing cells with caveolin-1 antisense oligonucleotides resulted in stimulation of ERK activity, with amelioration of the neuritic alterations. Transduction of alpha-synuclein-overexpressing cells, with an adenoviral vector directing the expression of ERK, resulted in suppression of caveolin-1 expression and re-establishment of the normal patterns of neurite outgrowth. These results suggest that alpha-synuclein may also interfere with ERK signaling by dysregulating caveolin-1 expression. Thus, the caveolin-1/ERK pathway could be a therapeutic target for the alpha-synuclein-related neurodegenerative disorders.     

Double-stranded DNA stimulates the fibrillation of alpha-synuclein in vitro and is associated with the mature fibrils: an electron microscopy study

Filamentous aggregates formed by alpha-synuclein are a prominent and presumably key etiological factor in Parkinson's and other neurodegenerative diseases characterized by motor disorders. Numerous studies have demonstrated that various environmental and intracellular factors affect the fibrillation properties of alpha-synuclein, e.g. by accelerating the process of assembly. Histones, the major component and constituent of chromatin, interact specifically with alpha-synuclein and enhance its fibrillation significantly. Here, we report that another component of chromatin, double-stranded DNA (dsDNA), either linear or supercoiled, also interacts with wild-type alpha-synuclein, leading to a significant stimulation of alpha-synuclein assembly into mature fibrils characterized by a reduced lag phase. In general, the morphology of the fibrils remains unchanged in the presence of linear dsDNA. Electron microscopy reveals that DNA forms various types of complexes upon association with the fibrils at their surface without distortion of the double-helical structure. The existence of these complexes was confirmed by the electrophoresis, which also demonstrated that a fraction of the associated DNA was resistant to digestion by restriction endonucleases. Fibrils assembled from the alpha-synuclein mutants A30P and A53T and the C-terminally truncated variants (encoding amino acid residues 1-108 or 1-124) also form complexes with linear dsDNA. Possible mechanisms and implications of dsDNA-alpha-synuclein interactions are discussed.     

Accumulation,localisation, and toxic effects of cadmium in the liverwort Lunularia cruciata

Summary. Accumulation, tissue and intracellular localisation, and toxic effects of cadmium were investigated in the liverwort Lunularia cruciata. The results of analyses carried out by atomic absorption spectrometry on single plants showed that the cadmium accumulation was dose- and time-dependent. Cadmium localisation was assessed by X-ray scanning electron microscopy microanalysis in gemmalings and in the different tissues of the thallus and by X-ray transmission electron microscopy microanalysis at the cellular level. The metal preferentially accumulated in the hyaline parenchyma and at the base of the gemma cups. Inside the cell, cadmium accumulated in the vacuoles and the cell wall. Metal accumulation was accompanied by a concomitant increase in sulphur content within the vacuoles of stressed cells. Gel-permeation chromatography showed that most of the cadmium was associated with a low-molecular-mass fraction eluting at a ratio of elution volume to void volume corresponding to that of phytochelatins. The excess of sulphur deposited in the vacuoles may well have been caused by the stress-induced synthesis of phytochelatins. At the ultrastructural level, sublethal concentrations of cadmium caused alterations of the fine structure of the cells, inducing marked alterations of the chloroplast structure. Cadmium also induced a dose-dependent inhibition of apical thallus growth and gemma germination.Correspondence and reprints: Department of Plant Biology, University Federico 11, via Foria 223, 80132 Naples, Italy.     

Ultrastructure and distribution of sensilla on the antennae of female fig wasp Eupristina sp. (Hymenoptera: Agaonidae)

In the species‐specific and obligate mutualism between the fig (Moraceae: Ficus spp.) and its pollinator (Hymenoptera: Agaonidae), the continuity of lifecycle of both partners completely depends on the female pollinator's ability to detect receptive figs. To better understand the chemical location mechanism, we examined the antennae and their sensilla of the female fig pollinator Eupristina sp. using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The antennae of female Eupristina sp. are geniculated, and in total, there were seven types of sensilla found on the antennae: two types of multiporous placoid sensilla (type 1 is sausage‐like and type 2 is rounded), sensilla trichodea (ST), basiconic sensilla (BS), chaetica sensilla (ChS), coeloconic sensilla (CoS), and one specialized sensillum classified as sensillum obscurum (SO). We described external morphology, abundance, distribution, ultrastructure and discussed putative functions. We inferred from their ultrastructures as chemoreceptors that two types of multiporous placoid sensilla, BS and CoS, were innervated by sensory neurons. The aporous type ST, ChS, and SO were not innervated by dendrites which may function as mechanoreceptor/proprioceptor. These results were also discussed in relation to the interaction between Eupristina sp. and its host fig.