Localization of α-synuclein in teleost central nervous system: immunohistochemical and Western blot evidence by 3D5 monoclonal antibody in the common carp, Cyprinus carpio

Alpha synuclein (α-syn) is a 140 amino acid vertebrate-specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinson's disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α-syn aggregates suggested a role for α-syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α-syns, we investigated this proteins in the CNS of the common carp Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α-syn. The distribution of α-syn was analyzed by semiquantitative Western blot, immunohistochemistry and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α-syn. The distribution of 3D5 immunoreactivity was also compared with that of ChAT, TH and 5HT by double immunolabelings. Results show that α-syn-like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. Present findings indicate that α-syn-like proteins may be expressed in few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be co-expressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α-syn expression in cholinergic systems and suggest that α-syn modulates similar molecular pathways in phylogenetically distant vertebrates. This article is protected by copyright. All rights reserved

Hippocampal neuronal cells that accumulate α-synuclein fragments are more vulnerable to Aβ oligomer toxicity via mGluR5--implications for dementia with Lewy bodies.

BackgroundIn dementia with Lewy bodies (DLB) abnormal interactions between α-synuclein (α-syn) and beta amyloid (Aβ) result in selective degeneration of neurons in the neocortex, limbic system and striatum. However, factors rendering these neurons selectively vulnerable have not been fully investigated. The metabotropic glutamate receptor 5 (mGluR5) has been shown to be up regulated in DLB and might play a role as a mediator of the neurotoxic effects of Aβ and α-syn in vulnerable neuronal populations. In this context, the main objective of the present study was to investigate the role of mGluR5 as a mediator of the neurotoxic effects of α-syn and Aβ in the hippocampus.ResultsWe generated double transgenic mice over-expressing amyloid precursor protein (APP) and α-syn under the mThy1 cassette and investigated the relationship between α-syn cleavage, Aβ, mGluR5 and neurodegeneration in the hippocampus. We found that compared to the single tg mice, the α-syn/APP tg mice displayed greater accumulation of α-syn and mGluR5 in the CA3 region of the hippocampus compared to the CA1 and other regions. This was accompanied by loss of CA3 (but not CA1) neurons in the single and α-syn/APP tg mice and greater loss of MAP 2 and synaptophysin in the CA3 in the α-syn/APP tg. mGluR5 gene transfer using a lentiviral vector into the hippocampus CA1 region resulted in greater α-syn accumulation and neurodegeneration in the single and α-syn/APP tg mice. In contrast, silencing mGluR5 with a lenti-shRNA protected neurons in the CA3 region of tg mice. In vitro, greater toxicity was observed in primary hippocampal neuronal cultures treated with Aβ oligomers and over-expressing α-syn; this effect was attenuated by down-regulating mGluR5 with an shRNA lentiviral vector. In α-syn-expressing neuronal cells lines, Aβ oligomers promoted increased intracellular calcium levels, calpain activation and α-syn cleavage resulting in caspase-3-dependent cell death. Treatment with pharmacological mGluR5 inhibitors such as 2-Methyl-6-(phenylethynyl)pyridine (MPEP) and 3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP) attenuated the toxic effects of Aβ in α-syn-expressing neuronal cells.ConclusionsTogether, these results support the possibility that vulnerability of hippocampal neurons to α-syn and Aβ might be mediated via mGluR5. Moreover, therapeutical interventions targeting mGluR5 might have a role in DLB

Striatal dopamine transmission is subtly modified in human A53Tα-synuclein overexpressing mice

Mutations in, or elevated dosage of, SNCA, the gene for α-synuclein (α-syn), cause familial Parkinson's disease (PD). Mouse lines overexpressing the mutant human A53Tα-syn may represent a model of early PD. They display progressive motor deficits, abnormal cellular accumulation of α-syn, and deficits in dopamine-dependent corticostriatal plasticity, which, in the absence of overt nigrostriatal degeneration, suggest there are age-related deficits in striatal dopamine (DA) signalling. In addition A53Tα-syn overexpression in cultured rodent neurons has been reported to inhibit transmitter release. Therefore here we have characterized for the first time DA release in the striatum of mice overexpressing human A53Tα-syn, and explored whether A53Tα-syn overexpression causes deficits in the release of DA. We used fast-scan cyclic voltammetry to detect DA release at carbon-fibre microelectrodes in acute striatal slices from two different lines of A53Tα-syn-overexpressing mice, at up to 24 months. In A53Tα-syn overexpressors, mean DA release evoked by a single stimulus pulse was not different from wild-types, in either dorsal striatum or nucleus accumbens. However the frequency responsiveness of DA release was slightly modified in A53Tα-syn overexpressors, and in particular showed slight deficiency when the confounding effects of striatal ACh acting at presynaptic nicotinic receptors (nAChRs) were antagonized. The re-release of DA was unmodified after single-pulse stimuli, but after prolonged stimulation trains, A53Tα-syn overexpressors showed enhanced recovery of DA release at old age, in keeping with elevated striatal DA content. In summary, A53Tα-syn overexpression in mice causes subtle changes in the regulation of DA release in the striatum. While modest, these modifications may indicate or contribute to striatal dysfunction

[2.2](4,7)Isobenzofuranophanes - Synthesis, Characterisation and Reactivity

The isomeric Diels-Alder adducts 3, obtained by cycloaddition of tetraphenylcyclopentadienone to the 4,5:12,13-bis-(oxanorbornadieno)[2.2]paracyclophanes syn,syn- and anti,-syn-2[Note ][The stereochemical descriptors syn and anti refer to the orientation of the oxygen bridge in the oxabicyclo[2.2.1]heptadiene subunits with respect to the [2.2]paracyclophaneskeleton.], yield the unstable isobenzofuranophane 4 by consecutive extrusion of carbon monoxide and tetraphenylbenzene when heated to 180°C. The molecular ion of 4 was observed in the EI mass spectrum. The stable tetraphenyl-substituted analogue 10 was synthesized independently from the previously unknown 4,5,12,13-tetrabenzoyl[2.2]paracyclophane (9). UV/Vis as well as fluorescence spectra and an X-ray crystal structure analysis of 9 are reported

Lipid peroxidation is essential for α-synuclein-induced cell death.

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α-synuclein (α-Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α-Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co-cultures of neurons and astrocytes. We found that oligomeric but not monomeric α-Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre-incubation of cells with isotope-reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of oligomeric α-Syn on lipid peroxidation. Inhibition of lipid peroxidation with D-PUFAs further protected cells from cell death induced by oligomeric α-Syn. Thus, lipid peroxidation induced by misfolding of α-Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease. We have found that aggregated α-synuclein-induced production of reactive oxygen species (ROS) that subsequently stimulates lipid peroxidation and cell death in neurons and astrocytes. Specific inhibition of lipid peroxidation by incubation with reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of α-synuclein on lipid peroxidation and cell death

Alpha- synuclein immunoreactivity in the enteric nervous system of human small intestine

Alpha-synuclein (α-syn) is a 140 amino acid protein, belonging to the synuclein family, expressed in mammalian neurons. Structural alterations of α-syn as well as its overexpression have been related to the onset and the progression of several human neurodegenerative diseases, as Parkinson’s diseases (PD). Indeed, α-syn aggregates are the main component of the Lewy bodies (Lbs), considered as pathological hallmarks of neurodegenerative diseases [1-2], known as synucleinopathies. PD is a multicentric neurodegenerative process that affects several neuronal structures in the central and peripheral nervous system, among which is the enteric nervous system (ENS). Remarkably, recent reports have shown that the lesions in the ENS occurred at very early stage of the disease, even before the involvement of the central nervous system. So, the ENS could be critical in the pathophysiology of PD [3-4] and the pathological alterations within the ENS could be involved in the gastrointestinal dysfunction frequently encountered by parkinsonian patients. Although at present Lbs, as well as α-syn pathological aggregates, have been evidenced throughout the autonomic nervous system projecting to the gut of patients affected by PD or other neurodegenerative diseases, however data on the distribution of α-syn in human normal ENS are lacking. Our study focused on the immunohistochemical distribution of α-syn in the ENS of proximal tract of human normal small intestine. Surgical specimens of duodenum and proximal jejunum, collected from patients submitted to a pancreaticoduodenectomy, were fixed and paraffin embedded. Intestinal slices underwent immunohistochemical procedure using monoclonal anti α-syn antibody. Alpha-syn immunoreactive (ir) structures were detected along both myenteric and submucosal plexuses as well as in the circular and longitudinal muscular layers. We found perivascular α-syn-ir fibers in the submucosa and a dense ir periglandular network projecting up to the axis of the villi in the mucosa. The immunohistochemical distribution pattern of α-syn has been compared with that of major enteric neurotransmitters. Our preliminary observations confirm a physiological role of α-syn in the ENS, and may contribute to clarify its role in the peripheral nervous system. References [1] Spillantini et al, Nature 1997; 388:839-40. [2] Arima et al, Brain Res 1998; 808:93-100. [3] Braak et al, Neurosci Lett 2006; 396:67-72. [4] Wakabayashi et al, Acta Neuropathol 2010; 120:1-12

The interplay of intrinsic disorder and macromolecular crowding on {\alpha}-synuclein fibril formation

{\alpha}-synuclein ({\alpha}-syn) is an intrinsically disordered protein which is considered to be one of the causes of Parkinson's disease. This protein forms amyloid fibrils when in a highly concentrated solution. The fibril formation of {\alpha}-syn is induced not only by increases in {\alpha}-syn concentration but also by macromolecular crowding. In order to investigate the coupled effect of the intrinsic disorder of {\alpha}-syn and macromolecular crowding, we construct a lattice gas model of {\alpha}-syn in contact with a crowding agent reservoir based on statistical mechanics. The main assumption is that {\alpha}-syn can be expressed as coarse-grained particles with internal states coupled with effective volume; and disordered states are modeled by larger particles with larger internal entropy than other states. Thanks to the simplicity of the model, we can exactly calculate the number of conformations of crowding agents, and this enables us to prove that the original grand canonical ensemble with a crowding agent reservoir is mathematically equivalent to a canonical ensemble without crowding agents. In this expression, the effect of macromolecular crowding is absorbed in the internal entropy of disordered states; it is clearly shown that the crowding effect reduces the internal entropy. Based on Monte Carlo simulation, we provide scenarios of crowding-induced fibril formation. We also discuss the recent controversy over the existence of helically folded tetramers of {\alpha}-syn, and suggest that macromolecular crowding is the key to resolving the controversy.Comment: 11 pages, 14 figure

Long Term Potentiation (LTP) and Long Term Depression (LTD) Cause Differential Spatial Redistribution of the Synaptic Vesicle Protein Synaptophysin in the Middle Molecular Layer of the Dentate Gyrus in Rat Hippocampus

The presynaptic modifications that accompany long-term changes in synaptic plasticity are still not fully understood. Synaptophysin is a major synaptic vesicle protein involved in neurotransmitter release. We have used quantitative electron microscopy to study synaptophysin (Syn) immunolabelling in the hippocampus of adult rats 24h after induction in vivo of long term potentiation (LTP), and long term depression (LTD). Electrodes were implanted chronically in hippocampus with stimulation at either the medial (MPP) or lateral perforant path (LPP). 24h following induction of LTP or LTD rats were rapidly perfusion fixed and hippocampal tissue processed to electron microscopy via freeze substitution method. Anti-synaptophysin post-embedding immunolabelling was performed and tissue was imaged in the middle molecular layer (MML) of the dentate gyrus. There was a significant decrease in number of Syn labelled vesicles per unit area of bouton after LTP, but not LTD. An analysis of the spatial distribution of Syn labelled synaptic vesicles showed an increase in nearest neighbour distances, more so in the LTP than the LTD group, which is consistent with the overall decrease of Syn after LTP. These data are in agreement with the suggestion that Syn is involved in clathrin-dependent and “kiss and run” endocytosis which occurs perisynaptically. Thus, an increase in release of neurotransmitter and in consequence endocytosis would be consistent with an increased active zone distance for vesicles containing Syn

The New World species of Ataenius Harold, 1867 : 5. Revision of the A. strigatus group (Scarabaeidae: Aphodiinae: Eupariini)

The strigatus group of the New World species of Ataenius Harold is revised. Seventeen species are recognized including two species described as new: Ataenius ecruensis sp. nov. from the United States and A. oaxacaensis sp. nov. from Mexico. Fifteen previously used names are considered valid, three new synonyms are proposed: A. liogaster Bates (= A. edwardsi Chapin syn. nov. = A. hoguei Cartwright and Spangler syn. nov.), A. wenzelii Horn (= A. rudellus Fall, syn. nov.). New state records are presented for A. spretulus (Haldeman) (Washington) and A. cognatus (LeConte) (Indiana, Missouri, and Mississippi). The taxa are diagnosed, keyed and illustrated; available biological information and distribution data are given