Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

BACKGROUND: The pathology of Parkinson's disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated alpha-synuclein are demonstrated in the signature inclusions of Parkinson's disease. However, whether the nitration of alpha-synuclein is relevant to the pathogenesis of PD is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, effect of nitrated alpha-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-alpha-synuclein intracellular. We provide evidence to show that the nitrated alpha-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated alpha-synuclein. Moreover, we show that administration of nitrated alpha-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D(2)R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated alpha-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified alpha-synuclein had significantly less severe behavioral effects. CONCLUSIONS/SIGNIFICANCE: Our results provide evidence that alpha-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD

Etudes des relations structure-activité et pharmacomodulation de ligands sérotoninergiques

CAEN-BU Médecine pharmacie (141182102) / SudocLYON1-BU Santé (693882101) / SudocSudocFranceF

Interplays between the bacterial endosymbiont Candidatus Midichloria mitochondrii and its arthropod host, the European tick Ixodes ricinus

Ixodes ricinus est la tique la plus répandue en Europe occidentale et le principal vecteur de Borrelia burgdorferi s.l. responsable de la maladie de Lyme. Outre les pathogènes, les tiques, à l'instar des nombreux animaux, hébergent de nombreux micro-organismes non-pathogènes, dont l'effet sur leur hôte s'étend le long d'un continuum allant du mutualisme au parasitisme. Au sein de cette communauté microbienne associée à I. ricinus, l'Alphaproteobactérie (Rickettsiales) Midichloria mitochondrii est la plus répandue au sein des différentes populations de tiques. Cet endosymbiote, capable de résider à l'intérieur des mitochondries des cellules de tiques, est particulièrement abondant dans les ovaires des femelles adultes où la bactérie est systématiquement transmise à la descendance et est suspectée d'être un symbiote mutualiste obligatoire. Cependant, l'existence même des lignées de tiques dépourvues du symbiote remet en cause le caractère obligatoire de cette symbiose. Ainsi, l'étude de l'interaction M. mitochondrii-I. ricinus est d'intérêt majeur afin d'établir si la bactérie est essentielle à la valeur adaptative de la tique, ce qui constituerait un potentiel levier d'action de lutte anti-vectorielle se basant sur une lutte antisymbiotique. Dans un premier temps, la dynamique de M. mitochondrii entre les nymphes mâles et femelles d’I. ricinus a été étudiée. Dans un second temps, un traitement antibiotique (tétracycline) visant à supprimer la bactérie de son hôte ainsi que l'hypothèse d'un apport de vitamines B par M. mitochondrii à la tique ont été testés sans permettre d'établir di conclusions. Dans un troisième temps, une étude du transcriptome de tiques hébergeant ou non M. mitochondrii a permis d'identifier des gènes différentiellement exprimés pouvant être impliqués dans l'interaction tique-bactérie. La nature de la relation M. mitochondrii-I. ricinus apparaît donc encore plus complexe que celle observée chez d'autres couples tiques-endosymbiotes.Ixodes ricinus is the most common tick in Western Europe and the main vector of Borrelia burgdorferi s.l., the agent of Lyme disease. In addition to pathogens, ticks, like many animais, harbour numerous non-pathogenic microorganisms, whose effect on their host extends along a continuum from mutualism to parasitism. Within this microbial community associated with I. ricinus, the Alphaproteobacterium (Rickettsiales) Midichloria mitochondrii is the most widespread in the different tick populations. This endosymbiont, capable ofresiding within the mitochondria of tick cells, is particularl) abundant in the avaries of adult females where the bacterium is systematically transmitted to the offspring and is suspected to be an obligate mutualistic symbiont. However, the very existence of tick lines without the symbiont calls into question the obligatory nature ofthis symbiosis. Thus, the study of the M mitochondrii-I. ricinus interaction is of major interest in order to establish whether the bacterium is essential to tick fitness, which would constitute a potential lever for vector contrai based on antisymbiotic contrai. Firstly, the dynamics of M mitochondrii between male and female nymphs of I. ricinus were studied. In a second step, an tetracycline-based antibiotic treatment aimed at removing the bacterium from its host as well as the hypothesis of a B vitamins provision by M mitochondrii to the tick were tested without making it possible to draw any conclusions. Thirdly, the analysis of the transcriptome ofticks harbouring or not M mitochondrii made it possible to identify differentially expressed genes that could be involved in the tick-bacteria interaction. The nature of the M mitochondrii-I. ricinus relationship thus appears even more complex than that observed in other tick-endosymbiont pairs

Symbiont dynamics during the blood meal of Ixodes ricinus nymphs differ according to their sex

Ticks harbour rich and diverse microbiota and, among the microorganisms associated with them, endosymbionts are the subject of a growing interest due to their crucial role in the biology of their arthropod host. Midichloria mitochondrii is the main endosymbiont of the European tick Ixodes ricinus and is found in abundance in all I. ricinus females, while at a much lower density in males, where it is even absent in 56 % of the individuals. This endosymbiont is also known to increase in numbers after the blood meal of larvae, nymphs or females. Because of this difference in the prevalence of M. mitochondrii between the two sexes, surveying the density of these bacteria in nymphs that will become either females or males could help to understand the behaviour of Midichloria in its arthropod host. To this aim, we have set up an experimental design by building 3 groups of unfed nymphs based on their scutum and hypostome lengths. After engorgement, weighing and moulting of a subset of the nymphs, a significant difference in sex-ratio among the 3 groups was observed. In parallel, Midichloria load in individual nymphs was quantified by qPCR both before and after engorgement. No difference in either body mass or Midichloria load was observed at the unfed stage, but following engorgement, both features were significantly different between each size group. Our results demonstrate that symbiont dynamics during nymphal engorgement is different between the two sexes, resulting in a significantly higher Midichloria load in nymphs that will become females. The consequences of those findings on our understanding of the interplay between the endosymbiont and its arthropod host are discussed

Sequence diversity and evolution of a group of iflaviruses associated with ticks

We studied a group of tick-associated viruses with characteristics of members of the family Iflaviridae, a family of viruses frequently found in arthropods. Our aim was to gain insight into the evolutionary dynamics of this group of viruses, which may be linked to the biology of ticks. We explored assembled RNA-Seq data sets for different species of ticks. We identified members of five different iflavirus species, four of them novel, and discovered nine new genome sequences, including variants. Five variants represented a virus species associated with Ixodes ricinus. Unexpectedly, a sequence found in the Ixodes scapularis cell line ISE6 was nearly identical to the sequences of I. ricinus variants, suggesting a contamination of this cell line by I. ricinus material. Analysing patterns of substitutions between these variants, we detected a strong excess of synonymous mutations, suggesting evolution under strong positive selection. The phylogenies of the viruses and of their tick hosts were not congruent, suggesting recurrent host changes across tick genera during their evolution. Overall, our work constitutes a step in the understanding of the interactions between this family of viruses and ticks

Sequence diversity and evolution of a group of iflaviruses associated with ticks

International audienceWe studied a group of tick-associated viruses with characteristics of members of the family Iflaviridae , a family of viruses frequently found in arthropods. Our aim was to gain insight into the evolutionary dynamics of this group of viruses, which may be linked to the biology of ticks. We explored assembled RNA-Seq data sets for different species of ticks. We identified members of five different iflavirus species, four of them novel, and discovered nine new genome sequences, including variants. Five variants represented a virus species associated with Ixodes ricinus . Unexpectedly, a sequence found in the Ixodes scapularis cell line ISE6 was nearly identical to the sequences of I. ricinus variants, suggesting a contamination of this cell line by I. ricinus material. Analysing patterns of substitutions between these variants, we detected a strong excess of synonymous mutations, suggesting evolution under strong positive selection. The phylogenies of the viruses and of their tick hosts were not congruent, suggesting recurrent host changes across tick genera during their evolution. Overall, our work constitutes a step in the understanding of the interactions between this family of viruses and ticks

Digging deep into intramitochondrial symbiosis: dual transcriptomics of the hard tick Ixodes ricinus and its bacterial symbiont Midichloria mitochondrii

INTRODUCTION. Ixodes ricinus is a hard tick, widespread in Europe and in the Mediterranean basin, that can act as vector of multiple diseases of human and veterinary importance. Midichloria mitochondrii is a bacterial symbiont of this tick, capable of living in the intermembrane space of mitochondria (Beninati et al., 2004, Appl. Environ. Microbiol., 70:2596\u20132602). The bacterium was found to be present in most tick individuals (Lo et al., 2006, Environ. Microbiol., 8:1280-1287) and to be vertically transmitted to the progeny (Sassera et al., 2008, Appl. Environ. Microbiol., 74:6138-6140). To investigate the relationship between host and symbiont we designed an ad-hoc protocol of dual RNA-Seq to sequence the transcriptomes of both organisms in different phases of tick engorgement. MATERIALS AND METHODS. Ticks were collected on roe deer, cut in half and stored in RNALater on site, in order to freeze the transcription patterns in each stage. Ticks were dissected in RNALater and total RNA was extracted from ovaries and salivary glands. A custom library construction kit was designed and used in order to preserve transcripts from both organisms and to limit the prevalence of rRNA sequences in the sample (normally over 85%). The kit protocol included the use of custom designed probes for rRNA depletion. Libraries were sequenced on Illumina machines and the resulting reads were assembled to obtain a reference transcriptome to be used for downstream analyses. RESULTS AND CONCLUSIONS. Ticks were subjected to a custom protocol for RNA sequencing which allowed to obtain an average of 40 million reads per sample and over 50% of the reads were transcripts of host or symbiont. After assembly, over 18,000 tick transcripts and over 1,200 bacterial transcripts were obtained. Differential expression analysis will be performed in the upcoming months; so far, the main result of this work is the development of a sound protocol for dual RNA-seq in this system

Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (·NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and ·NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg2+, and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by ·NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to ·NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions