Reduced expression of exocytotic proteins caused by anti-cholinesterase pesticides in Brachionus calyciflorus (Rotifera: Monogononta) / Redução da expressão de proteínas de exocitose causados por pesticidas anti-colinesterase em Brachionus calyciflorus (Rotifera: Monogononta)

AbstractThe organophosphate and carbamate pesticides methyl-parathion and carbaryl have a common action mechanism: they inhibit acetylcholinesterase enzyme by blocking the transmission of nerve impulses. However, they can alter the expression of exocytotic membrane proteins (SNARE), by modifying release of neurotransmitters and other substances. This study evaluated the adverse effects of the pesticides methyl-parathion and carbaryl on expression of SNARE proteins: Syntaxin-1, Syntaxin-4 and SNAP-23 in freshwater rotifer Brachionus calyciflorus. Protein expression of these three proteins was analyzed before and after exposure to these two pesticides by Western Blot. The expression of Syntaxin-1, Syntaxin-4 and SNAP-23 proteins in B. calyciflorussignificantly decreases with increasing concentration of either pesticides. This suggests that organophosphates and carbamates have adverse effects on expression of membrane proteins of exocytosis by altering the recognition, docking and fusion of presynaptic and vesicular membranes involved in exocytosis of neurotransmitters. Our results demonstrate that the neurotoxic effect of anticholinesterase pesticides influences the interaction of syntaxins and SNAP-25 and the proper assembly of the SNARE complex.

ResumoOs pesticidas organofosforados e carbamatos metil- paration e carbaril tem um mecanismo de ação comum: eles inibem a enzima acetilcolinesterase, bloqueando a transmissão dos impulsos nervosos. No entanto, eles podem alterar a expressão de proteínas de membrana de exocitose (SNARE), através da modificação da libertação de neurotransmissores e outras substâncias. Este estudo avaliou os efeitos adversos dos pesticidas metil- paration e carbaril sobre a expressão de proteínas SNARE: Sintaxina -1, Sintaxina-4 e SNAP-23 em rotíferos de água doce Brachionus calyciflorus. A expressão destas três proteínas foi analisada antes e depois da exposição a estes dois pesticidas por Western Blot. A expressão das proteínas Sintaxina-1, Sintaxina-4 e SNAP-23 em B. calyciflorus diminui significativamente com o aumento da concentração de ambos os pesticidas. Isto sugere que os organofosfatos e carbamatos têm efeitos adversos sobre a expressão de proteínas de membrana de exocitose, alterando o reconhecimento, de encaixe e fusão de membranas pré-sinápticas e vesiculares envolvidas na exocitose de neurotransmissores. Nossos resultados demonstram que o efeito neurotóxico de pesticidas anticolinesterásicos influencia a interação de sintaxinas e SNAP-25 e a montagem correta do complexo SNARE.

Insulin replacement restores the vesicular secretory apparatus in the diabetic rat lacrimal gland / Reposição de insulina restaura o mecanismo secretório da glândula lacrimal de ratos diabéticos

ABSTRACT Purpose: In the lacrimal gland (LG) acinar cells, signaling regulates the release of secretory vesicles through specific Rab and SNARE exocytotic proteins. In diabetes mellitus (DM), the LGs are dysfunctional. The aim of this work was to determine if secretory apparatus changes were associated with any effects on the secretory vesicles (SV) in diabetic rats as well as the expression levels of constituent Rab and members of the SNARE family, and if insulin supplementation reversed those changes. Methods: DM was induced in male Wistar rats with an intravenous dose of streptozotocin (60 mg/kg). One of the two diabetic groups was then treated every other day with insulin (1 IU). A third control group was injected with vehicle. After 10 weeks, Western blotting and RT-PCR were used to compared the Rab and SNARE secretory factor levels in the LGs. Transmission electron microscopy evaluated acinar cell SV density and integrity. Results: In the diabetes mellitus group, there were fewer and enlarged SV. The Rab 27b, Rab 3d, and syntaxin-1 protein expression declined in the rats with diabetes mellitus. Insulin treatment restored the SV density and the Rab 27b and syntaxin expression to their control protein levels, whereas the Vamp 2 mRNA expression increased above the control levels. Conclusions: Diabetes mellitus LG changes were associated with the declines in protein expression levels that were involved in supporting exocytosis and vesicular formation. They were partially reversed by insulin replacement therapy. These findings may help to improve therapeutic management of dry eye in diabetes mellitus. .

RESUMO Objetivo: Células acinares da glândula lacrimal (GL) sinalizam a regulação da liberação através de vesículas secretórias específicas Rab proteínas exocitóticas SNARE. No diabetes mellitus (DM), as glândulas lacrimais são disfuncionais. O objetivo deste trabalho foi determinar se em ratos diabéticos, alterações dos aparatos secretórios estão associados a efeitos sobre vesículas secretoras (VS) e sobre os níveis de expressão do constituinte Rab, bem como membros da família SNARE, e se a suplementação de insulina reverte as alterações. Métodos: DM foi induzido em ratos Wistar machos com uma dose intravenosa de estreptozotocina (60 mg/kg). Um dos dois grupos diabéticos foi então tratado a cada dois dias com insulina (1 UI). Um terceiro grupo controle foi injetado com o veículo. Após 10 semanas, western blot e RT-PCR comparou níveis de fatores secretórios de Rab e SNARE na glândula lacrimal. Microscopia eletrônica de transmissão (MET) avaliaram a densidade e integridade de VS de célula acinar. Resultados: No grupo diabetes mellitus , houve poucas e alargadas VS. Rab27b, Rab 3d e Sintaxina-1 diminuiu a expressão da proteína em ratos com Diabetes Mellitus. O tratamento com insulina restaurou a densidade das VS e expressão de Rab 27b e Sintaxina para seus níveis de proteína controle, enquanto a expressão de Vamp 2 RNAm aumentou em relação aos controles. Conclusões: Alterações na glândula lacrimal de diabetes mellitus estão associadas a reduções nos níveis de expressão de proteínas envolvidas no apoio a exocitose e formação vesicular. Eles são, em parte, revertida por terapia de reposição de insulina. Estes resultados podem ajudar a melhorar a conduta terapêutica do olho seco no diabetes mellitus. .

LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.