Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

Distribution and localization of abnormally expressed OPTN proteins in RGC5 retinal ganglion cells and their effects on subcellular morphology.

The objectives of this study were to investigate the distributions of abnormally expressed optineurin (OPTN) proteins in retinal ganglion cells (RGC5s) of transgenic rats and their effects on subcellular morphological structures. Green fluorescent protein labeled EGFP wild-type (OPTN(WT)), E50K mutant type (OPTN(E50K)), and OPTN siRNA (si-OPTN) eukaryotic expression plasmids were constructed and transfected into RGC5s. Intracellular structures were labeled with organelle specific fluorescent dyes. Construct localization and cell morphologies were visualized by confocal fluorescence microscopy. OPTN(WT) was observed to be distributed as fine punctate fluorescent particles in the cytoplasm around the nucleus, along with exhibiting nuclear expression. OPTNE50K exhibited similar distribution but with non-uniform fluorescence particle size. si-OPTN distribution was similar to that of EGFP: uniform across the cytoplasm and nucleus. Compared with the negative control group, OPTN(WT), and OPTN(E50K) and to a lesser degree pEGFP-transfected cells exhibited fracture and loss of myofilament proteins and mitochondrial swelling and cytoplasmic accumulation, along with abnormal lysosomal distribution and increased volume, and Golgi fragmentation. However, si- OPTN transfected cells exhibited no significant damage. Therefore, we demonstrated that the E50K mutation disrupts the uniformity of OPTN protein distribution upon exogenous overexpression. Furthermore, these results suggested that si-OPTN transfection, and thus potentially OPTN knockdown, did not impact subcellular morphology of RGC5 cells, whereas transfection, especially when combined with wild-type or mutant OPTN expression, led to substantial abnormalities in subcellular morphological structures. These findings lay a foundation for further research into the function of the OPTN protein.

Effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5.

The OPTN gene is thought to be associated with certain types of glaucoma and the function of the protein for which it encodes, optineurin, has been extensively researched, but with contradictory results. We explored the effects of abnormal optineurin expression on the survival of the rat retinal ganglion cell line RGC-5. Plasmids expressing wild-type (WT) or E50K mutant optineurin, or OPTN-specific double-hairpin small interfering RNA (si-RNA), were transfected into RGC-5 cells. The effects on cell survival were monitored by observation of cell morphology and propidium iodide and Hoechst 33342 fluorescent staining, while expression of optineurin was visualized by fluorescence microscopy. Abnormal optineurin expression influenced the survival of RGCs in vitro, as apoptosis was induced by increased WT and E50K mutant optineurin, while a reduction in apoptosis was observed in cells transfected with OPTN-siRNA. Similar results were also observed in transfected cells treated with apoptotic stimuli. Overexpression of WT and mutant E50K protein resulted in greater cell death, while downregulation decreased RGC-5 apoptosis.

Viroides, pequenos RNAs patogênicos capazes de replicação autônoma: modelos moleculares para o estudo de interações patógeno-hospedeiro e evolução / Viroids, small pathogenic RNAs capable of autonomous replication: molecular models for the study on host-pathogen interactions and evolution

Os viroides, apesar de serem constituídos por um pequeno RNA de fita simples, fortemente estruturado, circular, que não codifica proteínas, são capazes de se replicar de maneira autônoma em plantas superiores e causar doença interagindo diretamente com fatores do hospedeiro. Nesta revisão, serão apresentados e discutidos alguns dos mais recentes trabalhos envolvendo a interação de viroides com fatores do hospedeiro, incluindo aspectos relacionados à replicação, movimento e patogênese, além de suas características evolutivas. Nos últimos anos, alguns grupos de pesquisa têm se aventurado na busca por fatores do hospedeiro e mecanismos moleculares relacionados ao ciclo infeccioso dos viroides, tentando desvendar como esses pequenos RNAs interagem com o hospedeiro induzindo sintomas. Os viroides não codificam proteínas supressoras de silenciamento e, portanto, devem garantir sua existência utilizando estratégias baseadas em sua estrutura secundária, na compartimentalização em organelas, associação com fatores do hospedeiro e eficiência na replicação. A complexidade do ciclo infeccioso desses minúsculos RNAs indica que muitas interações desses patógenos com fatores do hospedeiro ainda devem ser identificadas.

Viroids are small, single-stranded, highly structured, circular RNAs that replicate autonomously in their hosts, without messenger RNA activity. Because they do not encode for proteins, viroids have to interact directly with host factors. This review presents recent progress in understanding the possible role of recently identified viroid-binding host proteins related to replication, trafficking and pathogenesis. It also discusses some aspects on viroid evolution. In recent years, efforts to understand how viroids replicate, cause disease and induce symptoms have prompted details on molecular mechanisms related to the viroid infectious cycle. Inasmuch as viroids lack protein-encoding capacity, including suppressors of gene silencing, their existence could be ensured by their compact conformation, compartimentalization in organelles, association with host factors or by their highly efficient replication. The complexity of the infectious cycle of these tiny pathogenic RNAs indicates that several interactions with host factors remain to be identified.

Myocilin and optineurin coding variants in Hispanics of Mexican descent with POAG.

Coding variants in both myocilin (MYOC) and optineurin (OPTN) are reported risk factors for primary open-angle glaucoma (POAG) in many populations. This study investigated the contribution of MYOC and OPTN coding variants in Hispanics of Mexican descent with and without POAG. We conducted a case-control study of unrelated POAG cases and nonglaucomatous controls in a population of Hispanics of Mexican descent. Ascertainment criteria for POAG included the presence of glaucomatous optic neuropathy with associated visual field loss and the absence of secondary causes of glaucoma. Controls had normal optic nerves, visual fields and intraocular pressure. All coding exons of MYOC and OPTN were sequenced. The data set consisted of 88 POAG cases and 93 controls. A novel nonsynonymous coding variant (R7H) in the first exon of MYOC was identified. Other identified variants in MYOC and OPTN have been previously described and do not seem to contribute to POAG risk. This is the first comprehensive study of MYOC and OPTN in Hispanics of Mexican descent with POAG. Neither MYOC nor OPTN sequence variants seem to have a major role in the etiology of POAG in this population.

Lack of association between optineurin gene variants T34T, E50K, M98K, 691_692insAG and R545Q and primary open angle glaucoma in Brazilian patients.

PURPOSE: To verify the frequencies of T34T, E50K, M98K, 691_692insAG, and R545Q variants in the optineurin (OPTN) gene in Brazilian subjects with primary open-angle glaucoma (POAG) and controls. PATIENTS AND METHODS: Ninety-nine patients with POAG and 100 normal controls were enrolled in this study. The frequency of alterations in the OPTN gene was analyzed by direct sequencing and enzymatic digestion of PCR products. RESULTS: None of the five alterations evaluated was significantly associated with POAG when compared to controls. However, the T34T silent change was present in greater frequency in POAG patients (37.37% vs. 23.00% in controls), while the R545Q change was more prevalent in controls (23.00% vs. 10.10% in POAG). The M98K and 691_692insAG presented with low frequencies in POAG patients (1.01% and 2.02%, respectively) and controls (2.00% and 2.00%, respectively). The E50K substitution was not observed. CONCLUSION: Our data show no association between the five evaluated variants and POAG in the Brazilian population.