Role of Microvesicles in the Spread of Herpes Simplex Virus 1 in Oligodendrocytic Cells

Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in the neurons of sensory ganglia. In some cases, the virus spreads into the central nervous system, causing encephalitis or meningitis. Cells infected with several different types of viruses may secrete microvesicles (MVs) containing viral proteins and RNAs. In some instances, extracellular microvesicles harboring infectious virus have been found. Here we describe the features of shedding microvesicles released by the human oligodendroglial HOG cell line infected with HSV-1 and their participation in the viral cycle. Using transmission electron microscopy, we detected for the first time microvesicles containing HSV-1 virions. Interestingly, the Chinese hamster ovary (CHO) cell line, which is resistant to infection by free HSV-1 virions, was susceptible to HSV-1 infection after being exposed to virus-containing microvesicles. Therefore, our results indicate for the first time that MVs released by infected cells contain virions, are endocytosed by naive cells, and lead to a productive infection. Furthermore, infection of CHO cells was not completely neutralized when virus-containing microvesicles were preincubated with neutralizing anti-HSV-1 antibodies. The lack of complete neutralization and the ability of MVs to infect nectin-1/HVEM-negative CHO-K1 cells suggest a novel way for HSV-1 to spread to and enter target cells. Taken together, our results suggest that HSV-1 could spread through microvesicles to expand its tropism and that microvesicles could shield the virus from neutralizing antibodies as a possible mechanism to escape the host immune response. IMPORTANCE Herpes simplex virus 1 (HSV-1) is a neurotropic pathogen that can infect many types of cells and establishes latent infections in neurons. Extracellular vesicles are a heterogeneous group of membrane vesicles secreted by most cell types. Microvesicles, which are extracellular vesicles which derive from the shedding of the plasma membrane, isolated from the supernatant of HSV-1-infected HOG cells were analyzed to find out whether they were involved in the viral cycle. The importance of our investigation lies in the detection, for the first time, of microvesicles containing HSV-1 virions. In addition, virus-containing microvesicles were endocytosed into CHO-K1 cells and were able to actively infect these otherwise nonpermissive cells. Finally, the infection of CHO cells with these virus-containing microvesicles was not completely neutralized by anti-HSV-1 antibodies, suggesting that these extracellular vesicles might shield the virus from neutralizing antibodies as a possible mechanism of immune evasion

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ

Role of MSK1 in the induction of NF-κB by the chemokine CX3CL1 in microglial cells

Microglial cells are essential mediators of neuroinflammatory processes involved in several pathologies. Moreover, the chemokine fractalkine (CX3CL1) is essential in the crosstalk between neurons and microglia. However, the exact roles of CX3CL1, CX3CL1 receptor (CX3CR1) and microglia signalling are not fully understood in neuroinflammation. In addition, the findings reported on this subject are controversial. In this work, we investigated whether CX3CL1 induced pro-inflammatory signalling activation through NF-κB pathway. We were able to show that CX3CL1 activates the pro-inflammatory pathway mediated by the transcription factor NF-κB as an early response in microglial cells. On the other side, CX3CR1-deficient microglia showed impaired NF-κB axis. Phospho-kinase assay proteome profiles indicated that CX3CL1 induced several kinases such as MAPK’s (ERK and JNK), SRC-family tyrosine kinases (YES, FGR, LCK and LYN) and most interesting and also related to NF-κB, the mitogen- and stress-activated kinase-1 (MSK1). Knockdown of MSK1 with short interfering RNAs decreased partially MSK1 protein levels (about 50%), enough to decrease the mRNA levels of Il-1β, Tnf-α and iNos triggered by stimulation with CX3CL1. These results indicate the relevance of CX3CL1 in the activation of the pro-inflammatory NF-κB signalling pathway through MSK1 in microglial cells.This work was supported by a Spanish Ministry of Economy and Competitiveness (Grants refs. SAF2016-76520-R).Peer reviewe

Implications of CB2 cannabinoid receptors in the neurodegenerative process associated to tauopathies

Resumen del trabajo presentado al 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Santander del 10 al 13 de septiembre de 2018.[Background] Tauopathies are a group of neurodegenerative diseases characterized by the aggregation of TAU protein for which there is no effective treatment yet. This is the reason why new approaches are now being investigated, such as the endocannabinoid system. In Alzheimer’s disease, it has been shown that the levels of type-2 cannabinoid receptors (CB2) are increased in the glial cells that surround amyloid plaques. Nevertheless, the effects that TAU accumulation has on CB2 expression and its implication in the neurodegenerative process are still unknown.[Results] We have seen an increase in CB2 expression in hippocampal neurons of 12 month-old transgenic mice overexpressing hTAUP301S protein. This alteration is an early event on the pathology, as CB2 levels are also increased in another animal model overexpressing hTAUP301L where there is not neuronal death. Moreover, CB2 expression in these mice can be modulated by dimethyl fumarate (DMF) treatment, an NRF2 inductor with neuroprotective effects. These results were reproduced in vitro when hTAUP301L was overexpressed in HT22 cells. Finally, we found an antioxidant response element on CNR2 promoter and our analysis in vitro point out at the possibility that CB2 expression could be modulated by NRF2.[Conclusions] Overexpression of hTAU protein increases CB2 levels in neurons and can be modulated by DMF treatment.Peer reviewe

Impaired signaling of Transcription factors NF-ƘB and NRF2 in CX3CR1-deficient microglia leads to altered neuroinflammation and phagocytosis: implications in tauopathies

Resumen del trabajo presentado al 5th Symposium on Biomedical Research: "Advances and Perspectives In Pharmacology, Drug Toxicity and Pharmacogenetics", celebrado en Madrid del 15 al 16 de marzo de 2018.[Introduction]: TAU protein aggregation is the main characteristic of a group of age-related neurodegenerative diseases called tauopathies. One of the key hallmarks associated with neurodegeneration is the presence of low-grade chronic inflammation, indicating a crosstalk between damaged neuron and glial cells. Previously we have shown that TAUP301L overexpressing neurons released CX3CL1 that activates anti-inflammatory NRF2 signalling in microglial cells in vitro and in vivo. However, the potential role of CX3CR1 in the context of TAU pathology and its implication neuroinflammation are poorly described.[Material and Methods]: Pro-inflammatory markers in immortalized microglia cells (IMG) treated with CX3CL1 were analysed. We also studied mRNA expression levels of NF-ƘB, anti-inflammatory Nrf2 signalling and TAM receptors (TYRO3, AXL and MER) in CX3CR1-deficient primary microglia cells. Finally, the effect of sulforaphane (SFN), a NRF2 inducer, was examined on neuroinflammation in Cx3cr1+/+ and Cx3cr1-/- mice stereotaxically injected in the right hippocampus with an adeno-associated vector expressing human TAUP301L and treated daily with SFN (50mg/kg, i.p) during three weeks.[Results]: In this study we show that CX3CL1 treatment induced NF-ƘB-p65 and pro-inflammatory cytokines expression. On the other hand, CX3CR1-deficient primary microglia cells present impaired NF-ƘB mRNA expression levels and decreased anti-inflammatory NRF2 signalling, suggesting a dual role of CX3CL1/CX3CR1 axis in neuroinflammation. Lack of CX3CR1 microglia exhibit decreased mRNA expression levels of TAM receptors (TYRO3, AXL and MER) that functionally results in a deficiency in phagocytosis. SFN treatment reverses astrogliosis in Cx3cr1+/+ and Cx3cr1-/-, whereas at microglial level we did not see any improvement in the Cx3cr1-/- mice.[Conclusions]: These findings suggest that CX3CR1-NRF2 axis activation is essential in the modulation of microglial activation associated with tauopathy, and that the associated polymorphisms of CX3CR1 must be taken into account in the design of pharmacological strategies for the treatment of taupathies.Peer reviewe

MGMT genomic rearrangements contribute to chemotherapy resistance in gliomas.

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.We would like to acknowledge Claudia Savini, Susana Garcia, and Hector Peinado for the help and discussion on the exosome isolation and analysis. We thank MCarmen Martin Guijarro and Francisco Jose Moya for performing the FISH staining. We thank Manuel Perez and Gadea Mata for their assistance with the high-throughput microscopy analysis. We also thank Alvaro Ucero for the help with the isolation of the blood from the mice. We are very grateful to Anne Harttrampf and Lilianne Massade for sharing treatment information of the medulloblastoma patient, in which they identified the ASAP2-MGMT fusion. This research was supported by funds from the Seve Ballesteros Foundation and the Asociacion Espanola Contra el Cancer (AECC) to M.S. This work was also supported by Natural Science Foundation of China (NSFC)/Research Grants Council (RGC) Joint Research Scheme (81761168038 to TJ and N_HKUST606/17 to J.W.), RGC-ECS grant 26102719, the NSFC grant (No. 31922088), ITC grant (ITCPD/17-9), Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (ZYLX201708), Beijing Municipal Administration of Hospitals' Mission Plan (SML20180501), Beijing Nova Program (Z171100001117022) and Beijing Talents Foundation from Organization department of Municipal committee of the CPC (2017000021223ZK32).S