Projects / Programmes
Exosome-associated Nef released from HIV infected cells contributes importantly to the development of neuroAIDS
| Code |
Science |
Field |
Subfield |
| 3.01.00 |
Medical sciences |
Microbiology and immunology |
|
| Code |
Science |
Field |
| B510 |
Biomedical sciences |
Infections |
| Code |
Science |
Field |
| 3.01 |
Medical and Health Sciences |
Basic medicine |
HIV-1, neuro AIDS, Nef, exosome, nanoparticle, trafficking, biomarkers
Researchers (29)
Organisations (5)
Abstract
In the era of highly active antiretroviral therapy (HAART), neurological manifestations of AIDS remain an insurmountable problem. Clinical symptoms of NeuroAIDS include HIV-associated dementia (HAD), mild cognitive impairment (MCMD) and vacuolar myelopathy. Whereas central nervous system (CNS) of children seems to be even more vulnerable to HIV, around 10% and 30% of HIV-infected adults develop HAD and MCMD, respectively. To combat NeuroAIDS, a thorough understanding of mechanisms implicated in HIV-associated neurodegeneration is needed. Additionally, discovering biomarkers for the risk and progression of NeuroAIDS could improve the treatment of infected individuals.
It is known that HIV enters CNS early in infection via hematopoetic cells that infect neural cells. The main targets are microglia and astrocytes, capable of supporting full or only limited viral replication, respectively. In pediatric autopsied brains as well as in in vitro infected cells, the NEgative Factor (Nef) is the primary HIV protein expressed in astrocytes. It has been implicated in NeuroAIDS, but the mechanism of Nef-induced neurodegeneration remains unclear. Indeed, Nef affects the viability and metabolism of microglia, astrocytes, oligodendrocytes and neurons. It also increases the migration and recruitment of leukocytes into the brain. Previously, we demonstrated that Nef, expressed from transfected or HIV-infected primary T cells, stimulates its own export via exosomes (vesicles 40-100 nm in diameter), which causes activation-induced cell death of resting lymphocytes. Others confirmed our results and demonstrated the presence of Nef-exosomes even in the blood of HAART-treated patients.
Thus, the hypothesis of this proposal is that Nef-exosomes, released from HIV-infected microglia and astrocytes, form a pool of bioactive vesicles capable of interfering with the viability and differentiation of surrounding CNS cells. Our long-term objective is to understand this role for Nef and to assess Nef-exosomes in the cerebrospinal fluid (CSF) and/or blood as biomarkers for NeuroAIDS. In particular, we will demonstrate the clinical relevance of Nef-exosomes in the CNS. To this end we will first optimize methods for isolating exosomes. Later, we will isolate and quantify exosomes from the CSF and blood of healthy and HIV-infected people and determine their levels of Nef. For exosome quantification we will develop a new approach based on the AF4-UV-MALS-QELS-RI technique. We will also measure Nef-exosomes released from transfected or infected microglia and astrocytes. This release will be inhibited by siRNAs targeting cellular trafficking pathways and with mutant Nef proteins. Later, we will detect Nef-exosome targets in the CNS (astrocytes, microglia, oligodendrocytes, neurons and neural stem cells) and follow their internalization by immunofluorescent microscopy . In the end, we will expose these cells to Nef-exosomes and evaluate their effect on apoptosis, proliferation and differentiation by FACS and immunofluorescent microscopy. We will use recombinant Nef proteins as a control. In the end, we will develop minimally invasive methods for following the onset and development of NeuroAIDS. To analyze the CSF for such biomarkers, we will isolate exosomes from the CSF of HIV-infected people and perform LC-MS/MS as well as miRNA expression profiling. Relevant biomarker for NeuroAIDS, identified by above approaches, will also be evaluated on blood samples corresponding to the CSF of infected people.
The findings of this proposal will contribute to the understanding of NeuroAIDS and physiological role/s of exosomes. It will also characterize effects of Nef on cells of the CNS, which will identify new strategies to inhibit and/or prevent their sequelae. It will also introduce a new method in AIDS diagnostics, where Nef-exosomes will serve as biomarkers for following this disease.
Significance for science
Extracellular vesicles are a heterogeneous population of membrane vesicles, which are shed from cells in vitro or are released into various body fluids in vivo, like blood plasma, cerebrospinal fluid, urine, and others. According to their size and site of formation, EVs are subdivided into exosomes (30 to 150 nm in diameter), microvesicles (100 - 1000 nm in diameter) and apoptotic bodies. Extracellular vesicles consist of a lipid bilayer membrane that surrounds a small amount of cytosol, and they contain various typical proteins, lipids and nucleic acids, which mirror the composition of the cell of origin. Extracellular vesicles have an important role in physiological processes and various pathological conditions, and thus have promising potential for human diagnostics and therapeutic applications. In the project J3-5499 we were focusing on the role of extracellular vesicles, which are released from HIV-1 infected cells through the action of viral protein Nef, on HIV (neuro)pathogenesis. We tried to uncover the mechanisms behind extracellular vesicle formation and the toxic effects they exert on target cells. Additionally we explored the usage of our findings in HIV diagnostics, with the goal to improve the quality of life of infected individuals. Our key contributions to science in the fields of extracellular vesicles and HIV pathogenesis are listed below: 1- We established a novel method to determine the size and concentration of vesicles in a population based on Asymmetric-Flow Field-Flow Fractionation (AF4) technique combined with a multi-detection system, which has filled the gap in available methods used for vesicle characterization. Advantages of the AF4 technique in comparison with existing methods (electron microscopy, NTA analysis, flow cytometry) are: a simple analysis of the entire population of vesicles, the determined size (radius) of the vesicle corresponds to the size in the physiological environment, we can detect even the smallest vesicles and are able to analyze vesicle populations that are heterogeneous in size. 2- We were the first to show that the commonly-used method for labeling vesicles with a fluorescent dye PKH is unsuitable for functional studies of the vesicles. The dye namely forms numerous micelles even in the absence of vesicles, which are similar in size to vesicles. Since in the presence of vesicles, the dye forms even more micelles, we cannot infer on the ratio between micelles and vesicles in the labelled vesicle sample based on the control sample. Importantly, we showed that micelles are taken up by cells with same efficiency as labelled vesicles. It is therefore crucial to separate micelles from labelled vesicles in a sucrose gradient based on their buoyant density, before using them in functional studies. 3- We were the first to show that extracellular vesicles can originate from a CD81 immunopositive organelle, which is similar in morphology to the multivesicular body, but originates from plasma membrane and not from endosomal membranes. Until now, only two possible intracellular sources of extracellular vesicles were described: fusion of multivesicular bodies with the plasma membrane or formation of vesicles with outward invagination of the plasma membrane. 4- We were the first to show that Nef is present in the plasma of half of HIV infected individuals, in which effective antiretroviral therapy maintains the level of plasma HIV RNA below the clinically detectable level. This implies that translationally active viral reservoirs, which do not produce viruses but release Nef, are present in the body. Nef is a known HIV pathogenic factor, which might contribute to inflammation and to non-AIDS related disorders, observed in effectively treated individuals. Plasma Nef could therefore serve as a biomarker to identify individuals, which would benefit from the change in the therapy. Because of Nef's toxicity, Nef-exosomes could also serve as a good therapeutic target.
Significance for the country
We have contributed importantly to the following areas important for the development of Slovenia: 1- The research findings described in section B.9.1. have contributed importantly to the development of new methodologies for the analysis of extracellular vesicles, to unraveling the mechanism of the formation of extracellular vesicles within the cell and to the understanding of HIV-1 pathogenesis in successfully treated HIV-infected individuals. These findings contributed importantly to the development of Slovenian science, both in general and specifically in the newly developing research field of extracellular vesicles. Our research results were presented at numerous international conferences and in the context of lectures given at foreign institutions, thus contributing to the recognition of Slovenian science in the international scientific environment. 2- In the long-term, we contributed importantly to the improvement of the treatment and thus the quality of life of individuals infected with HIV-1, both in Slovenia and elsewhere in the world. We have namely shown that Nef is present in the plasma of half of HIV infected individuals, in which effective antiretroviral therapy maintains the level of plasma HIV RNA below the clinically detectable level. Nef is a known HIV pathogenic factor, with many described negative effects. Nef might therefore contribute to inflammation and to non-AIDS related disorders, observed in effectively treated individuals. As part of the project, we have optimized the assay to detect Nef in plasma, which could be used to identify those patients which require the change of therapy. We have also shown that Nef-exosomes could serve as a good therapeutic target. 3- We have also contributed importantly to the improvement of teaching and training of personnel. Most of the co-workers on the project are involved in teaching at both, the undergraduate (Faculty of Medicine, Biotechnical Faculty, Faculty of Health Sciences) and postgraduate level (Doctoral Programme in Biomedicine, Jožef Stefan International Postgraduate School). We also participated in the organization of the international workshop Pharmacogenomics: From Research to Clinic (2015), and have organized a symposium Extracellular vesicles – role in pathogenesis and clinical potential (2016), where we educated the undergraduate, graduate and postdoctoral students, established researchers and clinics about extracellular vesicles. We continue to educate them by sending monthly news about the progress in the field of extracellular vesicles, and we also wrote a review article on this topic in the Slovenian language, which was published in Medicinski razgledi. Co-workers on the project are also mentors/supervisors to a number of students working on research projects, Bachelor degree, master degree and PhD thesis. Some of them were recognized for their work with various awards: student's Prešernova nagrada, Krkina nagrada, Lapanjetovo priznanje and L'Oréal-UNESCO For Women in Science.
Most important scientific results
Annual report
2014,
2015,
final report
Most important socioeconomically and culturally relevant results
Annual report
2014,
2015,
final report
