Extracellular vesicles for clinical diagnostics of nervous system diseases

In the last decade there has emerged a new dimension in molecular studies which can be applied to gliomas (brain tumors). Extracellular vesicles (EVs), small structures containing genetic materials, are now known to be produced by glioma cells. These EVs, often many hundreds in number, are released by glioma cells into the blood and fluids surrounding the brain. Originally, these EVs were described as "cell dust in human plasma". There is general consensus that EVs contain gene products including proteins, RNA and DNA. As these products originate from tumor they can be harvested to provide systems that mirror glioma development and progression. My objective has been to study the role of EVs in human blood and other fluids; to identify these EVs and to measure them in order to develop a non-invasive biomarker for detection of glioma. The studies that I have performed have explored the role of these EVs in cancer development and have reviewed the EV-gene products that might be measured to help patients. I then concentrated on developing techniques to find these EVs in small amounts of human fluids and then explored one gene product seen in gliomas. I have worked to improve the way in which we find these EV gene products with the eventual goal of providing diagnosis and care of brain tumor patients without resorting to surgery.There is increasing evidence that extracellular vesicles (EVs) isolated from biofluids will be an important contribution in medicine to develop a non-invasive ways to diagnose and monitor disease. This thesis describes some of the groundwork in development of this new biomarker resource that has so far primarily focused on brain cancer, but the knowledge gained here should have universal applications for all types of diseases

Untethering the Nuclear Envelope and Cytoskeleton: Biologically Distinct Dystonias Arising from a Common Cellular Dysfunction

Most cases of early onset DYT1 dystonia in humans are caused by a GAG deletion in the TOR1A gene leading to loss of a glutamic acid (ΔE) in the torsinA protein, which underlies a movement disorder associated with neuronal dysfunction without apparent neurodegeneration. Mutation/deletion of the gene (Dst) encoding dystonin in mice results in a dystonic movement disorder termed dystonia musculorum, which resembles aspects of dystonia in humans. While torsinA and dystonin proteins do not share modular domain architecture, they participate in a similar function by modulating a structural link between the nuclear envelope and the cytoskeleton in neuronal cells. We suggest that through a shared interaction with the nuclear envelope protein nesprin-3α, torsinA and the neuronal dystonin-a2 isoform comprise a bridge complex between the outer nuclear membrane and the cytoskeleton, which is critical for some aspects of neuronal development and function. Elucidation of the overlapping roles of torsinA and dystonin-a2 in nuclear/endoplasmic reticulum dynamics should provide insights into the cellular mechanisms underlying the dystonic phenotype

Heparin affinity purification of extracellular vesicles

Extracellular vesicles (EVs) are lipid membrane vesicles released by cells. They carry active biomolecules including DNA, RNA, and protein which can be transferred to recipient cells. Isolation and purification of EVs from culture cell media and biofluids is still a major challenge. The most widely used isolation method is ultracentrifugation (UC) which requires expensive equipment and only partially purifies EVs. Previously we have shown that heparin blocks EV uptake in cells, supporting a direct EV-heparin interaction. Here we show that EVs can be purified from cell culture media and human plasma using ultrafiltration (UF) followed by heparin-affinity beads. UF/heparin-purified EVs from cell culture displayed the EV marker Alix, contained a diverse RNA profile, had lower levels of protein contamination, and were functional at binding to and uptake into cells. RNA yield was similar for EVs isolated by UC. We were able to detect mRNAs in plasma samples with comparable levels to UC samples. In conclusion, we have discovered a simple, scalable, and effective method to purify EVs taking advantage of their heparin affinity

La Vigie marocaine

08 avril 19361936/04/08 (A27,N8879)-1936/04/08

Glioma diagnostics and biomarkers: an ongoing challenge in the field of medicine and science

Glioma is the most common brain tumor. For the more aggressive form, glioblastoma, standard treatment includes surgical resection, irradiation with adjuvant temozolomide and, on recurrence, experimental chemotherapy. However, the survival of patients remains poor. There is a critical need for minimally invasive biomarkers for diagnosis and as measures of response to therapeutic interventions. Glioma shed extracellular vesicles (EVs), which invade the surrounding tissue and circulate within both the cerebrospinal fluid and the systemic circulation. These tumor-derived EVs and their content serve as an attractive source of biomarkers. In this review, we discuss the current state of the art of biomarkers for glioma with emphasis on their EV derivatio

Heparin blocks transfer of extracellular vesicles between donor and recipient cells

Extracellular vesicles (EVs) have been implicated in tumorigenesis. Biomolecules which can block EV binding and uptake into recipient cells may be of therapeutic value as well as enhance understanding of EV biology. Here, we show that heparin interacts with uptake of tumor-derived as well as non-tumor-derived EVs into recipient cells. Incubation of glioma cell-derived EVs with heparin resulted in micron-sized structures observed by transmission electron microscopy, with EVs clearly visible within these structures. Inclusion of heparin greatly diminished transfer of labeled EVs from donor to recipient tumor cells. We also show a direct interaction between heparin and EVs using confocal microscopy. We found that the block in EV uptake was at the level of cell binding and not internalization. Finally, incubation of glioma-derived EVs containing EGFRvIII mRNA with heparin reduced transfer of this message to recipient cells. The effect of heparin on EVs uptake may provide a unique tool to study EV function. It may also foster research of heparin or its derivatives as a therapeutic for disease in which EVs play a rol

Microfluidic platform to evaluate migration of cells from patients with DYT1 dystonia

Microfluidic platforms for quantitative evaluation of cell biologic processes allow low cost and time efficient research studies of biological and pathological events, such as monitoring cell migration by real-time imaging. In healthy and disease states, cell migration is crucial in development and wound healing, as well as to maintain the body's homeostasis. The microfluidic chambers allow precise measurements to investigate whether fibroblasts carrying a mutation in the TOR1A gene, underlying the hereditary neurologic disease--DYT1 dystonia, have decreased migration properties when compared to control cells. We observed that fibroblasts from DYT1 patients showed abnormalities in basic features of cell migration, such as reduced velocity and persistence of movement. The microfluidic method enabled us to demonstrate reduced polarization of the nucleus and abnormal orientation of nuclei and Golgi inside the moving DYT1 patient cells compared to control cells, as well as vectorial movement of single cells. We report here different assays useful in determining various parameters of cell migration in DYT1 patient cells as a consequence of the TOR1A gene mutation, including a microfluidic platform, which provides a means to evaluate real-time vectorial movement with single cell resolution in a three-dimensional environmen

RT-qPCR analysis of expression of selected proteases and protease inhibitors in U87-MG and U373 GBM cells, NHA cells and GBM tissues and non-malignant brain <i>(in vivo)</i>.

<p>(A) Upregulated expression of seven genes (<i>TFRC</i>, <i>CTSK</i>, <i>GFPT2</i>, <i>ERAP2</i>, <i>GPR56</i>, <i>CD74</i>, <i>PI3</i>) as determined by microarray data was validated in GBM cells in comparison to NHA cells by RT-qPCR, using GAPDH as reference gene. (B) Additional RT-qPCR analysis of expression of the <i>CTSK</i> gene using GBM tissues and cell lines with reference genes <i>TBP</i> and <i>HPRT1</i> in comparison to NHA cells (NAtotRNA) and non-malignant brain (HBrefRNA). The experiments were performed in triplicate (except for 8 repetitions of GBM tissue and commercial RNA from NHA and normal brain, used in experiment B). Error bars represent standard deviation; * p-value<0.05, ** p-value<0.01, *** p-value<0.001.</p

Scheme of cellular processes and activities involving overexpressed protease and protease inhibitor genes in GBM.

<p>The overexpressed protease and inhibitor genes in GBM tissues and cells were queried by the Biomine search engine which identified processes and activities ascribed with KEGG and GO identifiers (in circles) in which selected genes (in bold caption) are involved.</p