Proteome integral solubility alteration high-throughput proteomics assay identifies Collectin-12 as a non-apoptotic microglial caspase-3 substrate.

Caspases are a family of proteins mostly known for their role in the activation of the apoptotic pathway leading to cell death. In the last decade, caspases have been found to fulfill other tasks regulating the cell phenotype independently to cell death. Microglia are the immune cells of the brain responsible for the maintenance of physiological brain functions but can also be involved in disease progression when overactivated. We have previously described non-apoptotic roles of caspase-3 (CASP3) in the regulation of the inflammatory phenotype of microglial cells or pro-tumoral activation in the context of brain tumors. CASP3 can regulate protein functions by cleavage of their target and therefore could have multiple substrates. So far, identification of CASP3 substrates has been performed mostly in apoptotic conditions where CASP3 activity is highly upregulated and these approaches do not have the capacity to uncover CASP3 substrates at the physiological level. In our study, we aim at discovering novel substrates of CASP3 involved in the normal regulation of the cell. We used an unconventional approach by chemically reducing the basal level CASP3-like activity (by DEVD-fmk treatment) coupled to a Mass Spectrometry screen (PISA) to identify proteins with different soluble amounts, and consequently, non-cleaved proteins in microglia cells. PISA assay identified several proteins with significant change in their solubility after DEVD-fmk treatment, including a few already known CASP3 substrates which validated our approach. Among them, we focused on the Collectin-12 (COLEC12 or CL-P1) transmembrane receptor and uncovered a potential role for CASP3 cleavage of COLEC12 in the regulation of the phagocytic capacity of microglial cells. Taken together, these findings suggest a new way to uncover non-apoptotic substrates of CASP3 important for the modulation of microglia cell physiology.

Reference and Ghost Proteins Identification in Rat C6 Glioma Extracellular Vesicles.

Extracellular vesicles (EVs) mediate intercellular communication and regulate a broad range of biological processes. Novel therapeutic strategies have emerged based on the use of EVs as biological nanoparticles. To separate isolated EVs from protein aggregates and the external part of EVs membrane proteins, we performed a Trypsin/Lys C digestion treatment of EVs pellets, followed by Amicon filtration. After these steps, all the fractions have been subjected to proteomic analyses. Comparison between 6 h Trypsin/Lys C treatment or non-treated EVs revealed a quantitative variation of the surface proteins. Some surface proteins have been demasked after 6 h enzymatic digestion like CD81, CD82, Ust, Vcan, Lamp 1, Rab43, Annexin A2, Synthenin, and VSP37b. Moreover, six ghost proteins have also been identified and one corresponds to a long noncoding RNA. We thus demonstrate the presence of ghost proteins in EVs produced by glioma cells that can contribute to tumorigenesis.

Location of neonatal microglia drives small extracellular vesicles content and biological functions in vitro.

Combining proteomics and systems biology approaches, we demonstrate that neonatal microglial cells derived from two different CNS locations, cortex and spinal cord, and cultured in vitro displayed different phenotypes upon different physiological or pathological conditions. These cells also exhibited greater variability in terms of cellular and small extracellular vesicles (sEVs) protein content and levels. Bioinformatic data analysis showed that cortical microglia exerted anti-inflammatory and neurogenesis/tumorigenesis properties, while the spinal cord microglia were more inflammatory. Interestingly, while both sEVs microglia sources enhanced growth of DRGs processes, only the spinal cord-derived sEVs microglia under LPS stimulation significantly attenuated glioma proliferation. These results were confirmed using the neurite outgrowth assay on DRGs cells and glioma proliferation analysis in 3D spheroid cultures. Results from these in vitro assays suggest that the microglia localized at different CNS regions can ensure different biological functions. Together, this study indicates that neonatal microglia locations regulate their physiological and pathological functional fates and could affect the high prevalence of brain vs spinal cord gliomas in adults.

SETD2 mutation in renal clear cell carcinoma suppress autophagy via regulation of ATG12.

Inactivating mutations in the SETD2 gene, encoding for a nonredundant histone H3 methyltransferase and regulator of transcription, is a frequent molecular feature in clear cell renal cell carcinomas (ccRCC). SETD2 deficiency is associated with recurrence of ccRCC and bears low prognostic values. Targeting autophagy, a conserved catabolic process with critical functions in maintenance of cellular homeostasis and cell conservation under stress condition, is emerging as a potential therapeutic strategy to combat ccRCC. Epigenetics-based pathways are now appreciated as key components in the regulation of autophagy. However, whether loss of function in the SETD2 histone modifying enzyme occurring in ccRCC cells may impact on their ability to undergo autophagy remained to be explored. Here, we report that SETD2 deficiency in RCC cells is associated with the aberrant accumulation of both free ATG12 and of an additional ATG12-containing complex, distinct from the ATG5-ATG12 complex. Rescue of SETD2 functions in the SETD2 deficiency in RCC cells, or reduction of SETD2 expression level in RCC cells wild type for this enzyme, demonstrates that SETD2 deficiency in RCC is directly involved in the acquisition of these alterations in the autophagic process. Furthermore, we revealed that deficiency in SETD2, known regulator of alternative splicing, is associated with increased expression of a short ATG12 spliced isoform at the depend of the canonical long ATG12 isoform in RCC cells. The defect in the ATG12-dependent conjugation system was found to be associated with a decrease autophagic flux, in accord with the role for this ubiquitin-like protein conjugation system in autophagosome formation and expansion. Finally, we report that SETD2 and ATG12 gene expression levels are associated with favorable respective unfavorable prognosis in ccRCC patients. Collectively, our findings bring further argument for considering the SETD2 gene status of ccRCC tumors, when therapeutic interventions, such as targeting the autophagic process, are considered to combat these kidney cancers.

Spinal Cord Injury: Animal Models, Imaging Tools and the Treatment Strategies.

Spinal cord injury (SCI) often leads to irreversible neuro-degenerative changes with life-long consequences. While there is still no effective therapy available, the results of past research have led to improved quality of life for patients suffering from partial or permanent paralysis. In this review we focus on the need, importance and the scientific value of experimental animal models simulating SCI in humans. Furthermore, we highlight modern imaging tools determining the location and extent of spinal cord damage and their contribution to early diagnosis and selection of appropriate treatment. Finally, we focus on available cellular and acellular therapies and novel combinatory approaches with exosomes and active biomaterials. Here we discuss the efficacy and limitations of adult mesenchymal stem cells which can be derived from bone marrow, adipose tissue or umbilical cord blood and its Wharton's jelly. Special attention is paid to stem cell-derived exosomes and smart biomaterials due to their special properties as a delivery system for proteins, bioactive molecules or even genetic material.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Paclitaxel Treatment and Proprotein Convertase 1/3 (PC1/3) Knockdown in Macrophages is a Promising Antiglioma Strategy as Revealed by Proteomics and Cytotoxicity Studies.

High grade gliomas are the most common brain tumors in adult. These tumors are characterized by a high infiltration in microglial cells and macrophages. The immunosuppressive tumor environment is known to orient immune cells toward a pro-tumoral and anti-inflammatory phenotype. Therefore, the current challenge for cancer therapy is to find a way to reorient macrophages toward an antitumoral phenotype. Previously, we demonstrated that macrophages secreted antitumoral factors when they were invalidated for the proprotein converstase 1/3 (PC1/3) and treated with LPS. However, achieving an activation of macrophages via LPS/TLR4/Myd88-dependent pathway appears yet unfeasible in cancer patients. On the contrary, the antitumor drug Paclitaxel is also known to activate the TLR4 MyD88-dependent signaling pathway and mimics LPS action. Therefore, we evaluated if PC1/3 knock-down (KD) macrophages could be activated by Paclitaxel and efficient against glioma. We report here that such a treatment of PC1/3 KD macrophages drove to the overexpression of proteins mainly involved in cytoskeleton rearrangement. In support of this finding, we found that these cells exhibited a Ca2+ increase after Paclitaxel treatment. This is indicative of a possible depolymerization of microtubules and may therefore reflect an activation of inflammatory pathways in macrophages. In such a way, we found that PC1/3 KD macrophages displayed a repression of the anti-inflammatory pathway STAT3 and secreted more pro-inflammatory cytokines. Extracellular vesicles isolated from these PC1/3 KD cells inhibited glioma growth. Finally, the supernatant collected from the coculture between glioma cells and PC1/3 KD macrophages contained more antitumoral factors. These findings unravel the potential value of a new therapeutic strategy combining Paclitaxel and PC1/3 inhibition to switch macrophages toward an antitumoral immunophenotype.

Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury.

It was recently shown that the conditioned medium (CM) of mesenchymal stem cells can enhance viability of neural and glial cell populations. In the present study, we have investigated a cell-free approach via CM from rat bone marrow stromal cells (MScCM) applied intrathecally (IT) for spinal cord injury (SCI) recovery in adult rats. Functional in vitro test on dorsal root ganglion (DRG) primary cultures confirmed biological properties of collected MScCM for production of neurosphere-like structures and axon outgrowth. Afterwards, rats underwent SCI and were treated with IT delivery of MScCM or vehicle at postsurgical Days 1, 5, 9, and 13, and left to survive 10 weeks. Rats that received MScCM showed significantly higher motor function recovery, increase in spared spinal cord tissue, enhanced GAP-43 expression and attenuated inflammation in comparison with vehicle-treated rats. Spared tissue around the lesion site was infiltrated with GAP-43-labeled axons at four weeks that gradually decreased at 10 weeks. Finally, a cytokine array performed on spinal cord extracts after MScCM treatment revealed decreased levels of IL-2, IL-6 and TNFα when compared to vehicle group. In conclusion, our results suggest that molecular cocktail found in MScCM is favorable for final neuroregeneration after SCI.

Brain-Cortex Microglia-Derived Exosomes: Nanoparticles for Glioma Therapy.

The function and integrity of the nervous system require interactive exchanges among neurons and glial cells. Exosomes and other extracellular vesicles (EVs) are emerging as a key mediator of intercellular communication, capable of transferring nucleic acids, proteins and lipids influencing numerous functional and pathological aspects of both donor and recipient cells. The immune response mediated by microglia-derived exosomes is most prominently involved in the spread of neuroinflammation, neurodegenerative disorders, and brain cancer. Therefore, in the present study we describe a reproducible and highly efficient method for yielding purified primary microglia cells, followed by exosome isolation and their characterization. An in vitro biological assay demonstrates that microglia-derived exosomes tested on a 3D spheroid glioma culture were able to inhibit tumor invasion in time course. These results evidence that brain microglia-derived exosomes could be used as nanotherapeutic agents against glioma cells.

Proprotein convertase 1/3 inhibited macrophages: A novel therapeutic based on drone macrophages.

We demonstrated here thanks to proteomic, that proprotein convertase 1/3 knockdown macrophages present all the characteristic of activated pro-inflammatory macrophages. TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and antitumor factors. We can control their activation by controlling one enzyme, PC1/3. In a tumor context, PC1/3 inhibition in macrophages may reactivate them and lead to a cytokine storm after stimulation "at distance" with a TLR ligand. Therefore, we name these proprotein convertase inhibited macrophages the "drone macrophages". They constitute an innovative cell therapy to treat efficiently tumors.