Multi-Omics Data Integration Reveals Sex-Dependent Hippocampal Programming by Maternal High-Fat Diet during Lactation in Adult Mouse Offspring.

Early-life exposure to high-fat diets (HF) can program metabolic and cognitive alterations in adult offspring. Although the hippocampus plays a crucial role in memory and metabolic homeostasis, few studies have reported the impact of maternal HF on this structure. We assessed the effects of maternal HF during lactation on physiological, metabolic, and cognitive parameters in young adult offspring mice. To identify early-programming mechanisms in the hippocampus, we developed a multi-omics strategy in male and female offspring. Maternal HF induced a transient increased body weight at weaning, and a mild glucose intolerance only in 3-month-old male mice with no change in plasma metabolic parameters in adult male and female offspring. Behavioral alterations revealed by a Barnes maze test were observed both in 6-month-old male and female mice. The multi-omics strategy unveiled sex-specific transcriptomic and proteomic modifications in the hippocampus of adult offspring. These studies that were confirmed by regulon analysis show that, although genes whose expression was modified by maternal HF were different between sexes, the main pathways affected were similar with mitochondria and synapses as main hippocampal targets of maternal HF. The effects of maternal HF reported here may help to better characterize sex-dependent molecular pathways involved in cognitive disorders and neurodegenerative diseases.

Fallopian tube lesions as potential precursors of early ovarian cancer: a comprehensive proteomic analysis.

Ovarian cancer is the leading cause of death from gynecologic cancer worldwide. High-grade serous carcinoma (HGSC) is the most common and deadliest subtype of ovarian cancer. While the origin of ovarian tumors is still debated, it has been suggested that HGSC originates from cells in the fallopian tube epithelium (FTE), specifically the epithelial cells in the region of the tubal-peritoneal junction. Three main lesions, p53 signatures, STILs, and STICs, have been defined based on the immunohistochemistry (IHC) pattern of p53 and Ki67 markers and the architectural alterations of the cells, using the Sectioning and Extensively Examining the Fimbriated End Protocol. In this study, we performed an in-depth proteomic analysis of these pre-neoplastic epithelial lesions guided by mass spectrometry imaging and IHC. We evaluated specific markers related to each preneoplastic lesion. The study identified specific lesion markers, such as CAVIN1, Emilin2, and FBLN5. We also used SpiderMass technology to perform a lipidomic analysis and identified the specific presence of specific lipids signature including dietary Fatty acids precursors in lesions. Our study provides new insights into the molecular mechanisms underlying the progression of ovarian cancer and confirms the fimbria origin of HGSC.

Heimdall, an alternative protein issued from a ncRNA related to kappa light chain variable region of immunoglobulins from astrocytes: a new player in neural proteome.

The dogma "One gene, one protein" is clearly obsolete since cells use alternative splicing and generate multiple transcripts which are translated into protein isoforms, but also use alternative translation initiation sites (TISs) and termination sites on a given transcript. Alternative open reading frames for individual transcripts give proteins originate from the 5'- and 3'-UTR mRNA regions, frameshifts of mRNA ORFs or from non-coding RNAs. Longtime considered as non-coding, recent in-silico translation prediction methods enriched the protein databases allowing the identification of new target structures that have not been identified previously. To gain insight into the role of these newly identified alternative proteins in the regulation of cellular functions, it is crucial to assess their dynamic modulation within a framework of altered physiological modifications such as experimental spinal cord injury (SCI). Here, we carried out a longitudinal proteomic study on rat SCI from 12 h to 10 days. Based on the alternative protein predictions, it was possible to identify a plethora of newly predicted protein hits. Among these proteins, some presented a special interest due to high homology with variable chain regions of immunoglobulins. We focus our interest on the one related to Kappa variable light chains which is similarly highly produced by B cells in the Bence jones disease, but here expressed in astrocytes. This protein, name Heimdall is an Intrinsically disordered protein which is secreted under inflammatory conditions. Immunoprecipitation experiments showed that the Heimdall interactome contained proteins related to astrocyte fate keepers such as "NOTCH1, EPHA3, IPO13" as well as membrane receptor protein including "CHRNA9; TGFBR, EPHB6, and TRAM". However, when Heimdall protein was neutralized utilizing a specific antibody or its gene knocked out by CRISPR-Cas9, sprouting elongations were observed in the corresponding astrocytes. Interestingly, depolarization assays and intracellular calcium measurements in Heimdall KO, established a depolarization effect on astrocyte membranes KO cells were more likely that the one found in neuroprogenitors. Proteomic analyses performed under injury conditions or under lipopolysaccharides (LPS) stimulation, revealed the expression of neuronal factors, stem cell proteins, proliferation, and neurogenesis of astrocyte convertor factors such as EPHA4, NOTCH2, SLIT3, SEMA3F, suggesting a role of Heimdall could regulate astrocytic fate. Taken together, Heimdall could be a novel member of the gatekeeping astrocyte-to-neuroprogenitor conversion factors.

Astrocytes express aberrant immunoglobulins as putative gatekeeper of astrocytes to neuronal progenitor conversion.

Using multi-omics analyses including RNAseq, RT-PCR, RACE-PCR, and shotgun proteomic with enrichment strategies, we demonstrated that newborn rat astrocytes produce neural immunoglobulin constant and variable heavy chains as well as light chains. However, their edification is different from the ones found in B cells and they resemble aberrant immunoglobulins observed in several cancers. Moreover, the complete enzymatic V(D)J recombination complex has also been identified in astrocytes. In addition, the constant heavy chain is also present in adult rat astrocytes, whereas in primary astrocytes from human fetus we identified constant and variable kappa chains as well as the substitution lambda chains known to be involved in pre-B cells. To gather insights into the function of these neural IgGs, CRISPR-Cas9 of IgG2B constant heavy chain encoding gene (Igh6), IgG2B overexpression, proximal labeling of rat astrocytes IgG2B and targets identification through 2D gels were performed. In Igh6 KO astrocytes, overrepresentation of factors involved in hematopoietic cells, neural stem cells, and the regulation of neuritogenesis have been identified. Moreover, overexpression of IgG2B in astrocytes induces the CRTC1-CREB-BDNF signaling pathway known to be involved in gliogenesis, whereas Igh6 KO triggers the BMP/YAP1/TEAD3 pathway activated in astrocytes dedifferentiation into neural progenitors. Proximal labeling experiments revealed that IgG2B is N-glycosylated by the OST complex, addressed to vesicle membranes containing the ATPase complex, and behaves partially like CD98hc through its association with LAT1. These experiments also suggest that proximal IgG2B-LAT1 interaction occurs concomitantly with MACO-1 and C2CD2L, at the heart of a potentially novel cell signaling platform. Finally, we demonstrated that these chains are synthesized individually and associated to recognize specific targets. Indeed, intermediate filaments Eif4a2 and Pdia6 involved in astrocyte fate constitute targets for these neural IgGs. Taken together, we hypothese that neural aberrant IgG chains may act as gatekeepers of astrocytes' fate.

Establishment and characterization of canine mammary tumoroids for translational research.

BACKGROUND: Cancer heterogeneity is a main obstacle for the development of effective therapies, as its replication in in vitro preclinical models is challenging. Around 96% of developed drugs are estimated to fail from discovery to the clinical trial phase probably because of the unsuitability and unreliability of current preclinical models (Front Pharmacol 9:6, 2018; Nat Rev Cancer 8: 147-56, 2008) in replicating the overall biology of tumors, for instance the tumor microenvironment. Breast cancer is the most frequent cancer among women causing the greatest number of cancer-related deaths. Breast cancer can typically be modeled in vitro through the use of tumoroids; however, current approaches using mouse tumoroids fail to reproduce crucial aspect of human breast cancer, while access to human cells is limited and the focus of ethical concerns. New models of breast cancer, such as companion dogs, have emerged given the resemblance of developed spontaneous mammary tumors to human breast cancer in many clinical and molecular aspects; however, they have so far failed to replicate the tumor microenvironment. The present work aimed at developing a robust canine mammary tumor model in the form of tumoroids which recapitulate the tumor diversity and heterogeneity. RESULTS: We conducted a complete characterization of canine mammary tumoroids through histologic, molecular, and proteomic analysis, demonstrating their strong similarity to the primary tumor. We demonstrated that these tumoroids can be used as a drug screening model. In fact, we showed that paclitaxel, a human chemotherapeutic, could kill canine tumoroids with the same efficacy as human tumoroids with 0.1 to 1 µM of drug needed to kill 50% of the cells. Due to easy tissue availability, canine tumoroids can be produced at larger scale and cryopreserved to constitute a biobank. We have demonstrated that cryopreserved tumoroids keep the same histologic and molecular features (ER, PR, and HER2 expression) as fresh tumoroids. Furthermore, two cryopreservation techniques were compared from a proteomic point of view which showed that tumoroids made from frozen material allowed to maintain the same molecular diversity as from freshly dissociated tumor. CONCLUSIONS: These findings revealed that canine mammary tumoroids can be easily generated and may provide an adequate and more reliable preclinical model to investigate tumorigenesis mechanisms and develop new treatments for both veterinary and human medicine.

Contribution of heterozygous PCSK1 variants to obesity and implications for precision medicine: a case-control study.

BACKGROUND: Rare biallelic pathogenic mutations in PCSK1 (encoding proprotein convertase subtilisin/kexin type 1 [PC1/3]) cause early-onset obesity associated with various endocrinopathies. Setmelanotide has been approved for carriers of these biallelic mutations in the past 3 years. We aimed to perform a large-scale functional genomic study focusing on rare heterozygous variants of PCSK1 to decipher their putative impact on obesity risk. METHODS: This case-control study included all participants with overweight and obesity (ie, cases) or healthy weight (ie, controls) from the RaDiO study of three community-based and one hospital-based cohort in France recruited between Jan 1, 1995, and Dec 31, 2000. In adults older than 18 years, healthy weight was defined as BMI of less than 25·0 kg/m2, overweight as 25·0-29·9 kg/m2, and obesity as 30·0 kg/m2 or higher. Participants with type 2 diabetes had fasting glucose of 7·0 mmol/L or higher or used treatment for hyperglycaemia (or both) and were negative for islet or insulin autoantibodies. Functional assessment of rare missense variants of PCSK1 was performed. Pathogenicity clusters of variants were determined with machine learning. The effect of each cluster of PCSK1 variants on obesity was assessed using the adjusted mixed-effects score test. FINDINGS: All 13 coding exons of PCSK1 were sequenced in 9320 participants (including 7260 adults and 2060 children and adolescents) recruited from the RaDiO study. We detected 65 rare heterozygous PCSK1 variants, including four null variants and 61 missense variants that were analysed in vitro and clustered into five groups (A-E), according to enzymatic activity. Compared with the wild-type, 15 missense variants led to complete PC1/3 loss of function (group A; reference) and rare exome variant ensemble learner (REVEL) led to 15 (25%) false positives and four (7%) false negatives. Carrying complete loss-of-function or null PCSK1 variants was significantly associated with obesity (six [86%] of seven carriers vs 1518 [35%] of 4395 non-carriers; OR 9·3 [95% CI 1·5-177·4]; p=0·014) and higher BMI (32·0 kg/m2 [SD 9·3] in carriers vs 27·3 kg/m2 [6·5] in non-carriers; mean effect π 6·94 [SE 1·95]; p=0·00029). Clusters of PCSK1 variants with partial or neutral effect on PC1/3 activity did not have an effect on obesity or overweight and on BMI. INTERPRETATION: Only carriers of heterozygous, null, or complete loss-of-function PCSK1 variants cause monogenic obesity and, therefore, might be eligible for setmelanotide. In silico tests were unable to accurately detect these variants, which suggests that in vitro assays are necessary to determine the variant pathogenicity for genetic diagnosis and precision medicine purposes. FUNDING: Agence Nationale de la Recherche, European Research Council, National Center for Precision Diabetic Medicine, European Regional Development Fund, Hauts-de-France Regional Council, and the European Metropolis of Lille.

Spatial analysis of the glioblastoma proteome reveals specific molecular signatures and markers of survival.

Molecular heterogeneity is a key feature of glioblastoma that impedes patient stratification and leads to large discrepancies in mean patient survival. Here, we analyze a cohort of 96 glioblastoma patients with survival ranging from a few months to over 4 years. 46 tumors are analyzed by mass spectrometry-based spatially-resolved proteomics guided by mass spectrometry imaging. Integration of protein expression and clinical information highlights three molecular groups associated with immune, neurogenesis, and tumorigenesis signatures with high intra-tumoral heterogeneity. Furthermore, a set of proteins originating from reference and alternative ORFs is found to be statistically significant based on patient survival times. Among these proteins, a 5-protein signature is associated with survival. The expression of these 5 proteins is validated by immunofluorescence on an additional cohort of 50 patients. Overall, our work characterizes distinct molecular regions within glioblastoma tissues based on protein expression, which may help guide glioblastoma prognosis and improve current glioblastoma classification.

Therapeutic anti-glioma effect of the combined action of PCSK inhibitor with the anti-tumoral factors secreted by Poly (I:C)-stimulated macrophages.

Macrophages plasticity is a key feature in cancer progression. Neoplastic cells can alter their immune functions and orient them into a pro-tumoral phenotype. In this context, we developed a new therapeutic strategy to switch macrophages phenotype and reactivate their anti-tumoral functions. We showed a dual activity of a proprotein convertases inhibitor as anti-glioma drug and anti-tumoral macrophages' reactivation drug. Proprotein convertases are proteases that cleave proteins into functional proteins. Several of their substrates are involved in tumorigenesis and immunosuppression. We combine here proprotein convertases inhibitor with Poly (I:C), a TLR3 ligand, to increase the anti-tumoral activity of macrophages. With mass spectrometry-based proteomics, system biology, combined with biological assays, we established that a stimulation of macrophages with Poly (I:C) increased their secretion of pro-inflammatory cytokines and anti-tumoral factors. 3D invasion assay showed the efficacy of these anti-tumoral factors against mixed glioma cells and macrophages spheroids. Besides, immunofluorescence and proliferation assays showed an additive effect of the proprotein convertases inhibitor and the anti-tumoral factors secreted by Poly (I:C)-treated macrophages on both anti-glioma activity and macrophages anti-tumoral orientation directly in tumor microenvironment, leading to an innovative glioma therapy.

On-tissue spatially resolved glycoproteomics guided by N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape.

Here, we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of matrix-assisted laser desorption ionization (MALDI) N-glycan mass spectrometry imaging (MSI) and spatially resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, thus directly linking glycan imaging with intact glycopeptide identification. In total, our spatially resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slices and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.

Surfaceome Proteomic of Glioblastoma Revealed Potential Targets for Immunotherapy.

Glioblastoma (GBM) is the most common and devastating malignant brain tumor in adults. The mortality rate is very high despite different treatments. New therapeutic targets are therefore highly needed. Cell-surface proteins represent attractive targets due to their accessibility, their involvement in essential signaling pathways, and their dysregulated expression in cancer. Moreover, they are potential targets for CAR-based immunotherapy or mRNA vaccine strategies. In this context, we investigated the GBM-associated surfaceome by comparing it to astrocytes cell line surfaceome to identify new specific targets for GBM. For this purpose, biotinylation of cell surface proteins has been carried out in GBM and astrocytes cell lines. Biotinylated proteins were purified on streptavidin beads and analyzed by shotgun proteomics. Cell surface proteins were identified with Cell Surface Proteins Atlas (CSPA) and Gene Ontology enrichment. Among all the surface proteins identified in the different cell lines we have confirmed the expression of 66 of these in patient's glioblastoma using spatial proteomic guided by MALDI-mass spectrometry. Moreover, 87 surface proteins overexpressed or exclusive in GBM cell lines have been identified. Among these, we found 11 specific potential targets for GBM including 5 mutated proteins such as RELL1, CYBA, EGFR, and MHC I proteins. Matching with drugs and clinical trials databases revealed that 7 proteins were druggable and under evaluation, 3 proteins have no known drug interaction yet and none of them are the mutated form of the identified proteins. Taken together, we discovered potential targets for immune therapy strategies in GBM.

Toward High Spatially Resolved Proteomics Using Expansion Microscopy.

Expansion microscopy (EM) is an emerging approach for morphological examination of biological specimens at nanoscale resolution using conventional optical microscopy. To achieve physical separation of cell structures, tissues are embedded in a swellable polymer and expanded several fold in an isotropic manner. This work shows the development and optimization of physical tissue expansion as a new method for spatially resolved large-scale proteomics. Herein we established a novel method to enlarge the tissue section to be compatible with manual microdissection on regions of interest and MS-based proteomic analysis. A major issue in expansion microscopy is the loss of protein information during the mechanical homogenization phase due to the use of proteinase K. For isotropic expansion, different homogenization agents were investigated, both to maximize protein identification and to minimize protein diffusion. Best results were obtained with SDS for homogenization. Using our modified protocol, we were able to enlarge a tissue section more than 3-fold and identified up to 655 proteins from 1 mm in size after expansion, equivalent to 330 µm in their real size corresponding thus to an average of 260 cells. This approach can be performed easily without any expensive sampling instrument. We demonstrated the compatibility of sample preparation for expansion microscopy and proteomic study in a spatial context.

In-depth proteomics analysis of sentinel lymph nodes from individuals with endometrial cancer.

Endometrial cancer (EC) is one of the most common gynecological cancers worldwide. Sentinel lymph node (SLN) status could be a major prognostic factor in evaluation of EC, but several prospective studies need to be performed. Here we report an in-depth proteomics analysis showing significant variations in the SLN protein landscape in EC. We show that SLNs are correlated to each tumor grade, which strengthens evidence of SLN involvement in EC. A few proteins are overexpressed specifically at each EC tumor grade and in the corresponding SLN. These proteins, which are significantly variable in both locations, should be considered potential markers of overall survival. Five major proteins for EC and SLN (PRSS3, PTX3, ASS1, ALDH2, and ANXA1) were identified in large-scale proteomics and validated by immunohistochemistry. This study improves stratification and diagnosis of individuals with EC as a result of proteomics profiling of SLNs.

Path to Clonal Theranostics in Luminal Breast Cancers.

Integrating tumor heterogeneity in the drug discovery process is a key challenge to tackle breast cancer resistance. Identifying protein targets for functionally distinct tumor clones is particularly important to tailor therapy to the heterogeneous tumor subpopulations and achieve clonal theranostics. For this purpose, we performed an unsupervised, label-free, spatially resolved shotgun proteomics guided by MALDI mass spectrometry imaging (MSI) on 124 selected tumor clonal areas from early luminal breast cancers, tumor stroma, and breast cancer metastases. 2868 proteins were identified. The main protein classes found in the clonal proteome dataset were enzymes, cytoskeletal proteins, membrane-traffic, translational or scaffold proteins, or transporters. As a comparison, gene-specific transcriptional regulators, chromatin related proteins or transmembrane signal receptor were more abundant in the TCGA dataset. Moreover, 26 mutated proteins have been identified. Similarly, expanding the search to alternative proteins databases retrieved 126 alternative proteins in the clonal proteome dataset. Most of these alternative proteins were coded mainly from non-coding RNA. To fully understand the molecular information brought by our approach and its relevance to drug target discovery, the clonal proteomic dataset was further compared to the TCGA breast cancer database and two transcriptomic panels, BC360 (nanoString®) and CDx (Foundation One®). We retrieved 139 pathways in the clonal proteome dataset. Only 55% of these pathways were also present in the TCGA dataset, 68% in BC360 and 50% in CDx. Seven of these pathways have been suggested as candidate for drug targeting, 22 have been associated with breast cancer in experimental or clinical reports, the remaining 19 pathways have been understudied in breast cancer. Among the anticancer drugs, 35 drugs matched uniquely with the clonal proteome dataset, with only 7 of them already approved in breast cancer. The number of target and drug interactions with non-anticancer drugs (such as agents targeting the cardiovascular system, metabolism, the musculoskeletal or the nervous systems) was higher in the clonal proteome dataset (540 interactions) compared to TCGA (83 interactions), BC360 (419 interactions), or CDx (172 interactions). Many of the protein targets identified and drugs screened were clinically relevant to breast cancer and are in clinical trials. Thus, we described the non-redundant knowledge brought by this clone-tailored approach compared to TCGA or transcriptomic panels, the targetable proteins identified in the clonal proteome dataset, and the potential of this approach for drug discovery and repurposing through drug interactions with antineoplastic agents and non-anticancer drugs.

Epigenetic Studies Revealed a Ghost Proteome in PC1/3 KD Macrophages under Antitumoral Resistance Induced by IL-10.

Our previous investigation on macrophages has allowed us to show that the inhibition of the enzyme proprotein convertase (PC1/3) controls the activation of macrophages. We demonstrated that PC1/3 knockdown (KD) in macrophages exhibits an increased secretion of proinflammatory and antitumoral factors. In this biological context, we assessed the presence of histone modifications and the presence and contribution of a "ghost proteome" in these macrophages. We identified a set of alternative proteins (AltProts) that have a key role in the regulation of various signaling pathways. In this study, to further investigate the underlying mechanisms involved in the resistance of PC1/3-KD macrophages to anti-inflammatory stimuli, we have conducted a proteomic system biology study to assess the epigenome variation, focusing on histone modifications. Results from our study have indicated the presence of significant variations in histone modifications along with the identification of 28 AltProts, which can be correlated with antitumoral resistance under IL-10 stimulation. These findings highlight a key role of altered epigenome histone modifications in driving resistance and indicate that like the reference proteins, AltProts can have a major impact in the field of epigenetics and regulation of gene expression, as shown in our results.

The Role of a Proprotein Convertase Inhibitor in Reactivation of Tumor-Associated Macrophages and Inhibition of Glioma Growth.

Tumors are characterized by the presence of malignant and non-malignant cells, such as immune cells including macrophages, which are preponderant. Macrophages impact the efficacy of chemotherapy and may lead to drug resistance. In this context and based on our previous work, we investigated the ability to reactivate macrophages by using a proprotein convertases inhibitor. Proprotein convertases process immature proteins into functional proteins, with several of them having a role in immune cell activation and tumorigenesis. Macrophages were treated with a peptidomimetic inhibitor targeting furin, PC1/3, PC4, PACE4, and PC5/6. Their anti-glioma activity was analyzed by mass spectrometry-based proteomics and viability assays in 2D and 3D in vitro cultures. Comparison with temozolomide, the drug used for glioma therapy, established that the inhibitor was more efficient for the reduction of cancer cell density. The inhibitor was also able to reactivate macrophages through the secretion of several immune factors with antitumor properties. Moreover, two proteins considered as good glioma patient survival indicators were also identified in 3D cultures treated with the inhibitor. Finally, we established that the proprotein convertases inhibitor has a dual role as an anti-glioma drug and anti-tumoral macrophage reactivation drug. This strategy could be used together with chemotherapy to increase therapy efficacy in glioma.

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.

Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in Experimental Model of Traumatic Brain Injury Detecting Acylcarnitines as Injury Related Markers.

Identifying new lipid markers linked to traumatic brain injury (TBI) is of major importance in characterizing their central role in the regeneration process and inflammatory response in such an injury model. In the present study, an advanced lipidomics analysis using high spectral resolution matrix-assisted laser desorption/ionization-mass spectrometry imaging was performed on different brain regions in an experimental rat model of moderate controlled cortical impact (CCI) while considering different time points (1 day, 3 days, 7 days, and 10 days) assessing the acute and subacute phase after injury. Our results revealed a new family of lipids, the acylcarnitines, as TBI-lipid related markers, with maximum expression at 3 days after impact and main colocalization within resident microglia of the brain. Furthermore, our experiments highlighted the upregulation of these acylcarnitine lipids, secreted by microglia, in the ipsilateral substantia nigra, the main region in the brain affected in Parkinson's disease (PD).

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.