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LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from 3-, 6-, 12-, and 18-months-old mice and Apoe-KO mice at 3 months followed by label-free quantification (LFQ)

ABSTRACT: Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry based proteomics. In this experiment, first we have compared MACS sorted BECs across multiple time points between 3 and 18 months of age. Using unsupervised cluster analysis, we found a segregation of age-related protein dynamics with biological functions including a downregulation of vesicle-mediated transport. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe. Therefor in our next proteomics experiment we compared BECs from 3-months-old Apoe-KO and WT mice and found 111 and 103 proteins to be up- and downregulated, respectively. Comparing the BEC proteomic signature of young Apoe-KO mice with the signature of aged (18-months-old) WT mice we found a positive correlation suggesting an accelerating effect of Apoe deficiency on BEC aging.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Endothelial Cell

SUBMITTER: Stephan Mueller

LAB HEAD: Stefan F. Lichtenthaler

PROVIDER: PXD044996 | Pride | 2024-02-15

REPOSITORIES: Pride

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Project description:Age-related decline in brain endothelial cell (BEC) function critically contributes to cerebrovascular and neurodegenerative disease. Comprehensive atlases of the BEC transcriptome have become available but results from proteomic profiling are lacking. To gain insights into endothelial pathways affected by aging, we developed a magnetic-activated cell sorting (MACS)-based mouse BEC enrichment protocol compatible with high-resolution mass-spectrometry and analysed the profiles of protein abundance changes across multiple time points between 3 and 18 months of age and identified Arf6 as one of the most prominently downregulated vesicle-mediated transport protein during BEC aging. To understand the role of ARF6 in human ECs, first we compared GFP-AAV treated human iECs differentiated from ARF6-KO vs WT iPSCs, next we compared ARF6-GFP-AAV vs GFP-AAV treated iECs differentiated from WT iPSCs. During the ARF6-KO vs WT iEC comparison we found 983 vs 741 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly downregulated proteins revealed mRNA processing among the most significantly affected biological processes. During the ARF6-GFP-AAV vs GFP-AAV treated WT iEC comparison we found 1106 vs 1218 proteins significantly down- and upregulated, respectively. Enrichment analyses of significantly upregulated proteins revealed endocytic recycling, retrograde transport (endosome to Golgi), and ER-Golgi vesicle-mediated transport among the most significantly affected biological processes. Specifically, ARF6 and its binding-protein GGA2, DNM1L and several subunits of the Conserved oligomeric Golgi complex (COG) were upregulated. Our approach uncovered changes not picked up by transcriptomic studies such as accumulation of vesicle cargo and receptor ligands including Apoe, a major regulator of brain lipid metabolism. Proteomic analysis of BECs from Apoe deficient mice revealed a signature of accelerated aging. To explore the role of APOE in human endothelial cells, in this experiment we compared human iECs differentiated from APOE-KO and WT iPSCs and found 326 significantly altered proteins. Enrichment analyses of significantly downregulated proteins revealed vesicle-mediated transport and vesicle fusion to be among the most significantly affected biological processes. Specifically, among the 34 significantly altered vesicle-mediated transport proteins 26 were downregulated. Accordingly, we found reduced levels of endocytosis of FM1-43FX in newly formed vesicles in APOE-KO iECs.

Project description:Preclinical studies of primary cancer cells are always done after tumors are removed from patients or animals at ambient atmospheric oxygen (O2, ~21%). However, O2 concentrations in organs are in the ~3-10% range, with most tumors in an hypoxic or 1-2% O2 environment in vivo. Although effects of O2 tension on tumor cell characteristics in vitro have been studied, these studies are done only after tumors are first collected and processed in ambient air. Similarly, sensitivity of primary cancer cells to anti-cancer agents is routinely examined at ambient O2. Here, using both mouse models and human cancers, we demonstrate that tumors collected, processed and propagated at physiologic (physioxia) O2 compared to ambient air display very distinct differences in key signaling networks including Lgr5/Wnt, Yap, and Nrf2/Keap1, nuclear reactive oxygen species levels, alternative splicing, and sensitivity to several targeted therapies including PIK3CAalpha-specific and EGFR inhibitors. Significance: Extra-physiologic oxygen shock/stress (EPHOSS), as noted in cells collected/processed under ambient air, has been demonstrated to have significant impact on numbers and engrafting ability of hematopoietic stem cells. We report deleterious effects of EPHOSS on cancer cell behavior and EPHOSS-mediated effects on cancer cells give misleading information on signaling pathway activation that could severely impact the relevance of these findings. Cancer cells under EPHOSS show higher proliferation rate compared to cells under physioxia and thus are sensitive to anti-proliferative agents. Thus, drugs that show effectiveness on cancer cells collected in ambient air and subjected to EPHOSS may not be effective or as relevant in vivo, results that could partially explain the limited clinical translation of laboratory findings. Evaluating cell signaling and effects of drugs on cancer cells under physiologic O2 prior to in vivo studies could substantially reduce cost and aid in drug discovery relevant to the actual physioxia/pathological status of the tumor cells in vivo.

Project description:Human APOE genotype is intricately involved in the homeostasis of the central nervous system, which is particularly evident from its association with neurodegenerative disease. Compared to APOE3, APOE4 is associated with greater cognitive decline in aging, poorer outcomes following stroke and traumatic brain injury, and is a major genetic risk factor for Alzheimer’s disease. Human APOE genotypes can differentially modulate neuronal function via both direct and indirect pathways. Of the indirect pathways, increasing evidence supports that APOE4-associated neurovascular dysfunction plays a significant role in neurodegeneration. For example, deficits in cerebral blood flow, neuronal-vascular coupling and blood-brain barrier integrity are observed with APOE4 both during aging and in Alzheimer’s disease patients; data that has been recapitulated in mouse models. Therefore, the identification of cellular mechanistic pathways that contribute to APOE-modulated vascular function is important for fundamental and disease-focused research. Highly specialized brain endothelial cells (BECs) are essential for virtually all neurovascular functions and, therefore, the influence of apoE on the cerebrovasculature likely involves alterations in brain endothelial cells function, directly or via supporting cells. ApoE synthesis has been demonstrated in multiple cell types in both the brain (e.g. astrocytes, pericytes and neurons) and periphery (e.g. hepatocytes and macrophages). Current research has largely focused on the effects of apoE produced by supporting cell types, particularly astrocytes and pericytes, on BEC function. However, less explored is whether BECs produce apoE to modulate their function. Data from limited studies on apoE production by BECs and APOE genotype-specific functional differences are conflicting. Evaluation of whether the APOE genotype of BECs modulates their function is important, as this could represent a novel mechanisms that contributes to altered cerebrovascular function in aging and AD. The goal of this study was to evaluate the role of human APOE genotype in modulating the phenotype and/or function of brain endothelial cells in vitro. To this end, we utilized primary brain endothelial cells isolated from APOE-targeted replacement mice that express human APOE3 or APOE4 under the endogenous mouse APOE promoter. BECs are highly specialized and functionally complex to maintain homeostasis of the central nervous system and APOE genotype could theoretically alter levels of any number of molecules and associated functions. Thus, without a priori knowledge we initially evaluated the extent that APOE modulated the transcriptomic profile of brain endothelial cells.

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	ApoE_03M_4.raw	Raw
	ApoE_03M_5.raw	Raw

Dataset Information

LC-MS/MS analysis of isolated murine brain endothelial cells (BECs) from 3-, 6-, 12-, and 18-months-old mice and Apoe-KO mice at 3 months followed by label-free quantification (LFQ)

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