SPATIAL TRANSCRIPTOMICS IDENTIFY HOMOLOGOUS MECHANISMS IN THE PATHOPHYSIOLOGY OF HUMAN AND E4FAD MOUSE CEREBRAL MICROBLEEDS
INTRODUCTION: Cerebral microbleeds (CMBs) are age-related vascular lesions, predisposing patients to a lifetime risk of stroke and cognitive decline. Little is known about the cellular architecture of the CMB lesional milieu as well as CMB pathobiological mechanisms. Understanding common histopathologic and transcriptomic signatures across heterogenous CMBs can aid in biomarker feature selection.  METHODS: These knowledge gaps were first addressed through the morphological and quantitative assessment of human CMB autopsy tissue and E4FAD mouse CMB tissue for cell types previously implicated in neurovascular diseases. Following, assessment of human and mouse lesional differentially expressed genes (DEGs) and pathway enrichment discovered important common and differential mechanisms in CMB pathogenesis. These findings facilitated identification of circulating proteins, which were quantified and underwent feature selection to identify the strongest diagnostic biomarker candidate molecules. In addition, a circulating DE miRNome found mechanistically-relevant homologous miRNA between human subjects and E4FAD mice as candidates.  RESULTS: Histopathologic studies identified significant increases in normalized counts of astrocytes and macrophages in human CMBs, as well as astrocytes and microglia in E4FAD murine CMBs. Spatial transcriptomics identified 781 human CMB DEGs and 448 mouse CMB DEGs, with 47 common DEGs (p<0.05, FDR-corrected). Pathway enrichment identified 110 common pathways related to vascular permeability, endothelial dysfunction, and inflammation (p<0.05, FDR-corrected). A supervised literature search then found 10 blood circulating proteins that had previously been assessed in CMB patients and healthy controls. Protein levels were assessed, and elastic net feature selection identified 7 circulating protein biomarker candidates. Through miRNome sequencing, two miRNAs (p<0.1, FDR-corrected) were discovered common between human CMB patients and E4FAD mice with mechanistic ties to IPA pathways enriched from the respective transcriptomes.  CONCLUSION: This is the first report to characterize the CMB microenvironment, provide mechanistic insight into its pathobiology, and identify biologically plausible circulating diagnostic biomarker features. Future studies should identify specific mechanisms related to lesion genesis, maturation, and progression to hemorrhagic stroke. In addition, studies aimed at validation of these features, and model validation in large multi-site cohorts are needed. The framework applied here paves the way for biomarker discovery in CMBs and other cerebrovascular diseases across varying contexts of use. Overall design: 12 microRNA samples from mouse blood plasma.Obtained from 6 E4FAD mice (5xFAD+/-; human APOE4+/+, C57BL/6 background) emulating Cerebral Microbleeds (CMB) and 6 wild-type mice (C57BL/6 background).