Meningeal lymphatics-microglia axis regulates synaptic physiology [egfp/vegfc]

Microglia are thought to originate exclusively from primitive macrophage progenitors in the yolk sac (YS) and persist throughout life without contributions from definitive hematopoiesis. Here, using Ms4a3-Cre lineage tracing, pharmacological manipulation, and RNA-sequencing, we elucidated the presence and unique features of monocyte-derived macrophages (MDMs) in the brain parenchyma at baseline and during microglia repopulation. Using parabiosis and skull transplantation, we show that MDMs derived from both peripheral blood and skull bone marrow can repopulate microglia-depleted brains, and they display distinct transcriptional profiles compared to YS-derived microglia. Blood-derived macrophages possess a disease-associated microglia (DAM)-like phenotype, while skull-derived macrophages mirror injury-responsive microglia (IRM) and exhibit significant interactions with T cells. Repetitive microglia depletion and subsequent MDM engraftment in 5xFAD mice led to increased amyloid burden, suggesting differential responses of resident microglia and MDMs towards amyloid pathology. Our results reveal the heterogeneous origins and functions of microglia during cell turnover and neurodegeneration, offering insights that may guide microglia-targeted immunomodulatory strategies for neurological disorders. The subject AAV1-CMV-EGFP/VEGF-C-administrated 19-mo C57BL/6NCrL mice were cardio-perfused with 30 ml of 5 U/ml Heparin-PBS after 4-weeks from i.c.m. administration of AAV. Brains were harvested, located in heparin-PBS andcortices were dissected from 2 and 3 mice, respectively. Pulled Brains are minced with the sharp razor in RPMI 1640 medium with 2% FBS and digested by collagenase VIII and DNase (in 5 ml RPMI 1640 + 2 % FBS) for 45 minutes and titrated with P1000 pipettes once at 30 minutes after digestion starts, and one more at 45 minutes after. Samples were strained through a 70-um strainer, and 5 ml RPMI 1640 + 10% FBS medium was added to stop digestion and spun down in 450g for 5 minutes. Pellets were resuspended in 14% BSA and spun down in 800 g for 10 minutes with slow acceleration/declaration. Supernatnace was removed carefully, and the pellet was resuspended in MACS buffer (0.5% BSA, 2 mM EDTA containing PBS), followed by spinning down (450g, 5 mins). 50 ul of CD45-bead (Miltenyi, 130-052-301) was added with 450 ul MACS buffer, and CD45+ cells were prepared according to the manufacturer's protocol.