Age-related alterations in meningeal immunity drive impaired CNS lymphatic drainage [bulk LECs]

The meningeal lymphatic network—housed within the dural meninges surrounding the brain— is critical for cerebrospinal fluid (CSF) drainage. Through continuous brain interstitial fluid (ISF) mixing with CSF via the glymphatic system, this lymphatic network facilitates the removal of central nervous system (CNS) waste. During aging and in Alzheimer's disease (AD), attenuated meningeal lymphatic drainage promotes the buildup of toxic misfolded proteins—including amyloid beta—in the CNS. Alleviating this age-related meningeal lymphatic dysfunction represents a promising therapeutic strategy to alleviate AD pathology. However, the mechanisms underlying this lymphatic decline remain elusive. Here we demonstrate that age-related alterations in meningeal immunity contribute to meningeal lymphatic impairment. Single-cell RNA-sequencing of dural lymphatic endothelial cells in aged mice demonstrated a response signature to the cytokine IFN?, which was elevated in the aged dura due to meningeal T cell accumulation. Chronic elevation of IFN? in the meninges of young mice via AAV-mediated overexpression altered lymphatic adherans junctions and impaired CSF drainage to deep cervical lymph nodes—comparable to the deficits observed in aged mice. Direct disruption of lymphatic junctions via CSF-delivered VE-Cadherin disrupting antibodies was sufficient to phenocopy impairments in CSF drainage. Therapeutically, IFN? neutralization in aged mice alleviated age-related impairments in meningeal lymphatic function. These data suggest manipulation of meningeal immunity as a viable therapeutic target to normalize CSF drainage in aged mice and alleviate the pathology in AD mice associated with impaired waste removal. Overall design: C57BL/6 mouse primary lymphatic endothelial cells were purchased from Cell Biologics. Cells were maintained in T25 tissue culture flasks with gelatin-based coating solution (Cell Biologics) and Complete Human Endothelial Cell Medium (Cell Biologics). Cells were passaged with Trypsin/EDTA 0.25% Solution (Cell Biolgics) and plated at 15,000 cells/cm2 surface area in coated 6 well plates or 6 well chamber slides. Cells were grown ~72-96 hours until a confluent monolayer was formed. At completion, RNA was extracted using the RNAqueous™-Micro Total RNA Isolation Kit and DNAase performed using the Invitrogen Ambion TURBO DNA free Kit. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.