Age-related alterations in meningeal immunity drive impaired CNS lymphatic drainage [FACS 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: Mice were given a lethal dose of anesthetics via i.p. Euthasol (10% v/v) and transcardial perfusion performed with 0.025% heparin in PBS. Mice were decapitated immediately posterior to the occipital bone, and overlying skin and muscle was removed from the skull. Meninges were peeled from the skull cap using fine forceps, and placed in ice-cold DMEM for the entirety of collection. Meninges were then digested for 15 minutes at 37 °C with constant agitation using 1 mL of pre-warmed digestion buffer (DMEM, with 2% FBS, 1 mg/mL collagenase VIII (Sigma Aldrich), and 0.5 mg/mL DNase I (Sigma Aldrich)), filtered through a 70 µm cell strainer, and neutralized with 1 mL of complete medium (DMEM with 10% FBS). An additional 2 mL of FACS buffer was added, samples were centrifuged at 400 × g for five minutes, and resuspended in FACS buffer and kept on ice. Cells were resuspended in FACS buffer with 1:100 CD16/32 Fc block. They were stained with CD31, CD45, Podoplanin and Lyve1, washed, and resuspended in DAPI-containing FACS buffer. Single meningeal lymphatic endothelial cells were sorted as Live (DAPI-) CD45- CD31+ PDPN+ Lyve1+ cells using a FACS Aria II. Individual cells were sorted into 96-well plates containing 2 µL of 10X RNA lysis buffer (Takara) and 5% RNAse out (Takara) and rapidly frozen over dry ice. Library preparation was performed with 2 µL of single cell lysates arrayed in 96-well PCR plates. ds-cDNA was prepared using a protocol adapted from the Takara-Clontech SMARTer methods and scaled to a 5µL reaction volume. This method introduces a unique barcode upstream of the polyA tail using a modified oligo-dT primer. Briefly, 0.5µLof the Takara dilution buffer with 5% RNase inhibitor and 0.25µL of 25µM FACSseq barcode primer was added to the lysate and heated to 72 °C for 3 minutes. Then, 2.25 µL of the reverse transcription master mix was added to each well with 1 µL 5X first strand buffer, 0.125µL 100 mM DTT, 0.25 µL 20mM dNTPs, 0.25 µL, 50µM FACSseq TSO primer, 0.125 µLRNase inhibitor, and 0.5 µL SMARTscribe reverse transcriptase (Takara). The reaction was incubated at 42 °C for 90 minutes, 70 °C for 10minutes, then a 4 °C hold. All wells from the plate were then pooled and purified with Ampure XP beads (Beckman Coulter) with a 1X ratio. cDNA was eluted in 39µLwater. cDNA was amplified using 5 µL10X PCR buffer, 2 µL10mM dNTPs, 2 µL 12µM FACSseq206TSO PCR primer, 2 µL 50X Advantage 2 Polymerase (Takara). PCR conditions were 95 °C for 1 minute, 16 cycles of 95 °C for 15 seconds, 65 °C for 30 seconds, 68 °C for 6 minutes, 1 cycle of 72 °C for 10 minutes, followed by a 4 °C hold. cDNA was purified with 1.2XAmpure bead cleanup, measured with 209 Qubit dsDNA assay, and visualized on bioanalyzer. cDNA was fragmented using a Covaris E220 210 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, had an A base added to the 3' ends, and had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 16 cycles using a standard Illumina i7 primer to introduce an index sequence and FACSseq Lib PCR 1.0 specific to fragments containing the cell barcode added during cDNA synthesis. Fragments were sequenced on an Illumina NextSeq using paired end reads with 25 cycles for read 1, 7 cycles for the i7 index, and 100 cycles for the paired read. The sequencing run was performed with a custom sequencing primer for read 1 to read the 10bp barcode unique to each cell. The i7 index allows for multiple plates to be sequenced together. Read 2 contains the mRNA sequences. Two plates (S1 and S2) for each age time point were sequenced in separate sequencing runs.