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 IFNγ, which was elevated in the aged dura due to meningeal T cell accumulation. Chronic elevation of IFNγ 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, IFNγ 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. 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.

脑膜淋巴管网络——坐落于脑周围的硬脑膜（dural meninges）中——对脑脊液（cerebrospinal fluid, CSF）的引流至关重要。通过胶质淋巴系统（glymphatic system）介导的脑间质液（brain interstitial fluid, ISF）与脑脊液持续混合，该淋巴管网络可促进中枢神经系统（central nervous system, CNS）代谢废物的清除。在衰老及阿尔茨海默病（Alzheimer’s disease, AD）进程中，脑膜淋巴引流减弱会促进中枢神经系统内毒性错误折叠蛋白（包括β淀粉样蛋白（amyloid beta））的蓄积。缓解此类年龄相关性脑膜淋巴功能障碍，是减轻阿尔茨海默病病理损伤的极具前景的治疗策略。然而，该淋巴管功能衰退的潜在机制仍未阐明。

本研究证实，年龄相关的脑膜免疫改变可导致脑膜淋巴管功能受损。对衰老小鼠硬脑膜淋巴管内皮细胞开展单细胞RNA测序（single-cell RNA-sequencing），结果显示其存在细胞因子干扰素γ（IFNγ）的应答特征；而由于脑膜T细胞蓄积，衰老硬脑膜中的IFNγ水平显著升高。通过腺相关病毒（AAV）介导的过表达，在年轻小鼠脑膜中长期升高IFNγ水平，可改变淋巴管黏着连接（adherens junctions），并损害脑脊液向颈深淋巴结的引流，该表型与衰老小鼠中观察到的功能缺陷高度相似。通过脑脊液递送的血管内皮钙黏蛋白（VE-Cadherin）干扰抗体直接破坏淋巴管连接，足以模拟脑脊液引流受损的表型。在治疗层面，对衰老小鼠进行IFNγ中和可缓解年龄相关性脑膜淋巴功能损伤。上述数据表明，调控脑膜免疫可作为可行的治疗靶点，以恢复衰老小鼠的脑脊液引流功能，并减轻与废物清除受损相关的阿尔茨海默病模型小鼠的病理改变。

小鼠经腹腔注射（intraperitoneal, i.p.）致死剂量的麻醉剂Euthasol（体积分数10%），随后经心脏灌注含0.025%肝素的磷酸盐缓冲液（PBS）。在枕骨后方断头处死小鼠，去除颅骨表面的皮肤与肌肉。使用精细镊子将脑膜从颅盖骨上剥离，全程将样本置于冰预冷的杜氏改良伊格尔培养基（DMEM）中。随后将脑膜置于1mL预热的消化缓冲液（含2%胎牛血清（FBS）、1mg/mL VIII型胶原酶（Sigma Aldrich）及0.5mg/mL脱氧核糖核酸酶I（DNase I，Sigma Aldrich）的DMEM）中，于37℃恒温振荡消化15分钟。将消化液经70μm细胞筛过滤，并用1mL完全培养基（含10%胎牛血清的DMEM）终止消化。再加入2mL流式细胞术缓冲液（FACS buffer），将样本以400×g离心5分钟，重悬于流式缓冲液后置于冰上。

将细胞重悬于含1:100稀释的CD16/32 Fc封闭抗体的流式缓冲液中，随后用CD31、CD45、Podoplanin及Lyve1抗体进行染色，洗涤后重悬于含DAPI的流式缓冲液中。使用FACSAria II流式细胞仪，将活细胞（DAPI阴性）、CD45阴性、CD31阳性、PDPN阳性、Lyve1阳性的单个脑膜淋巴管内皮细胞分选至含2μL 10×RNA裂解缓冲液（Takara）及5%核糖核酸酶抑制剂（RNAse out，Takara）的96孔板中，随后置于干冰上快速冷冻。

使用2μL单细胞裂解液，于96孔PCR板中开展文库制备。采用优化自Takara-Clontech SMARTer技术的方法制备双链cDNA（ds-cDNA），并将反应体系缩至5μL。该方法使用修饰后的寡聚dT引物，在polyA尾上游引入唯一的条形码（barcode）。具体步骤为：向裂解液中加入0.5μL含5%核糖核酸酶抑制剂的Takara稀释缓冲液，以及0.25μL 25μM FACSseq条形码引物，于72℃加热3分钟。随后向每孔加入2.25μL逆转录预混液，其中含1μL 5×第一链缓冲液、0.125μL 100mM二硫苏糖醇（DTT）、0.25μL 20mM dNTPs、0.25μL 50μM FACSseq TSO引物、0.125μL核糖核酸酶抑制剂及0.5μL SMARTscribe逆转录酶（Takara）。将反应体系于42℃孵育90分钟，70℃孵育10分钟，随后维持在4℃。

将96孔板的所有孔内液体混合，使用1×比例的AMPure XP磁珠（Beckman Coulter）进行纯化，用39μL水洗脱cDNA。使用5μL 10×PCR缓冲液、2μL 10mM dNTPs、2μL 12μM FACSseq206TSO PCR引物、2μL 50×Advantage 2聚合酶（Takara）对cDNA进行扩增。PCR反应条件为：95℃预变性1分钟，16个循环（95℃ 15秒、65℃ 30秒、68℃ 6分钟），72℃延伸10分钟，随后维持在4℃。使用1.2×AMPure磁珠纯化扩增后的cDNA，通过Qubit dsDNA 209检测试剂盒定量，并使用生物分析仪进行电泳验证。

使用Covaris E220 210超声破碎仪对cDNA进行片段化，参数设置为：入射峰值功率18，占空比20%，每循环脉冲数50，超声时长120秒。将cDNA末端补平，在3'端加A尾，并连接Illumina测序接头。将连接后的片段使用标准Illumina i7引物进行16个循环的扩增，以引入索引序列，以及与cDNA合成阶段添加的细胞条形码对应的FACSseq Lib PCR 1.0引物。使用Illumina NextSeq测序平台对片段进行测序，采用双端测序模式：读段1（read 1）为25个循环，i7索引为7个循环，读段2（read 2）为100个循环。测序时为读段1使用定制测序引物，以读取每个细胞特有的10bp条形码。i7索引可实现多块板同时测序。读段2包含mRNA序列。每个年龄时间点设置两块板（S1和S2），分别在不同的测序运行中完成测序。