Abstract WP411: Identification and Characterization of Human Meningeal Lymphatics

The potential existence and roles of the meningeal lymphatic system in normal and pathological brain function have been a long-standing enigma. Recent evidence suggests that meningeal lymphatic vessels are present in both the mouse and human brain; in mice, they seem to play a role in clearing toxic amyloid-beta peptides, which have been connected with Alzheimer disease (AD). Here, we review the evidence linking the meningeal lymphatic system with human AD. Novel findings suggest that the recently described meningeal lymphatic vessels could be linked to, and possibly drain, the efferent paravascular glial lymphatic (glymphatic) system carrying cerebrospinal fluid, after solute and immune cell exchange with brain interstitial fluid. In so doing, the glymphatic system could contribute to the export of toxic solutes and immune cells from the brain (an exported fluid we wish to describe as glymph, similarly to lymph) to the meningeal lymphatic system; the latter, by being connected with downstream anatomic regions, carries the glymph to the conventional cervical lymphatic vessels and nodes. Thus, abnormal function in the meningeal lymphatic system could, in theory, lead to the accumulation, in the brain, of amyloid-beta, cellular debris, and inflammatory mediators, as well as immune cells, resulting in damage of the brain parenchyma and, in turn, cognitive and other neurologic dysfunctions. In addition, we provide novel insights into APOE4—the leading genetic risk factor for AD—and its relation to the meningeal lymphatic system. In this regard, we have reanalyzed previously published RNA-Seq data to show that induced pluripotent stem cells (iPSCs) carrying the APOE4 allele (either as APOE4 knock-in or stemming from APOE4 patients) express lower levels of (a) genes associated with lymphatic markers, and (b) genes for which well-characterized missense mutations have been linked to peripheral lymphedema. Taking into account this evidence, we propose a new conceptual framework, according to which APOE4 could play a novel role in the premature shrinkage of meningeal lymphatic vessels (meningeal lymphosclerosis), leading to abnormal meningeal lymphatic functions (meningeal lymphedema), and, in turn, reduction in the clearance of amyloid-beta and other macromolecules and inflammatory mediators, as well as immune cells, from the brain, exacerbation of AD manifestations, and progression of the disease. Altogether, these findings and their potential interpretations may herald novel diagnostic tools and therapeutic approaches in patients with AD.

In recent years, increasing evidence has suggested that meningeal lymphatic drainage plays a significant role in central nervous system (CNS) diseases. Studies have indicated that CNS diseases and conditions associated with meningeal lymphatic drainage dysfunction include neurodegenerative diseases, stroke, infections, traumatic brain injury, tumors, functional cranial disorders, and hydrocephalus. However, the understanding of the regulatory and damage mechanisms of meningeal lymphatics under physiological and pathological conditions is currently limited. Given the importance of a profound understanding of the interplay between meningeal lymphatic drainage and CNS diseases, this review covers seven key aspects: the development and structure of meningeal lymphatic vessels, methods for observing meningeal lymphatics, the function of meningeal lymphatics, the molecular mechanisms of meningeal lymphatic injury, the relationships between meningeal lymphatic vessels and CNS diseases, potential regulatory mechanisms of meningeal lymphatics, and conclusions and outstanding questions. We will explore the relationship between the development, structure, and function of meningeal lymphatics, review current methods for observing meningeal lymphatic vessels in both animal models and humans, and identify unresolved key points in meningeal lymphatic research. The aim of this review is to provide new directions for future research and therapeutic strategies targeting meningeal lymphatics by critically analyzing recent advancements in the field, identifying gaps in current knowledge, and proposing innovative approaches to address these gaps.

Meningeal lymphatic vessels (MLVs) have been identified to associate with various neurological diseases, such as traumatic brain injury (TBI), Alzheimer's disease (AD), Parkinson's disease, multiple sclerosis, and brain tumors. Damage to MLVs can exacerbate the pathological progression of these diseases and significantly impede therapeutic efficacy. Therefore, targeted repair of the damaged MLVs has emerged as an innovative strategy for treating these central nervous system (CNS) diseases. In this study, we find that inorganic Cu2-xSe nanoparticles, rather than conventional endogenous vascular endothelial growth factor-C (VEGF-C), can repair the damaged MLVs to restore their structure and functions. These nanoparticles not only promote the growth and development of lymphatic vessels but also enhance the drainage capacity of impaired MLVs, thereby facilitating the transport of immune cells and macromolecules through these vessels. Unlike the conventional repair of damaged MLVs, this is an instance where inorganic nanoparticles have been explored to stimulate the expression of VEGF-C and its receptor VEGFR3, thereby promoting the structural and functional recovery of these vessels. The enhanced drainage function of MLVs mediated by Cu2-xSe nanoparticles significantly alleviates the symptoms of pre-formed fibrils (PFFs)-induced Parkinson's disease in mice. Collectively, our findings demonstrate that inorganic nanoparticles can promote the growth and development of meningeal lymphatics like VEGF-C, providing a cost-effective and innovative strategy for repairing damaged MLVs to boost the therapeutic efficacy of CNS diseases.

The meningeal lymphatic system is critical for proper brain waste clearance and maintenance of the local meningeal neuroimmune niche. Meningeal lymphatic function is impaired during aging and neurodegenerative diseases and is associated with the development of cognitive impairment. There is a bidirectional communication between meningeal lymphatic function and dural T cells, whereby the drainage of T cells into the deep cervical lymph nodes depends on functional lymphatic vessels, and meningeal lymphatic function is itself modulated by T cell derived cytokines. We have previously shown that IL-17A producing T cells accumulate in the dura during deoxycorticosterone acetate (DOCA)-salt hypertension and contribute to the impairment of neurovascular coupling and cognitive function. However, the mechanisms by which these T cells accumulate in the dura remain to be determined. Here, we tested the hypothesis that the meningeal lymphatic vessels are impaired during DOCA-salt hypertension, thus contributing to dural T cell accumulation. 12-week old C57BL6 male mice were implanted with a s.c. DOCA pellet and received 0.9% NaCl in the drinking water for 21 days (n=4). Control mice underwent a sham surgery (n=4). Blood pressure was monitored by tail-cuff plethysmography. Meningeal lymphatic vessels were assessed by immunohistochemistry using the lymphatic vessel marker LYVE1. In a separate group of mice, we assessed meningeal lymphatic function by delivering Alexa647-Ovalbumin or IgG-RPE into the cisterna magna and measuring fluorescence in the deep cervical lymph nodes one hour after injection. DOCA-salt mice developed increased systolic blood pressure (control: 117.7 ± 4.9 vs DOCA: 141.5 ± 2.9mmHg). We observed no difference in the CD31+ dural sinus area between control and DOCA-salt (control: 11.482 ± 0.718 vs DOCA: 11.816 ± 0.662 mm 2 ). We found a significant increase in LYVE1+ area surrounding the dural sinuses in DOCA-salt (control: 0.508 ± 0.039 vs DOCA: 0.816 ± 0.105 mm 2 ; p=0.0329 by unpaired two-tailed t-test) and an increase in LYVE1+/CD31+ % area (control: 4.476 ± 0.462 vs DOCA: 6.840 ± 0.609 %; p=0.0213 by unpaired two-tailed t-test) which was not different between the sagittal sinus or the transverse sinus. Contrary to our hypothesis, our data suggests that the meningeal lymphatic vessels are expanded, not retracted, during DOCA-salt hypertension. The interaction between meningeal lymphatics and dura immunity in hypertension remains to be determined.

Here we show the interaction between the meningeal lymphatic system and the blood-brain barrier (BBB) function. In normal state, the meningeal lymphatic vessels are invisible on optical coherent tomography (OCT), while during the opening of the BBB, meningeal lymphatic vessels are clearly visualized by OCT in the area of cerebral venous sinuses. These results give a significant impulse in the new application of OCT for the study of physiology of meningeal lymphatic system as well as sheds light on novel strategies in the prognosis of the opening of the BBB related with many central nervous system diseases, such as stroke, brain trauma, Alzheimers disease, etc.

Highly developed meningeal lymphatics remove waste products from the brain. Disruption of meningeal lymphatic vessels in a mouse model of amyloid pathology (5XFAD) accelerates the accumulation of amyloid plaques in the meninges and brain, and causes learning and memory deficits, suggesting that clearance of toxic wastes by lymphatic vessels plays a key role in neurodegenerative diseases. Here, we discovered that DSCR1 (Down syndrome critical region 1, known also as RCAN1, regulator of calcineurin 1) facilitates the drainage of waste products by increasing the coverage of dorsal meningeal lymphatic vessels. Furthermore, upregulation of DSCR1 in 5XFAD mice diminishes Aβ pathology in the brain and improves memory defects. Surgical ligation of cervical lymphatic vessels afferent to dcLN blocks the beneficial effects of DSCR1 on Aβ accumulation and cognitive function. Interestingly, intracerebroventricular delivery of AAV1-DSCR1 to 5XFAD mice is sufficient to rebuild the meningeal lymphatic system and re-establish cognitive performance. Collectively, our data indicate that DSCR1 facilitates the growth of dorsal meningeal lymphatics to improve drainage efficiency and protect against Alzheimer's disease (AD) pathologies, further highlighting that improving meningeal lymphatic function is a feasible treatment strategy for AD. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Meningeal Lymphatic System: A New Player in Neurophysiology

The considerable metabolic activity of the central nervous system (CNS) requires an efficient system of tissue drainage and detoxification. The CNS is however devoid of lymphatic vessels, a vasculature ensuring interstitial fluid drainage and immune survey in other organs. A unique system of drainage has recently been identified between the cerebrospinal fluid (CSF), brain interstitial fluids and meningeal lymphatic vessels. This system is coupling a cerebral "glymphatic" flow with a meningeal lymphatic vasculature. The "glymphatic" system includes perivascular spaces and astrocytes, and drains interstitial fluids, from and towards the CSF. Meningeal lymphatic vessels are functionally linked to the cerebral "glymphatic" efflux by clearing intracerebral macromolecules and antigens towards the peripheral lymphatic system. The "glymphatic"-"meningeal lymphatics" system is potentially offering new therapeutic targets to improve cerebral drainage and immune survey in human CNS diseases.

Characterization of dural sinus-associated lymphatic vasculature in human Alzheimer’s dementia subjects

The recent discovery of meningeal lymphatic vessels (MLVs) has revolutionized our understanding of immune regulation within the central nervous system (CNS), overturning the long-standing view of the brain as an immune-privileged organ. Glioblastoma (GBM), the most aggressive primary brain tumor, remains therapeutically intractable due to its highly immunosuppressive microenvironment and poor response to conventional and immune-based therapies. Emerging evidence suggests that MLVs play a crucial role in CNS immune surveillance, cerebrospinal fluid drainage, and solute clearance, all of which are directly linked to GBM pathophysiology. This review is motivated by the urgent need to explore novel therapeutic strategies that address GBM's immune escape and therapeutic resistance. We comprehensively analyze the bidirectional interactions between MLVs and GBM, including their role in antigen transport, T cell activation, and tumor dissemination. Furthermore, we evaluate the therapeutic potential of targeting MLVs through lymphangiogenic stimulation or as alternative routes for immune modulation and drug delivery. These approaches offer promising avenues to enhance anti-tumor immunity and may pave the way for next-generation treatment paradigms in GBM.

Central nervous system lymphatic drainage system (CNSLDS) is composed of glymphatic system, perivascular lymphatic drainage pathways and meningeal lymphatic vessels. Based on new findings of structures and functions of CNSLDS, CNSLDS is one of the mechanisms for promoting the clearance of β-amyloid (Aβ). CNSLDS functions physiologically as a route of drainage for Aβ from glymphatic system or perivascular lymphatic drainage pathways to meningeal lymphatic vessels, and the meningeal lymphatic vessels helps Aβ drainage to the nearby lymph nodes. In this review, we summarize the key component elements (structure and function) in the clearance of Aβ during the CNS lymphatic drainage. Also, we highlight their potential roles in the pathogenesis of Alzheimer's disease and their clinical importance in diagnosis and treatment of neurologic diseases associated with Aβ, including Alzheimer's disease. Key words: Central nervous system; Lymphatic drainage system; Glymphatic system; Meningeal lymphatic vessel; β-amyloid protein

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the leading cause of dementia. Recent research highlights meningeal lymphatics as key regulators in neurological diseases, suggesting that enhancing their drainage function could be a potential therapeutic strategy for AD. Our proof-of-concept study demonstrated that cranial bone transport can improve meningeal lymphatic drainage function and promote ischemic stroke recovery. This study defined cranial bone maneuver (CBM) technique. After osteotomy, a small circular bone flap was made and attached to an external fixator for subsequent maneuver in a controlled fashion for a defined period using 5xFAD mice. CBM treatment improved memory functions, reduced amyloid deposits, and promoted meningeal lymphatic drainage function. CBM induced cascades of inflammatory and lymphangiogenic processes in skull and meninges. Meningeal lymphatics are indispensable elements for the therapeutic effects of CBM. CBM might be a promising innovative therapy for AD management, warranting further clinical investigation. Cranial bone maneuver (CBM) alleviated memory deficits and amyloid depositions. CBM promoted meningeal lymphangiogenesis and lymphatic drainage function. The beneficial effects of CBM lasted for a long time following the CBM procedures. CBM induced cascades of inflammatory and lymphangiogenic processes in the meninges. Meningeal lymphatic vessels are indispensable elements for CBM therapeutic effects.

Recently, the presence of lymphatics has been demonstrated and characterized in the dura mater, which is in contrast to the well-accepted view indicating the lack of a classical lymphatic drainage system of the central nervous system (CNS). Moreover, the role of meningeal lymphatics in the pathogenesis of Alzheimer's disease and multiple sclerosis was suggested. However, the possible regulators of the developmental program and function of meningeal lymphatics remain unclear. Here, we aimed at characterizing the lymph flow dependence of the developmental program and function of the meningeal lymphatics. First, we demonstrated that lymphatics present in the dura mater are involved in the uptake and transport of macromolecules from the CNS. Meningeal lymphatics develop during the postnatal period which process involves the maturation of the vessels. The formation of mature meningeal lymphatics coincides with the increase of the drainage of macromolecules from the CNS to the deep cervical lymph nodes. Importantly, the structural remodeling and maturation of meningeal lymphatics is impaired in Plcγ2−/− mice with reduced lymph flow. Furthermore, macromolecule uptake and transport by the meningeal lymphatics are also affected in Plcγ2−/− mice. Collectively, lymph flow-induced mechanical forces are required for the postnatal formation of mature and functional meningeal lymphatic vessels. Defining lymph flow-dependence of the development and function of meningeal lymphatics may lead to better understanding of the pathogenesis of neurological diseases including Alzheimer's disease and multiple sclerosis.

Meningeal lymphatic vessels form a relationship between the nervous system and periphery, which is relevant in both health and disease. Meningeal lymphatic vessels not only play a key role in the drainage of brain metabolites but also contribute to antigen delivery and immune cell activation. The advent of novel genomic technologies has enabled rapid progress in the characterization of myeloid and lymphoid cells and their interactions with meningeal lymphatic vessels within the central nervous system. In this review, we provide an overview of the multifaceted roles of meningeal lymphatic vessels within the context of the central nervous system immune network, highlighting recent discoveries on the immunological niche provided by meningeal lymphatic vessels. Furthermore, we delve into the mechanisms of crosstalk between meningeal lymphatic vessels and immune cells in the central nervous system under both homeostatic conditions and neurodegenerative diseases, discussing how these interactions shape the pathological outcomes. Regulation of meningeal lymphatic vessel function and structure can influence lymphatic drainage, cerebrospinal fluid-borne immune modulators, and immune cell populations in aging and neurodegenerative disorders, thereby playing a key role in shaping meningeal and brain parenchyma immunity.

Comparison of neuropsychological profiles in patients with Alzheimer's disease and mixed dementia