Leptomeningeal Cells Research Articles

Single-cell transcriptomic profiling of the hypothalamic median eminence during aging

Aging is a slow and progressive natural process that compromises the normal functions of cells, tissues, organs, and systems. The aging of the hypothalamic median eminence (ME), a structural gate linking neural and endocrine systems, may impair hormone release, energy homeostasis, and central sensing of circulating molecules, leading to systemic and reproductive aging. However, the molecular and cellular features of ME aging remain largely unknown. Here, we describe the transcriptional landscape of young and middle-aged mouse ME at single-cell resolution, revealing the common and cell type-specific transcriptional changes with age. The transcriptional changes in cell-intrinsic programs, cell-cell crosstalk, and cell-extrinsic factors highlight five molecular features of ME aging and also implicate several potentially druggable targets at cellular, signaling, and molecular levels. Importantly, our results suggest that vascular and leptomeningeal cells may lead the asynchronized aging process among diverse cell types and drive local inflammation and cellular senescence via a unique secretome. Together, our study uncovers how intrinsic and extrinsic features of each cell type in the hypothalamic ME are changed by the aging process, which will facilitate our understanding of brain aging and provide clues for efficient anti-aging intervention at the middle-aged stage.

Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.

Porphyromonas Gingivalis Infection Induces Synaptic Failure via Increased IL-1β Production in Leptomeningeal Cells.

Studies have reported that synaptic failure occurs before the Alzheimer's disease (AD) onset. The systemic Porphyromonas gingivalis (P. gingivalis) infection is involved in memory decline. We previously showed that leptomeningeal cells, covering the brain, activate glial cells by releasing IL-1β in response to systemic inflammation. In the present study, we focused on the impact of leptomeningeal cells on neurons during systemic P. gingivalis infection. The responses of leptomeningeal cells and cortical neurons to systemic P. gingivalis infection were examined in 15-month-old mice. The mechanism of IL-1β production by P. gingivalis infected leptomeningeal cells was examined, and primary cortical neurons were treated with P. gingivalis infected leptomeningeal cells condition medium (Pg LCM). Systemic P. gingivalis infection increased the expression of IL-1β in leptomeninges and reduced the synaptophysin (SYP) expression in leptomeninges proximity cortex in mice. Leptomeningeal cells phagocytosed P. gingivalis resulting in lysosomal rupture and cathepsin B (CatB) leakage. Leaked CatB mediated NLRP3 inflammasome activation inducing IL-1β secretion in leptomeningeal cells. Pg LCM decreased the expression of synaptic molecules, including SYP, which was inhibited by an IL-1 receptor antagonist pre-treatment. These observations demonstrate that P. gingivalis infection is involved in synaptic failure by inducing CatB/NLRP3 inflammasome-mediated IL-1β production in leptomeningeal cells. The periodontal bacteria-induced synaptic damage may accelerate the onset and cognitive decline of AD.

Neuronal VCP loss of function recapitulates FTLD-TDP pathology.

SUMMARYThe pathogenic mechanism by which dominant mutations in VCP cause multisystem proteinopathy (MSP), a rare neurodegenerative disease that presents as fronto-temporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), remains unclear. To explore this, we inactivate VCP in murine postnatal forebrain neurons (VCP conditional knockout [cKO]). VCP cKO mice have cortical brain atrophy, neuronal loss, autophago-lysosomal dysfunction, and TDP-43 inclusions resembling FTLD-TDP pathology. Conditional expression of a single disease-associated mutation, VCP-R155C, in a VCP null background similarly recapitulates features of VCP inactivation and FTLD-TDP, suggesting that this MSP mutation is hypomorphic. Comparison of transcriptomic and proteomic datasets from genetically defined patients with FTLD-TDP reveal that progranulin deficiency and VCP insufficiency result in similar profiles. These data identify a loss of VCP-dependent functions as a mediator of FTLD-TDP and reveal an unexpected biochemical similarity with progranulin deficiency.

Role of perivascular and meningeal macrophages in outcome following experimental subarachnoid hemorrhage

The distribution and clearance of erythrocytes after subarachnoid hemorrhage (SAH) is poorly understood. We aimed to characterize the distribution of erythrocytes after SAH and the cells involved in their clearance. To visualize erythrocyte distribution, we injected fluorescently-labelled erythrocytes into the prechiasmatic cistern of mice. 10 minutes after injection, we found labelled erythrocytes in the subarachnoid space and ventricular system, and also in the perivascular spaces surrounding large penetrating arterioles. 2 and 5 days after SAH, fluorescence was confined within leptomeningeal and perivascular cells. We identified the perivascular cells as perivascular macrophages based on their morphology, location, Iba-1 immunoreactivity and preferential uptake of FITC-dextran. We subsequently depleted meningeal and perivascular macrophages 2 days before or 3 hours after SAH with clodronate liposomes. At day 5 after SAH, we found increased blood deposition in mice treated prior to SAH, but not those treated after. Treatment post-SAH improved neurological scoring, reduced neuronal cell death and perivascular inflammation, whereas pre-treatment only reduced perivascular inflammation. Our data indicate that after SAH, erythrocytes are distributed throughout the subarachnoid space extending into the perivascular spaces of parenchymal arterioles. Furthermore, meningeal and perivascular macrophages are involved in erythrocyte uptake and play an important role in outcome after SAH.

Mutant Cx30-A88V mice exhibit hydrocephaly and sex-dependent behavioral abnormalities, implicating a functional role for Cx30 in the brain.

ABSTRACTConnexin 30 (Cx30; also known as Gjb6 when referring to the mouse gene) is expressed in ependymal cells of the brain ventricles, in leptomeningeal cells and in astrocytes rich in connexin 43 (Cx43), leading us to question whether patients harboring GJB6 mutations exhibit any brain anomalies. Here, we used mice harboring the human disease-associated A88V Cx30 mutation to address this gap in knowledge. Brain Cx30 levels were lower in male and female Cx30A88V/A88V mice compared with Cx30A88V/+ and Cx30+/+ mice, whereas Cx43 levels were lower only in female Cx30 mutant mice. Characterization of brain morphology revealed a disrupted ependymal cell layer, significant hydrocephalus and enlarged ventricles in 3- to 6-month-old adult male and female Cx30A88V/A88V mice compared with Cx30A88V/+ or Cx30+/+ sex-matched littermate mice. To determine the functional significance of these molecular and morphological changes, we investigated a number of behavioral activities in these mice. Interestingly, only female Cx30A88V/A88V mice exhibited abnormal behavior compared with all other groups. Cx30A88V/A88V female mice demonstrated increased locomotor and exploratory activity in both the open field and the elevated plus maze. They also exhibited dramatically reduced ability to learn the location of the escape platform during Morris water maze training, although they were able to swim as well as other genotypes. Our findings suggest that the homozygous A88V mutation in Cx30 causes major morphological changes in the brain of aging mice, possibly attributable to an abnormal ependymal cell layer. Remarkably, these changes had a more pronounced consequence for cognitive function in female mice, which is likely to be linked to the dysregulation of both Cx30 and Cx43 levels in the brain.

Generation of CSF1-Independent Ramified Microglia-Like Cells from Leptomeninges In Vitro.

Although del Río-Hortega originally reported that leptomeningeal cells are the source of ramified microglia in the developing brain, recent views do not seem to pay much attention to this notion. In this study, in vitro experiments were conducted to determine whether leptomeninges generate ramified microglia. The leptomeninges of neonatal rats containing Iba1+ macrophages were peeled off the brain surface. Leptomeningeal macrophages strongly expressed CD68 and CD163, but microglia in the brain parenchyma did not. Leptomeningeal macrophages expressed epidermal growth factor receptor (EGFR) as revealed by RT-PCR and immunohistochemical staining. Cells obtained from the peeled-off leptomeninges were cultured in a serum-free medium containing EGF, resulting in the formation of large cell aggregates in which many proliferating macrophages were present. In contrast, colony-stimulating factor 1 (CSF1) did not enhance the generation of Iba1+ cells from the leptomeningeal culture. The cell aggregates generated ramified Iba1+ cells in the presence of serum, which express CD68 and CD163 at much lower levels than primary microglia isolated from a mixed glial culture. Therefore, the leptomeningeal-derived cells resembled parenchymal microglia better than primary microglia. This study suggests that microglial progenitors expressing EGFR reside in the leptomeninges and that there is a population of microglia-like cells that grow independently of CSF1.

DDEL-03. LONG-TERM INTRAVENTRICULAR THERAPY ALTERNATING ETOPOSIDE AND LIPOSOMAL CYTARABINE: EXPERIENCE IN 75 CHILDREN AND ADOLESCENTS WITH MALIGNANT BRAIN TUMORS

BACKGROUNDMalignant brain tumors of childhood carry a high risk for leptomeningeal dissemination and tumor cells floating in the CSF are often not amenable to systemic and/or antiangiogenic chemotherapy. We report on our experience with an intraventricular therapy consisting of alternating cycles of liposomal cytarabine and etoposide.PATIENTS AND METHODSBetween 2004 and 2017, 75 patients aged 0.6 to 22 years (median 11) with various malignant brain tumors received intraventricular etoposide 0.25mg (<1year) - 0.5mg on five consecutive days alternating with liposomal cytarabine at a dose of 25mg (<3 years) - 50mg via an Ommaya reservoir.RESULTS5533 doses of etoposide (5–277/patient, median 141) corresponding to 1–56 five-day-cycles/patient alternating with 534 doses of liposomal cytarabine (1–21/patient, median 11) were administered. Treatment was given over a period of 1 – 146 months (median 73.5). Toxicities did occur but were infrequent and mostly mild. Since all patients received some sort of concurrent anti-cancer therapy, the efficacy of intrathecal therapy cannot be assessed independently. However, 29/75 patients are still alive, and none of the patients had tumor cells in the CSF at their last evaluation.CONCLUSIONIn conclusion, alternating intraventricular liposomal cytarabine and etoposide produced responses and proved to be an important adjunct for patients receiving drugs with a low penetrance into the CSF. Since production of liposomal cytarabine was discontinued in 2017 it remains to be determined whether substitution of the slow release formulation by aqueous cytarabine on days 1, 4, 8, and 11 may produce similar results.

Coniferyl Aldehyde Inhibits the Inflammatory Effects of Leptomeningeal Cells by Suppressing the JAK2 Signaling

Background The brain is in many ways an immunologically and pharmacologically privileged site because of the blood-brain barrier (BBB). But for chronic peripheral inflammation, inflammatory signals can be transmitted from the peripheral system into the central nervous system (CNS) through multiple channels and result in neuroinflammation. Leptomeningeal cells that form the BBB can trigger one signaling pathway by releasing cytokines to transmit inflammatory signals. Besides, the Janus kinase (JAK) family may have a certain function in the activation of leptomeninges. In the present study, we try to use coniferyl aldehyde (CA), a natural anti-inflammatory phenolic compound, to inhibit this inflammatory process and elucidate the underlying molecular mechanisms. Results Secretion of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) significantly increased after incubation with P. gingivalis. Moreover, TNF-α, IL-1β, and IL-6 levels were upregulated, and the JAK2 signaling was enhanced in leptomeningeal cells in a conditioned medium from activated macrophages, which leads to the immune response in microglia. However, this inflammatory effect of leptomeningeal cells was reversed by CA administration, accompanied by the decreased immune response in microglia. The western blot assay revealed that JAK2 phosphorylation was suppressed in leptomeningeal cells treated with CA. Conclusions This study demonstrates that activated macrophages by P. gingivalis markedly induce the release of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) from leptomeningeal cells, thereby activating the JAK2 signaling pathway and subsequently enhancing immune responses in microglia in the CNS. CA effectively inhibits the inflammatory effect of leptomeningeal cells via suppressing the JAK2 signaling pathway.

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection.

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

Leptomeningeal metastatic cells adopt two phenotypic states.

BackgroundLeptomeningeal metastasis (LM), or spread of cancer cells into the cerebrospinal fluid (CSF), is characterized by a rapid onset of debilitating neurological symptoms and markedly bleak prognosis. The lack of reproducible in vitro and in vivo models has prevented the development of novel, LM‐specific therapies. Although LM allows for longitudinal sampling of floating cancer cells with a spinal tap, attempts to culture patient‐derived leptomeningeal cancer cells have not been successful.AimWe, therefore, employ leptomeningeal derivatives of human breast and lung cancer cell lines that reproduce both floating and adherent phenotypes of human LM in vivo and in vitro.Methods and ResultsWe introduce a trypsin/EDTA‐based fractionation method to reliably separate the two cell subsets and demonstrate that in vitro cultured floating cells have decreased proliferation rate, lower ATP content, and are enriched in distinct metabolic signatures. Long‐term fractionation and transcriptomic analysis suggest high degree plasticity between the two phenotypes in vitro. Floating cells colonize mouse leptomeninges more rapidly and associate with shortened survival. In addition, patients harboring LM diagnosed with CSF disease alone succumbed to the disease earlier than patients with adherent (MRI positive) disease.ConclusionTogether, these data support mechanistic evidence of a metabolic adaptation that allows cancer cells to thrive in their natural environment but leads to death in vitro.

Ultrastructural and Molecular Characterization of Platelet-derived growth factor Beta-Positive Leptomeningeal Cells in the Adult Rat Brain.

The leptomeninges, referring to the arachnoid and pia mater and their projections into the perivascular compartments in the central nervous system, actively participate in diverse biological processes including fluid homeostasis, immune cell infiltrations, and neurogenesis, yet their detailed cellular and molecular identities remain elusive. This study aimed to characterize platelet-derived growth factor beta (PDGFR-β)-expressing cells in the leptomeninges in the adult rat brain using light and electron microscopy. PDGFR-β+ cells were observed in the inner arachnoid, arachnoid trabeculae, pia mater, and leptomeningeal sheath of the subarachnoid vessels, thereby forming a cellular network throughout the leptomeninges. Leptomeningeal PDGFR-β+ cells were commonly characterized by large euchromatic nuclei, thin branching processes forming web-like network, and the expression of the intermediate filaments nestin and vimentin. These cells were typical of active fibroblasts with a well-developed rough endoplasmic reticulum and close spatial correlation with collagen fibrils. Leptomeningeal PDGFR-β+ cells ensheathing the vasculature in the subarachnoid space joined with pial PDGFR-β+ cells upon entering the cortical parenchyma, yet perivascular PDGFR-β+ cells in these penetrating vessels underwent abrupt changes in their morphological and molecular characteristics: they became more flattened with loss of immunoreactivity for nestin and vimentin and deficient collagen deposition, which was indicative of inactive fibroblasts termed fibrocytes. In the cortical parenchyma, PDGFR-β immunoreactivity was almost exclusively localized to larger caliber vessels, and significantly decreased in capillary-like microvessels. Collectively, our data identify PDGFR-β as a novel cellular marker for leptomeningeal fibroblasts comprising the leptomeninges and perivascular adventitial cells of the subarachnoid and penetrating large-sized cortical vasculatures.

Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-β, the toxic driver of Alzheimer’s disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

Tmem119-EGFP and Tmem119-CreERT2 Transgenic Mice for Labeling and Manipulating Microglia.

Microglia are specialized brain-resident macrophages with important functions in health and disease. To improve our understanding of these cells, the research community needs genetic tools to identify and control them in a manner that distinguishes them from closely related cell types. We have targeted the recently discovered microglia-specific Tmem119 gene to generate knock-in mice expressing EGFP (JAX#031823) or CreERT2 (JAX#031820) for the identification and manipulation of microglia, respectively. Genetic characterization of the locus and qPCR-based analysis demonstrate correct positioning of the transgenes and intact expression of endogenous Tmem119 in the knock-in mouse models. Immunofluorescence analysis further shows that parenchymal microglia, but not other brain macrophages, are completely and faithfully labeled in the EGFP-line at different time points of development. Flow cytometry indicates highly selective expression of EGFP in CD11b+CD45lo microglia. Similarly, immunofluorescence and flow cytometry analyses using a Cre-dependent reporter mouse line demonstrate activity of CreERT2 primarily in microglia upon tamoxifen administration with the caveat of activity in leptomeningeal cells. Finally, flow cytometric analyses reveal absence of EGFP expression and minimal activity of CreERT2 in blood monocytes of the Tmem119-EGFP and Tmem119-CreERT2 lines, respectively. These new transgenic lines extend the microglia toolbox by providing the currently most specific genetic labeling and control over these cells in the myeloid compartment of mice.

Social defeat stress induces phosphorylation of extracellular signal-regulated kinase in the leptomeninges in mice.

AimsAnimal studies using various stress models have shown that excessive environmental stress induces depression? and anxiety?like behaviors through inflammatory responses in the brain and periphery. Although the leptomeningeal cells have multiple functions related to inflammatory responses in the brain, whether environmental stress influences the leptomeninges remains unknown. In this study, we aimed to examine phosphorylation of the extracellular signal‐regulated kinase (ERK) in the leptomeninges.MethodsWe subjected C57BL/6 male mice to a single episode of social defeat stress and analyzed the expression of phosphorylated ERK in the leptomeninges by immunohistochemistry.ResultsSocial defeat stress in mice induced phosphorylation of ERK in the leptomeninges, adjacent to vascular endothelial cells and the glia limitans. This ERK phosphorylation was maintained for at least one hour after the stress.ConclusionsThis study shows the effect of environmental stress on the leptomeninges for the first time and paves the way for elucidating its functional role in stress‐induced changes in neural functions.

Single-cell RNA sequencing of the mammalian pineal gland identifies two pinealocyte subtypes and cell type-specific daily patterns of gene expression.

The vertebrate pineal gland is dedicated to the production of the hormone melatonin, which increases at night to influence circadian and seasonal rhythms. This increase is associated with dramatic changes in the pineal transcriptome. Here, single-cell analysis of the rat pineal transcriptome was approached by sequencing mRNA from ~17,000 individual pineal cells, with the goals of profiling the cells that comprise the pineal gland and examining the proposal that there are two distinct populations of pinealocytes differentiated by the expression of Asmt, which encodes the enzyme that converts N-acetylserotonin to melatonin. In addition, this analysis provides evidence of cell-specific time-of-day dependent changes in gene expression. Nine transcriptomically distinct cell types were identified: ~90% were classified as melatonin-producing α- and β-pinealocytes (1:19 ratio). Non-pinealocytes included three astrocyte subtypes, two microglia subtypes, vascular and leptomeningeal cells, and endothelial cells. α-Pinealocytes were distinguished from β-pinealocytes by ~3-fold higher levels of Asmt transcripts. In addition, α-pinealocytes have transcriptomic differences that likely enhance melatonin formation by increasing the availability of the Asmt cofactor S-adenosylmethionine, resulting from increased production of a precursor of S-adenosylmethionine, ATP. These transcriptomic differences include ~2-fold higher levels of the ATP-generating oxidative phosphorylation transcriptome and ~8-fold lower levels of the ribosome transcriptome, which is expected to reduce the consumption of ATP by protein synthesis. These findings suggest that α-pinealocytes have a specialized role in the pineal gland: efficiently O-methylating the N-acetylserotonin produced and released by β-pinealocytes, thereby improving the overall efficiency of melatonin synthesis. We have also identified transcriptomic changes that occur between night and day in seven cell types, the majority of which occur in β-pinealocytes and to a lesser degree in α-pinealocytes; many of these changes were mimicked by adrenergic stimulation with isoproterenol. The cellular heterogeneity of the pineal gland as revealed by this study provides a new framework for understanding pineal cell biology at single-cell resolution.

MASP‐3: a new leptomeningeal protein in the lectin pathway

IntroductionMASP‐3, a protein of the innate immune response is attracting increasing interest, in particularly in relation to its regulatory function in the lectin pathway.ObjectiveProvide data that discriminate the source of MASP‐3 in cerebrospinal fluid.Materials and methods60 serum and cerebrospinal fluid samples from normal controls without organic disease with normal barrier function were obtained. MASP‐3 was assayed in samples of cerebrospinal fluid and serum with an ELISA, coated with anti MASP‐3 antibodies. It takes into account three variables: the molecular size‐dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MASP‐3 concentration correlation with the albumin CSF/serum quotient (Qalb), i.e., with CSF flow rate.ResultsMASP‐3 concentration in CSF was lower than its serum concentration but MASP‐3 concentration in CSF was at least three‐fold higher than expected for a molecular‐size dependent passage from blood. The smaller inter‐individual variation of MASP‐3 concentrations in CSF of the control group (CV =0,396) compared to the MBL concentrations in serum (CV=0.413) indicate an independent source of MASP‐3 in CSF. The absolute MASP‐3 concentration in CSF increases with increasing Qalb. Among brain‐derived proteins in CSF only the leptomeningeal proteins showed a (linear) increase with decreasing CSF flow rate in comparison to neuronal and glial proteins that are invariant to changes of Qalb.ConclusionsMASP‐3 in CSF is predominantly brain‐derived and all results pointed to the leptomeningeal cells as the source of the protein.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gene expression of A6-like subgroup of ATP-binding cassette transporters in mouse brain parenchyma and microvessels.

The A-subclass of ATP-binding cassette (ABC) transporters is a highly conserved superfamily of potent lipid transporters. Although the ABCA1-like subgroup of ABCA1-4, and A7 have been shown to mediate the transport of endogenous lipids, the roles of the ABCA6-like subgroup transporters, which have been identified as a unique gene cluster on human chromosome 17q24 (ABCA5, A6, A8, A9, and A10) and mouse chromosome 11 (Abca5, a6, a8a, a8b, and a9), remains largely unknown. The purpose of the present study was to clarify the spatial and temporal expression profiles of Abca6-like subgroup transporters in embryonic and postnatal mouse brains by a combination of in situ hybridization and quantitative polymerase chain reaction (PCR) using magnetically isolated brain vascular endothelial cells. In embryonic brains, the transcripts of Abca5, a8a and a8b were detected predominantly in the mantle zone, where postmitotic neurons differentiate. At the postnatal stages, they were expressed in various nuclei and neuronal layers. Abca9 mRNA was detected diffusely in the embryonic and postnatal brains and sequential and/or strong spotted signals were detected in the leptomeninges on the brain surface. PCR detected expression of Abca8a and Abca9 mRNAs in isolated vascular endothelial cells. Expression signals for Abca6 mRNA were hardly observed at any stages examined. These distinct spatio-temporal expression patterns of Abca6-like subgroup transporters may reflect their functional significance and diversity to regulate lipid transport, particularly in neurons, leptomeningeal cells, and vascular endothelial cells.

Leptomeninges-Derived Induced Pluripotent Stem Cells and Directly Converted Neurons From Autopsy Cases With Varying Neuropathologic Backgrounds.

Patient-specific stem cell technology from skin and other biopsy sources has transformed in vitro models of neurodegenerative disease, permitting interrogation of the effects of complex human genetics on neurotoxicity. However, the neuropathologic changes that underlie cognitive and behavioral phenotypes can only be determined at autopsy. To better correlate the biology of derived neurons with age-related and neurodegenerative changes, we generated leptomeningeal cell lines from well-characterized research subjects that have undergone comprehensive postmortem neuropathologic examinations. In a series of proof of principle experiments, we reprogrammed autopsy leptomeningeal cell lines to human-induced pluripotent stem cells (hiPSCs) and differentiated these into neurons. We show that leptomeningeal-derived hiPSC lines can be generated from fresh and frozen leptomeninges, are pluripotent, and retain the karyotype of the starting cell population. Additionally, neurons differentiated from these hiPSCs are functional and produce measurable Alzheimer disease-relevant analytes (Aβ and Tau). Finally, we used direct conversion protocols to transdifferentiate leptomeningeal cells to neurons. These resources allow the generation of in vitro models to test mechanistic hypotheses as well as diagnostic and therapeutic strategies in association with neuropathology, clinical and cognitive data, and biomarker studies, aiding in the study of late-onset Alzheimer disease and other age-related neurodegenerative diseases.

Intrathecal antibody distribution in the rat brain: surface diffusion, perivascular transport and osmotic enhancement of delivery.

It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain-CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally-infused antibodies exhibited size-dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full-length IgG could be enhanced by intrathecal co-infusion of hyperosmolar mannitol. Pores or stomata present on CSF-facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain-CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies. The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using exvivo fluorescence and invivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by ∼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.