Not So Fast—The Brain Has Three Meningeal Membranes After All

Earlier this year, a high-profile Science paper caused a stir with its claim that the brain has a fourth, previously unrecognized, meningeal membrane. Dubbed SLYM and containing Prox1+ cells, this membrane was proposed to divide the subarachnoid space into two compartments, according to researchers led by Kjeld Møllgård and Maiken Nedergaard at the University of Copenhagen, Denmark. Now, two new papers challenge this view, arguing that Prox1+ cells instead compose the inner layer of the arachnoid mater, with no intervening space.

  • Prox1+ fibroblasts form part of the inner layer of the arachnoid mater.
  • They are glued to arachnoid barrier cells via adherens junctions.
  • Tracer injections show the arachnoid mater acts as the sole barrier membrane.

In today’s Neuron, researchers led by Christer Betsholtz and Johanna Andrae at Uppsala University and Urban Lendahl at the Karolinska Institute, all in Sweden, describe RNA-Seq studies of mouse meningeal fibroblasts that identified six different expression profiles. Transgenic reporter labeling showed Prox1+ cells touching arachnoid barrier cells. Zooming in, electron microscopy revealed abundant adherens junctions connecting these layers.

Likewise, in the September 20 Nature Communications, researchers led by Britta Engelhardt at the University of Bern, Switzerland, detailed experiments with transgenic mice that express a fluorescent marker in their meninges. Live imaging of injected tracers showed that the arachnoid mater acted as the sole meningeal barrier. The two groups collaborated, with several authors appearing on both papers.

Some scientists said the new data settle the matter. “These two studies contradict the existence of SLYM. Together, using a state-of-the-art spectrum of techniques, they are very convincing,” Roxana Carare at the University of Southampton, U.K., wrote to Alzforum. Julie Siegenthaler at the University of Colorado Anschutz Medical Campus, Aurora, said she is now confident that Prox1+ cells are part of the arachnoid mater.

At the same time, researchers foresaw fresh questions about how the meninges work to control access to the brain. “The studies … add new valuable insight regarding the molecular and functional organization of the CNS meninges—and lay ground for further debate and controversy,” Per Kristian Eide at the University of Oslo, Norway, wrote to Alzforum. Siegenthaler noted implications for future studies of immune cell surveillance and cerebrospinal fluid flow. “The impact of these papers for the meninges biology field goes far beyond addressing recent controversies … The tools, datasets, and knowledge gained from these two studies will be vital for advancing studies on functional relevance of meningeal layer subtypes,” she wrote (comments below).

Meningeal Map. Transcriptome profiling reveals six distinct fibroblast profiles in the leptomeninges. Two (gold and tan) are in the pia mater, four (mauve, pink, red, and dark blue) in the arachnoid mater. Prox1+ cells (pink) make up the arachnoid’s inner layer. Dural fibroblasts (aqua) lie above the arachnoid, embedded in a collagen matrix (dots). [Courtesy of Pietilä et al., Neuron, 2023.]

Traditionally, neurologists have recognized three meningeal layers: the outer dura mater, middle arachnoid mater, and inner pia mater. The dura mater does not have a blood-brain barrier, and immune cells gather there. Below it, the arachnoid mater forms a tight blood-CSF barrier that protects the brain. Beneath that, the subarachnoid space provides room for blood vessels, and the thin pia mater covers the brain parenchyma. The arachnoid and pia mater together are collectively called the leptomeninges.

Nedergaard’s paper made a splash with its claim that SLYM formed a separate, impermeable membrane dividing the subarachnoid space. As evidence, the authors showed live imaging of fluorescent tracer injections that appeared to reveal distinct spaces above and below the Prox1+ cells (Jan 2023 news and extensive commentary below). The paper generated intense controversy on Alzforum and in Science magazine, where several groups submitted critical eLetters (Science, scroll to bottom). In a nutshell, commenters believed that tracers appearing above the Prox1+ cells had filled a subdural space created by the experimental manipulations, and did not define a separate subarachnoid space.

In particular, Betsholtz doubted that Møllgård and Nedergaard’s experiments defined a new subarachnoid compartment, because he already had data that Prox1+ cells tightly hugged the inner surface of the arachnoid mater. The goal of his lab’s study, which represents years of work, was to create an atlas of meningeal cell types and their locations as a foundational body of knowledge for further research on the arachnoid. His lab's paper was initially submitted before Møllgård et al. was published.

Joint first authors Riikka Pietilä, Liqun He, and Elisa Vázquez-Liébanas at Uppsala and Francesca Del Gaudio at Karolinska analyzed single-cell RNA-Seq data from 1,341 fibroblasts isolated from mouse leptomeninges, 4,061 from dura, and 407 from vascular fragments. They found six distinct profiles, which they named brain fibroblast (BFB) 1-6. BFB1 fibroblasts further divided into two subtypes, a and b. The transcriptomes closely matched previous RNA-Seq data from mouse brain (Saunders et al., 2018; Dani et al., 2021). 

How did these expression profiles relate to meningeal layers? The authors selected marker genes for each transcriptome and used a combination of in situ hybridization, immuno-electron microscopy, and transgenic reporter mice to localize the fibroblasts in cerebral cortical sections. They found that BFB1a fibroblasts resided around blood vessels in the parenchyma, while BFB1b fibroblasts made up the pia mater. The closely related BFB2 and BFB3 fibroblasts made up the inner arachnoid layer, with the latter expressing Prox1. BFB4 were the arachnoid barrier cells, and BFB5, just above them, were dural border cells. BFB6 fibroblasts were found in the choroid plexus.

Stitched Together Transmission electron micrograph of the arachnoid mater shows a variety of cellular connections, including gap (gj), adherens (aj), tight (tj), and tripartite cellular junctions (tcj), as well as focal adhesions (fa), holding together the layers of the arachnoid mater. Basement membrane (bm) and collagen (c) also connect layers; asterisks mark the boundary between inner and outer arachnoid layers. [Courtesy of Pietilä et al., Neuron, 2023.]

In all experiments, Prox1+ fibroblasts were connected to other arachnoid mater cells. Transmission electron microscopy showed them forming adherens junctions with arachnoid barrier cells (see image above). The visual presence of these junctions was further supported by expression of VE-cadherin and connexins, components of these junctions, in BFB2, BFB3, and BFB4 transcriptomes from these layers of the arachnoid. Betsholtz told Alzforum that all these data make it unlikely that there is an intervening space between barrier and Prox1+ cells that collapsed during postmortem tissue preparation, as had been claimed in the controversy following Møllgård et al., 2023 (see commentary). Instead, the arachnoid layers are glued together via different types of junction to form a single membrane.

Stuck Like Glue. Electron microscopy of mouse arachnoid mater revealed that dural border cells (top) are loosely associated with the arachnoid membrane, while the double layers of arachnoid barrier cells (middle) are bound to each other via tight junctions (tj), and are fastened to the inner arachnoid (bottom) via adherens junctions (aj). [Courtesy of Pietilä et al., Neuron, 2023.]

To get a closer look at Prox1+ cells, Pietilä and colleagues used Prox1-EGFP reporter mice, and immunostained cortical sections for a new arachnoid barrier cell marker they had identified, the membrane-bound enzyme dipeptidyl peptidase IV (DPP4). Not only did this show the two layers closely apposed, it also revealed that Prox1+ cells are discontinuous, possibly intermixing with BFB2 fibroblasts to form the inner arachnoid layer, Betsholtz said.

Double Layer. Arachnoid barrier cells overlay each other in a double layer (false color reveals individual cells). Where three cells meet (arrows, inset far right), tripartite junctions hold them together. Arrowheads mark caveolae. [Courtesy of Pietilä et al., Neuron, 2023.]

The study offered other insights into the composition of the arachnoid mater. For example, it showed that arachnoid barrier cells form a double layer, with cell-to-cell contacts on the lower layer covered by the body of a third cell on the upper layer. The whole is sewn together with tricellular junctions, a type of tight junction (see image above). This arrangement enables the barrier cells to form an impermeable shield, Betsholtz noted. “We learned something new about the arachnoid barrier,” he told Alzforum.

In addition, Betsholtz noted that the data on membrane-bound transporters and enzymes may inform studies of how material passes into and out of the brain. A spate of recent papers proposed various types of exchange of fluids, molecules, and cells across the arachnoid mater (Oct 2019 news; Jan 2020 news; Jun 2021 news; Jul 2021 news).

Single Barrier. The arachnoid mater (green, AM) forms an impermeable barrier between blood (gray) in the dura mater (blue), and CSF (red) in the subarachnoid space (SAS). The pial membrane (green) defines the floor of the SAS, and surrounds blood vessels (BV) that cross this space. [Courtesy of Mapunda et al., Nature Commun, 2023.]

For their part, Engelhardt and colleagues explored how the arachnoid barrier works. While using transgenic vascular-endothelial (VE)-cadherin-GFP mice to study endothelial cells, they were surprised to find that the marker also lit up the leptomeninges. In fact, as shown by Betsholtz’s RNA-Seq data, VE-cadherin is expressed by BFB1, BFB2, BFB3, BFB4, and BFB5 fibroblasts, that is, by all cells in the pia and arachnoid. Because the marker visualized both the pia and the arachnoid mater, it defined both the floor and ceiling of the subarachnoid space, Engelhardt told Alzforum. This makes these mice useful for functional studies, she noted.

Joint first authors Josephine Mapunda and Javier Pareja injected fluorescent tracers into these mice and followed their path by two-photon live imaging. They observed that tracers injected into the bloodstream appeared in the dural membrane, but were stopped by the top green fluorescent layer representing the arachnoid mater. Tracers injected into the cerebrospinal fluid, on the other hand, filled the subarachnoid space between the arachnoid and pia mater, but did not cross into the dura (see image above). Together, these tracers showed the existence of a single barrier between blood and CSF—the arachnoid.

Double Layer. Immunostaining for the tight junction protein E-cadherin (pink) that marks arachnoid barrier cells reveals a continuous layer, in close contact with VE-cadherin reporter cells (green). Asterisks denote blood vessels traveling through the subarachnoid space. [Courtesy of Mapunda et al., Nature Commun, 2023.]

Live imaging, as used by both the Engelhardt and Nedergaard labs, has a low resolution of two to three microns. Hence it does not permit scientists to pinpoint cellular layers that are but a micron thick. For this reason, Mapunda, Pareja, and colleagues followed up with immunostaining to fill in details about the arachnoid barrier. In the VE-cadherin reporter mice, immunostaining for E-cadherin, a tight junction protein expressed by arachnoid barrier cells, revealed the two markers forming contiguous layers. The VE-cadherin+ cells hugged the E-cadherin+ layer, exactly as Betsholtz and colleagues found (see image above).

Arachnoid Architecture. The barrier cells of the outer layer (magenta) are in close contact with cells that express both Prox1 (white) and VE-cadherin (green, yellow arrowhead). VE-cadherin is expressed more strongly by vascular endothelial cells (white arrowhead). Asterisk marks blood vessel lumen, nuclei are blue. [Courtesy of Mapunda et al., Nature Commun, 2023.]

To further test the relationship between these cells, Engelhardt and colleagues crossed VE-cadherin-GFP mice with Prox1 reporter mice. The two signals overlaid each other in the arachnoid mater. Once again, immunostaining for E-cadherin revealed Prox1+ cells clinging to arachnoid barrier cells (see image above).

Besides repudiating the existence of a SLYM layer separating the subarachnoid space, the study produced new insights into the barrier properties of the arachnoid and pia mater. For example, after inducing inflammation in transgenic mice, the authors watched via live imaging as T cells crawled along the pia mater. Occasionally, a T cell would flatten and cross the membrane, suggesting these cells needed to find specific sites that allowed them to transmigrate into the brain. “This has functional consequences for immune surveillance in the subarachnoid space,” Engelhardt told Alzforum.

Siegenthaler noted that the two papers together offer a much clearer picture of the molecular and cellular anatomy of the leptomeninges than scientists previously had. Even so, she noted that there is still more to learn about how the meninges are structured to control CSF flow and exit from the central nervous system. “All of these studies have stimulated a lot of interest in this important structure, and, hopefully, there will be more groups engaging in the meninges biology field,” she wrote.

When asked to comment on these new studies, Nedergaard wrote two sentences to Alzforum. “I am pleased to see that these authors have confirmed our finding of SLYM—a Prox1+ meningeal membrane below the arachnoid barrier layer. Their in vivo studies also confirm the separation of the two meningeal layers as we originally reported.” If this comment appears confusing, that might be because it is. Mapunda/Pareja et al. and Pietilä et al. report the opposite.—Madolyn Bowman Rogers

Comments

    • User Profile Image Roxana Carare
      University of Southampton School of Medicine
    • Posted:

    The study from Britta Englehardt’s group provides a very clear analysis of the morphological, molecular, and functional characteristics of the arachnoid. It also challenges the recently described impermeable SLYM. The present study convincingly shows that Prox1-tdTomato+ cells are located within the VE-cadherin layers of inner arachnoid and they did not form a continuous layer. The present study uses double reporter mice (VE-cadherin-GFP crossed with Prox1-tdTomato mice), allowing thus a very clear and accurate analysis in vivo as well as ex vivo.

    Using transcriptomics as well as electron microscopy, Pietilä et al. provide further evidence that SLYM is not molecularly and morphologically defined as a different entity.

    These two studies are indeed contradicting the existence of SLYM. Together, using a very convincing state-of-the-art spectrum of techniques, they are very convincing. The significance of the studies is multiple, in the context of the fact that the leptomeninges are important in drug delivery to the brain as well as in the pathogenesis of dilated perivascular spaces, a major feature of vascular cognitive disorders and even amyloid related imaging abnormalities (ARIA).

    View all comments by Roxana Carare

  2. These two studies add new valuable insight regarding the molecular and functional organization of the CNS meninges—and lay ground for further debate and controversy.

    Since the discovery in 2015 of functional meningeal lymphatic vessels capable of draining substances from cerebrospinal fluid (CSF) (Louveau et al., 2015; Aspelund et al., 2015), emerging evidence suggests a crucial role of meninges in CNS immune surveillance and clearance of substances from subarachnoid CSF (Da Mesquita, 2022; Da Mesquita et al., 2018; Da Mesquita et al., 2021; Cugurra et al., 2021; Mazzitelli et al., 2022; Ding et al., 2021; Rustenhoven and Kipnis, 2022). The existence of two-way passage of cells and substances between CSF of the subarachnoid space and dura mater/skull bone marrow obviously has major implications for our understanding of normal CNS function, and for evolvement of diseases such as neurodegenerative disease, neuro-inflammatory disease, or consequences of traumatic brain injury or stroke.

    The two reports touch upon important research questions that need to be resolved, namely relating to the functional organization of the subarachnoid space, and the barrier function of the arachnoid membrane, primarily the outer arachnoid barrier cell layer.

    In the pioneering work by Pietilä and collaborators, the authors challenge the traditional, rather simplistic view of the meningeal organization; here, they report on six different single-cell transcriptomic variants of meningeal fibroblasts (type 5 in dura border cells, type 4 in arachnoid barrier cells, types 3-2 in inner arachnoid cells, type 1 in pia, and a sixth type in parenchymal perivascular fibroblasts). This molecular differentiation of fibroblasts in the various cell layers suggests a complex functional organization of the meninges. These results should inspire further work into molecular organization of the arachnoid, as the output might have obvious clinical relevance, given the role of fibroblasts in fibrotic reactions after inflammation and trauma. Other possible areas include events following subarachnoid hemorrhage and neuroinflammation associated with tumors or neurodegeneration. 

    Earlier this year, a fourth meningeal layer between the pia and outer arachnoid barrier cell layer, referred to as the subarachnoid lymphatic-like membrane (SLYM), was reported (Møllgård et al., 2023). With their experimental setup, Pietilä et al. found no evidence for this fourth membrane; they rather suggest their observations support the concept of “a single SAS bordered by pial cells on the brain side and by the arachnoid on the skull side.” This view compares with a traditional concept that the subarachnoid space is one compartment with no directionality of CSF flow. In my view, the authors are wrong at this point, and I don’t agree that their findings justify such a generalized view. Further research using a variety of methodologies, including both the macro- and micro-perspective, is required. The subarachnoid space is functionally compartmentalized. However, to which degree the previously reported SLYM captures this needs to be further explored.  

    Regarding the barrier function of the arachnoid, Pietilä et al. report that the arachnoid barrier cell layer consists of a two-layer cell arrangement where cells connect by tight junctions, adherens junction and tricellular junctions. Such an organization might give an anatomical basis for an impermeable arachnoid membrane. A traditional view states that the arachnoid is impermeable to CSF causing the CSF to be contained within the subarachnoid space (Weller et al., 2018), because cells of the outer barrier layer of the arachnoid are joined by tight junctions that were considered to establish a barrier to the passage of CSF out of the subarachnoid space.

    The study by Mapunda et al. supports this traditional view. They boldly state that the recent studies reporting on two-way passage of cells and substances between the subarachnoid space and dura mater/skull bone marrow have “omitted consideration of the barrier properties of the arachnoid mater, which forms a barrier between the dura mater and the SAS.” In support of their view, they used two-photon imaging in vascular endothelial (VE) cadherin-green fluorescent protein (GFP) knock-in mice to explore the barrier properties of arachnoid and pia to substances and cells. Cadherin is a cell adhesion molecule enabling cells to adhere. They identified a VE-cadherin-GFP+ cellular layer both just beneath the dura and at a location corresponding to the pia, and in trabecula crossing the subarachnoid space. From this, they claim that the previously reported SLYM (Møllgård et al., 2023), characterized by positive PROX-1 expressing meningeal cells, is instead part of the inner arachnoid cell layer without any barrier function, and state that the lack of simultaneous visualization of arachnoid membrane with PROX-1 expression caused a wrong interpretation of a fourth meningeal layer.

    All experimental setups have limitations, including the setup of Mapunda et al.; caution should therefore be made when criticizing previous reports about crosstalk of cells and substances between CSF and dura mater/skull bone marrow across the arachnoid. However, given the possible implications, this is a field for further intensive research.

    Lastly, Mapunda et al. found that the arachnoid barrier layer was impermeable to both low molecular weight molecules (tracers 3kDa) and large molecular weight tracers (40-66 kDa tracers), leading the authors to conclude that the outer arachnoid barrier layer due to its tight barrier properties is impermeable also to low molecular weight substances. In my view, this conclusion underlines the need for addressing research questions from different perspectives, including the macro perspective.

    In this regard, we have sstudied how a CSF tracer, the magnetic resonance imaging (MRI) contrast agent gadobutrol (a hydrophilic 604 molecular weight substance), injected to the intrathecal space enriches the subarachnoid space and passes further to the inside of the dura nearby the superior sagittal sinus, denoted the parasagittal dura (PSD) (Ringstad and Eide, 2020), and even passes to the skull bone marrow (Ringstad and Eide, 2022) and enriches extracranial lymph nodes (Eide et al., 2018). As can be seen in Figure 1 to the left, the parasagittal dura (PSD; yellow color) is irregular. We further showed that the volume of PSD shows a high degree of inter-individual variation (Melin et al., 2023). These observations clearly show that the arachnoid is not impermeable to substances in the CSF, at least not to low molecular weight substances. Therefore, human tracer studies do not support the traditional view of an impermeable arachnoid membrane. However, given the irregular form of PSD, the anatomical region of dura mater studied in experimental setups would have major impact on the observations.

     

    Figure. 1. In humans, a CSF tracer injected to the intrathecal space enriches the subarachnoid CSF space, as well as the dura mater itself nearby the superior sagittal sinus, denoted the parasagittal dura (PSD), demonstrating direct passage from the CSF (shown as turquoise color in the image to the left), via arachnoid to the dura mater (yellow color). As indicated in the graph to the right, enrichment of the PSD starts after a few hours and peaks after several hours (about 24 hours). The CSF tracer is an MRI contrast agent, gadobutrol, which is a hydrophilic 604 MW molecule. [Courtesy of Ringstad and Eide, Nature Communications, 2020.] 

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    Aspelund A, Antila S, Proulx ST, Karlsen TV, Karaman S, Detmar M, Wiig H, Alitalo K. A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules. J Exp Med. 2015 Jun 29;212(7):991-9. Epub 2015 Jun 15 PubMed.

    Da Mesquita S. Charting the meningeal lymphatic network. J Exp Med. 2022 Aug 1;219(8) Epub 2022 Jul 5 PubMed.

    Da Mesquita S, Louveau A, Vaccari A, Smirnov I, Cornelison RC, Kingsmore KM, Contarino C, Onengut-Gumuscu S, Farber E, Raper D, Viar KE, Powell RD, Baker W, Dabhi N, Bai R, Cao R, Hu S, Rich SS, Munson JM, Lopes MB, Overall CC, Acton ST, Kipnis J. Publisher Correction: Functional aspects of meningeal lymphatics in ageing and Alzheimer's disease. Nature. 2018 Dec;564(7734):E7. PubMed.

    Da Mesquita S, Papadopoulos Z, Dykstra T, Brase L, Farias FG, Wall M, Jiang H, Kodira CD, de Lima KA, Herz J, Louveau A, Goldman DH, Salvador AF, Onengut-Gumuscu S, Farber E, Dabhi N, Kennedy T, Milam MG, Baker W, Smirnov I, Rich SS, Dominantly Inherited Alzheimer Network, Benitez BA, Karch CM, Perrin RJ, Farlow M, Chhatwal JP, Holtzman DM, Cruchaga C, Harari O, Kipnis J. Meningeal lymphatics affect microglia responses and anti-Aβ immunotherapy. Nature. 2021 May;593(7858):255-260. Epub 2021 Apr 28 PubMed.

    Cugurra A, Mamuladze T, Rustenhoven J, Dykstra T, Beroshvili G, Greenberg ZJ, Baker W, Papadopoulos Z, Drieu A, Blackburn S, Kanamori M, Brioschi S, Herz J, Schuettpelz LG, Colonna M, Smirnov I, Kipnis J. Skull and vertebral bone marrow are myeloid cell reservoirs for the meninges and CNS parenchyma. Science. 2021 Jun 3; PubMed.

    Mazzitelli JA, Smyth LC, Cross KA, Dykstra T, Sun J, Du S, Mamuladze T, Smirnov I, Rustenhoven J, Kipnis J. Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels. Nat Neurosci. 2022 May;25(5):555-560. Epub 2022 Mar 17 PubMed.

    Ding XB, Wang XX, Xia DH, Liu H, Tian HY, Fu Y, Chen YK, Qin C, Wang JQ, Xiang Z, Zhang ZX, Cao QC, Wang W, Li JY, Wu E, Tang BS, Ma MM, Teng JF, Wang XJ. Impaired meningeal lymphatic drainage in patients with idiopathic Parkinson's disease. Nat Med. 2021 Mar;27(3):411-418. Epub 2021 Jan 18 PubMed.

    Rustenhoven J, Kipnis J. Brain borders at the central stage of neuroimmunology. Nature. 2022 Dec;612(7940):417-429. Epub 2022 Dec 14 PubMed.

    Møllgård K, Beinlich FR, Kusk P, Miyakoshi LM, Delle C, Plá V, Hauglund NL, Esmail T, Rasmussen MK, Gomolka RS, Mori Y, Nedergaard M. A mesothelium divides the subarachnoid space into functional compartments. Science. 2023 Jan 6;379(6627):84-88. Epub 2023 Jan 5 PubMed.

    Weller RO, Sharp MM, Christodoulides M, Carare RO, Møllgård K. The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS. Acta Neuropathol. 2018 Mar;135(3):363-385. Epub 2018 Jan 24 PubMed.

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    View all comments by Per Kristian Eide

  3. Both these papers were submitted prior to the Science publication by Dr. Maiken Naadergard’s group reporting the fourth layer; I expect that both papers were in active revision. Therefore, the authors took an opportunity to address some of the controversial aspects of the Science paper with the tools and data that they uniquely had at their disposal due to their extensive ongoing work in this area. Therefore, these papers are not "reactive" but rather asked the question, “Can we find evidence in our ongoing studies that supports or does not support the claims of a functionally distinct fourth layer?”

    Building off the molecular marker Prox1 reported in the Science study and using some of the same technical approaches (Prox1-RFP, 2-photon live imaging, histology), both papers studied the identity and positioning of the Prox1+ layer, identified as a defining characteristic of the fourth layer. They conclude that Prox1+ cells are part of what is historically considered the arachnoid mater. They were unable, using 2-photon in vivo live imaging, electron microscopy and immunofluorescence, to detect a separate space created by Prox1+ cells reported in the Science paper.

    Notably, the single cell profiling by the Betsholtz’s group shows that tight junction expression and accessory proteins is limited to arachnoid barrier cells, supporting that arachnoid barrier cells are most likely the only meningeal cell layer with size-restrictive barrier properties.

    The Engelhardt group’s paper shows Prox1+ cells express adherens junction protein VE-Cadherin; however, pial cells also express VE-cadherin, form a near continuous sheet connected by adherens junctions yet the pial layer lacks size restrictive barrier properties. Collectively, these two papers provide important information about the Prox1+ arachnoid cell layer that allows scientists both in and outside the field to better evaluate the validity of the conclusions of the paper reporting a fourth meningeal layer.

    While the authors of these two papers were unable to find evidence of CSF-containing space between the Prox1+ layer and the arachnoid barrier layer, the experiment that would test this definitively is combined use of transgenic mouse lines that independently mark the E-cadherin+ arachnoid barrier and Prox1 layer for use in 2-photon studies, also ideally the dura. Further, combining tracer studies with transmission electron microscopy and immunogold to detect Prox1+ cells would permit high-resolution detection of where exactly CSF moves in or around cell layers and spaces in the leptomeninges, including Prox1+ cells. I think to better understand the functional relevance of Prox1+ arachnoid layer, or any meningeal layer, ablation studies to disrupt the layers would be ideal. 

    Both papers take different technical approaches to molecularly and functionally define the leptomeninges, an understudied structure with clear functional roles in CNS development, adult CNS function and a well-known site of neuroinflammation and barrier breakdown in disease states. The impact of these papers for the meninges biology field goes far beyond addressing recent controversies. In the paper by the Engelhardt group, the authors illustrate the utility of transgenic mouse lines to visualize meningeal cell layers and the cellular boundaries created by different layers. Their findings generated important new knowledge on immune cell surveillance, in particular around the pia, and movement of molecules in the leptomeninges. In the paper by the Betsholtz group, not only does this provide unprecedented insight into the molecular identity of leptomeningeal fibroblasts but also their positioning within the leptomeninges, including important new data on the organization of tricellular junctions in arachnoid barrier layer. The tools, datasets and knowledge gained from these two studies will be vital for advancing studies on functional relevance of meningeal layer subtypes.

    As I outlined in my rebuttal to the Science paper soon after it was published, I strongly suspected at the time that Prox1+ cells were most likely inner arachnoid cells based on extensive histological and electron microscopy data of leptomeninges cellular architecture that show the arachnoid has multiple cell layers, all connected by a variety of junctions (tight, adherens, gap). I had major doubts about this layer having barrier properties on its own, mostly due to the lack of tight junctions protein expression. But at the time, the characterization of Prox1-expressing cells was very limited. Now we have a lot more information because of these papers by the Engelhardt and Betsholtz groups and a much better picture of the molecular and cellular anatomy of the leptomeninges. I am now confident in my original suspicion that the Prox1+ cell layer is part of what is historically considered the arachnoid mater and lacks the functional components to be a barrier layer. I am also confident in my opinion that there are not large, previously unknown spaces in the leptomeninges nor a bifurcation of the subarachnoid space.

    That said, there is much more to learn about meninges structure and function. There is a lot more to learn about how the meningeal layers, defined both as the three main layers and as sublayers of the arachnoid and dura, are compartmentalized and structured to control CSF flow and exit from the CNS. All of these studies have stimulated a lot of interest in this important structure, and, hopefully, there will be more groups engaging in the meninges biology field. 

    View all comments by Julie Siegenthaler

  4. The meninges, a multilayered membrane structure that covers the surface of the brain and spinal cord, acts as a barrier to prevent uncontrolled molecular exchange and contributes to immune surveillance of the central nervous system. However, the cellular and molecular composition of the meningeal layers and their accessibility to immune cells are not well understood. These two studies took different approaches to shed light on (1) the anatomical structure and barrier properties of brain and spinal cord meningeal layers to CSF prefusion and immune cell infiltration using in vivo imaging in VE-cadherin GFP reporter mice (Mapunda et al.) and (2) the identification of transcriptionally distinct fibroblasts and their cellular localization in the individual layers of the meninges (R. Pietilä et al.).

    Traditionally, the meninges are thought to consist of three layers, the dura, arachnoid, and pia matter. Interestingly, both papers challenge the claims of a recently described fourth meningeal layer consisting of Prox1-positive cells subdividing the arachnoid space, described by Møllgård et al. Mapunda et al. provide evidence using imaging of VE-cadherin/Prox1 double reporter mice that Prox1-positive cells do not form a separate barrier within the subarachnoid space, but in fact are an integral and continuous part of the arachnoid mater. Pietilä et al. found transcriptomic and in situ hybridization evidence of tight and adherens junctions between the cells of the inner arachnoid layer, consistent with Mapunda et al.’s observations of tight and adherins junctions in VE-cadherin reporter mice. Pietilä et al. also explored the location of Prox1 mRNA, finding it specifically expressed in a subset of fibroblast-like cells (BFB3). Both studies mapped the location of Prox1-positive cells to the inner arachnoid layer. These observations argue against the existence of a fourth meningeal layer, and instead suggest a single coherent arachnoid cellular layer.

    These studies highlight that the anatomy and function of the meningeal layers may be highly complex, and do not rule out the possibility of structural, functional, or transcriptomic changes in disease or inflammatory conditions. Indeed, Mapunda et al. observed changes in the depth of the subarachnoid space in mice with experimental autoimmune encephalomyelitis (EAE), and Pietilä et al. observed ultrastructural changes in the meninges after experimental traumatic brain injury (TBI). Thus, upregulated inflammation in the CNS reported with various conditions including meningitis, Alzheimer’s disease and stroke, TBI, as well as potential crosstalk from systematic conditions, may affect the meningeal layer structure, integrity and functionality.

    Both manuscripts provide important contributions to our understanding of the transcriptomic, anatomical and functional composition of the meninges. Future studies should more thoroughly investigate the leptomeningeal barrier properties to physiological molecules such as cytokines, interactions with immune cells of the skull, meninges, and CNS. Understanding these functions may also aid in developing new methods of drug delivery into the CNS. Future research may further provide new insights into the debate around the meningeal layers and the role of Prox1-positive cells within the meninges.

    View all comments by Kassandra Kisler View all comments by Ruslan Rust View all comments by Berislav Zlokovic
    • User Profile Image Rupal Mehta
      Department of Pathology, RUSH Medical College
    • Posted:

    These new works further elucidate the molecular anatomy of mouse meninges, focusing on dorsal brain regions. The thorough analysis by Pietilä et al. combines histologic and transcriptomic analyses and elucidates intercellular junctional protein expression among the various meningeal cell types and layers, documenting the multilaminar arachnoid barrier makeup and structure. Mapunda and Pareja et al. present additional in vivo data that depicts in detail the meningeal layers in situ. The resolution of the live 2p images approximates ex vivo data. The VE-cadherin marker in these studies is important, as it will allow for better correlation of future in vivo and ex vivo evidence which has been a limitation of prior studies.

    The current studies also highlight the importance of traditional and advanced histology techniques and careful interpretation for elucidation of meningeal structure and function. Undervaluation of histology data and interpretation is a mistake as histological and physiological evidence must be correlated to definitively and comprehensively conclude routes of intracranial fluid movement in vivo. 

    I must admit that I agree with these authors as well as with Dr. Julie Siegenthaler and others who have alluded earlier to the fact that data reported by Møllgård et al. failed to clearly depict the E-cadherin-positive arachnoid barrier cell layer. Thus, the interpretation of a double SAS compartment is highly problematic and unproven in the study by Møllgård and colleagues. Upon my interpretation​, I cannot exclude that the "outer subarachnoid space” ​reported in Figure 2A of that study ​is actually artifactual subdural space infiltration by experimentally administered tracer. This may not necessarily indicate that some amount of fluid does not naturally transit across the subdural space, but I do not feel the Møllgård​ et al. study has definitively proven that, either, as the ​technique was nonphysiological and molecular anatomy data ​in that study was lacking.

    View all comments by Rupal Mehta
    • User Profile Image Rashi Mehta
      Rockefeller Neuroscience Institute of West Virginia University
    • Posted:

    These studies lay a groundwork for analysis of leptomeningeal structure and function as well as alterations in aging and disease. As these works focus primarily on dorsal cerebral cortical brain regions of mice, additional investigations are needed to further map the meninges across various brain regions and species. It will be very interesting to see what is learned next using these novel approaches in mice and to understand how new knowledge of rodent leptomeninges will inform on neurofluid physiology and mechanisms of disease in humans.

    View all comments by Rashi Mehta

  7. Mapunda et al. used two-photon imaging with VE-cadherin-GFP knock-in mice and discovered that VE-cadherin, previously considered a marker for vascular endothelial cells, is actually expressed in leptomeninges as well and borders the subarachnoid space filled with CSF. Furthermore, they demonstrated that Prox1-positive cells are indeed part of the VE-cadherin-positive arachnoid mater.

    The paper by Pietila et al., through single-cell RNA sequencing analysis, examined the transcriptome of meningeal fibroblasts and revealed six distinct groups of brain and leptomeningeal fibroblast transcriptomes referred to as BFB1-6, with Prox1 aligning with BFB3, representing the inner arachnoid. Both papers confirm the alignment between Prox1-positive meninges and arachnoid markers.

    These new two papers have shed light on the molecular anatomies of leptomeninges, which were previously poorly characterized. It is now expected that further investigations into the functional roles of leptomeninges under physiological and pathological conditions will become feasible.

    View all comments by Kaoru Yamada View all comments by Takeshi Iwatsubo
    • User Profile Image Ashutosh Kumar
      All India Institute of Medical Sciences-Patna
    • Posted:

    A team of human anatomists, clinicians, and basic scientists have come together to systematically examine the controversy of intermediate leptomeningeal layer or SLYM in the brain. A preprint of the document is at this open-access link: https://osf.io/5mhtu/?view_only=e5a2bf6004dd498db87587ec8c32df9e.

    View all comments by Ashutosh Kumar

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References

News Citations

  1. And Then There Were Four: A New Meningeal Membrane Discovered
  2. Do Immune Cells in the Meninges Help with … Memory?
  3. Attack of the Clones? Memory CD8+ T Cells Stalk the AD, PD Brain
  4. Private Stock—Brain Taps Skull Bone Marrow for Immune Cells
  5. More Evidence for Meningeal B Cells

Paper Citations

  1. Saunders A, Macosko EZ, Wysoker A, Goldman M, Krienen FM, de Rivera H, Bien E, Baum M, Bortolin L, Wang S, Goeva A, Nemesh J, Kamitaki N, Brumbaugh S, Kulp D, McCarroll SA. Molecular Diversity and Specializations among the Cells of the Adult Mouse Brain. Cell. 2018 Aug 9;174(4):1015-1030.e16. PubMed.
  2. Dani N, Herbst RH, McCabe C, Green GS, Kaiser K, Head JP, Cui J, Shipley FB, Jang A, Dionne D, Nguyen L, Rodman C, Riesenfeld SJ, Prochazka J, Prochazkova M, Sedlacek R, Zhang F, Bryja V, Rozenblatt-Rosen O, Habib N, Regev A, Lehtinen MK. A cellular and spatial map of the choroid plexus across brain ventricles and ages. Cell. 2021 Apr 27; PubMed.

External Citations

  1. Science

Further Reading

Primary Papers

  1. Pietilä R, Del Gaudio F, He L, Vázquez-Liébanas E, Vanlandewijck M, Muhl L, Mocci G, Bjørnholm KD, Lindblad C, Fletcher-Sandersjöö A, Svensson M, Thelin EP, Liu J, van Voorden AJ, Torres M, Antila S, Xin L, Karlström H, Storm-Mathisen J, Bergersen LH, Moggio A, Hansson EM, Ulvmar MH, Nilsson P, Mäkinen T, Andaloussi Mäe M, Alitalo K, Proulx ST, Engelhardt B, McDonald DM, Lendahl U, Andrae J, Betsholtz C. Molecular anatomy of adult mouse leptomeninges. Neuron. 2023 Dec 6;111(23):3745-3764.e7. Epub 2023 Sep 29 PubMed.
  2. Mapunda JA, Pareja J, Vladymyrov M, Bouillet E, Hélie P, Pleskač P, Barcos S, Andrae J, Vestweber D, McDonald DM, Betsholtz C, Deutsch U, Proulx ST, Engelhardt B. VE-cadherin in arachnoid and pia mater cells serves as a suitable landmark for in vivo imaging of CNS immune surveillance and inflammation. Nat Commun. 2023 Sep 20;14(1):5837. PubMed.

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