The discovery of a fourth meninges: Potential implications for brain disorders

A new, breakthrough discovery by Møllgård and colleagues (2023) of a fourth meningeal membrane has (yet again) reshaped our fundamental understanding of the brain. Møllgård and colleagues term the layer subarachnoid lymphatic-like membrane (SLYM) – an immunologically active membrane situated in the subarachnoid space. Conventional teaching states that only three meningeal layers (pia, arachnoid, and dura mater) enclose the brain. Now, textbooks may have to be rewritten after the authors’ recent Science publication. The discovery adds to a long list of important findings in neuroimmunology over the last decades, which have challenged our idea of the brain as an “immune privileged” site (Rustenhoven and Kipnis, 2022). Classically, the blood–brain barrier (BBB) has been thought to protect the brain from the systemic immune response; however, we now know that the BBB does not provide complete immune privilege. Increased BBB permeability has been demonstrated in a range of conditions, including head trauma, severe systemic inflammation, multiple sclerosis and delirium (Elwood et al., 2017). Furthermore, immunocompetent cells termed microglia resides inside the brain, and interactions between the brain and peripheral immune cells have been extensively demonstrated (Dantzer, 2018). At the borders of the brain, the meninges have been shown to play a key role in CNS immunity (Rustenhoven and Kipnis, 2022). A few years ago, a delicate system of lymphatic vessels was discovered in the walls of the venous sinuses of the skull (Louveau et al., 2015). In the brain itself, researchers have identified the so-called “glymphatic” system, which facilitate flow of cerebrospinal fluid (CSF) (Iliff et al., 2012). More recently, it was uncovered how brain injury can stimulate production of immune cells in the skull bone marrow, which provides a reservoir of immune cells close to the brain (Mazzitelli et al., 2022) – and conversely, how depletion of specific immune cells in the skull and meninges directly modulates anxiety-like behavior, thereby linking meningeal immunity and psychopathology (Alves de Lima et al., 2020). While most research has focused on the potential disease-causing role of immune cells in the CNS, pioneering work has also demonstrated how a subset of T cells play an important role in brain homeostasis, neuronal repair and cognitive functioning (Schwartz and Raposo, 2014). In their recent paper, Møllgård and colleagues (2023) discovered the novel membrane using genetically modified mice expressing a florescent protein, which enabled identification of lymphatic tissue. When visualizing the subarachnoid space using two photon imaging, the authors noted an unknown, thin, continuous monolayer situated in the subarachnoid space between the pia and arachnoid mater (Fig. 1). The membrane expressed a lymphatic marker, podoplanin (PDPN), and a meningeal marker, CRABP2. Moving to humans, the authors confirmed the existence of a PDPNand CRABP2-positive membrane above the pia mater. PDPN is also a marker used to identify mesothelial tissue, which implies that SLYM provides the brain with a mesothelium that “cushions” the brain and reduce friction between brain and skull. Next, the authors used fluorescent microspheres to show that SLYM functions as a barrier, effectively dividing the CSF in the subarachnoid space in an outer and inner compartment, with the inner compartment being in close proximity to the brain surface. They confirmed that molecules >3 kDa (kilo Dalton) cannot cross SLYM, making the membrane impermeable to proteins and peptides such as immunoglobulins, amyloid beta, and tau (at least under normal conditions). In addition, a high number of leukocytes – comparable to that in dura mater – was visualized in SLYM, suggesting that the membrane plays a key role in CNS immunity. However, the origin and function of these immune cells remain unknown. Interestingly, it appeared that the number of immune cells in SLYM increased with the age of the mice and in response to immunological stress through stimulation with LPS (lipopolysaccharide, a bacterial antigen). The findings by Møllgård and colleagues (2023) need to be confirmed in future, independent experiments and many questions remain to be answered. For example, it is yet unknown whether SLYM extends into the vertebrae, how the division into an inner and outer

pioneering work has also demonstrated how a subset of T cells play an important role in brain homeostasis, neuronal repair and cognitive functioning (Schwartz and Raposo, 2014).
In their recent paper, Møllgård and colleagues (2023) discovered the novel membrane using genetically modified mice expressing a florescent protein, which enabled identification of lymphatic tissue. When visualizing the subarachnoid space using two photon imaging, the authors noted an unknown, thin, continuous monolayer situated in the subarachnoid space between the pia and arachnoid mater (Fig. 1). The membrane expressed a lymphatic marker, podoplanin (PDPN), and a meningeal marker, CRABP2. Moving to humans, the authors confirmed the existence of a PDPN-and CRABP2-positive membrane above the pia mater. PDPN is also a marker used to identify mesothelial tissue, which implies that SLYM provides the brain with a mesothelium that "cushions" the brain and reduce friction between brain and skull. Next, the authors used fluorescent microspheres to show that SLYM functions as a barrier, effectively dividing the CSF in the subarachnoid space in an outer and inner compartment, with the inner compartment being in close proximity to the brain surface. They confirmed that molecules >3 kDa (kilo Dalton) cannot cross SLYM, making the membrane impermeable to proteins and peptides such as immunoglobulins, amyloid beta, and tau (at least under normal conditions). In addition, a high number of leukocytescomparable to that in dura materwas visualized in SLYM, suggesting that the membrane plays a key role in CNS immunity. However, the origin and function of these immune cells remain unknown. Interestingly, it appeared that the number of immune cells in SLYM increased with the age of the mice and in response to immunological stress through stimulation with LPS (lipopolysaccharide, a bacterial antigen).
The findings by Møllgård and colleagues (2023) need to be confirmed in future, independent experiments and many questions remain to be answered. For example, it is yet unknown whether SLYM extends into the vertebrae, how the division into an inner and outer compartment impact overall CSF drainage and flow, and how SLYM relates to the glymphatic system. However, a key strength of the study is that the authors moved from mouse to human to confirm the presence of a previously unknown membrane in the subarachnoid space. The existence of SLYM in humans indicate that the membrane will most likely play a significant role in brain functioning and associated disorders. The observation that SLYM is immunologically active (and responds to systemic infections) adds to the complexity of the immune-CNS interface and gives us a new framework for investigating important, clinical questions. Severe and repeating infections increase the risk of a range of mental disorders, including schizophrenia and mood disorders (Benros et al., 2011;Benros et al., 2013). An interesting hypothesis is that activation of SLYMthrough psychological stress and acute or latent infections (such as herpes or toxoplasmosis) -may induce a chronic, lowgrade inflammatory state that affect brain function in some patients. Similarly, the thickening of SLYM with age could implicate the membrane in neurodegenerative disorders, such as Alzheimer's, while the "cushioning" effect of the membrane and its inflammatory properties suggest a role in the pathophysiology of traumatic brain injury. Moreover, recent years have seen growing interest in disorders such as autoimmune encephalitis, in which patients experience symptoms ranging from motor disturbances to psychosis due to autoantibodies directed against brain tissue. These disorders are typically diagnosed by measuring antibodies in CSF from a spinal tab. However, if SLYM confines immunoglobulins to the inner compartment of the subarachnoid space, might they be accessible to our diagnostic tools at all? Or are they only accessible in severe cases, with potential spillover through SLYM?
The paper by Møllgård and colleagues (2023) will undoubtably stimulate further studiesand if technical advances allow in vivo visualization of SLYM in humans, this could have important implications for our understanding of brain disorders and, potentially, inspire treatment strategies that may target the new-found structure.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
No data was used for the research described in the article.