A novel GFAP frameshift variant identified in a family with optico-retinal dysplasia and vision impairment.

Gain-of-function variants in GFAP leads to protein aggregation and is the cause of the severe neurodegenerative disorder Alexander Disease (AxD), while loss of GFAP function has been considered benign. Here, we investigated a six-generation family, where multiple individuals presented with gliosis of the optic nerve head and visual impairment. Whole genome sequencing (WGS) revealed a frameshift variant in GFAP (c.928dup, p.(Met310Asnfs*113)) segregating with disease. Analysis of human embryonic tissues revealed strong expression of GFAP in retinal neural progenitors. A zebrafish model verified that c.928dup does not result in extensive GFAP protein aggregation and zebrafish gfap loss-of-function mutants showed vision impairment and retinal dysplasia, characterized by a significant loss of Müller glia cells and photoreceptor cells. Our findings show how different mutational mechanisms can cause diverging phenotypes and reveal a novel function of GFAP in vertebrate eye development.

Response to Commentary on "Structural characterization of SLYM - a 4th meningeal membrane" by Julie Siegenthaler and Christer Betsholtz.

Histological studies have for decades documented that each of the classical meningeal membranes contains multiple fibroblast layers with distinct cellular morphology. Particularly, the sublayers of the arachnoid membranes have received attention due to their anatomical complexity. Early studies found that tracers injected into the cerebrospinal fluid (CSF) do not distribute freely but are restricted by the innermost sublayer of the arachnoid membrane. The existence of restrictions on CSF movement and the subdivision of the subarachnoid space into several distinct compartments have recently been confirmed by in vivo 2-photon studies of rodents, as well as macroscopic imaging of pigs and magnetic resonance imaging of human brain. Based on in vivo imaging and immunophenotyping characterization, we identified the structural basis for this compartmentalization of the subarachnoid space, which we term 'Subarachnoid lymphatic-like membrane', SLYM. The SLYM layer engages the subarachnoid vasculature as it approaches the brain parenchyma, demarcating a roof over pial perivascular spaces. Functionally, the separation of pial periarterial and perivenous spaces in the larger subarachnoid space is critical for the maintenance of unidirectional glymphatic clearance. In light of its close apposition to the pial surface and to the brain perivascular fluid exit points, the SLYM also provides a primary locus for immune surveillance of the brain. Yet, the introduction of SLYM, in terms of its anatomic distinction and hence functional specialization, has met resistance. Its critics assert that SLYM has been described in the literature by other terms, including the inner arachnoid membrane, the interlaminate membrane, the outer pial layer, the intermediate lamella, the pial membrane, the reticular layer of the arachnoid membrane or, more recently, BFB2-3. We argue that our conception of SLYM as an anatomically and functionally distinct construct is both necessary and warranted since its functional roles are wholly distinct from those of the overlying arachnoid barrier layer. Our terminology also lends clarity to a complex anatomy that has hitherto been ill-described. In that regard, we also note the lack of specificity of DPP4, which has recently been introduced as a 'selected defining marker' of the arachnoid barrier layer. We note that DPP4 labels fibroblasts in all meningeal membranes as well as in the trabecula arachnoides and the vascular adventitial layers, thus obviating its utility in meningeal characterization. Instead, we report a set of glymphatic-associated proteins that serve to accurately specify SLYM and distinguish it from its adjacent yet functionally distinct membranes.

Trigeminal ganglion neurons are directly activated by influx of CSF solutes in a migraine model.

Classical migraine patients experience aura, which is transient neurological deficits associated with cortical spreading depression (CSD), preceding headache attacks. It is not currently understood how a pathological event in cortex can affect peripheral sensory neurons. In this study, we show that cerebrospinal fluid (CSF) flows into the trigeminal ganglion, establishing nonsynaptic signaling between brain and trigeminal cells. After CSD, ~11% of the CSF proteome is altered, with up-regulation of proteins that directly activate receptors in the trigeminal ganglion. CSF collected from animals exposed to CSD activates trigeminal neurons in naïve mice in part by CSF-borne calcitonin gene-related peptide (CGRP). We identify a communication pathway between the central and peripheral nervous system that might explain the relationship between migrainous aura and headache.

Modeling of brain efflux: Constraints of brain surfaces.

Fluid efflux from the brain plays an important role in solute waste clearance. Current experimental approaches provide little spatial information, and data collection is limited due to short duration or low frequency of sampling. One approach shows tracer efflux to be independent of molecular size, indicating bulk flow, yet also decelerating like simple membrane diffusion. In an apparent contradiction to this report, other studies point to tracer efflux acceleration. We here develop a one-dimensional advection-diffusion model to gain insight into brain efflux principles. The model is characterized by nine physiological constants and three efflux parameters for which we quantify prior uncertainty. Using Bayes' rule and the two efflux studies, we validate the model and calculate data-informed parameter distributions. The apparent contradictions in the efflux studies are resolved by brain surface boundaries being bottlenecks for efflux. To critically test the model, a custom MRI efflux assay measuring solute dispersion in tissue and release to cerebrospinal fluid was employed. The model passed the test with tissue bulk flow velocities in the range 60 to 190 [Formula: see text]m/h. Dimensional analysis identified three principal determinants of efflux, highlighting brain surfaces as a restricting factor for metabolite solute clearance.

Glucagon does not directly stimulate pituitary secretion of ACTH, GH or copeptin.

Glucagon is best known for its contribution to glucose regulation through activation of the glucagon receptor (GCGR), primarily located in the liver. However, glucagon's impact on other organs may also contribute to its potent effects in health and disease. Given that glucagon-based medicine is entering the arena of anti-obesity drugs, elucidating extrahepatic actions of glucagon are of increased importance. It has been reported that glucagon may stimulate secretion of arginine-vasopressin (AVP)/copeptin, growth hormone (GH) and adrenocorticotrophic hormone (ACTH) from the pituitary gland. Nevertheless, the mechanisms and whether GCGR is present in human pituitary are unknown. In this study we found that intravenous administration of 0.2 mg glucagon to 14 healthy subjects was not associated with increases in plasma concentrations of copeptin, GH, ACTH or cortisol over a 120-min period. GCGR immunoreactivity was present in the anterior pituitary but not in cells containing GH or ACTH. Collectively, glucagon may not directly stimulate secretion of GH, ACTH or AVP/copeptin in humans but may instead be involved in yet unidentified pituitary functions.

Structural characterization of SLYM-a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Structural characterization of SLYM - a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Structural characterization of SLYM - a 4 th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4 th meningeal membrane, S ubarachnoid Ly mphatic-like M embrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

Delivery of gene therapy through a cerebrospinal fluid conduit to rescue hearing in adult mice.

Inner ear gene therapy has recently effectively restored hearing in neonatal mice, but it is complicated in adulthood by the structural inaccessibility of the cochlea, which is embedded within the temporal bone. Alternative delivery routes may advance auditory research and also prove useful when translated to humans with progressive genetic-mediated hearing loss. Cerebrospinal fluid flow via the glymphatic system is emerging as a new approach for brain-wide drug delivery in rodents as well as humans. The cerebrospinal fluid and the fluid of the inner ear are connected via a bony channel called the cochlear aqueduct, but previous studies have not explored the possibility of delivering gene therapy via the cerebrospinal fluid to restore hearing in adult deaf mice. Here, we showed that the cochlear aqueduct in mice exhibits lymphatic-like characteristics. In vivo time-lapse magnetic resonance imaging, computed tomography, and optical fluorescence microscopy showed that large-particle tracers injected into the cerebrospinal fluid reached the inner ear by dispersive transport via the cochlear aqueduct in adult mice. A single intracisternal injection of adeno-associated virus carrying solute carrier family 17, member 8 (Slc17A8), which encodes vesicular glutamate transporter-3 (VGLUT3), rescued hearing in adult deaf Slc17A8-/- mice by restoring VGLUT3 protein expression in inner hair cells, with minimal ectopic expression in the brain and none in the liver. Our findings demonstrate that cerebrospinal fluid transport comprises an accessible route for gene delivery to the adult inner ear and may represent an important step toward using gene therapy to restore hearing in humans.

The HOPX and BLBP landscape and gliogenic regions in developing human brain.

Outer radial glial cells (oRGs) give rise to neurons and glial cells and contribute to cell migration and expansion in developing neocortex. HOPX has been described as a marker of oRGs and possible actor in glioblastomas. Recent years' evidence points to spatiotemporal differences in brain development which may have implications for the classification of cell types in the central nervous system and understanding of a range of neurological diseases. Using the Human Embryonic/Fetal Biobank, Institute of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, HOPX and BLBP immunoexpression was investigated in developing frontal, parietal, temporal and occipital human neocortex, other cortical areas and brain stem regions to interrogate oRG and HOPX regional heterogeneity. Furthermore, usage of high-plex spatial profiling (Nanostring GeoMx® DSP) was tested on the same material. HOPX marked oRGs in several human developing brain regions as well as cells in known gliogenic areas but did not completely overlap with BLBP or GFAP. Interestingly, limbic structures (e.g. olfactory bulb, indusium griseum, entorhinal cortex, fimbria) showed more intense HOPX immunoreactivity than adjacent neocortex and in cerebellum and brain stem, HOPX and BLBP seemed to stain different cell populations in cerebellar cortex and corpus pontobulbare. DSP screening of corresponding regions indicated differences in cell type composition, vessel density and presence of apolipoproteins within and across regions and thereby confirming the importance of acknowledging time and place in developmental neuroscience.

A mesothelium divides the subarachnoid space into functional compartments.

The central nervous system is lined by meninges, classically known as dura, arachnoid, and pia mater. We show the existence of a fourth meningeal layer that compartmentalizes the subarachnoid space in the mouse and human brain, designated the subarachnoid lymphatic-like membrane (SLYM). SLYM is morpho- and immunophenotypically similar to the mesothelial membrane lining of peripheral organs and body cavities, and it encases blood vessels and harbors immune cells. Functionally, the close apposition of SLYM with the endothelial lining of the meningeal venous sinus permits direct exchange of small solutes between cerebrospinal fluid and venous blood, thus representing the mouse equivalent of the arachnoid granulations. The functional characterization of SLYM provides fundamental insights into brain immune barriers and fluid transport.

RRP7A links primary microcephaly to dysfunction of ribosome biogenesis, resorption of primary cilia, and neurogenesis.

Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. We identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, segregating with MCPH in a consanguineous family with 10 affected individuals. RRP7A is highly expressed in neural stem cells in developing human forebrain, and targeted mutation of Rrp7a leads to defects in neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in ribosomal RNA processing, primary cilia resorption, and cell cycle progression. Analysis of zebrafish embryos supported that the patient mutation in RRP7A causes reduced brain size, impaired neurogenesis and cell proliferation, and defective ribosomal RNA processing. These findings provide novel insight into human brain development and MCPH.

Aquaporin 1 and the Na+/K+/2Cl- cotransporter 1 are present in the leptomeningeal vasculature of the adult rodent central nervous system.

BACKGROUND: The classical view of cerebrospinal fluid (CSF) production posits the choroid plexus as its major source. Although previous studies indicate that part of CSF production occurs in the subarachnoid space (SAS), the mechanisms underlying extra-choroidal CSF production remain elusive. We here investigated the distributions of aquaporin 1 (AQP1) and Na+/K+/2Cl- cotransporter 1 (NKCC1), key proteins for choroidal CSF production, in the adult rodent brain and spinal cord. METHODS: We have accessed AQP1 distribution in the intact brain using uDISCO tissue clearing technique and by Western blot. AQP1 and NKCC1 cellular localization were accessed by immunohistochemistry in brain and spinal cord obtained from adult rodents. Imaging was performed using light-sheet, confocal and bright field light microscopy. RESULTS: We determined that AQP1 is widely distributed in the leptomeningeal vasculature of the intact brain and that its glycosylated isoform is the most prominent in different brain regions. Moreover, AQP1 and NKCC1 show specific distributions in the smooth muscle cell layer of penetrating arterioles and veins in the brain and spinal cord, and in the endothelia of capillaries and venules, restricted to the SAS vasculature. CONCLUSIONS: Our results shed light on the molecular framework that may underlie extra-choroidal CSF production and we propose that AQP1 and NKCC1 within the leptomeningeal vasculature, specifically at the capillary level, are poised to play a role in CSF production throughout the central nervous system.

Developmental differences in the expression of ABC transporters at rat brain barrier interfaces following chronic exposure to diallyl sulfide.

Many pregnant women and prematurely born infants require medication for clinical conditions including cancer, cardiac defects and psychiatric disorders. In adults drug transfer from blood into brain is mostly restricted by efflux mechanisms (ATP-binding cassette, ABC transporters). These mechanisms have been little studied during brain development. Here expression of eight ABC transporters (abcb1a, abcb1b, abcg2, abcc1, abcc2, abcc3, abcc4, abcc5) and activity of conjugating enzyme glutathione-s-transferase (GST) were measured in livers, brain cortices (blood-brain-barrier) and choroid plexuses (blood-cerebrospinal fluid, CSF, barrier) during postnatal rat development. Controls were compared to animals chronically injected (4 days, 200 mg/kg/day) with known abcb1a inducer diallyl sulfide (DAS). Results reveal both tissue- and age-dependent regulation. In liver abcb1a and abcc3 were up-regulated at all ages. In cortex abcb1a/b, abcg2 and abcc4/abcc5 were up-regulated in adults only, while in choroid plexus abcb1a and abcc2 were up-regulated only at P14. DAS treatment increased GST activity in livers, but not in cortex or choroid plexuses. Immunocytochemistry of ABC transporters at the CSF-brain interface showed that PGP and BCRP predominated in neuroepithelium while MRP2/4/5 were prominent in adult ependyma. These results indicate an age-related capacity of brain barriers to dynamically regulate their defence mechanisms when chronically challenged by xenobiotic compounds.

PDGF-B Is Required for Development of the Glymphatic System.

The glymphatic system is a highly polarized cerebrospinal fluid (CSF) transport system that facilitates the clearance of neurotoxic molecules through a brain-wide network of perivascular pathways. Herein we have mapped the development of the glymphatic system in mice. Perivascular CSF transport first emerges in hippocampus in newborn mice, and a mature glymphatic system is established in the cortex at 2 weeks of age. Formation of astrocytic endfeet and polarized expression of aquaporin 4 (AQP4) consistently coincided with the appearance of perivascular CSF transport. Deficiency of platelet-derived growth factor B (PDGF-B) function in the PDGF retention motif knockout mouse line Pdgfbret/ret suppressed the development of the glymphatic system, whose functions remained suppressed in adulthood compared with wild-type mice. These experiments map the natural development of the glymphatic system in mice and define a critical role of PDGF-B in the development of perivascular CSF transport.

Astrogliogenesis in human fetal brain: complex spatiotemporal immunoreactivity patterns of GFAP, S100, AQP4 and YKL-40.

The astroglial lineage consists of heterogeneous cells instrumental for normal brain development, function and repair. Unfortunately, this heterogeneity complicates research in the field, which suffers from lack of truly specific and sensitive astroglial markers. Nevertheless, single astroglial markers are often used to describe astrocytes in different settings. We therefore investigated and compared spatiotemporal patterns of immunoreactivity in developing human brain from 12 to 21 weeks post conception and publicly available RNA expression data for four established and potential astroglial markers - GFAP, S100, AQP4 and YKL-40. In the hippocampal region, we also screened for C3, a complement component highly expressed in A1-reactive astrocytes. We found diverging partly overlapping patterns of the established astroglial markers GFAP, S100 and AQP4, confirming that none of these markers can fully describe and discriminate different developmental forms and subpopulations of astrocytes in human developing brain, although AQP4 seems to be the most sensitive and specific marker for the astroglial lineage at midgestation. AQP4 characterizes a brain-wide water transport system in cerebral cortex with regional differences in immunoreactivity at midgestation. AQP4 distinguishes a vast proportion of astrocytes and subpopulations of radial glial cells destined for the astroglial lineage, including astrocytes determined for the future glia limitans and apical truncated radial glial cells in ganglionic eminences, devoid of GFAP and S100. YKL-40 and C3d, previously found in reactive astrocytes, stain different subpopulations of astrocytes/astroglial progenitors in developing hippocampus at midgestation and may characterize specific subpopulations of 'developmental astrocytes'. Our results clearly reflect that lack of pan-astrocytic markers necessitates the consideration of time, region, context and aim when choosing appropriate astroglial markers.

Comparison of Cultured Human Cardiomyocyte Clusters Obtained from Embryos/Fetuses or Derived from Human Embryonic Stem Cells.

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are used to study cardiogenesis and mechanisms of heart disease, and are being used in methods for toxiological screening of drugs. The phenotype of stem-cell-derived CMs should ideally resemble native CMs. Here, we compare embryonic/fetal CMs with hESC-derived CMs according to function and morphology. CM clusters were obtained from human embryonic/fetal hearts from elective terminated pregnancies before gestational week 12, and separated into atrial and ventricular tissues. Specific markers for embryonic CMs and primary cilia were visualized using immunofluorescence microscopy analysis. Contracting human embryonic cardiomyocyte (hECM) clusters morphologically and phenotypically resemble CMs in the embryonic/fetal heart. In addition, the contracting hECM clusters expressed primary cilia similar to that of cells in the embryonic/fetal heart. The electrophysiological characteristics of atrial and ventricular CMs were established by recording action potentials (APs) using sharp electrodes. In contrast to ventricular APs, atrial APs displayed a marked early repolarization followed by a plateau phase. hESC-CMs displayed a continuum of AP shapes. In all embryonic/fetal clusters, both atrial and ventricular, AP duration was prolonged by exposure to the KV11.1 channel inhibitor dofetilide (50 nM); however, the prolongation was not significant, possibly due to the relatively small number of experiments. This study provides novel information on APs and functional characteristics of atrial and ventricular CMs in first trimester hearts, and demonstrates that Kv11.1 channels play a functional role already at these early stages. These results provide information needed to validate methods being developed on the basis of in vitro-derived CMs from either hESC or iPSC, and although there was a good correlation between the morphology of the two types of CMs, differences in electrophysiological characteristics exist.

Neuroserpin expression during human brain development and in adult brain revealed by immunohistochemistry and single cell RNA sequencing.

Neuroserpin is a serine-protease inhibitor mainly expressed in the CNS and involved in the inhibition of the proteolytic cascade. Animal models confirmed its neuroprotective role in perinatal hypoxia-ischaemia and adult stroke. Although neuroserpin may be a potential therapeutic target in the treatment of the aforementioned conditions, there is still no information in the literature on its distribution during human brain development. The present study provides a detailed description of the changing spatiotemporal patterns of neuroserpin focusing on physiological human brain development. Five stages were distinguished within our examined age range which spanned from the 7th gestational week until adulthood. In particular, subplate and deep cortical plate neurons were identified as the main sources of neuroserpin production between the 25th gestational week and the first postnatal month. Our immunohistochemical findings were substantiated by single cell RNA sequencing data showing specific neuronal and glial cell types expressing neuroserpin. The characterization of neuroserpin expression during physiological human brain development is essential for forthcoming studies which will explore its involvement in pathological conditions, such as perinatal hypoxia-ischaemia and adult stroke in human.

Recent Developments in Understanding Barrier Mechanisms in the Developing Brain: Drugs and Drug Transporters in Pregnancy, Susceptibility or Protection in the Fetal Brain?

Efflux mechanisms situated in various brain barrier interfaces control drug entry into the adult brain; this review considers the effectiveness of these protective mechanisms in the embryo, fetus, and newborn brain. The longstanding belief that the blood-brain barrier is absent or immature in the fetus and newborn has led to many misleading statements with potential clinical implications. The immature brain is undoubtedly more vulnerable to damage by drugs and toxins; as is reviewed here, some developmentally regulated normal brain barrier mechanisms probably contribute to this vulnerability. We propose that the functional status of brain barrier efflux mechanisms should be investigated at different stages of brain development to provide a rational basis for the use of drugs in pregnancy and in newborns, especially in those prematurely born, where protection usually provided by the placenta is no longer present.