Fibrotic scar tissue limits central nervous system regeneration in adult mammals. The extent of fibrotic tissue generation and distribution of stromal cells across different lesions in the brain and ...spinal cord has not been systematically investigated in mice and humans. Furthermore, it is unknown whether scar-forming stromal cells have the same origin throughout the central nervous system and in different types of lesions. In the current study, we compared fibrotic scarring in human pathological tissue and corresponding mouse models of penetrating and non-penetrating spinal cord injury, traumatic brain injury, ischemic stroke, multiple sclerosis and glioblastoma. We show that the extent and distribution of stromal cells are specific to the type of lesion and, in most cases, similar between mice and humans. Employing in vivo lineage tracing, we report that in all mouse models that develop fibrotic tissue, the primary source of scar-forming fibroblasts is a discrete subset of perivascular cells, termed type A pericytes. Perivascular cells with a type A pericyte marker profile also exist in the human brain and spinal cord. We uncover type A pericyte-derived fibrosis as a conserved mechanism that may be explored as a therapeutic target to improve recovery after central nervous system lesions.
Aquaporin‐4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting brain microvessels. There is a rich literature on the role of AQP4 in experimental ...stroke. While its role in oedema formation following middle cerebral artery occlusion (MCAO) has been studied extensively, its specific impact on infarct volume remains unclear. This study investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery (dMCAO) occlusion. Compared to MCAO, this model induces smaller infarcts confined to neocortex, and less oedema. We show that AQP4 deletion significantly reduced infarct volume as assessed 1 week after dMCAO, suggesting that the role of AQP4 in stroke goes beyond its effect on oedema formation and dissolution. The reduction in infarct volume was associated with increased astrocyte reactivity in the peri‐infarct areas. No significant differences were observed in the number of microglia among the genotypes. These findings provide new insights in the role of AQP4 in ischaemic injury indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri‐infarct zone.
Key points
Aquaporin‐4 (AQP4) is the main water channel in brain and is enriched in perivascular astrocyte processes abutting microvessels.
A rich literature exists on the role of AQP4 in oedema formation following middle cerebral artery occlusion (MCAO).
We investigated the effects of total and partial AQP4 deletion on infarct volume in mice subjected to distal medial cerebral artery occlusion (dMCAO), a model inducing smaller infarcts confined to neocortex and less oedema compared to MCAO.
AQP4 deletion significantly reduced infarct volume 1 week after dMCAO, suggesting a broader role for AQP4 in stroke beyond oedema formation.
The reduction in infarct volume was associated with increased astrocyte reactivity in the peri‐infarct areas, while no significant differences were observed in the number of microglia among the genotypes.
These findings provide new insights into the role of AQP4 in stroke, indicating that AQP4 affects both infarct volume and astrocyte reactivity in the peri‐infarct zone.
figure legend Infarct size and astrocyte reactivity in wild‐type (WT) mice compared with AQP4 knockout (AQP4−/−) littermates, following distal medial cerebral artery occlusion. Genetic deletion of Aqp4 (AQP4−/−) results in reduced infarct size and heightened astrocyte reactivity, as evidenced by increased glial fibrillary acidic protein (GFAP) immunostaining in the infarct border zone.
Astrocyte endfeet are endowed with aquaporin‐4 (AQP4)‐based assemblies called orthogonal arrays of particles (OAPs) whose function is still unclear. To investigate the function of OAPs and of AQP4 ...tetramers, we have generated a novel “OAP‐null” mouse model selectively lacking the OAP forming M23‐AQP4 isoform. We demonstrated that AQP4 transcript levels were not reduced by using qPCR. Blue native (BN)/SDS‐PAGE and Western blot performed on OAP‐null brain and primary astrocyte cultures showed the complete depletion of AQP4 assemblies, the selective expression of M1‐AQP4‐based tetramers, and a substantial reduction in AQP4 total expression level. Fluorescence quenching and super‐resolution microscopy experiments showed that AQP4 tetramers were functionally expressed in astrocyte plasma membrane and their dimensions were reduced compared to wild‐type assemblies. Finally, as shown by light and electron microscopy, OAP depletion resulted in a massive reduction in AQP4 expression and a loss of perivascular AQP4 staining at astrocyte endfeet, with only sparse labeling throughout the brain areas analyzed. Our study relies on the unique property of AQP4 to form OAPs, using a novel OAP‐null mouse model for the first time, to show that (a) AQP4 assembly is essential for normal AQP4 expression level in the brain and (b) most of AQP4 is organized into OAPs under physiological conditions. Therefore, AQP4 tetramers cannot be used by astrocytes as an alternative to OAPs without affecting AQP4 expression levels, which is important in the physiological and pathological conditions in which OAP aggregation/disaggregation dynamics have been implicated.
Main Points
Orthogonal arrays of particle (OAP) assembly is essential for normal aquaporin‐4 expression level in the brain.
Most brain AQP4 is organized into OAPs under physiological conditions.
Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma ...membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.
Grant Support
This project was funded by the Research Council of Norway.
Background
Oxygen uptake through the gastrointestinal tract after oral administration of oxygenated water in humans is not ...well studied and is debated in the literature. Due to the paramagnetic properties of oxygen and deoxyhemoglobin, MRI as a technique might be able to detect changes in relaxometry values caused by increased oxygen levels in the blood.
Purpose
To assess whether oxygen dissolved in water is absorbed from the gastrointestinal tract and transported into the bloodstream after oral administration.
Study Type
A randomized, double‐blinded, placebo‐controlled crossover trial.
Population/Subjects
Thirty healthy male volunteers age 20–35.
Field Strength/Sequence
3T/Modified Look–Locker inversion recovery (MOLLI) T1‐mapping and multi fast field echo (mFFE) T2*‐mapping.
Assessment
Each volunteer was scanned in two separate sessions. T1 and T2* maps were acquired repeatedly covering the hepatic portal vein (HPV) and vena cava inferior (VCI, control vein) before and after intake of oxygenated or control water. Assessments were done by placing a region of interest in the HPV and VCI.
Statistical Test
A mixed linear model was performed to the compare control vs. oxygen group.
Results
Drinking caused a mean 1.6% 95% CI (1.1–2.0% P < 0.001) increase in T1 of HPV blood and water oxygenation attributed another 0.70% 95% confidence interval (CI) (0.07–1.3% P = 0.028) increase. Oxygenation did not change T1 in VCI blood. Mean T2* increased 9.6% 95% CI (1.7–17.5% P = 0.017) after ingestion of oxygenated water and 1.2% 95% CI (−4.3–6.8% P = 0.661) after ingestion of control water. The corresponding changes in VCI blood were not significant.
Data Conclusion
Ingestion of water caused changes in T1 and T2* of HPV blood compatible with dilution due to water absorption. The effects were enhanced by oxygen. Assessment of oxygen enrichment of HPV blood was not possible due to the dilution effect.
Level of Evidence
2
Technical Efficacy Stage
2 J. Magn. Reson. Imaging 2020;52:720–728.
The brain–blood interface holds the key to our understanding of how cerebral blood flow is regulated and how water and solutes are exchanged between blood and brain. The highly specialized astrocytic ...membranes that enwrap brain microvessels are salient constituents of the brain‐blood interface. These endfoot membranes contain a distinct set of molecules that is anchored to the subendothelial basal lamina forming an endfoot‐basal lamina junctional complex. Here we explore the mechanisms underpinning the formation of this complex. By use of a tailor made model system we show that endothelial cells promote AQP4 accumulation by exerting an inductive effect through extracellular matrix components such as agrin, as well as through a direct mechanical interaction with the endfoot processes. Through the compounds they secrete, the endothelial cells also increase AQP4 expression. The present data suggest that the highly specialized gliovascular interface is established through inductive processes that include both chemical and mechanical factors. GLIA 2015;63:2073–2091
Main points
Primary astrocytes regain their polarity in culture with brain endothelium.
Astrocyte polarization and membrane compartmentalization is governed by brain endothelial cells, extracellular matrix, soluble factors and direct mechanical interaction.
Proper function of the retina depends heavily on a specialized form of retinal glia called Müller cells. These cells carry out important homeostatic functions that are contingent on their polarized ...nature. Specifically, the Müller cell endfeet that contact retinal microvessels and the corpus vitreum show a tenfold higher concentration of the inwardly rectifying potassium channel Kir4.1 than other Müller cell plasma membrane domains. This highly selective enrichment of Kir4.1 allows K+ to be siphoned through endfoot membranes in a special form of spatial buffering. Here, we show that Kir4.1 is enriched in endfoot membranes through an interaction with β1‐syntrophin. Targeted disruption of this syntrophin caused a loss of Kir4.1 from Müller cell endfeet without affecting the total level of Kir4.1 expression in the retina. Targeted disruption of α1‐syntrophin had no effect on Kir4.1 localization. Our findings show that the Kir4.1 aggregation that forms the basis for K+ siphoning depends on a specific syntrophin isoform that colocalizes with Kir4.1 in Müller endfoot membranes.
Main Points
Inwardly rectifying K+ channel Kir4.1 is concentrated at the perivascular and subvitreal Müller cell endfeet.
Targeted deletion of β1‐syntrophin leads to significant loss of Kir4.1 at the Müller cell endfeet without changing its expression levels.
Brain function is inextricably coupled to water homeostasis. The fact that most of the volume between neurons is occupied by glial cells, leaving only a narrow extracellular space, represents an ...important challenge, as even small extracellular volume changes will affect ion concentrations and therefore neuronal excitability. Further, the ionic transmembrane shifts that are required to maintain ion homeostasis during neuronal activity must be accompanied by water. It follows that the mechanisms for water transport across plasma membranes must have a central part in brain physiology. These mechanisms are also likely to be of pathophysiological importance in brain oedema, which represents a net accumulation of water in brain tissue. Recent studies have shed light on the molecular basis for brain water transport and have identified a class of specialized water channels in the brain that might be crucial to the physiological and pathophysiological handling of water.
Evidence suggests that extracellular matrix molecules of perivascular basal laminae help orchestrate the molecular assemblies at the gliovascular interface. Specifically, laminin and agrin are ...thought to tether the dystrophin-associated protein (DAP) complex to the astrocytic basal lamina. This complex includes α-syntrophin (α-Syn), which is believed to anchor aquaporin-4 (AQP4) to astrocytic endfoot membrane domains. We have previously shown that the size of the perivascular AQP4 pool differs considerably between brain regions in an α-Syn-dependent manner. Also, both AQP4 and α-Syn occur at higher densities in endfoot membrane domains facing pericytes than in endfoot membrane domains facing endothelial cells. The heterogeneous distribution of AQP4 at the regional and capillary level has been attributed to a direct interaction between AQP4 and α-Syn. This would be challenged (1) if the microdistributions of laminin and agrin fail to align with those of DAP and AQP4 and (2) if targeted deletion of α-Syn leads to a loss of laminin and/or agrin. Here, we provide the first detailed and quantitative analysis of laminin and agrin in brain basal laminae of mice. We show that the microdistributions of these molecules vary in a fashion that is well aligned with the previously reported microdistribution of AQP4. We also demonstrate that the expression patterns of laminin and agrin are insensitive to targeted deletion of α-Syn, suggesting that α-Syn deletion affects AQP4 directly and not indirectly via laminin or agrin. These data fill remaining voids in the current model of how key molecules are assembled and tethered at the gliovascular interface.
Regulatory volume decrease (RVD) is a key mechanism for volume control that serves to prevent detrimental swelling in response to hypo-osmotic stress. The molecular basis of RVD is not understood. ...Here we show that a complex containing aquaporin-4 (AQP4) and transient receptor potential vanilloid 4 (TRPV4) is essential for RVD in astrocytes. Astrocytes from AQP4-KO mice and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress by increased intracellular Ca²⁺ and RVD. Coimmunoprecipitation and immunohistochemistry analyses show that AQP4 and TRPV4 interact and colocalize. Functional analysis of an astrocyte-derived cell line expressing TRPV4 but not AQP4 shows that RVD and intracellular Ca²⁺ response can be reconstituted by transfection with AQP4 but not with aquaporin-1. Our data indicate that astrocytes contain a TRPV4/AQP4 complex that constitutes a key element in the brain's volume homeostasis by acting as an osmosensor that couples osmotic stress to downstream signaling cascades.
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