Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood–brain barrier

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.NM has been funded by SNF grant 320030E-164245 and ERC Consolidator grant 2018 819717. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Benoît Zuber is supported by SNF grant 179520 and ERA-NET NEURON grant 185536. M.O. was supported by SNF grant PCEFP3_186993.S

Loss of claudin-3 impairs hepatic metabolism, biliary barrier function and cell proliferation in the murine liver.

BACKGROUND & AIMS Tight junctions in the liver are essential to maintain the blood-biliary-barrier, however the functional contribution of individual tight junction proteins to barrier- and metabolic homeostasis remains largely unexplored. Here, we describe the cell type specific expression of tight junction genes in the murine liver, and explore the regulation and functional importance of the transmembrane protein claudin-3 in liver metabolism, barrier function and cell proliferation. METHODS The cell type specific expression of hepatic tight junction genes is described using our mouse liver single cell sequencing dataset. Differential gene expression in Cldn3-/- and Cldn3+/+ livers was assessed in young and aged mice by RNA-seq and hepatic tissue was analysed for lipid content and bile acid composition. A surgical model of partial hepatectomy (PHx) was used to induce liver cell proliferation. RESULTS Claudin-3 is a highly expressed tight junction protein found in the liver and is expressed predominantly in hepatocytes and cholangiocytes. The histology of Cldn3-/- livers showed no overt phenotype, and the canalicular tight junctions appeared intact. Nevertheless, by RNAseq we detected a downregulation of metabolic pathways in the livers of Cldn3-/- young and aged mice as well as a decrease in lipid content and a weakened biliary-barrier for primary bile acids, such as TCA, TCDCA and TMCA. Coinciding with defects in the biliary barrier and lower lipid metabolism, there was a diminished hepatocyte proliferative response in Cldn3-/- mice following PHx. CONCLUSION Our data shows that in the liver, claudin-3 is necessary to maintain metabolic homeostasis, retention of bile acids, and optimal hepatocyte proliferation during liver regeneration

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spino-cerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory cir-cuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compro-mising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcit-able MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibi-tion of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level def-icits upstream of PNs are one of the main disease triggers in SCA1

Early molecular layer interneuron hyperactivity triggers Purkinje neuron degeneration in SCA1.

Toxic proteinaceous deposits and alterations in excitability and activity levels characterize vulnerable neuronal populations in neurodegenerative diseases. Using in vivo two-photon imaging in behaving spinocerebellar ataxia type 1 (Sca1) mice, wherein Purkinje neurons (PNs) degenerate, we identify an inhibitory circuit element (molecular layer interneurons [MLINs]) that becomes prematurely hyperexcitable, compromising sensorimotor signals in the cerebellum at early stages. Mutant MLINs express abnormally elevated parvalbumin, harbor high excitatory-to-inhibitory synaptic density, and display more numerous synaptic connections on PNs, indicating an excitation/inhibition imbalance. Chemogenetic inhibition of hyperexcitable MLINs normalizes parvalbumin expression and restores calcium signaling in Sca1 PNs. Chronic inhibition of mutant MLINs delayed PN degeneration, reduced pathology, and ameliorated motor deficits in Sca1 mice. Conserved proteomic signature of Sca1 MLINs, shared with human SCA1 interneurons, involved the higher expression of FRRS1L, implicated in AMPA receptor trafficking. We thus propose that circuit-level deficits upstream of PNs are one of the main disease triggers in SCA1

PolyGA targets the ER stress-adaptive response by impairing GRP75 function at the MAM in C9ORF72-ALS/FTD.

ER stress signaling is linked to the pathophysiological and clinical disease manifestations in amyotrophic lateral sclerosis (ALS). Here, we have investigated ER stress-induced adaptive mechanisms in C9ORF72-ALS/FTD, focusing on uncovering early endogenous neuroprotective mechanisms and the crosstalk between pathological and adaptive responses in disease onset and progression. We provide evidence for the early onset of ER stress-mediated adaptive response in C9ORF72 patient-derived motoneurons (MNs), reflected by the elevated increase in GRP75 expression. These transiently increased GRP75 levels enhance ER-mitochondrial association, boosting mitochondrial function and sustaining cellular bioenergetics during the initial stage of disease, thereby counteracting early mitochondrial deficits. In C9orf72 rodent neurons, an abrupt reduction in GRP75 expression coincided with the onset of UPR, mitochondrial dysfunction and the emergence of PolyGA aggregates, which co-localize with GRP75. Similarly, the overexpression of PolyGA in WT cortical neurons or C9ORF72 patient-derived MNs led to the sequestration of GRP75 within PolyGA inclusions, resulting in mitochondrial calcium (Ca2+) uptake impairments. Corroborating these findings, we found that PolyGA aggregate-bearing human post-mortem C9ORF72 hippocampal dentate gyrus neurons not only display reduced expression of GRP75 but also exhibit GRP75 sequestration within inclusions. Sustaining high GRP75 expression in spinal C9orf72 rodent MNs specifically prevented ER stress, normalized mitochondrial function, abrogated PolyGA accumulation in spinal MNs, and ameliorated ALS-associated behavioral phenotype. Taken together, our results are in line with the notion that neurons in C9ORF72-ALS/FTD are particularly susceptible to ER-mitochondrial dysfunction and that GRP75 serves as a critical endogenous neuroprotective factor. This neuroprotective pathway, is eventually targeted by PolyGA, leading to GRP75 sequestration, and its subsequent loss of function at the MAM, compromising mitochondrial function and promoting disease onset

Transluminal Pillars-Their Origin and Role in the Remodelling of the Zebrafish Caudal Vein Plexus.

Intussusceptive pillars, regarded as a hallmark of intussusceptive angiogenesis, have been described in developing vasculature of many organs and organisms. The aim of this study was to resolve the question about pillar formation and their further maturation employing zebrafish caudal vein plexus (CVP). The CVP development was monitored by in vivo confocal microscopy in high spatio-temporal resolution using the transgenic zebrafish model Fli1a:eGPF//Gata1:dsRed. We tracked back the formation of pillars (diameter ≤ 4 µm) and intercapillary meshes (diameter > 4 µm) and analysed their morphology and behaviour. Transluminal pillars in the CVP arose via a combination of sprouting, lumen expansion, and/or the creation of intraluminal folds, and those mechanisms were not associated directly with blood flow. The follow-up of pillars indicated that one-third of them disappeared between 28 and 48 h post fertilisation (hpf), and of the remaining ones, only 1/17 changed their cross-section area by >50%. The majority of the bigger meshes (39/62) increased their cross-section area by >50%. Plexus simplification and the establishment of hierarchy were dominated by the dynamics of intercapillary meshes, which formed mainly via sprouting angiogenesis. These meshes were observed to grow, reshape, and merge with each other. Our observations suggested an alternative view on intussusceptive angiogenesis in the CVP

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle‐Specific VEGF Expression

Vascular endothelial growth factor-A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle-specific VEGF knockout (mVEGF-KO, n = 7) mice and their wild-type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: -63.5%; soleus: -54.8%; P ≤ 0.05) in mVEGF-KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv-values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF-KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse-strains: Vv (-17.4%), absolute area size (-19.1%) and thickness (-19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF-KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF-KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239-2249, 2017. © 2017 Wiley Periodicals, Inc

Ultrastructure of Skeletal Muscles in Mice Lacking Muscle-Specific VEGF Expression.

Vascular endothelial growth factor-A (VEGF) influences several physiological processes including endothelial cell function, angiogenesis and maintenance of organ/tissue capillarity. While the functional aspects of VEGF were vigorously investigated, only little detail is known on structural integrity of skeletal muscle fibers and capillaries in mice lacking VEGF expression in their muscles. Therefore, we assessed systematically the architecture of the glycolytic plantaris and the oxidative soleus muscles obtained from muscle-specific VEGF knockout (mVEGF-KO, n = 7) mice and their wild-type (WT, n = 7) littermates by morphometry after transmission electron microscopy. The capillary/fiber ratio was lower (plantaris: -63.5%; soleus: -54.8%; P ≤ 0.05) in mVEGF-KO mice than in WT mice. In plantaris, quantification of volume density (Vv) of compartments revealed higher Vv of total mitochondria (+56.5%, P ≤ 0.05) as well as higher Vv-values for both intrafibrillar (+39%; P ≤ 0.05) and subsarcolemmal (+220%; P ≤ 0.05) mitochondrial pools in mVEGF-KO mice than WT mice. The capillary phenotype also differed (P ≤ 0.05) between the two mouse-strains: Vv (-17.4%), absolute area size (-19.1%) and thickness (-19.6%) of the endothelium layer were lower and Vv of capillary lumen (+15.1%) was higher in mVEGF-KO mice than in WT littermates. In soleus, mitochondrial Vv in fibers and the structural indicators specific to the capillary phenotype exhibited the same tendency in differences between the mouse strains without reaching statistical significance. Our morphometric analysis demonstrates that the lower capillary supply in plantaris of mVEGF-KO mice is accompanied by higher mitochondrial Vv in muscle fibers as well as lumen dilation and endothelium thinning of capillaries. These structural alterations were more pronounced in a glycolytic than an oxidative muscle. Anat Rec, 300:2239-2249, 2017. © 2017 Wiley Periodicals, Inc