Abundant evidence indicates extracellular vesicles (EVs) are mediators of intercellular communication. Previous reports from this group further showed that EVs from brain microvascular endothelial cells (BMEC) transferred the tight junction protein (TJ) claudin-5 (CLN-5) to leukocytes in vitro and during experimental autoimmune encephalomyelitis, leading us to hypothesize that such interaction might facilitate transendothelial migration (TEM) by a “zipper mechanism” whereby CLN-5 molecules on leukocyte-bound EVs temporarily replace those at interendothelial junctions. Such a mechanism is likely to be under strict spatiotemporal control. A corollary to this is that EVs are released from BMEC in a polar manner, such that only those released from the apical surface interact with leukocytes, while those from the basolateral surface bind perivascular and/or parenchymal targets. To assess polar EV release from BMEC and subsequent BMEC EV: leukocyte interactions during TEM using transwell assays, flow cytometry, nanoparticle tracking, super-resolution and live-cell time-lapse imaging were used. It was shown that EV release was highly polarized, as EVs accumulated predominantly in the chamber facing their membrane of origin. Supportive of polarized exosome release, apical and basolateral membrane material appeared segregated into discrete late endosomal populations. Consistent with a role in TEM, apical-derived BMEC EVs preferentially associated with adherent leukocytes, with TEM suppressed when EV release was inhibited. Polarized EV release was maintained under physiological flow, wherein CLN-5+ EVs exhibited predominant release at the apical surface, possibly reflecting their predisposition toward interacting with circulating immune cells. BMEC EVs are released in a polarized manner and those derived from the apical surface focally interact with adherent leukocytes. That inhibition of EV release further suppressed TEM suggests that BMEC EV: leukocyte binding supports the TEM process.
Idiopathic normal pressure hydrocephalus (iNPH) is an aging-associated neurological disorder characterized by ventricular enlargement and disturbances in brain fluid homeostasis, while the contribution of blood-brain barrier (BBB) abnormalities remains incompletely defined. Experimental models linking disease-associated genetic factors to regional BBB phenotypes remain limited. We generated a rat carrying a truncating Cwh43 allele using CRISPR/Cas9 genome editing. Ventricular size and motor performance were assessed, and BBB permeability was examined using intravenously administered tracers of 556 Da, 10 kDa, and 70 kDa. Junctional protein abundance, representative vascular ultrastructural features, vessel-associated VCAM-1 immunoreactivity, glial markers, and ventricular-interface phenotypes were evaluated using immunoblotting, immunofluorescence, and transmission electron microscopy. The mutant allele was defined as Cwh43 NM_001191677.1:c.1597del, predicting NP_001178606.1:p.(Met533Ter), and was associated with reduced Cwh43 transcript abundance. Heterozygous mutant rats developed ventricular enlargement and impaired rotarod performance. Repeated-measures analyses of the three tracer cohorts demonstrated significant overall genotype-associated BBB permeability phenotypes across a multiregional dataset spanning cortical, hippocampal, and cerebellar regions. These permeability-associated phenotypes occurred without a significant reduction in the measured full-length junctional proteins, while representative ultrastructural images showed identifiable vascular junctional contacts. Vessel-associated VCAM-1 immunoreactivity was increased in mutant rats, whereas whole-tissue VCAM-1 and the measured inflammasome-associated proteins did not show consistent genotype-dependent increases. Additional analysis identified reduced apical acetylated α-tubulin-positive coverage and mean fluorescence intensity along the lateral ventricular ependymal surface. In the adjacent periventricular region, normalized tracer values were higher in HZ rats across all three tracer cohorts, but none of the individual genotype comparisons remained significant after correction for the three-tracer family. Cwh43 p.(Met533Ter) rats exhibit reproducible multiregional BBB permeability-associated phenotypes together with associated vascular, glial, and ventricular-interface phenotypes. The present cross-sectional study does not establish their temporal ordering, primary cellular origin, or underlying molecular mechanism, but provides a framework for further investigation of barrier and ventricular-interface abnormalities in CWH43-related experimental models.
Blood-brain barrier (BBB) disruption and cortical thinning are both implicated in neurodegenerative cognitive impairment, yet their temporal relationship remains unresolved. Cross-sectional studies have yielded conflicting results, likely reflecting disease heterogeneity. We aimed to identify subtypes with distinct model-inferred sequences of Ktrans elevation and cortical thinning and to compare their clinical characteristics. This retrospective multi-center study included participants with normal cognition, mild cognitive impairment (MCI), and dementia recruited from two centers. BBB permeability (Ktrans) was derived from dynamic contrast-enhanced MRI, and cortical thickness was measured from structural MRI across six bilateral cortical regions. The Subtype and Stage Inference (SuStaIn) algorithm was used to infer subtypes and the relative ordering of imaging abnormalities from cross-sectional data. Subtype differences in cognition, cerebrospinal fluid biomarkers, APOE genotype, and small vessel disease markers were assessed. Among 216 participants (mean age 70.1 years; 64.4
Transthyretin (TTR) is a thyroid hormone (TH)-binding protein, that distributes TH through the cerebrospinal fluid to brain cells. Increasing evidence suggests additional cell-autonomous roles in the central nervous system. Here, we investigated Ttr expression and function in the murine subventricular zone, the largest postnatal neural stem cell niche. Public single-cell RNA sequencing datasets were re-analysed to assess Ttr expression across the neurogliogenic lineage. RNAscope and immunohistochemistry were used to evaluate messenger RNA and protein localization. Subventricular zone-derived neurospheres were exposed to exogenous TTR and analysed using single-cell RNA-sequencing and in vitro functional assays. Re-analysis of single-cell RNA-sequencing datasets revealed a transcriptional peak of Ttr at postnatal day 7 (P7) across the neurogliogenic lineage. RNAscope confirmed broad but weak mRNA expression in the subventricular zone, whereas immunohistochemistry failed to detect TTR protein, despite robust choroid plexus staining. In P7 subventricular zone-derived neurospheres, exogenous TTR induced a pronounced shift toward more differentiated populations (neuroblasts, oligodendrocyte precursor cells and committed oligodendrocyte precursors), while reducing progenitor proliferation, thereby activating mitochondrial programs, but repressing stemness and proliferation-related pathways. Lower TTR doses reproduced the anti-proliferative effects, and TTR treatment partially mimicked TH-induced outcomes, suggesting partial convergence between TTR- and TH-responsive pathways, despite minimal endogenous TH signalling activity. These findings uncover a developmental window of high Ttr expression without detectable protein and reveal that exogenous TTR is sufficient to regulate subventricular zone progenitor behaviour through signalling pathways that at least partially converge with TH responses, although the mechanisms remain unknown. This work expands the functional landscape of TTR and highlights potential relevance for neurodevelopment, as well as neurodegenerative and TH-related disorders.
Diffusion tensor image analysis along the perivascular space (DTI-ALPS) is increasingly used as an indirect MRI marker of glymphatic and perivascular fluid transport, yet its dependence on local anatomy remains uncertain. Because ALPS is sampled in periventricular white matter, ventricular enlargement may influence the metric independently of, or alongside, brain-fluid physiology. Endocrine ageing provides a biological test case as reproductive hormone changes occur alongside age-related ventricular enlargement. We examined whether associations between reproductive hormones and DTI-ALPS were independent of lateral ventricular (LV) volume. This cross-sectional cohort study used baseline data from female participants in the Human Connectome Project Aging and Aging Adult Brain Connectome studies. The primary outcome was the DTI-ALPS index; exposures were circulating estradiol, follicle-stimulating hormone (FSH), and luteinising hormone (LH). LV volume was derived from FreeSurfer segmentations. Four sequential linear regression models were fitted: unadjusted; adjusted for age; adjusted for age plus LV volume; and fully adjusted for age, LV volume, body mass index, study site, and hormone replacement therapy status. Potential effects of sleep quality, APOE ε4 status, and choroid plexus volume were explored as secondary analyses. The analysis included 784 females (mean age 60.0 years, SD 15.1). In unadjusted models, higher estradiol was associated with higher ALPS (B = 0.044, 95
