Matthew MacGregor Sharp

α-Dystrobrevin (α-DB) is a major component of the dystrophin-associated protein complex (DAPC). Knockout (KO) of α-DB in the brain is associated with astrocytic abnormalities and loss of neuronal GABA receptor clustering. Mutations in DAPC proteins are associated with altered dopamine signaling and cognitive and psychiatric disorders, including schizophrenia. This study tested the hypothesis that motivation and associated underlying biological pathways are altered in the absence of α-DB expression. Male wildtype and α-DB KO mice were tested for measures of motivation, executive function and extinction in the rodent touchscreen apparatus. Subsequently, brain tissues were evaluated for mRNA and/or protein levels of dysbindin-1, dopamine transporter and receptor 1 and 2, mu opioid receptor 1 (mOR1) and cannabinoid receptor 1 (CB1). α-DB KO mice had significantly increased motivation for the appetitive reward, while measures of executive function and extinction were unaffected. No differences were observed between wildtype and KO animals on mRNA levels of dysbindin-1 or any of the dopamine markers. mRNA levels of mOR1were significantly decreased in the caudate-putamen and nucleus accumbens of α-DB KO compared to WT animals, but protein levels were unaltered. However, CB1 protein levels were significantly increased in the prefrontal cortex and decreased in the nucleus accumbens of α-DB KO mice. Triple-labelling immunohistochemistry confirmed that changes in CB1 were not specific to astrocytes. These results highlight a novel role for α-DB in the regulation of appetitive motivation that may have implications for other behaviours that involve the dopaminergic and endocannabinoid systems.

The extracellular matrix (ECM) of the cerebral vasculature provides a pathway for the flow of interstitial fluid (ISF) and solutes out of the brain by intramural periarterial drainage (IPAD). Failure of IPAD leads to protein elimination failure arteriopathies such as cerebral amyloid angiopathy (CAA). The ECM consists of a complex network of glycoproteins and proteoglycans that form distinct basement membranes (BM) around different vascular cell types. Astrocyte endfeet that are localised against the walls of blood vessels are tethered to these BMs by dystrophin associated protein complex (DPC). Alpha-dystrobrevin (α-DB) is a key dystrophin associated protein within perivascular astrocyte endfeet; its deficiency leads to a reduction in other dystrophin associated proteins, loss of AQP4 and altered ECM. In human dementia cohorts there is a positive correlation between dystrobrevin gene expression and CAA. In the present study, we test the hypotheses that (a) the positive correlation between dystrobrevin gene expression and CAA is associated with elevated expression of α-DB at glial-vascular endfeet and (b) a deficiency in α-DB results in changes to the ECM and failure of IPAD. We used human post-mortem brain tissue with different severities of CAA and transgenic α-DB deficient mice. In human post-mortem tissue we observed a significant increase in vascular α-DB with CAA (CAA vrs. Old p < 0.005, CAA vrs. Young p < 0.005). In the mouse model of α-DB deficiency, there was early modifications to vascular ECM (collagen IV and BM thickening) that translated into reduced IPAD efficiency. Our findings highlight the important role of α-DB in maintaining structure and function of ECM, particularly as a pathway for the flow of ISF and solutes out of the brain by IPAD.

Drainage of interstitial fluid from the brain occurs via the intramural periarterial drainage (IPAD) pathways along the basement membranes of cerebral capillaries and arteries against the direction of blood flow into the brain. The cerebrovascular smooth muscle cells (SMCs) provide the motive force for driving IPAD, and their decrease in function may explain the deposition of amyloid-beta as cerebral amyloid angiopathy (CAA), a key feature of Alzheimer’s disease. The α-adrenoceptor subtype α_1A is abundant in the brain, but its distribution in the cerebral vessels is unclear. We analysed cultured human cerebrovascular SMCs and young, old and CAA human brains for (a) the presence of α_1A receptor and (b) the distribution of the α_1A receptor within the cerebral vessels. The α_1A receptor was present on the wall of cerebrovascular SMCs. No significant changes were observed in the vascular expression of the α_1A-adrenergic receptor in young, old and CAA cases. The pattern of vascular staining appeared less punctate and more diffuse with ageing and CAA. Our results show that the α_1A-adrenergic receptor is preserved in cerebral vessels with ageing and in CAA and is expressed on cerebrovascular smooth muscle cells, suggesting that vascular adrenergic receptors may hold potential for therapeutic targeting of IPAD.

White matter hyperintensities (WMH) occur in association with dementia but the aetiology is unclear. Here we test the hypothesis that there is a combination of impaired elimination of interstitial fluid from the white matter together with a degree of hypoxia in WMH. One of the mechanisms for the elimination of amyloid-β (Aβ) from the brain is along the basement membranes in the walls of capillaries and arteries (Intramural Peri-Arterial Drainage – IPAD). We compared the dynamics of IPAD in the grey matter of the hippocampus and in the white matter of the corpus callosum in 10 week old C57/B16 mice by injecting soluble Aβ as a tracer. The dynamics of IPAD in the white matter were significantly slower compared with the grey matter and this was associated with a lower density of capillaries in the white matter. Exposing cultures of smooth muscle cells to hypercapnia as a model of cerebral hypoperfusion resulted in a reduction in fibronectin and an increase in laminin in the extracellular matrix. Similar changes were detected in the white matter in human WMH suggesting that hypercapnia/hypoxia may play a role in WMH. Employing therapies to enhance both IPAD and blood flow in the white matter may reduce WMH in patients with dementia.

The accumulation of amyloid-β (Aβ) in the walls of capillaries and arteries as cerebral amyloid angiopathy (CAA) is part of the small vessel disease spectrum, related to a failure of elimination of Aβ from the brain. Aβ is eliminated along basement membranes in walls of cerebral capillaries and arteries (Intramural Peri-Arterial Drainage—IPAD), a pathway that fails with age and ApolipoproteinEε4 (ApoE4) genotype. IPAD is along basement membranes formed by capillary endothelial cells and surrounding astrocytes. Here, we examine (1) the composition of basement membranes synthesised by ApoE4 astrocytes; (2) structural differences between ApoE4 and ApoE3 astrocytes, and (3) how flow of Aβ affects Apo3/4 astrocytes. Using cultured astrocytes expressing ApoE3 or ApoE4, immunofluorescence, confocal, correlative light and electron microscopy (CLEM), and a millifluidic flow system, we show that ApoE4 astrocytes synthesise more fibronectin, possess smaller processes, and become rarefied when Aβ flows over them, as compared to ApoE3 astrocytes. Our results suggest that basement membranes synthesised by ApoE4 astrocytes favour the aggregation of Aβ, its reduced clearance via IPAD, thus promoting cerebral amyloid angiopathy.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

In the absence of lymphatics, fluid and solutes such as amyloid-β (Aβ) are eliminated from the brain along basement membranes in the walls of cerebral capillaries and arteries—the Intramural Peri-Arterial Drainage (IPAD) pathway. IPAD fails with age and insoluble Aβ is deposited as plaques in the brain and in IPAD pathways as cerebral amyloid angiopathy (CAA); fluid accumulates in the white matter as reflected by hyperintensities (WMH) on MRI. Within the brain, fluid uptake by astrocytes is regulated by aquaporin 4 (AQP4). We test the hypothesis that expression of astrocytic AQP4 increases in grey matter and decreases in white matter with onset of CAA. AQP4 expression was quantitated by immunocytochemistry and confocal microscopy in post-mortem occipital grey and white matter from young and old non-demented human brains, in CAA and in WMH. Results: AQP4 expression tended to increase with normal ageing but AQP4 expression in severe CAA was significantly reduced when compared to moderate CAA (p = 0.018). AQP4 expression tended to decline in the white matter with CAA and WMH, both of which are associated with impaired IPAD. Adjusting the level of AQP4 activity may be a valid therapeutic target for restoring homoeostasis in the brain as IPAD fails with age and CAA.

There are very few reliable methods in the literature to discern with certainty between cerebral arterioles and venules. Smooth muscle cells (SMC) and pericytes are present in both arterioles and venules, so immunocytochemistry for markers specific to intramural cells (IMC) is unreliable. This study employed transmission electron microscopy (TEM) and a canine brain to produce robust criteria for the correct identification of cerebral arterioles and venules based on lumen:vessel wall area, tested against the less accurate lumen diameter:vessel wall thickness. We first used morphology of IMC to identify two distinct groups of vessels; group 1 with morphology akin to venules and group 2 with morphology akin to arterioles. We then quantitatively assessed these vessels for lumen:vessel wall area ratio and lumen diameter:wall thickness ratio. After assessing 112 vessels, we show two distinct groups of vessels that can be separated using lumen:vessel wall area (group 1, 1.89 −10.96 vs. group 2, 0.27–1.57; p < 0.001) but not using lumen diameter:vessel wall thickness where a substantial overlap in ranges between groups occurred (group 1, 1.58–22.66 vs. group 2, 1.40–11.63). We, therefore, conclude that lumen:vessel wall area is a more sensitive and preferred method for distinguishing cerebral arterioles from venules. The significance of this study is wide, as cerebral small vessel disease is a key feature of vascular dementia and understanding the pathogenesis relies on correct identification of vessels.

Tracers injected into CSF pass into the brain alongside arteries and out again. This has been recently termed the “glymphatic system” that proposes tracers enter the brain along periarterial “spaces” and leave the brain along the walls of veins. The object of the present study is to test the hypothesis that: (1) tracers from the CSF enter the cerebral cortex along pial-glial basement membranes as there are no perivascular “spaces” around cortical arteries, (2) tracers leave the brain along smooth muscle cell basement membranes that form the Intramural Peri-Arterial Drainage (IPAD) pathways for the elimination of interstitial fluid and solutes from the brain. 2 μL of 100 μM soluble, fluorescent fixable amyloid β (Aβ) were injected into the CSF of the cisterna magna of 6–10 and 24–30 month-old male mice and their brains were examined 5 and 30 min later. At 5 min, immunocytochemistry and confocal microscopy revealed Aβ on the outer aspects of cortical arteries colocalized with α-2 laminin in the pial-glial basement membranes. At 30 min, Aβ was colocalised with collagen IV in smooth muscle cell basement membranes in the walls of cortical arteries corresponding to the IPAD pathways. No evidence for drainage along the walls of veins was found. Measurements of the depth of penetration of tracer were taken from 11 regions of the brain. Maximum depths of penetration of tracer into the brain were achieved in the pons and caudoputamen. Conclusions drawn from the present study are that tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways. The exit route is along IPAD pathways in which Aβ accumulates in cerebral amyloid angiopathy (CAA) in Alzheimer’s disease. Results from this study suggest that CSF may be a suitable route for delivery of therapies for neurological diseases, including CAA.