Quantitative proteomic profiling of white matter in cases of cer-ebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin


Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aβ) in the walls of cerebral arterioles, arteries and capillaries. Changes in the white matter in CAA are observed as hyperintensities and dilated perivascular spaces on MRI suggesting impairment of fluid drainage but the pathophysiology behind these changes is poorly understood. We tested the hypothesis that proteins associated with clearance of Aβ peptides are upregulated in the white matter in cases of CAA.

Methods: In this study, we compare the quantitative proteomic profile of white matter from post-mortem brains of patients with CAA and age-matched controls in order to gain insight into the cellular processes and key molecules involved in the pathophysiology of CAA.

Results: Our proteomic analysis resulted in the profiling of 3,734 proteins (peptide FDR p<0.05). Of these, 189 were differentially expressed in CAA vs. control. Bioinformatics analysis of these proteins showed significant enrichment of proteins related to cell adhesion | cell-matrix interaction, mitochondrial dysfunction and hypoxia. Upregulated proteins in CAA included EMILIN2, COL4A2, TLN1, CLU, HSPG2. Downregulated proteins included DSP, IDE, HBG1.

Conclusions: The present study reports an in-depth quantitative proteomic profiling of white matter from patients with CAA, highlighting extracellular matrix proteins and clusterin as key molecules in the pathophysiology of white matter changes in cases of CAA.

Manousopoulou A, Yuen HO, MacGregor Sharp M, Saito S, Aldea R, Mazer N, Garbis SD, Carare RO

Vascular α1A Adrenergic Receptors as a Potential Therapeutic Target for IPAD in Alzheimer’s Disease


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 α1A 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 α1A receptor and (b) the distribution of the α1A receptor within the cerebral vessels. The α1A receptor was present on the wall of cerebrovascular SMCs. No significant changes were observed in the vascular expression of the α1A-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 α1A-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.

Frost M, Keable A, Baseley D, Sealy A, Zbarcea DA, Gatherer M, Yuen HO, MacGregor Sharp M, Weller RO, Attems J, Smith C, Chiarot PR, Carare RO

Peri-arterial pathways for clearance of α-Synuclein and tau from the brain: Implications for the pathogenesis of dementias and for immunotherapy


Introduction: Accumulation of amyloid beta (Aβ), α-synuclein (αSyn), and tau in dementias indicates their age-related failure of elimination from the brain. Aβ is eliminated along basement membranes in walls of cerebral arterioles and leptomeningeal arteries (intramural peri-arterial drainage [IPAD]); IPAD is impaired with age. We test the hypothesis that αSyn and tau are also eliminated from the normal brain along IPAD pathways.

Methods: Soluble αSyn or tau was injected into mouse hippocampus. Animals were perfused 5 minutes to 7 days post-injection. Blood vessels were identified by ROX-SE for light-sheet and immunolabeling for confocal microscopy. IPAD was quantified by measuring the proportion of arterioles with αSyn/tau.

Results: αSyn and tau are eliminated from the brain by IPAD but with different dynamics.

Discussion: Age-related failure of IPAD may play a role in the pathogenesis of synucleinopathies and tauopathies. αSyn persists within IPAD at 24 hours, which may affect immunotherapy for αSyn.

Nimmo J, Johnston DA, Dodart JC, MacGregor Sharp M, Weller RO, Nicoll JAR, Verma A, Carare RO

Clearance of interstitial fluid (ISF) and CSF (CLIC) group-part of Vascular Professional Interest Area (PIA): Cerebrovascular disease and the failure of elimination of Amyloid-β from the brain and retina with age and Alzheimer’s disease-Opportunities for Therapy


Two of the key functions of arteries in the brain are (1) the well-recognized supply of blood via the vascular lumen and (2) the emerging role for the arterial walls as routes for the elimination of interstitial fluid (ISF) and soluble metabolites, such as amyloid beta (Aβ), from the brain and retina. As the brain and retina possess no conventional lymphatic vessels, fluid drainage toward peripheral lymph nodes is mediated via transport along basement membranes in the walls of capillaries and arteries that form the intramural peri-arterial drainage (IPAD) system. IPAD tends to fail as arteries age but the mechanisms underlying the failure are unclear. In some people this is reflected in the accumulation of Aβ plaques in the brain in Alzheimer’s disease (AD) and deposition of Aβ within artery walls as cerebral amyloid angiopathy (CAA). Knowledge of the dynamics of IPAD and why it fails with age is essential for establishing diagnostic tests for the early stages of the disease and for devising therapies that promote the clearance of Aβ in the prevention and treatment of AD and CAA. This editorial is intended to introduce the rationale that has led to the establishment of the Clearance of Interstitial Fluid (ISF) and CSF (CLIC) group, within the Vascular Professional Interest Area of the Alzheimer’s Association International Society to Advance Alzheimer’s Research and Treatment.

Carare RO, Aldea R, Agarwal N et al.

Demonstrating a reduced capacity for removal of fluid from cerebral white matter and hypoxia in areas of white matter hyperintensity associated with age and dementia


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.

MacGregor Sharp M, Saito S, Keable A, Gatherer M, Aldea R, Agarwal N, Simpson JE, Wharton SB, Weller RO, Carare RO 

ApoE4 Astrocytes Secrete Basement Membranes Rich in Fibronectin and Poor in Laminin Compared to ApoE3 Astrocytes


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.

Keable A, O’Neill R, MacGregor Sharp M, Gatherer M, Yeun HM, Johnston DA, Weller RO, Carare RO 

Neurofilaments: neurobiological foundations for biomarker applications


Interest in neurofilaments has risen sharply in recent years with recognition of their potential as biomarkers of brain injury or neurodegeneration in CSF and blood. This is in the context of a growing appreciation for the complexity of the neurobiology of neurofilaments, new recognition of specialized roles for neurofilaments in synapses and a developing understanding of mechanisms responsible for their turnover. Here we will review the neurobiology of neurofilament proteins, describing current understanding of their structure and function, including recently discovered evidence for their roles in synapses. We will explore emerging understanding of the mechanisms of neurofilament degradation and clearance and review new methods for future elucidation of the kinetics of their turnover in humans. Primary roles of neurofilaments in the pathogenesis of human diseases will be described. With this background, we then will review critically evidence supporting use of neurofilament concentration measures as biomarkers of neuronal injury or degeneration. Finally, we will reflect on major challenges for studies of the neurobiology of intermediate filaments with specific attention to identifying what needs to be learned for more precise use and confident interpretation of neurofilament measures as biomarkers of neurodegeneration.

Gafson AR, Barthélemy NR, Bomont P, Carare RO et al.

UK Consensus on Pre-Clinical Vascular Cognitive Impairment Functional Outcomes Assessment: Questionnaire and Workshop Proceedings


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.

McFall A, Hietamies TM, Bernard A et al.

Vital Functions Contribute to the Spread of Extracellular Fluids in the Brain: Comparison Between Life and Death


Vascular pulsations, contractions of vascular smooth muscle cells and breathing have been reported to foster movement and clearance of interstitial and cerebrospinal fluids from the brain. The aim of this study was to estimate the contribution of these vital functions. We compared the spread of an injected hydrophilic tracer (Fluoro-Emerald, a 10 kDa fluorescein-coupled dextran amine) in the brains of live anesthetized and sacrificed rats at 30 and 90 min after injection. To determine the overall pattern of distribution of tracers, we created 3D-reconstructions of the horizontal transections of the whole brain. Immunofluorescence staining with laminin and collagen IV was performed to determine the pattern of distribution of tracer in relation to the cerebrovascular basement membranes. We found that diffusion was widely restricted to the periventricular region in sacrificed rats with no spread to the contralateral hemisphere, while the bulk flow occurred along the vasculature and reached the surface and the contralateral hemisphere as soon as 30 min after injection in live anesthetized animals. The tracer appeared to be localized along the vascular basement membranes and along fiber tracts as reported previously. Thus, our data indicate that vital functions are essential for the remote movement of extracellular fluids within the cerebral parenchyma.

Piotrowska A, Winter K, Carare RO, Bechmann I.

The Pattern of AQP4 Expression in the Ageing Human Brain and in Cerebral Amyloid Angiopathy


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.

Owasil R, O’Neill R, Keable A, Nimmo J, MacGregor Sharp M et al.

Vasomotion Drives Periarterial Drainage of Aβ from the Brain

In this issue of Neuron, van Veluw et al. (2020) show that elimination of solutes from the brain along arterial walls is driven by low-frequency arteriolar oscillations and suggest that age-related reduction of this vasomotion may contribute to impaired clearance of Aβ.

Carare RO, Aldea R, Bulters D, Alzetani A, Birch AA, Richardson G, Weller RO


The Diverse Roles of TIMP-3: Insights Into Degenerative Diseases of the Senescent Retina and Brain


Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer’s disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer’s patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer’s-linked amyloid-beta (Aß) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of Aß. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs.

Dewing JM, Carare RO, Lotery AJ, Ratnayaka JA.

Multi-trait Genome-Wide Association Study Identifies New Loci Associated With Optic Disc Parameters


A new avenue of mining published genome-wide association studies includes the joint analysis of related traits. The power of this approach depends on the genetic correlation of traits, which reflects the number of pleiotropic loci, i.e. genetic loci influencing multiple traits. Here, we applied new meta-analyses of optic nerve head (ONH) related traits implicated in primary open-angle glaucoma (POAG); intraocular pressure and central corneal thickness using Haplotype reference consortium imputations. We performed a multi-trait analysis of ONH parameters cup area, disc area and vertical cup-disc ratio. We uncover new variants; rs11158547 in PPP1R36-PLEKHG3 and rs1028727 near SERPINE3 at genome-wide significance that replicate in independent Asian cohorts imputed to 1000 Genomes. At this point, validation of these variants in POAG cohorts is hampered by the high degree of heterogeneity. Our results show that multi-trait analysis is a valid approach to identify novel pleiotropic variants for ONH.

Bonnemaijer PWM, Leeuwen EMV, Iglesias AI, et al.

Solving an Old Dogma: Is it an Arteriole or a Venule?

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.

MacGregor Sharp M, Criswell TP, Dobson H,  Finucane C, Verma ACarare RO.

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Mazur C, Powers B, Zasadny K,  Sullivan JM, et al.

The spectrum of age-related small vessel diseases: potential overlap and interactions of amyloid and non-amyloid vasculopathies

Deep perforator arteriopathy (DPA) and cerebral amyloid angiopathy (CAA) are the commonest known cerebral small vessel diseases (CSVD), which cause ischaemic stroke, intracebral haemorrhage (ICH) and vascular cognitive impairment (VCI). While thus far mainly considered as separate entities, we here propose that DPA and CAA share similarities, overlap and interact, so that “pure” DPA or CAA are extremes along a continuum of age-related small vessel pathologies. We suggest blood-brain barrier (BBB) breakdown, endothelial damage and impaired perivascular β-amyloid (Aβ) drainage are hallmark common mechanisms connecting DPA and CAA. We also suggest a need for new biomarkers (e.g. high-resolution imaging) to deepen understanding of the complex relationships between DPA and CAA.

Schreiber S, Wilisch-Neumann A, Schreiber F,  Assmann A, Scheumann V, Perosa V, Jandke S, Mawrin C, Carare RO, Werring DJ.

Small Vessel Disease Pathological Changes in Neurodegenerative and Vascular Dementias concomitant with Autonomic Dysfunction

We performed a clinicopathological study to assess the burden of small vessel disease (SVD) type of pathological changes in elderly demented subjects, who had clinical evidence of autonomic dysfunction, either carotid sinus hypersensitivity or orthostatic hypotension or both or had exhibited unexpected repeated falls. Clinical and neuropathological diagnoses in 112 demented subjects comprised dementia with Lewy bodies (DLB), Parkinson’s disease with dementia (PDD), Alzheimer’s disease (AD), Mixed dementia (mostly AD-DLB) and vascular dementia (VaD). Of these, 12 DLB subjects had no recorded unexpected falls in life and therefore no evidence of concomitant autonomic dysfunction. A further 17 subjects were assessed as ageing controls without significant pathology or signs of autonomic dysfunction. We quantified brain vascular pathological changes and determined severities of neurodegenerative lesions including α-synuclein pathology. We found moderate-severe vascular changes and high vascular pathology scores (P<0.01) in all neurodegenerative dementias and as expected in VaD compared to similar age controls. Arteriolosclerosis, perivascular spacing and microinfarcts were frequent in the basal ganglia and frontal white matter (WM) across all dementias whereas small infarcts (<5 mm) were restricted to VaD. In a sub-set of demented subjects, we found that vascular pathology scores were correlated with WM hyperintensity volumes determined by MRI in life (P<0.02). Sclerotic index values were increased by ~50% in both the WM and neocortex in all dementias compared to similar age controls. We found no evidence for increased α-synuclein deposition in subjects with autonomic dysfunction. Our findings suggest greater SVD pathological changes occur in the elderly diagnosed with neurodegenerative dementias including DLB and who acquire autonomic dysfunction. SVD changes may not necessarily manifest in clinically overt symptoms but they likely confound motor or cognitive dysfunction. We propose autonomic dysfunction promotes chronic cerebral hypoperfusion to impact upon ageing-related neurodegenerative processes and characterise their end-stage clinical syndromes. This article is protected by copyright. All rights reserved.

 Hase Y, Polvikoski TM, Firbank MJ, Craggs LJ, Hawthorne E, Platten C, Stevenson W, Deramecourt V, Ballard C, Kenny RA, Perry RH, Ince P, Carare RO, Allan LM, Horsburgh K, Kalaria RN.

3D Reconstruction of the Neurovascular Unit Reveals Differential Loss of Cholinergic Innervation in the Cortex and Hippocampus of the Adult Mouse Brain

Increasing evidence supports a role for cerebrovasculature dysfunction in the etiology of Alzheimer’s disease (AD). Blood vessels in the brain are composed of a collection of cells and acellular material that comprise the neurovascular unit (NVU). The NVU in the hippocampus and cortex receives innervation from cholinergic neurons that originate in the basal forebrain. Death of these neurons and their nerve fibers is an early feature of AD. However, the effect of the loss of cholinergic innervation on the NVU is not well characterized. The purpose of this study was to evaluate the effect of the loss of cholinergic innervation of components of the NVU at capillaries, arteries and veins in the hippocampus and cortex. Adult male C57BL/6 mice received an intracerebroventricular injection of the immunotoxin p75NTR mu-saporin to induce the loss of cholinergic neurons. Quadruple labeling immunohistochemistry and 3D reconstruction were carried out to characterize specific points of contact between cholinergic fibers and collagen IV, smooth muscle cells and astrocyte endfeet. Innate differences were observed between vessels of the hippocampus and cortex of control mice, including a greater amount of cholinergic contact with perivascular astrocytes in hippocampal capillaries and a thicker basement membrane in hippocampal veins. Saporin treatment induced a loss of cholinergic innervation at the arterial basement membrane and smooth muscle cells of both the hippocampus and the cortex. In the cortex, there was an additional loss of innervation at the astrocytic endfeet. The current results suggest that cortical arteries are more strongly affected by cholinergic denervation than arteries in the hippocampus. This regional variation may have implications for the etiology of the vascular pathology that develops in AD.

Nizari S, Carare RO, Romero IA, Hawkes CA.

Dispersion in porous media in oscillatory flow between flat plates: applications to intrathecal, periarterial and paraarterial solute transport in the central nervous system

Background: As an alternative to advection, solute transport by shear-augmented dispersion within oscillatory cerebrospinal fluid flow was investigated in small channels representing the basement membranes located between cerebral arterial smooth muscle cells, the paraarterial space surrounding the vessel wall and in large channels modeling the spinal subarachnoid space (SSS).

METHODS: Geometries were modeled as two-dimensional. Fully developed flows in the channels were modeled by the Darcy-Brinkman momentum equation and dispersion by the passive transport equation. Scaling of the enhancement of axial dispersion relative to molecular diffusion was developed for regimes of flow including quasi-steady, porous and unsteady, and for regimes of dispersion including diffusive and unsteady.

RESULTS: Maximum enhancement occurs when the characteristic time for lateral dispersion is matched to the cycle period. The Darcy-Brinkman model represents the porous media as a continuous flow resistance, and also imposes no-slip boundary conditions at the walls of the channel. Consequently, predicted dispersion is always reduced relative to that of a channel without porous media, except when the flow and dispersion are both unsteady.

DISCUSSION/CONCLUSIONS: In the basement membranes, flow and dispersion are both quasi-steady and enhancement of dispersion is small even if lateral dispersion is reduced by the porous media to achieve maximum enhancement. In the paraarterial space, maximum enhancement Rmax = 73,200 has the potential to be significant. In the SSS, the dispersion is unsteady and the flow is in the transition zone between porous and unsteady. Enhancement is 5.8 times that of molecular diffusion, and grows to a maximum of 1.6E+6 when lateral dispersion is increased. The maximum enhancement produces rostral transport time in agreement with experiments.

Keith Sharp M, Carare RO, Martin BA.

Knockout of apolipoprotein A‐I decreases parenchymal and vascular β‐amyloid pathology in the Tg2576 mouse model of Alzheimer’s disease

Aims: Apolipoprotein A‐I (apoA‐I), the principal apolipoprotein associated with high density lipoproteins (HDL) in the periphery, is also found at high concentrations in the cerebrospinal fluid. Previous studies have reported either no impact or vascular‐specific effects of apoA‐I knockout on β‐amyloid (Aβ) pathology. However, the putative mechanism(s) by which apoA‐I may influence Aβ deposition is unknown. Methods We evaluated the effect of apoA‐I deletion on Aβ pathology, Aβ production and clearance from the brain in the Tg2576 mouse model of AD.

Results: Contrary to previous reports, deletion of the APOA1 gene significantly reduced concentrations of insoluble Aβ40 and Aβ42 and reduced plaque load in both the parenchyma and blood vessels of apoA‐I knockout x Tg2576 mice compared to Tg2576 animals. This was not due to decreased Aβ production or alterations in Aβ species. Levels of soluble clusterin/apoJ were significantly higher in neurons of apoA‐I KO mice compared to both wildtype and apoA‐I KO x Tg2576 mice. In addition, clearance of Aβ along intramural periarterial drainage pathways was significantly higher in apoA‐I KO mice compared to wildtype animals.

Conclusion: These data suggest that deletion of apoA‐I is associated increased clearance of Aβ and reduced parenchymal and vascular Aβ pathology in the Tg2576 model. These results suggest that peripheral dyslipidaemia can modulate the expression of apolipoproteins in the brain and may influence Aβ clearance and aggregation in AD. This article is protected by copyright. All rights reserved.

Contu L, Carare RO, Hawkes C.

Vascular dysfunction—The disregarded partner of Alzheimer’s disease

Increasing evidence recognizes Alzheimer’s disease (AD) as a multifactorial and heterogeneous disease with multiple contributors to its pathophysiology, including vascular dysfunction. The recently updated AD Research Framework put forth by the National Institute on Aging–Alzheimer’s Association describes a biomarker-based pathologic definition of AD focused on amyloid, tau, and neuronal injury. In response to this article, here we first discussed evidence that vascular dysfunction is an important early event in AD pathophysiology. Next, we examined various imaging sequences that could be easily implemented to evaluate different types of vascular dysfunction associated with, and/or contributing to, AD pathophysiology, including changes in blood-brain barrier integrity and cerebral blood flow. Vascular imaging biomarkers of small vessel disease of the brain, which is responsible for >50% of dementia worldwide, including AD, are already established, well characterized, and easy to recognize. We suggest that these vascular biomarkers should be incorporated into the AD Research Framework to gain a better understanding of AD pathophysiology and aid in treatment efforts.

Sweeney MD, Montagne A, Sagare AP, Nation DA, Schneider LS, Chui HC, et al

Cerebrovascular smooth muscle cells as the drivers of intramural periarterial drainage of the brain

The human brain is the organ with the highest metabolic activity but it lacks a traditional lymphatic system responsible for clearing waste products. We have demonstrated that the basement membranes of cerebral capillaries and arteries represent the lymphatic pathways of the brain along which intramural periarterial drainage (IPAD) of soluble metabolites occurs. Failure of IPAD could explain the vascular deposition of the amyloid-beta protein as cerebral amyloid angiopathy (CAA), which is a key pathological feature of Alzheimer’s disease. The underlying mechanisms of IPAD, including its motive force, have not been clarified, delaying successful therapies for CAA. Although arterial pulsations from the heart were initially considered to be the motive force for IPAD, they are not strong enough for efficient IPAD. This study aims to unravel the driving force for IPAD, by shifting the perspective of a heart-driven clearance of soluble metabolites from the brain to an intrinsic mechanisms of cerebral arteries (e.g. vasomotion-driven IPAD). We test the hypothesis that the cerebrovascular smooth muscle cells, whose cycles of contraction and relaxation generate vasomotion, are the drivers of IPAD. A novel multiscale model of arteries, in which we treat the basement membrane as a fluid-filled poroelastic medium deformed by the contractile cerebrovascular smooth muscle cells, is used to test the hypothesis. The vasomotion-induced intramural flow rates suggest that vasomotion-driven IPAD is the only mechanism postulated to date capable of explaining the available experimental observations. The cerebrovascular smooth muscle cells could represent valuable drug targets for prevention and early interventions in CAA.

Aldea R, Weller RO, Wilcock DM, Carare RO, Richardson G.


Military-related risk factors for dementia

In recent years, there has been growing discussion to better understand the pathophysiological mechanisms of traumatic brain injury and post-traumatic stress disorder and how they may be linked to an increased risk of neurodegenerative diseases including Alzheimer’s disease in veterans. Building on that discussion, and subsequent to a special issue of Alzheimer’s & Dementia published in June 2014, which focused on military risk factors, the Alzheimer’s Association convened a continued discussion of the scientific community on December 1, 2016. During this meeting, participants presented and evaluated progress made since 2012 and identified outstanding knowledge gaps regarding factors that may impact veterans’ risk for later life dementia. The following is a summary of the invited presentations and moderated discussions of both the review of scientific understanding and identification of gaps to inform further investigations.

Snyder HM, Carare RO, DeKosky ST, de Leon MJ, et al.

A control mechanism for intra-mural periarterial drainage via astrocytes: How neuronal activity could improve waste clearance from the brain

The mechanisms behind the clearance of soluble waste from deep within the parenchyma of the brain remain unclear. Experimental evidence reveals that one pathway for clearance of waste, termed intra-mural peri-arterial drainage (IPAD), is the rapid drainage of interstitial fluid along basement membranes (BM) of the smooth muscle cells of cerebral arteries; failure of IPAD is closely associated with the pathology of Alzheimer’s disease (AD), but its driving mechanism remains unclear. We have previously shown that arterial pulsations generated by the heart beat are not strong enough to drive IPAD. Here we present computational evidence for a mechanism for clearance of waste from the brain that is driven by functional hyperaemia, that is, the dilatation of cerebral arterioles as a consequence of increased nutrient demand from neurons. This mechanism is based on our model for the flow of fluid through the vascular BM. It accounts for clearance rates observed in mouse experiments, and aligns with pathological observations and recommendations to lower the individual risk of AD, such as mental and physical activity. Thus, our neurovascular hypothesis should act as the new working hypothesis for the driving force behind IPAD.

Diem KA, Carare RO, Weller RO, Bressloff NW.

Lymphatic Drainage of the CNS and Its Role in Neuroinflammation and Neurodegenerative Disease

CSF in ventricles and subarachnoid spaces and interstitial fluid (ISF) in the extracellular spaces of the CNS parenchyma both drain to lymph nodes but by largely separate routes. CSF drains along lymphatic vessels that allow traffic of antigen presenting cells (APC) to lymph nodes. ISF, on the other hand, drains to lymph nodes along 100–150 nm thick basement membranes in the intramural periarterial pathways that do not allow traffic of APC from the CNS parenchyma to lymph nodes. This is one factor that may account for immune privilege in the CNS; the other factor is the highly controlled entry of T lymphocytes into CNS tissues. Lymphatic drainage of CSF and ISF and draining lymph nodes play a role in neuroimmunological diseases. Lymphatic drainage of ISF maintains homeostasis of the CNS but fails with age and this is associated with failure of elimination and accumulation of amyloid β in the brain in Alzheimer’s disease. Understanding lymphatic drainage of the CNS may aid the development of therapies for neuroimmunological disorders and Alzheimer’s disease.

Weller RO, Carare RO.

The association between hypertensive arteriopathy and cerebral amyloid angiopathy in spontaneously hypertensive stroke-prone rats

We aimed to test the hypothesis that in spontaneously hypertensive stroke-prone rats (SHRSP), non-amyloid cerebral small vessel disease/hypertensive arteriopathy (HA) results in vessel wall injury that may promote cerebral amyloid angiopathy (CAA). Our study comprised 21 male SHRSP (age 17-44 weeks) and 10 age- and sex-matched Wistar control rats, that underwent two-photon (2PM) imaging of the arterioles in the parietal cortex using Methoxy-X04, Dextran and cerebral blood flow (CBF) measurements. Our data suggest that HA in SHRSP progresses in a temporal and age-dependent manner, starting from small vessel wall damage (stage 1A), proceeding to CBF reduction (stage 1B), non-occlusive (stage 2), and finally, occlusive thrombi (stage 3). Wistar animals also demonstrated small vessel wall damage, but were free of any of the later HA stages. Nearly half of all SHRSP additionally displayed vascular Methoxy-X04 positivity indicative of cortical CAA. Vascular β-amyloid deposits were found in small vessels characterized by thrombotic occlusions (stage 2 or 3). Post-mortem analysis of the rat brains confirmed the findings derived from intravital 2PM microscopy. Our data thus overall suggest that advanced HA may play a role in CAA development with the two small vessel disease entities might be related to the same pathological spectrum of the aging brain.

Solveig J, Garz C, Schwanke D, Sendtner M, Heinze HJ, Carare RO, Schreiber S

Convective influx/glymphatic system: tracers injected into the CSF enter and leave the brain along separate periarterial basement membrane pathways

Tracers injected into CSF pass into the brain alongside arteries and out again. This has been recently termed the “glymphaticsystem” 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 basementmembranes 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 basementmembranes. 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 enterand 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.

Albargothy NJ, Johnston DA, MacGregor Sharp M, Weller RO, Verma A, Hawkes CA, Carare RO.

Small vessels, dementia and chronic diseases – molecular mechanisms and pathophysiology

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

Horsburgh K, Wardlaw JM, van Agtmael T, Allan SM, Ashford MLJ, Bath Philip M, et al.

The meninges as barriers and facilitators for the movement of fluid, cells and pathogens related to the rodent and human CNS

Meninges that surround the CNS consist of an outer fibrous sheet of dura mater (pachymeninx) that is also the inner periosteum of the skull. Underlying the dura are the arachnoid and pia mater (leptomeninges) that form the boundaries of the subarachnoid space. In this review we (1) examine the development of leptomeninges and their role as barriers and facilitators in the foetal CNS. There are two separate CSF systems during early foetal life, inner CSF in the ventricles and outer CSF in the subarachnoid space. As the foramina of Magendi and Luschka develop, one continuous CSF system evolves. Due to the lack of arachnoid granulations during foetal life, it is most likely that CSF is eliminated by lymphatic drainage pathways passing through the cribriform plate and nasal submucosa. (2) We then review the fine structure of the adult human and rodent leptomeninges to establish their roles as barriers and facilitators for the movement of fluid, cells and pathogens. Leptomeningeal cells line CSF spaces, including arachnoid granulations and lymphatic drainage pathways, and separate elements of extracellular matrix from the CSF. The leptomeningeal lining facilitates the traffic of inflammatory cells within CSF but also allows attachment of bacteria such as Neisseria meningitidis and of tumour cells as CSF metastases. Single layers of leptomeningeal cells extend into the brain closely associated with the walls of arteries so that there are no perivascular spaces around arteries in the cerebral cortex. Perivascular spaces surrounding arteries in the white matter and basal ganglia relate to their two encompassing layers of leptomeninges. (3) Finally we examine the roles of ligands expressed by leptomeningeal cells for the attachment of inflammatory cells, bacteria and tumour cells as understanding these roles may aid the design of therapeutic strategies to manage developmental, autoimmune, infectious and neoplastic diseases relating to the CSF, the leptomeninges and the associated CNS.

Weller RO, MacGregor Sharp M, Christodoulides M, Carare RO, Møllgård K.

The fine anatomy of the perivascular compartment in the brain. Relevance to dilated perivascular spaces in cerebral amyloid angiopathy

Cerebral white matter hyperintensities (WMH) observed on magnetic resonance imaging (MRI), or low attenuation on computed tomographic scanning (CT), are the most frequent brain imaging finding in patients with small vessel disease or dementia. It has been assumed that WMH are due to arteriosclerosis or blood-brain barrier breakdown, though recently it was demonstrated that WMH have distinct molecular signatures in Alzheimer’s disease (AD) where markers of Wallerian degeneration are present, compared to normal ageing. Dilated perivascular spaces (PVS) are of particular interest for the study of interstitial fluid (ISF) dynamics because they are related to the intramural periarterial drainage (IPAD) of ISF and solutes along arterial basement membranes and can potentially be detected by MRI.

MacGregor Sharp M, Bulters D, Brandner S, Holton J, Verma A, Carare R.O, Werring D.


Inhibition of Aquaporin-4 Improves the Outcome of Ischaemic Stroke and Modulates Brain Paravascular Drainage Pathways

Pirici I, Balsanu T, Bogdan C, Margaritescu C, Divan T, Vitalie V, et al.

Editorial: Clearance Pathways for Amyloid-β. Significance for Alzheimer’s Disease and Its Therapy

Carare RO.

The perivascular pathways for influx of cerebrospinal fluid are most efficient in the midbrain

Dobson H, MacGregor Sharp M, Cumpsty R, Criswell TP, Wellman T, Finucane C, et al.

The structure of the perivascular compartment in the old canine brain: a case study

Criswell TP, MacGregor Sharp M, Dobson H, Finucane C, Weller RO, Verma A, et al

The increasing impact of cerebral amyloid angiopathy: essential new insights for clinical practice

Banerjee G, Carare R, Cordonnier C, Greenberg SM, Schneider JA, Smith EE, et al.

Arterial pulsations cannot drive intramural periarterial drainage: Significance for Aβ drainage

Diem A.K, MacGregor Sharp M, Gatherer M, Bressloff N.W, Carare R.O & Richardson G.

Hypercholesterolemia induced cerebral small vessel disease

Kraft P, Schuhmann MK, Garz C, Jandke S, Urlaub D, Mencl S, et al.

Loss of clusterin shifts amyloid deposition to the cerebrovasculature via disruption of perivascular drainage pathways

Wojtas AM, Kang SS, Olley BM, Gatherer M, Shinohara M, Lozano PA, et al.

Vascular basement membrane alterations and β-amyloid accumulations in an animal model of cerebral small vessel disease

Held F, Morris AWJ, Pirici D, Niklass S, MacGregor Sharp M, Garz C, et al.

The movers and shapers in immune privilege of the CNS

Engelhardt B, Vajkoczy P, Weller RO.

Quantitative assessment of cerebral basement membranes using electron microscopy

MacGregor Sharp M, Page A, Morris AWJ, Weller RO, Carare RO.

Investigating the Lymphatic Drainage of the Brain: Essential Skills and Tools

Albargothy JN, MacGregor Sharp M, Gatherer M, Morris AWJ, Weller RO, Hawkes CA, Carare RO.


Cerebrovascular pathology: the dark side of neurodegeneration.

Carare RO, Kalaria R.

Systems proteomic analysis reveals that Clusterin and Tissue Inhibitor of Metalloproteinases 3 increase in leptomeningeal arteries affected by cerebral amyloid angiopathy.

Manousopoulou A, Gatherer M, Smith C, Nicoll JA, Woelk CH, Johnson M, Kalaria R, Attems J, Garbis SD, Carare RO.

Vascular, glial, and lymphatic immune gateways of the central nervous system.

Engelhardt B, Carare RO, Bechmann B, Flügel A, Laman JD, Weller RO.

Pulsations with reflected boundary waves: a hydrodynamic reverse transport mechanism for perivascular drainage in the brain.

Coloma M, Schaffer JD, Carare RO, Chiarot PR, Huang P.

Vascular basement membranes as pathways for the passage of fluid into and out of the brain.

Morris AWJ, MacGregor Sharp M, Albargothy NJ, Hawkes CA, Verma A, Weller RO, Carare RO.

Hemisphere Asymmetry of Response to Pharmacologic Treatment in an Alzheimer’s Disease Mouse Model.

Manousopoulou A, Saito S, Yamamoto Y, Al-Daghri NM, Ihara M, Carare RO, Garbis SD.

Lymphatic Clearance of the Brain: Perivascular, Paravascular and Significance for Neurodegenerative Diseases.

Bakker E.N, Bacskai B.J, Arbel-Ornath M, Aldea R, Bedussi B, Morris A.W.J, Weller R.O, Carare R.O.

Increased Abeta pathology in aged Tg2576 mice born to mothers fed a high fat diet.

Nizari S, Carare RO, Hawkes CA.

A Simulation Model of Periarterial Clearance of Amyloid-beta from the Brain.

Diem AK, Tan M, Bressloff NW, Hawkes C, Morris AWJ, Weller RO, et al.


Chapter 19 – Pathophysiology of Lymphatic Drainage of the Central Nervous System: Implications for the Pathophysiology of Multiple Sclerosis.

Weller RO, Carare RO, Hawkes CA, Galea I.

Deposition of Amyloid β in the walls of human leptomeningeal arteries in relation to perivascular drainage pathways in cerebral amyloid angiopathy.

Keable A, Fenna K, Yuen HM, Johnston DA, Smyth NR, Smith C, Salman R, Samarasekera N, Nicoll JAR, Attems J, Kalaria RN, Weller RO, Carare RO.

Quantification of molecular interactions between apoE, Amyloid-beta (Aβ) and laminin: Relevance to accumulation of Aβ in Alzheimer’s disease.

Zekonyte J, Sakai K, Nicoll J, Weller RO, Carare RO.

Clearance systems in the brain-implications for Alzheimer disease.

Tarasoff-Conway JM, Carare RO, Osorio RS, Glodzik L, Butler T, Fieremans E, Axel L, Rusinek H, Nicholson C, Zlokovic BV, Frangione B, Blennow K, Ménard J, Zetterberg H, Wisniewski T, de Leon MJ.

Does the difference between PART and Alzheimer’s disease lie in the age-related changes in cerebral arteries that trigger the accumulation of Aβ and propagation of tau?

Weller RO, Hawkes CA, Carare RO, Hardy J.

Peristalsis with oscillating flow resistance: A mechanism for periarterial clearance of amyloid beta from the brain.

Sharp, MK, Diem AK, Weller RO, Carare RO.

White matter changes in dementia: role of impaired drainage of interstitial fluid.

Weller RO, Hawkes CA, Kalaria RN, Werring DJ, Carare RO.

Are you also what your mother eats: Distinct proteomic portrait as a result of maternal high-fat diet in the cerebral cortex of the adult mouse.

Manousopoulou A, Woo J, Hawkes CA, Johnston H, Singhania A, Woelk CH, Garbis SD, Carare RO.

Are the effects of APOE ϵ4 on cognitive function in nonclinical populations age- and gender-dependent?

Rusted J & Carare RO.


Prenatal high fat diet alters the cerebrovasculature and clearance of beta-amyloid in adult offspring.

Hawkes CA, Gentleman S, Nicoll JAR, Carare RO.

The cerebrovascular basement membrane: role in the clearance of β-amyloid and Cerebral Amyloid Angiopathy.

Morris A.W, Carare RO, Schreiber S, Hawkes CA.

Failure of perivascular drainage of beta-amyloid in cerebral amyloid angiopathy.

Hawkes CA, Jayakody N, Johnston DA, Bechmann I, Carare RO.

Impact of N-acetylcysteine on cerebral parenchymal Aβ plaques and kidney damage in spontaneously hypertensive stroke-prone rats.

Bueche CZ, Garz C, Stanaszek L, Niklass S, Kropf S, Bittner D, Härtig W, Reymann KG, Heinze HJ, Carare RO, Schreiber S.

Aß immunotherapy for Alzheimer’s disease: effects on apoE and cerebral vasculopathy.

Sakai K, Boche D, Carare RO, Johnston D, Holmes C, Love S, Nicoll JAR.

Amyloid and tau in the brain in sporadic Alzheimer’s disease: defining the chicken and the egg.

Hawkes C, Carare RO, Weller RO.

Hypertension drives parenchymal ß-amyloid accumulation in the brain parenchyma.

Bueche CZ, Hawkes C, Garz C, Vielhaber S, Attems J, Knight RT, Reymann K, Heinze H, Carare R, Schreiber S.


MK886 reduces cerebral amyloid angiopathy severity in TgCRND8 mice.

Hawkes CA, Shaw JE, Brown MS, Anthony P, McLaurin J, Carare RO.

Immune complex formation impairs the elimination of solutes from the brain: implications for immunotherapy in Alzheimer’s disease.

Carare RO, Teeling J, Hawkes CA, Püntener U, Weller RO, Nicoll JAR, Perry V.

Regional differences in the morphological and functional effects of aging on cerebral basement membranes and perivascular drainage of amyloid-β from the mouse brain.

Hawkes CA, Gatherer M, Sharp MM, Dorr A, Yuen HM, Kalaria R, Weller RO, Carare RO.

Apolipoprotein E epsilon 4 (apoE4) may impair perivascular elimination of amyloid-beta (A beta).

Hawkes CA, Zekonyte J, Howard K, Nicoll JAR, Weller RO, Carare RO.