What is IPAD?

Apart from the blood, there are two fluids associated with the brain: cerebrospinal fluid (CSF) and interstitial fluid (ISF). CSF drains through arachnoid villi into the blood and via routes adjacent to olfactory nerves into the nasal mucosa, cervical and dural lymphatics….
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Strategies for improving IPAD?

Phosphodiesterase III is the major cAMP-hydrolyzing PDE uniquely expressed in vascular smooth muscle cells; PDE IIIA isoforms are also involved in cardiovascular function by regulating vascular smooth muscle growth and phenotypic changes…..
Read more
What is IPAD?

Intramural periarterial drainage

Apart from the blood, there are two fluids associated with the brain: cerebrospinal fluid (CSF) and interstitial fluid (ISF). CSF drains through arachnoid villi into the blood and via routes adjacent to olfactory nerves into the nasal mucosa, cervical and dural lymphatics (1). This route permits the drainage of antigen presenting cells from the subarachnoid space into the lymphatic system.

The brain parenchyma is not endowed with traditional lymphatic vessels. For the last 50 years different physiological studies have shown that interstitial fluid drains from the brain along perivascular pathways into cervical lymphatics (2). Using refined injection techniques and confocal microscopy, our group has demonstrated that drainage of interstitial fluid and solutes from the brain occurs along 100-150 nm-wide basement membranes (BM) in the walls of cerebral capillaries and arteries. Older experimental studies suggest that only 10-15% of solutes draining by this route escape into the CSF (3). We have demonstrated that injection of soluble Aβ into the brain parenchyma of young mice results in its rapid elimination along the BM of capillaries and arteries as intramural periarterial drainage (IPAD) (4).

Our theoretical modelling studies suggest that the motive force for perivascular lymphatic drainage is derived from vascular smooth muscle contractions and biochemical interactions with basement membranes (5) (6) (7). With increasing age and arteriosclerosis, cerebral arteries become stiffer (8) with reduced contractility of arterial smooth muscle cells. Motive force declines reducing efficiency of lymphatic drainage of the brain as shown in aged mice (4). Our working hypothesis is that the deposition of amyloid plaques in the human brain with age and Alzheimer’s disease reflects a failure of elimination of Aβ from the brain. Several mechanisms for the elimination of Aβ from the brain have been defined. These include degradation by enzymes such as neprilysin (9), receptor-mediated absorption into the blood (10), passage into the CSF (11) and perivascular lymphatic drainage (12). Reduction in neprilysin activity and failure of absorption of Aβ into the blood with age (9) (10) may divert more Aβ along perivascular lymphatic drainage pathways (9) (13).

As arteries age, IPAD becomes less efficient [4] and Aβ is deposited in basement membranes of arteries and capillaries as cerebral amyloid angiopathy (CAA), which further impairs perivascular lymphatic drainage (14). APOE ε4 is also associated with impaired perivascular lymphatic drainage as demonstrated in mice expressing human ApoE ε4 (15).

As a result of the failure of elimination of Aβ from the brain associated with ageing of cerebral arteries and CAA there is loss of homoeostasis of the extracellular environment in the brain as reflected in the rise of soluble Aβ in Alzheimer’s disease (16). It is likely that there is also failure of elimination of soluble metabolites other than Aβ adding further to the loss of homoeostasis of the neuronal environment. The association of CAA with accumulation of fluid in the subcortical white matter reported after recent therapeutic trials in Alzheimer’s disease suggests that drainage of fluid is ultimately impaired (17) (18).

We are working in an interdisciplinary manner to demonstrate that changes in extracellular matrix and artery walls due to age, genotype, diet or different patterns of innervation or branching of blood vessels could have a marked effect upon the extracellular environment of brain tissue leading especially to failure of elimination of Aβ from the extracellular space but also to failure of elimination of other metabolites and loss of homeostasis.  By clarifying the exact factors that are responsible for efficient drainage along basement membranes of capillaries and arteries we are identifying new therapeutic targets for cerebral amyloid angiopathy and Alzheimer’s disease.

 

Strategies for improving IPAD

Agents that improve contractility of vascular smooth muscle cells

Phosphodiesterase III is the major cAMP-hydrolyzing PDE uniquely expressed in vascular smooth muscle cells; PDE IIIA isoforms are also involved in cardiovascular function by regulating vascular smooth muscle growth and phenotypic changes. Cilostazol is a selective inhibitor of PDE III that increases cAMP in vascular cells and has multiple effects on the vasculature such as vasodilatation, anti-oxidation, anti-inflammation, regulation of smooth muscle cells, increase in cerebral haemodynamics and arterial elasticity with maintenance of microvascular integrity, as reviewed in (19). Cognition is significantly improved in experimental models and in humans receiving Cilostazol (20)(21)(22)(23). Administration of Cilostazol significantly improves IPAD and the brains of mice treated with Cilostazol show effects upon extracellular matrix, with upregulation of the anti-fibrillogenic glycoproteins (24)(25).

Using chaperones for efficient transport along the IPAD pathways

Clusterin (Apolipoprotein J) is a multifunctional protein that reduces the aggregation and toxicity of Aβ and appears to be beneficial in atherosclerosis (26)(27). We recently demonstrated that in APP/PS1 mouse models of Alzheimer’s disease, crossed with clusterin knockout mice, result in disappearance of Aβ plaques but an increase in severity of CAA. These findings suggest that clusterin is required for efficient chaperoning of solubilized proteins from plaques along IPAD (28). Administration of clusterin as a preventative therapy when the integrity and function of smooth muscle cells and basement membranes are not compromised may yield positive results for the prevention or delay in onset of symptoms of CAA and Alzheimer’s disease. Taxifolin is flavonoid that appears to maintain amyloid in its soluble forms more amenable for clearance (29) We are investigating whether Taxifolin facilitates IPAD.

Agents acting upon the innervation of smooth muscle cells

Experimental work is ongoing in this area. Results suggest that agents such as Prazosin, an alpha(1)-adrenoceptor antagonist, acting upon cholinergic or adrenergic innervation of cerebral arteries result in improvements of IPAD and in reduction of CAA in transgenic mouse models of Alzheimer’s disease (30).

references

1.
Kida S, Pantazis A, Weller R. CSF drains directly from the subarachnoid space into nasal lymphatics in the rat. Anatomy, histology and immunological significance. Neuropathol Appl Neurobiol. 1993;19(6):480-488. [PubMed]
2.
Bradbury M, Cserr H, Westrop R. Drainage of cerebral interstitial fluid into deep cervical lymph of the rabbit. Am J Physiol. 1981;240(4):F329-36. [PubMed]
3.
Szentistványi I, Patlak C, Ellis R, Cserr H. Drainage of interstitial fluid from different regions of rat brain. Am J Physiol. 1984;246(6 Pt 2):F835-44. [PubMed]
4.
Hawkes C, Härtig W, Kacza J, et al. Perivascular drainage of solutes is impaired in the ageing mouse brain and in the presence of cerebral amyloid angiopathy. Acta Neuropathol. 2011;121(4):431-443. [PubMed]
5.
Schley D, Carare-Nnadi R, Please CP, Perry VH, Weller RO. Mechanisms to explain the reverse perivascular transport of solutes out of the brain. J. 2006;238(4):962-974. doi: 10.1016/j.jtbi.2005.07.005
6.
Arbel-Ornath M, Hudry E, Eikermann-Haerter K, et al. Interstitial fluid drainage is impaired in ischemic stroke and Alzheimer’s disease mouse models. Acta Neuropathol. 2013;126(3):353-364. [PubMed]
7.
Diem A, MacGregor S, Gatherer M, Bressloff N, Carare R, Richardson G. Arterial Pulsations cannot Drive Intramural Periarterial Drainage: Significance for Aβ Drainage. Front Neurosci. 2017;11:475. [PMC]
8.
Weller R, Boche D, Nicoll J. Microvasculature changes and cerebral amyloid angiopathy in Alzheimer’s disease and their potential impact on therapy. Acta Neuropathol. 2009;118(1):87-102. [PubMed]
9.
Miners J, Van H, Chalmers K, Wilcock G, Love S, Kehoe P. Decreased expression and activity of neprilysin in Alzheimer disease are associated with cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2006;65(10):1012-1021. [PubMed]
10.
Zlokovic B. Clearing amyloid through the blood-brain barrier. J Neurochem. 2004;89(4):807-811. [PubMed]
11.
Iliff J, Wang M, Liao Y, et al. A paravascular pathway facilitates CSF flow through the brain parenchyma and the clearance of interstitial solutes, including amyloid β. Sci Transl Med. 2012;4(147):147ra111. [PubMed]
12.
Weller R, Djuanda E, Yow H, Carare R. Lymphatic drainage of the brain and the pathophysiology of neurological disease. Acta Neuropathol. 2009;117(1):1-14. [PubMed]
13.
Shibata M, Yamada S, Kumar S, et al. Clearance of Alzheimer’s amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J Clin Invest. 2000;106(12):1489-1499. [PubMed]
14.
Hawkes C, Jayakody N, Johnston D, Bechmann I, Carare R. Failure of perivascular drainage of β-amyloid in cerebral amyloid angiopathy. Brain Pathol. 2014;24(4):396-403. [PubMed]
15.
Hawkes C, Sullivan P, Hands S, Weller R, Nicoll J, Carare R. Disruption of Arterial Perivascular Drainage of Amyloid-β from the Brains of Mice Expressing the Human APOE ε4 Allele. PLoS One. 2012;7(7):e41636. [PMC]
16.
Tomic J, Pensalfini A, Head E, Glabe C. Soluble fibrillar oligomer levels are elevated in Alzheimer’s disease brain and correlate with cognitive dysfunction. Neurobiol Dis. 2009;35(3):352-358. [PubMed]
17.
Roher A, Kuo Y, Esh C, et al. Cortical and leptomeningeal cerebrovascular amyloid and white matter pathology in Alzheimer’s disease. Mol Med. 2003;9(3-4):112-122. [PubMed]
18.
Weller R, Hawkes C, Kalaria R, Werring D, Carare R. White matter changes in dementia: role of impaired drainage of interstitial fluid. Brain Pathol. 2015;25(1):63-78. [PubMed]
19.
Saito S, Ihara M. New therapeutic approaches for Alzheimer’s disease and cerebral amyloid angiopathy. Front Aging Neurosci. 2014 Oct 20;6:290. [PubMed]
20.
Saito S, Kojima S, Oishi N, Kakuta R, Maki T, Yasuno F, et al. A multicenter, randomized, placebo-controlled trial for cilostazol in patients with mild cognitive impairment: The COMCID study protocol. Alzheimers Dement (N Y). 2016 Oct 27;2(4):250–7. [PMC]
21.
Kitamura A, Manso Y, Duncombe J, Searcy J, Koudelka J, Binnie M, et al. Long-term cilostazol treatment reduces gliovascular damage and memory impairment in a mouse model of chronic cerebral hypoperfusion. Sci Rep. 2017 Jun 27;7(1):4299. [PubMed]
22.
Yanai S, Ito H, Endo S. Long-term cilostazol administration prevents age-related decline of hippocampus-dependent memory in mice. Neuropharmacology. 2018 Feb 1;129:57–68. [PubMed]
23.
Yanai S, Toyohara J, Ishiwata K, Ito H, Endo S. Long-term cilostazol administration ameliorates memory decline in senescence-accelerated mouse prone 8 (SAMP8) through a dual effect on cAMP and blood-brain barrier. Neuropharmacology. 2017 Apr 1;116:247–59. [PubMed]
24.
Maki T, Okamoto Y, Carare R, Hase Y, Hattori Y, Hawkes C, et al. Phosphodiesterase III inhibitor promotes drainage of cerebrovascular β-amyloid. Ann Clin Transl Neurol. 2014 Jul 8;1(8):519–33. [PMC]
25.
Manousopoulou A, Saito S, Yamamoto Y, Al-Daghri N, Ihara M, Carare R, et al. Hemisphere Asymmetry of Response to Pharmacologic Treatment in an Alzheimer’s Disease Mouse Model. J Alzheimers Dis. 2016 Mar 15;51(2):333–8. [PMC]
26.
Bielicki J, Zhang H, Cortez Y, Zheng Y, Narayanaswami V, Patel A, et al. A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice. J Lipid Res. 2010 Jun 1;51(6):1496–503. [PubMed]
27.
Narayan P, Orte A, Clarke R, Bolognesi B, Hook S, Ganzinger K, et al. The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β(1-40) peptide. Nat Struct Mol Biol. 2011 Dec 18;19(1):79–83. [PubMed]
28.
Wojtas AM, Kang SS, Olley BM, Gatherer M, Shinohara M, Lozano PA, et al. Loss of clusterin shifts amyloid deposition to the cerebrovasculature via disruption of perivascular drainage pathways. P [Internet]. 2017 Jul 12;114(33):E6962–71. Available from: http://dx.doi.org/10.1073/pnas.1701137114
29.
Saito S, Yamamoto Y, Maki T, Hattori Y, Ito H, Mizuno K, et al. Taxifolin inhibits amyloid-β oligomer formation and fully restores vascular integrity and memory in cerebral amyloid angiopathy. Acta Neuropathol Commun. 2017 Apr 4;5:26. [PMC]
30.
Katsouri L, Vizcaychipi M, McArthur S, Harrison I, Suárez-Calvet M, Lleo A, et al. Prazosin, an α(1)-adrenoceptor antagonist, prevents memory deterioration in the APP23 transgenic mouse model of Alzheimer’s disease. Neurobiol Aging. 2013 Apr 1;34(4):1105–15. [PubMed]

Overview

Investigating Immunisation Strategies for the Treatment of Synucleinopathies

Next generation immunisation strategies have enabled the manufacture of highly efficacious vaccines to treat major global diseases which are currently untreatable. United Neuroscience (UNS), a biotechnological company, has aimed to overcome the current vaccine challenges in the field of neurodegenerative disease by designing highly targeted vaccines which elicit a protective immune response. Synucleinopathies comprise a group of neurodegenerative diseases that are characterised by primary alpha-synuclein (α-Syn) pathology such as Dementia with Lewy Bodies (DLB), Parkinson’s disease (PD) and Multiple systems atrophy (MSA). The central role of α-Syn in the pathogenesis of these diseases highlights it as a promising target for therapy. In this study we aim to test the effects of novel α-Syn vaccines developed by UNS on preventing the onset and progression of neurodegeneration in mouse models of these synucleinopathies. In order to investigate this, we first need to understand the pathway along which α-Syn is naturally cleared from the brain and we can then establish how immunotherapy modulates this process and evaluate the neuroprotective effects of this as a treatment.

Funding
United Neuroscience project grant – “Pathology and mechanisms of immunization in neurodegenerative diseases” £240,000
Overview

Failure of drainage of fluid from the brain along the walls of blood vessels in vascular dementia

Cerebral small vessel disease (SVD) is a key feature of vascular dementia, radiologically defined by the presence of white matter hyperintensities, lacunar infarcts, microbleeds and perivascular spaces.  Cerebral arteriolosclerosis resulting in loss of elasticity and segmental disorganisation of the arterial wall leads to damage of the deep white matter.  The primary functions of penetrating and perforating cerebral arteries supplying blood and drainage of fluid and solutes from the parenchyma along IPAD pathways are impaired.  In this project, using animal models and post-mortem brain tissue from stroke survivors with SVD (CogFAST study) along with light sheet 3D microscopy and post-mortem MRI, we will assess the immunocytochemical pattern of distribution of AQP4, α-dystrobrevin and β-dystroglycan.  We will then test the hypotheses that 1) disruption in the anchoring system of the basement membranes such as that observed in α-dystrobrevin knock-out mice and 2) disruption of gliovascular end feet tracked by aquaporin 4 (AQP4) knock-out mice there is failure of perivascular clearance of fluid from the deep gray matter and the corpus callosum.  Our aim is to demonstrate that failure of perivascular drainage of fluid from the brain is a mechanism underlying SVD and this could be targeted therapeutically.

Funding
Stroke Association Priority Programme Award (vascular dementia) – “Failure of drainage of fluid from the brain along the walls of blood vessels in vascular dementia“. £245,198.00
Key publications
Apr 2018
Small vessels, dementia and chronic diseases – molecular mechanisms and pathophysiology.
Horsburgh K, Wardlaw JM, van Agtmael T, Allan SM, Ashford MLJ, Bath Philip M, et al.

 

Feb 2018
The fine anatomy of the perivascular compartment in the brain. Relevance to dilated perivascular spaces in cerebral amyloid angiopathy.
MacGregor Sharp M, Bulters D, Brandner S, Holton J, Verma A, Carare R.O, Werring D.

Overview

In vivo MRI imaging of the motive force driving intramural perivascular clearance

Based on results from mathematical modelling we now know that the motive force for IPAD is provided by spontaneous vasomotion resulting from the intrinsic contractions of pericytes and cerebrovascular smooth muscle cells and not by the pulsations derived from cardiac cycle (Dr Alexandra Diem, Prof Neil Bressloff). Collaborations are in place with senior neuroradiologists in University College London and Leiden, The Netherlands to demonstrate in vivo in real time using MRI, the features of the motive force for efficient IPAD clearance and correlate this with findings in different stages of Alzheimer’s disease and mild cognitive impairment. This could be the first marker for impaired clearance of cerebral interstitial fluid in humans.

Funding
EPSRC Doctoral Prize
Key publications
Mar 2016
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.

Overview

Development of an in vitro perivascular clearance system

Using a novel Quasi Vivo in vitro system developed by Kirkstall Ltd and mouse astrocytes that express humanised ApoE, (collaboration with David Holtzman, Washington University, USA), we aim to test the hypothesis that flow of Aβ over astrocytes expressing different forms of ApoE results in morphological alterations to the astrocytes expressing ApoE4, compared to those expressing ApoE2 or ApoE3.

Using coated coverslips, the astrocytes are plated and left to adhere for 24 hours before being loaded into the QV500 chamber or 24 well plate for static experiments. A solution of astrocyte growth medium supplemented with 100nM Aβ 1-40 is circulated around the system for 24 hours. The coverslip is removed and fixed in 4% PFA, immunostained and examined by confocal microscopy. We have already optimised the system for testing the activity of Quasi Vivo and concluded that the Quasi Vivo system has no significant toxic effect on the growth or viability of the cells when cells grown under flow were compared with static controls. There appears to be a decrease in cell number when Aβ is applied to ApoE4 astrocytes under flow. We have also shown that there are morphological changes to ApoE4 astrocytes when Aβ is applied that are further enhanced when combined with flow. These changes are not seen in ApoE2 or ApoE3 astrocytes. We also observed that Aβ appears to be concentrated where there are clusters of cells, though this has only been seen in ApoE3 astrocytes. This work suggests that the dynamics of interactions between Aβ and astrocytes are dependent on their APOE genotype and this is likely to contribute to the reduced clearance of Aβ in APOE4 individuals.

Funding
BBSRC CASE PhD Studentship with Kirkstall Ltd – “Development of an in vitro perivascular clearance system“. £95,042
Overview

Does maternal high fat diet lead to dementia?

In this project we test the hypothesis that exposure to a high fat diet during development and early life, leads to the remodelling of the neurovascular unit and reduces the efficiency of Aβ clearance from the brain, leading to increased CAA severity. A mouse model of pre- and postnatal high fat diet exposure will be established by feeding female mice (C57Bl/6), either a standard (21% kcal fat) or high fat (45% kcal fat) diet for 4 weeks before conception and during gestation and lactation. At weaning, male and female offspring will be fed either a normal or high fat to generate 4 groups of experimental mice. We will create a separate group of female pregnant mice that will be treated with Metformin to assess whether this therapeutic agent is effective in halting the pathological process. We aim to demonstrate that simple measures like improving the diet of pregnant mothers and managing hypercholesterolaemia and diabetes will prevent or delay the onset of dementia.

Funding
Rosetrees Trust Project grant – “The effect of maternal high fat on the clearance of interstitial fluid from the brain“. £25,000.
Overview

Innervation of cerebral arteries is key to maintenance of efficient clearance and flow

This project, funded by Alzheimer’s Research UK in collaboration with Dr Cheryl Hawkes (Open University), tests the hypothesis that loss of perivascular innervation by cholinergic neurons leads to dysfunctional regulation of vascular tone, thereby reducing the motive force for perivascular drainage of Aβ leading to a worsening of cerebral amyloid angiopathy.

Funding
Alzheimer’s Research UK – Co-PI “Targeting perivascular innervation and vascular tone for improved clearance of ß-amyloid from the brain“. £88,440

Overview

What is IPAD?

Intramural periarterial drainage

Apart from the blood, there are two fluids associated with the brain: cerebrospinal fluid (CSF) and interstitial fluid (ISF). CSF drains through arachnoid villi into the blood and via routes adjacent to olfactory nerves into the nasal mucosa, cervical and dural lymphatics (1). This route permits the drainage of antigen presenting cells from the subarachnoid space into the lymphatic system.

The brain parenchyma is not endowed with traditional lymphatic vessels. For the last 50 years different physiological studies have shown that interstitial fluid drains from the brain along perivascular pathways into cervical lymphatics (2). Using refined injection techniques and confocal microscopy, our group has demonstrated that drainage of interstitial fluid and solutes from the brain occurs along 100-150 nm-wide basement membranes (BM) in the walls of cerebral capillaries and arteries. Older experimental studies suggest that only 10-15% of solutes draining by this route escape into the CSF (3). We have demonstrated that injection of soluble Aβ into the brain parenchyma of young mice results in its rapid elimination along the BM of capillaries and arteries as intramural periarterial drainage (IPAD) (4).

Our theoretical modelling studies suggest that the motive force for perivascular lymphatic drainage is derived from vascular smooth muscle contractions and biochemical interactions with basement membranes (5) (6) (7). With increasing age and arteriosclerosis, cerebral arteries become stiffer (8) with reduced contractility of arterial smooth muscle cells. Motive force declines reducing efficiency of lymphatic drainage of the brain as shown in aged mice (4). Our working hypothesis is that the deposition of amyloid plaques in the human brain with age and Alzheimer’s disease reflects a failure of elimination of Aβ from the brain. Several mechanisms for the elimination of Aβ from the brain have been defined. These include degradation by enzymes such as neprilysin (9), receptor-mediated absorption into the blood (10), passage into the CSF (11) and perivascular lymphatic drainage (12). Reduction in neprilysin activity and failure of absorption of Aβ into the blood with age (9) (10) may divert more Aβ along perivascular lymphatic drainage pathways (9) (13).

As arteries age, IPAD becomes less efficient [4] and Aβ is deposited in basement membranes of arteries and capillaries as cerebral amyloid angiopathy (CAA), which further impairs perivascular lymphatic drainage (14). APOE ε4 is also associated with impaired perivascular lymphatic drainage as demonstrated in mice expressing human ApoE ε4 (15).

As a result of the failure of elimination of Aβ from the brain associated with ageing of cerebral arteries and CAA there is loss of homoeostasis of the extracellular environment in the brain as reflected in the rise of soluble Aβ in Alzheimer’s disease (16). It is likely that there is also failure of elimination of soluble metabolites other than Aβ adding further to the loss of homoeostasis of the neuronal environment. The association of CAA with accumulation of fluid in the subcortical white matter reported after recent therapeutic trials in Alzheimer’s disease suggests that drainage of fluid is ultimately impaired (17) (18).

We are working in an interdisciplinary manner to demonstrate that changes in extracellular matrix and artery walls due to age, genotype, diet or different patterns of innervation or branching of blood vessels could have a marked effect upon the extracellular environment of brain tissue leading especially to failure of elimination of Aβ from the extracellular space but also to failure of elimination of other metabolites and loss of homeostasis.  By clarifying the exact factors that are responsible for efficient drainage along basement membranes of capillaries and arteries we are identifying new therapeutic targets for cerebral amyloid angiopathy and Alzheimer’s disease.

 

Strategies for improving IPAD

Agents that improve contractility of vascular smooth muscle cells

Phosphodiesterase III is the major cAMP-hydrolyzing PDE uniquely expressed in vascular smooth muscle cells; PDE IIIA isoforms are also involved in cardiovascular function by regulating vascular smooth muscle growth and phenotypic changes. Cilostazol is a selective inhibitor of PDE III that increases cAMP in vascular cells and has multiple effects on the vasculature such as vasodilatation, anti-oxidation, anti-inflammation, regulation of smooth muscle cells, increase in cerebral haemodynamics and arterial elasticity with maintenance of microvascular integrity, as reviewed in (19). Cognition is significantly improved in experimental models and in humans receiving Cilostazol (20)(21)(22)(23). Administration of Cilostazol significantly improves IPAD and the brains of mice treated with Cilostazol show effects upon extracellular matrix, with upregulation of the anti-fibrillogenic glycoproteins (24)(25).

Using chaperones for efficient transport along the IPAD pathways

Clusterin (Apolipoprotein J) is a multifunctional protein that reduces the aggregation and toxicity of Aβ and appears to be beneficial in atherosclerosis (26)(27). We recently demonstrated that in APP/PS1 mouse models of Alzheimer’s disease, crossed with clusterin knockout mice, result in disappearance of Aβ plaques but an increase in severity of CAA. These findings suggest that clusterin is required for efficient chaperoning of solubilized proteins from plaques along IPAD (28). Administration of clusterin as a preventative therapy when the integrity and function of smooth muscle cells and basement membranes are not compromised may yield positive results for the prevention or delay in onset of symptoms of CAA and Alzheimer’s disease. Taxifolin is flavonoid that appears to maintain amyloid in its soluble forms more amenable for clearance (29) We are investigating whether Taxifolin facilitates IPAD.

Agents acting upon the innervation of smooth muscle cells

Experimental work is ongoing in this area. Results suggest that agents such as Prazosin, an alpha(1)-adrenoceptor antagonist, acting upon cholinergic or adrenergic innervation of cerebral arteries result in improvements of IPAD and in reduction of CAA in transgenic mouse models of Alzheimer’s disease (30).

Active projects

Overview

Investigating Immunisation Strategies for the Treatment of Synucleinopathies

Next generation immunisation strategies have enabled the manufacture of highly efficacious vaccines to treat major global diseases which are currently untreatable. United Neuroscience (UNS), a biotechnological company, has aimed to overcome the current vaccine challenges in the field of neurodegenerative disease by designing highly targeted vaccines which elicit a protective immune response. Synucleinopathies comprise a group of neurodegenerative diseases that are characterised by primary alpha-synuclein (α-Syn) pathology such as Dementia with Lewy Bodies (DLB), Parkinson’s disease (PD) and Multiple systems atrophy (MSA). The central role of α-Syn in the pathogenesis of these diseases highlights it as a promising target for therapy. In this study we aim to test the effects of novel α-Syn vaccines developed by UNS on preventing the onset and progression of neurodegeneration in mouse models of these synucleinopathies. In order to investigate this, we first need to understand the pathway along which α-Syn is naturally cleared from the brain and we can then establish how immunotherapy modulates this process and evaluate the neuroprotective effects of this as a treatment.

Funding
United Neuroscience project grant – “Pathology and mechanisms of immunization in neurodegenerative diseases” £240,000
Overview

Failure of drainage of fluid from the brain along the walls of blood vessels in vascular dementia

Cerebral small vessel disease (SVD) is a key feature of vascular dementia, radiologically defined by the presence of white matter hyperintensities, lacunar infarcts, microbleeds and perivascular spaces.  Cerebral arteriolosclerosis resulting in loss of elasticity and segmental disorganisation of the arterial wall leads to damage of the deep white matter.  The primary functions of penetrating and perforating cerebral arteries supplying blood and drainage of fluid and solutes from the parenchyma along IPAD pathways are impaired.  In this project, using animal models and post-mortem brain tissue from stroke survivors with SVD (CogFAST study) along with light sheet 3D microscopy and post-mortem MRI, we will assess the immunocytochemical pattern of distribution of AQP4, α-dystrobrevin and β-dystroglycan.  We will then test the hypotheses that 1) disruption in the anchoring system of the basement membranes such as that observed in α-dystrobrevin knock-out mice and 2) disruption of gliovascular end feet tracked by aquaporin 4 (AQP4) knock-out mice there is failure of perivascular clearance of fluid from the deep gray matter and the corpus callosum.  Our aim is to demonstrate that failure of perivascular drainage of fluid from the brain is a mechanism underlying SVD and this could be targeted therapeutically.

Funding
Stroke Association Priority Programme Award (vascular dementia) – “Failure of drainage of fluid from the brain along the walls of blood vessels in vascular dementia“. £245,198.00
Key publications
Apr 2018
Small vessels, dementia and chronic diseases – molecular mechanisms and pathophysiology.
Horsburgh K, Wardlaw JM, van Agtmael T, Allan SM, Ashford MLJ, Bath Philip M, et al.

 

Feb 2018
The fine anatomy of the perivascular compartment in the brain. Relevance to dilated perivascular spaces in cerebral amyloid angiopathy.
MacGregor Sharp M, Bulters D, Brandner S, Holton J, Verma A, Carare R.O, Werring D.

Overview

In vivo MRI imaging of the motive force driving intramural perivascular clearance

Based on results from mathematical modelling we now know that the motive force for IPAD is provided by spontaneous vasomotion resulting from the intrinsic contractions of pericytes and cerebrovascular smooth muscle cells and not by the pulsations derived from cardiac cycle (Dr Alexandra Diem, Prof Neil Bressloff). Collaborations are in place with senior neuroradiologists in University College London and Leiden, The Netherlands to demonstrate in vivo in real time using MRI, the features of the motive force for efficient IPAD clearance and correlate this with findings in different stages of Alzheimer’s disease and mild cognitive impairment. This could be the first marker for impaired clearance of cerebral interstitial fluid in humans.

Funding
EPSRC Doctoral Prize
Key publications
Mar 2016
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.

Overview

Development of an in vitro perivascular clearance system

Using a novel Quasi Vivo in vitro system developed by Kirkstall Ltd and mouse astrocytes that express humanised ApoE, (collaboration with David Holtzman, Washington University, USA), we aim to test the hypothesis that flow of Aβ over astrocytes expressing different forms of ApoE results in morphological alterations to the astrocytes expressing ApoE4, compared to those expressing ApoE2 or ApoE3.

Using coated coverslips, the astrocytes are plated and left to adhere for 24 hours before being loaded into the QV500 chamber or 24 well plate for static experiments. A solution of astrocyte growth medium supplemented with 100nM Aβ 1-40 is circulated around the system for 24 hours. The coverslip is removed and fixed in 4% PFA, immunostained and examined by confocal microscopy. We have already optimised the system for testing the activity of Quasi Vivo and concluded that the Quasi Vivo system has no significant toxic effect on the growth or viability of the cells when cells grown under flow were compared with static controls. There appears to be a decrease in cell number when Aβ is applied to ApoE4 astrocytes under flow. We have also shown that there are morphological changes to ApoE4 astrocytes when Aβ is applied that are further enhanced when combined with flow. These changes are not seen in ApoE2 or ApoE3 astrocytes. We also observed that Aβ appears to be concentrated where there are clusters of cells, though this has only been seen in ApoE3 astrocytes. This work suggests that the dynamics of interactions between Aβ and astrocytes are dependent on their APOE genotype and this is likely to contribute to the reduced clearance of Aβ in APOE4 individuals.

Funding
BBSRC CASE PhD Studentship with Kirkstall Ltd – “Development of an in vitro perivascular clearance system“. £95,042
Overview

Does maternal high fat diet lead to dementia?

In this project we test the hypothesis that exposure to a high fat diet during development and early life, leads to the remodelling of the neurovascular unit and reduces the efficiency of Aβ clearance from the brain, leading to increased CAA severity. A mouse model of pre- and postnatal high fat diet exposure will be established by feeding female mice (C57Bl/6), either a standard (21% kcal fat) or high fat (45% kcal fat) diet for 4 weeks before conception and during gestation and lactation. At weaning, male and female offspring will be fed either a normal or high fat to generate 4 groups of experimental mice. We will create a separate group of female pregnant mice that will be treated with Metformin to assess whether this therapeutic agent is effective in halting the pathological process. We aim to demonstrate that simple measures like improving the diet of pregnant mothers and managing hypercholesterolaemia and diabetes will prevent or delay the onset of dementia.

Funding
Rosetrees Trust Project grant – “The effect of maternal high fat on the clearance of interstitial fluid from the brain“. £25,000.
Overview

Innervation of cerebral arteries is key to maintenance of efficient clearance and flow

This project, funded by Alzheimer’s Research UK in collaboration with Dr Cheryl Hawkes (Open University), tests the hypothesis that loss of perivascular innervation by cholinergic neurons leads to dysfunctional regulation of vascular tone, thereby reducing the motive force for perivascular drainage of Aβ leading to a worsening of cerebral amyloid angiopathy.

Funding
Alzheimer’s Research UK – Co-PI “Targeting perivascular innervation and vascular tone for improved clearance of ß-amyloid from the brain“. £88,440