Targeting lysyl-oxidase (LOX) may facilitate intramural periarterial drainage for the treatment of Alzheimer's disease.

Alzheimer's disease is the commonest form of dementia. It is likely that a lack of clearance of amyloid beta (Aß) results in its accumulation in the parenchyma as Aß oligomers and insoluble plaques, and within the walls of blood vessels as cerebral amyloid angiopathy (CAA). The drainage of Aß along the basement membranes of blood vessels as intramural periarterial drainage (IPAD), could be improved if the driving force behind IPAD could be augmented, therefore reducing Aß accumulation. There are alterations in the composition of the vascular basement membrane in Alzheimer's disease. Lysyl oxidase (LOX) is an enzyme involved in the remodelling of the extracellular matrix and its expression and function is altered in various disease states. The expression of LOX is increased in Alzheimer's disease, but it is unclear whether this is a contributory factor in the impairment of IPAD in Alzheimer's disease. The pharmacological inhibition of LOX may be a strategy to improve IPAD and reduce the accumulation of Aß in the parenchyma and within the walls of blood vessels.

The α-dystrobrevins play a key role in maintaining the structure and function of the extracellular matrix-significance for protein elimination failure arteriopathies.

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.

Quantitative proteomic profiling of white matter in cases of cerebral amyloid angiopathy reveals upregulation of extracellular matrix proteins and clusterin.

Aims: Cerebral amyloid angiopathy (CAA) is the accumulation of amyloid beta (Aß) 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 Aß 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.

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

Although there are no conventional lymphatic vessels in the brain, fluid and solutes drain along basement membranes (BMs) of cerebral capillaries and arteries towards the subarachnoid space and cervical lymph nodes. Convective influx/glymphatic entry of the cerebrospinal fluid (CSF) into the brain parenchyma occurs along the pial-glial BMs of arteries. This project tested the hypotheses that pial-glial BM of arteries are thicker in the midbrain, allowing more glymphatic entry of CSF. The in vivo MRI and PET images were obtained from a 4.2-year-old dog, whereas the post-mortem electron microscopy was performed in a 12-year-old dog. We demonstrated a significant increase in the thickness of the pial-glial BM in the midbrain compared with the same BM in different regions of the brain and an increase in the convective influx of fluid from the subarachnoid space. These results are highly significant for the intrathecal drug delivery into the brain, indicating that the midbrain is better equipped for convective influx/glymphatic entry of the CSF.

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

Dilatation of periarteriolar spaces in MRI of the ageing human brains occurs in white matter (WM), basal ganglia and midbrain but not in cerebral cortex. Perivenous collagenous occurs in periventricular but not in subcortical WM.Here we test the hypotheses that (a) the capacity for dilatation of periarteriolar spaces correlates with the anatomical distribution of leptomeningeal cells coating intracerebral arteries and (b) the regional development of perivenous collagenous in the WM correlates with the population of intramural cells in the walls of veins.The anatomical distribution of leptomeningeal and intramural cells related to cerebral blood vessels is best documented by electron microscopy, requiring perfusion-fixed tissue not available in human material. We therefore analysed perfusion-fixed brain from a 12-year-old Beagle dog as the canine brain represents the anatomical arrangement in the human brain. Results showed regional variation in the arrangement of leptomeningeal cells around blood vessels. Arterioles are enveloped by one complete layer of leptomeninges often with a second incomplete layer in the WM. Venules showed incomplete layers of leptomeningeal cells. Intramural cell expression was higher in the post-capillary venules of the subcortical WM when compared with periventricular WM, suggesting that periventricular collagenosis around venules may be due to a lower resistance in the venular walls. It appears that the regional variation in the capacity for dilatation of arteriolar perivascular spaces in the white WM may be related to the number of perivascular leptomeningeal cells surrounding vessels in different areas of the brain.

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

In this chapter we describe in detail the surgical and imaging techniques employed for the study of the anatomical routes of drainage of cerebrospinal fluid (CSF) and interstitial fluid (ISF) from the brain. The types of tracers, sites of injection, and volumes injected are crucial. For example, when testing the drainage of ISF from the parenchyma, volumes larger than 0.5 µL result in spillage of ISF into the ventricular CSF.

Quantitative Assessment of Cerebral Basement Membranes Using Electron Microscopy.

In this chapter we describe in detail the tissue processing techniques we employ for the study of cerebral tissue by transmission electron microscopy (TEM). In particular, we explain a technique that enables quantification of changes in cerebral basement membranes at the ultrastructural level. This is significant, as age related pathological conditions affecting the brain are often accompanied by ultrastructural changes in the cerebral vasculature.Briefly, experimental mice are fixed by perfusion and their brains removed. Brains are then vibratomed into 100 µm slices with regions of interest microdissected and processed for TEM following a protocol optimized for the preservation of cerebral tissue. Changes in the thickness of cerebral basement membranes are then quantified using novel software. Some prior knowledge of general TEM specimen preparation and sectioning will be useful when performing this protocol.

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

In the absence of conventional lymphatics, drainage of interstitial fluid and solutes from the brain parenchyma to cervical lymph nodes is along basement membranes in the walls of cerebral capillaries and tunica media of arteries. Perivascular pathways are also involved in the entry of CSF into the brain by the convective influx/glymphatic system. The objective of this study is to differentiate the cerebral vascular basement membrane pathways by which fluid passes out of the brain from the pathway by which CSF enters the brain. Experiment 1: 0.5 µl of soluble biotinylated or fluorescent Aß, or 1 µl 15 nm gold nanoparticles was injected into the mouse hippocampus and their distributions determined at 5 min by transmission electron microscopy. Aß was distributed within the extracellular spaces of the hippocampus and within basement membranes of capillaries and tunica media of arteries. Nanoparticles did not enter capillary basement membranes from the extracellular spaces. Experiment 2: 2 µl of 15 nm nanoparticles were injected into mouse CSF. Within 5 min, groups of nanoparticles were present in the pial-glial basement membrane on the outer aspect of cortical arteries between the investing layer of pia mater and the glia limitans. The results of this study and previous research suggest that cerebral vascular basement membranes form the pathways by which fluid passes into and out of the brain but that different basement membrane layers are involved. The significance of these findings for neuroimmunology, Alzheimer's disease, drug delivery to the brain and the concept of the Virchow-Robin space are discussed.