Alzheimer’s disease (AD) is the most common form of dementia, with prevalence estimated currently to stand at around 24 million, a figure thought to quadruple by the year 2050 due to our ageing population¹. Neuropathologically, AD is characterised by the formation of two species of toxic protein aggregates in the brain: extracellular accumulation of amyloid-β (Aβ) in the form of plaques, and intracellular accumulation of hyperphosphorylated tau in the form of neurofibrillary tangles (NFTs)². Therefore failure of the clearance mechanisms of Aβ and tau from the brain parenchyma are becoming increasingly recognised in the pathogenesis of AD³.

A recently described mechanism of solute clearance from the brain is referred to as the ‘glymphatic’ clearance pathway, based on its appropriation of the lymphatic function of interstitial protein management, and its dependence upon glial water transport⁴. In this brain-wide pathway, cerebrospinal fluid (CSF) enters the brain along para-arterial routes via convective bulk flow, where it exchanges with interstitial fluid (ISF) and is cleared from the brain along para-venous routes, taking interstitial solutes with it⁴. Recently this pathway has been imaged using contrast-enhanced MRI and two photon microscopy, demonstrating that Aβ is indeed a substrate for ‘glymphatic’ clearance in mice⁵, and that tau follows the same para-venous route of clearance in the mouse brain⁶, hence the implication of impairment of this ‘glymphatic’ clearance pathway in AD pathogenesis.

Therefore in this study we sought to quantify the extent of ‘glymphatic’ clearance in the brain of an animal model of tauopathy using contrast-enhanced MRI and intracortical injection of tau to determine the effects of pathological tau accumulation on ‘glymphatic’ inflow and tau clearance. Furthermore, it is known that astrocytic expression of the water channel, aquaporin-4, and its polarisation to astrocytic endfeet surrounding blood vessels in the brain is crucially involved in facilitation of ‘glymphatic’ clearance. Hence molecular and cellular expression analysis of this channel in animal brains was additionally utilised to help elucidate the mechanism by which ‘glymphatic’ clearance is achieved and/or is impaired in this model.

Generation of homozygous rTg4510 transgenic mice has been reported previously⁷. Glymphatic inflow in the brains of 8.5 month old rTg4510 and litter-matched wildtype mice was captured using contrast-enhanced MRI. Briefly, an intrathecal cannula was surgically implanted into the cisterna magna of mice (see figure 1A). After baseline T1-weighted MR images were acquired, low molecular weight paramagnetic contrast agent Gadolinium (Magnevist®, 21mM Gd-DTPA), was infused intrathecally via the implanted cannula (0.6 µl/min, total infusion time, 50mins) and subsequent T1-weighted MR images acquired every 8mins for 120mins. 3D region-of-interest analysis of brain regions was then performed to determine the infiltration of Gadolinium-tagged into the brain parenchyma.

Additionally glymphatic clearance of tau from the cortex in these animals was quantified using intracortical injection of tau and its subsequent detection in extracted CSF. Briefly, tau was extracted from the brain of a late stage rTg4510 animal, and prepared for injection into either the rostral or caudal cortex. 60mins post injection, CSF was extracted from the cisterna magna and containing tau quantified using an ELISA.

Histological examination and laser capture microdissection of astrocytes surrounding blood vessels, and quantification of their aquaporin-4 expression was additionally performed to help understand the involvement of this water channel in mediating changes in glymphatic function in this animal model of tauopathy.

Glymphatic inflow of MR contrast agent was significantly impaired in the caudal cortex of rTg4510 mice compared to wildtype animals (see figure 1), in line with reduced tau clearance and pathological accumulation of NFTs. Aquaporin-4 expression levels and the extent of astrocytic aquaporin-4 polarisation in this region suggest an involvement of this glial water channel in mediating glymphatic impairment in this animal model of tauopathy (see figure 2).Aβ and tau are both known to be cleared from the brain via the recently described ‘glymphatic’ clearance pathway^5,6. Based on these previous findings we therefore hypothesised that an impairment of ‘glymphatic’ clearance occurs during AD development leading to accumulation of these toxic proteins in the brain. Here we demonstrate that pathological accumulation of tau in the rTg4510 animal model of tauopathy is associated with impaired ‘glymphatic’ clearance from the brain. Expression levels and polarisation of astrocytic aquaporin-4 highlight a possible role for this protein in impairment of ‘glymphatic’ clearance in this model. This is the first investigation of glymphatic clearance in a tau model and warrants further investigation of the mechanisms involved. Furthermore, this study provides proof-of-concept that manipulation of the glymphatic clearance pathway may harbour new avenues of therapeutic intervention for AD.