Glymphatic fluid transport is suppressed by the aquaporin-4 inhibitor AER-271

Abstract

The glymphatic system transports cerebrospinal fluid (CSF) into the brain via arterial perivascular spaces and removes interstitial fluid from the brain along perivenous spaces and white matter tracts. This directional fluid flow supports the clearance of metabolic wastes produced by the brain. Glymphatic fluid transport is facilitated by aquaporin-4 (AQP4) water channels, which are enriched in the astrocytic vascular endfeet comprising the outer boundary of the perivascular space. Yet, prior studies of AQP4 function have relied on genetic models, or correlated altered AQP4 expression with glymphatic flow in disease states. Herein, we sought to pharmacologically manipulate AQP4 function with the inhibitor AER-271 to assess the contribution of AQP4 to glymphatic fluid transport in mouse brain. Administration of AER-271 inhibited glymphatic influx as measured by CSF tracer infused into the cisterna magna and inhibited increases in the interstitial fluid volume as measured by diffusion-weighted MRI. Furthermore, AER-271 inhibited glymphatic efflux as assessed by an in vivo clearance assay. Importantly, AER-271 did not affect AQP4 localization to the astrocytic endfeet, nor have any effect in AQP4 deficient mice. Since acute pharmacological inhibition of AQP4 directly decreased glymphatic flow in wild-type but not in AQP4 deficient mice, we foresee AER-271 as a new tool for manipulation of the glymphatic system in rodent brain.

Keywords: aquaporin-4; astrocyte endfeet; brain edema; fluorescent microscopy; glymphatic system.

Figure 1:. AER-271 protects from water intoxication.

(A) Schematic for hypo-osomotic induction of brain swelling via water intoxication (WI). WI induces an acute drop in plasma osmolality, resulting in increased water influx to the brain across astrocytic endfeet, facilitated by AQP4 water channels. (B) Experimental scheme. AER-271 is given to 5 months old wild-type (WT) mice at 5mg/kg administered intraperitoneally 20minutes prior to WI challenge. (C) Survival curves for mice treated with AER-271 or vehicle after acute WI (WT, males n=12 vehicle, 13 AER-271, p=.031 Mantel-Cox test). (D) Survival curves for mice treated with TGN-020 or vehicle control after acute WI, TGN-020 was included at T=0 because it is already its active form. (WT, males n=8 vehicle, 10 TGN-020, p=.034 Mantel-Cox test). (E) Survival curves for male and female mice after acute WI with no drug administration (p=.008 Mantel-Cox test, n= 13 female, 12 male)

Figure 2:. AER-271 reduces glymphatic influx

(A) Experimental schema for AER-271 treatment and cisterna magna tracer infusions under ketamine/xylazine anesthesia. (B) Representative images of BSA-647 CSF tracer influx in whole brain of vehicle- and AER-271-treated mice. CSF tracer is visible surrounding the circle of Willis (bottom) and middle cerebral arteries (MCA, Top) (scale bar = 1 mm). (C) Representative coronal sections depicting BSA-647 CSF tracer influx. White arrows emphasize the reduced influx in ventral cortical areas. (scale bar = 1 mm) (D) Total fluorescence present in whole brain surfaces for ventral (left, p=.024), dorsal (center, p=.050), and MCA PVSs (right, p=.191). (E) Total fluorescence signal across 6 brain sections extending from 1.2 mm+ to −1.8 mm relative to bregma (p=.01) and mean pixel intensity (p=.033). (F) Anterior sections from each mouse brain were divided into subregions, and tracer signal intensity analyzed for each subregion, showing significantly less tracer in ventral cortex of AER-271 treated mice (p=.005). (G) Posterior sections divided into subregions and tracer intensity analyzed for each subregion, showing no significant differences within each subregion. (F-G) D CTX = dorsal cortex, L CTX = lateral cortex, V CTX = ventral cortex, CPu= caudate-putamen, HIP=hippocampus, TH=thalamus, HY=hypothalamus. All statistical tests were unpaired two-tailed t-tests. *-p<.05, **-p<.01. All plots depict the mean and standard deviation of each group, where each point represents one animal (n=5 Vehicle, n=7 AER-271).

Figure 3:. Diffusion-weighted MRI in vivo: AER-271 inhibits brain-wide ADC in WT animals.

(A) Experimental timeline for DWI experiment. (B) Box and whisker plots for the group–wise mean ADC values at the baseline and (C) the relative ADC changes from the baseline DWI at ~30 and ~75 minutes post AER-271 and saline (vehicle) injection, measured in 19 parenchymal ROIs in AQP4 KO (n=5 vehicle, 5 AER-271) and WT (n=6 vehicle and n=6 AER-271) mice, by means of DWI acquired in 6 diffusion-encoding directions. (D) ROI-wise correlation plots for the mean ΔADC vs. mean pixel intensity values of BSA-647 tracer, considering both saline and AER-271 injected mice jointly. Legend: ns - not significant, * - p<.05, ** - p<.01, *** - p<.001, **** - p<.0001 from 2-way ANOVA with Bonferroni’s post-hoc (B-C). Correlation plots show the respective regression lines along with semi-transparent areas marking 95% confidence intervals of the fitting. Correlations were considered significant for Person’s r or Spearman’s rho>0.5 with p<0.05, and non-zero regression slope.

Figure 4:. AER-271 reduces glymphatic efflux

(A) Schematic showing striatal cannula implantation, recovery, and in vivo DB53 measurement. (B) Representative coronal sections (+0.6 mm AP bregma) depicting DB53 infusion in striatum (scale bar = 1 mm). (C) DB53 infusion volume measured as total area covered by DB53. (Left) DB53 area covered in individual coronal sections. (Right) Mean area covered by DB53 across 6 coronal sections, where each data point is one animal. (D) Representative images of DB53 fluorescence in mouse femoral vein after DB53 striatal infusion (scale bar= 500 μm). (E) Fluorescent intensity of DB53 in femoral vein plotted over time (solid lines, mean ± SEM plotted for each group, dotted lines; mean temporal derivative of DB53 for each group). Results for the entire tracer circulation time (0–120 min;left), and inset focusing on 60–120 min (right). (F) Venous DB53 intensity at 0 minutes (left) and 120 minutes (right) after striatal infusion of DB53 tracer (120 min, p=.037; n=6 Vehicle, 6 AER-271). Legend: ns - not significant, * - p<.05, by means of unpaired two-tailed t-test.

Figure 5:. AER-271 suppression of glymphatic influx is Aquaporin 4 dependent.

(A) Schematic showing AQP4 genetic knock-out strategy and representative immunohistochemistry images demonstrating the absence of AQP4 in AQP4 deficient mice (AQP4KO). (Green=glial fibrillary acid protein, Magenta = aquaporin 4, Scale bar = 25 μm) (B) Schematic showing AER-271 or vehicle treatment prior to cisterna magna tracer infusion in AQP4KO mice. (C) Whole mount brain images showing total BSA-647 CSF tracer distribution along the dorsal brain surface (Top) and ventral surface (Bottom). (Scale bar = 1 mm) (D) (Top) Representative coronal sections showing BSA-647 influx in AQP4KO mice. (Bottom) Representative images of BSA-647 influx in wild-type mice (WT, same mice as Figure 2C). Equivalent anterior sections shown (+0.6 mm bregma, Scale bar= 1 mm). (E) (Left) Total fluorescence intensity across the ventral brain surface, (Center) dorsal surface, and (Right) middle cerebral arteries in AQP4KO mice treated with either AER-271 or vehicle. (F) (Left) Total CSF tracer fluorescence in AQP4KO mouse coronal sections. (Right) Mean pixel intensity of CSF tracer in AQP4KO mice. (G) Total coronal section tracer fluorescence from WT mice (same data as Figure 2E; circles) plotted together with AQP4KO mice (same data as Panel F right; triangles). WT vehicle-treated mice had significantly more tracer than vehicle-treated AQP4KO mice (p=.001), WT AER-271 treated mice had more influx than vehicle (p=.017) and AER-271 treated (p=.026) AQP4KO mice. (H) Representative confocal images of AQP4 immunostaining from WT mice treated with vehicle or AER-271 (Same mice as Figure 2 B–G, Scale bar = 25 μm). (I) (Left) Intensity plots taken perpendicular to small vessels ( <6 μm), showing mean ± SD (Vehicle n= 5 mice, 123 vessels; AER-271 n=7 mice, 168 vessels). (Right) Mean AQP4 polarization, where each point represents mean AQP4 polarization measurement for all vessels of individual animals (p=.578). All plots depict mean ± SD, with each individual point representing one animal. All statistical tests were unpaired two-tailed t-tests, α=.05. Legend: ns - not significant, * - p<.05, ** - p<.01.