DOI: 10.5061/dryad.gb5mkkx43
title: Data from: Selective removal of astrocytic PERK protects against glymphatic impairment and decreases toxic aggregation of β-amyloid and tau

description: Dysfunction of the glymphatic system, a brain-wide waste clearance
 network, is strongly linked to Alzheimer’s disease (AD) and the
 accumulation of β-amyloid (Aβ) and tau proteins. Here, we identify an
 astrocytic signaling pathway that can be targeted to preserve glymphatic
 function and mitigate neurotoxic protein buildup. Analysis of astrocytes
 from both human AD brains and two transgenic mouse models (5XFAD and PS19)
 reveals robust activation of the protein kinase R-like endoplasmic
 reticulum (ER) kinase (PERK)-α subunit of eukaryotic initiation factor 2
 (eIF2α) branch of the unfolded protein response. Chronic PERK activation
 suppresses astrocytic protein synthesis and, through casein kinase 2
 (CK2)-dependent mechanisms, disrupts the perivascular localization of
 aquaporin-4 (AQP4), a water channel essential for glymphatic flow.
 Importantly, astrocyte-specific PERK deletion or pharmacological
 inhibition restores AQP4 localization, enhances glymphatic clearance,
 reduces Aβ and tau pathology, and improves cognitive performance in mice.
 These findings highlight the critical role of the astrocytic PERK-CK2-AQP4
 axis in glymphatic dysfunction and AD pathogenesis, positioning this
 pathway as a promising therapeutic target.
description: # Data from: Selective removal of astrocytic PERK protects against
 glymphatic impairment and decreases toxic aggregation of β-amyloid and tau
 Dataset DOI:
 [10.5061/dryad.gb5mkkx43](https://doi.org/10.5061/dryad.gb5mkkx43) ##
 Description of the data and file structure This dataset contains the
 individual source data supporting figures and analyses in the associated
 article (doi: 10.1016/j.neuron.2025.04.027). ### Files and variables
 ###### **File: SourceData_Fig_1.xlsx** Description: Source data for Figure
 1, panels C–F. Sheet Fig. 1C: quantification of Eif2ak3 puncta number per
 S100β^+^ astrocyte in the cortex of 6-month-old WT and 5XFAD mice (n = 5
 mice per group). Sheet Fig. 1D: quantification of indicated proteins (fold
 change) in cortical astrocytes isolated from WT and 5XFAD mice (n = 5 mice
 per group). Sheet Fig. 1E: quantification of p-PERK fluorescence intensity
 (fold change) in GFAP^+^ astrocytes from WT and 5XFAD mice (n = 5 mice per
 group). Sheet Fig. 1F: quantification of p-PERK fluorescence intensity
 (fold change) in GFAP^+^ astrocytes from control and AD subjects (n = 5
 samples per group). ###### **File: SourceData_Fig_2.xlsx** Description:
 Source data for Figure 2, panels B, C, D, F–H. Sheet Fig. 2B: Western blot
 analysis of puromycin-labeled proteins (fold change) in isolated cortical
 astrocytes from WT, cKO, 5XFAD, and 5XFAD;cKO mice. Sheet Fig. 2C:
 quantification of perivascular AQP4 expression (arbitrary units) and AQP4
 polarization. Sheet Fig. 2D: Western blot analysis of AQP4 protein levels
 (fold change) in isolated cerebral vessels from WT, cKO, 5XFAD, and
 5XFAD;cKO mice. Sheet Fig. 2F: quantification of average fluorescence
 intensity (arbitrary units) in periarteriolar spaces in WT, cKO, 5XFAD,
 and 5XFAD;cKO mice. Sheet Fig. 2G: quantification of fluorescence
 intensity (arbitrary units) in the somatosensory cortex of WT, cKO, 5XFAD,
 and 5XFAD;cKO mice. Sheet Fig. 2H: quantification of tracer fluorescence
 intensity (arbitrary units) in dCLNs of WT, cKO, 5XFAD, and 5XFAD;cKO
 mice. ###### **File: SourceData_Fig_3.xlsx** Description: Source data for
 Figure 3, panels B–E, G, and I. Sheet Fig. 3B: ELISA quantification of
 Aβ~40~ and Aβ~42~ levels (ng/mL) in cortical lysates (n = 6 mice per
 group), measured in TBS, TBSX, and GDN fractions. Sheet Fig. 3C: ELISA
 quantification of Aβ~40~ and Aβ~42~ levels (ng/mL) in hippocampal lysates
 (n = 6 mice per group), measured in TBS, TBSX, and GND fractions. Sheet
 Fig. 3D: quantification of Aβ burden (fold change) in the cortex and
 hippocampus of 5XFAD and 5XFAD;cKO mice. Sheet Fig. 3E: quantification of
 Aβ plaque load (fold change) in the cortex and hippocampus of 5XFAD and
 5XFAD;cKO mice. Sheet Fig. 3G: escape latency (s) across four training
 days (days 1–4) in the Barnes maze and percentage of time spent in the
 target quadrant during the probe test (day 5). Sheet Fig. 3I: fear
 response (%) on day 0, day 1, and day 7 in WT and 5XFAD mice with or
 without astrocytic PERK deletion. ###### **File: SourceData_Fig_4.xlsx**
 Description: Source data for Figure 4, panels B–F. Sheet Fig. 4B: Western
 blot analysis of puromycin-labeled proteins (fold change) in isolated
 cortical astrocytes from vehicle- or GSK-treated 5XFAD mice (n = 6 mice
 per group). Sheet Fig. 4C: quantification of perivascular AQP4 expression
 (arbitrary units) and AQP4 polarization in GSK- versus vehicle-treated
 5XFAD mice (n = 6 mice per group). Sheet Fig. 4D: quantification of
 fluorescence intensity (arbitrary units) in periarteriolar spaces in 5XFAD
 mice treated with vehicle or GSK. Sheet Fig. 4E: quantification of Aβ
 burden (fold change) in the cortex and hippocampus of 5XFAD mice treated
 with vehicle or GSK. Sheet Fig. 4F: freezing response (%) on day 1 and day
 7 post-conditioning in GSK- or vehicle-treated 5XFAD mice. ###### **File:
 SourceData_Fig_5.xlsx** Description: Source data for Figure 5, panels A,
 C–G. Sheet Fig. 5A: Western blot analysis of indicated proteins (fold
 change) in cortical astrocytes isolated from 8-month-old WT and PS19 mice
 (n = 5 mice per group). Sheet Fig. 5C: Western blot analysis of
 puromycin-labeled proteins (fold change) in cortical astrocytes isolated
 from WT, PS19, and PERK-deleted PS19 mice (n = 5 mice per group). Sheet
 Fig. 5D: quantification of perivascular AQP4 expression (arbitrary units)
 and AQP4 polarization. Sheet Fig. 5E: Western blot analysis of AQP4
 protein levels (fold change) in cerebral vessels isolated from WT, PS19,
 and PERK-deleted PS19 mice (n = 5 mice per group). Sheet Fig. 5F:
 quantification of tracer fluorescence intensity (arbitrary units) in
 periarteriolar spaces. Sheet Fig. 5G: quantification of tracer
 fluorescence intensity (arbitrary units) in the somatosensory cortex.
 ###### **File: SourceData_Fig_6.xlsx** Description: Source data for Figure
 6, panels A–F. Sheet Fig. 6A: quantification of AT8-positive area (fold
 change) in the piriform cortex and hippocampus. Sheet Fig. 6B:
 quantification of MC1-positive area (fold change) in the piriform cortex
 and hippocampus. Sheet Fig. 6C: quantification of ThioS-positive area
 (fold change). Sheet Fig. 6D: ELISA analysis of human tau (ng/mL) in
 cortical lysates from PS19 mice with or without astrocytic PERK deletion
 (n = 6 mice per group), measured in RAB, RIPA, and formic acid fractions.
 Sheet Fig. 6E: escape latency (s) across four training days (days 1–4) in
 the Barnes maze and percentage of time spent in the target quadrant during
 the probe test (day 5). Sheet Fig. 6F: freezing response (%) on day 1 and
 day 7 post-conditioning in WT, PS19, and PS19;cKO mice. ###### **File:
 SourceData_Fig_7.xlsx** Description: Source data for Figure 7, panels A–C,
 E, and G–J. Sheet Fig. 7A: quantification of CK2α immunoreactivity (fold
 change) in cortical astrocytes of WT, 5XFAD, and 5XFAD;cKO mice. Sheet
 Fig. 7B: quantification of CK2α immunoreactivity (fold change) in cortical
 astrocytes of WT, PS19, and PS19;cKO mice. Sheet Fig. 7C: quantification
 of CK2α immunoreactivity (fold change) in GFAP-positive astrocytes from
 control and AD subjects. Sheet Fig. 7E: quantification of perivascular
 AQP4 expression (arbitrary units) and polarization following vehicle or
 CK2 administration. Sheet Fig. 7G: quantification of perivascular AQP4
 expression (arbitrary units) and polarization in vehicle- versus
 CX-4945-treated 5XFAD mice. Sheet Fig. 7H: quantification of FITC-dextran
 tracer intensity in periarteriolar spaces following intracisternal
 injection of vehicle or CX-4945. Sheet Fig. 7I: quantification of Aβ
 plaque burden in the cortex and hippocampus of vehicle- and
 CX-4945-treated 5XFAD mice (n = 6 mice per group). Sheet Fig. 7J: freezing
 response (%) on day 1 and day 7 post-conditioning in vehicle- and
 CX-4945-treated 5XFAD mice.