DOI: 10.5061/dryad.mkkwh71dq
title: Astrocytic PERK deficiency drives prefrontal circuit dysfunction and depressive-like behaviors

description: Major depressive disorder (MDD) is associated with dysfunction in
 prefrontal cortex (PFC) circuits, yet the glial mechanisms underlying
 these abnormalities remain unclear. Here, downregulation of the
 endoplasmic reticulum (ER) stress sensor PERK in PFC astrocytes is
 identified as a mechanistic contributor to depression‐related phenotypes.
 PERK expression is markedly reduced in PFC astrocytes from individuals
 with MDD and in two chronic‐stress mouse models. Astrocyte‐specific PERK
 deletion in stress‐naïve mice is sufficient to induce robust
 depressive‐like behaviors and widespread PFC circuit pathology, including
 dendritic spine loss, pyramidal neuron hypoactivity, and weakened
 functional connectivity. Mechanistically, PERK‐deficient astrocytes
 display reduced Nrf2 abundance, dysregulated ER and cytosolic Ca2+
 dynamics, and decreased expression of the synaptogenic protein
 thrombospondin‐1 (TSP1). Restoring astrocytic TSP1 via a blood‐brain
 barrier‐penetrant adeno‐associated virus rescues PFC circuit function and
 reverses depressive‐like behaviors. These findings establish astrocytic
 PERK deficiency as a sufficient driver of synaptic and network dysfunction
 underlying depressive phenotypes and highlight astrocyte‐directed TSP1
 augmentation as a potential therapeutic strategy for MDD.
description: Detailed descriptions of data collection and analysis methods are
 provided in the associated article.
description: # Astrocytic PERK deficiency drives prefrontal circuit dysfunction and
 depressive-like behaviors Dataset DOI:
 [10.5061/dryad.mkkwh71dq](https://doi.org/10.5061/dryad.mkkwh71dq) ##
 Description of the data and file structure This dataset contains the
 individual source data supporting figures and analyses in the associated
 article. ### Files and variables **File:** SourceData_Fig_1.xlsx
 **Description:** Source data for Figure 1, panels C, D, F–H, and J–K.
 Sheet Fig. 1C: quantification of *GFAP* puncta number per astrocyte and
 *EIF2AK3* puncta number per *GFAP*^+^ astrocyte in control and MDD
 samples. Sheet Fig. 1D: quantification of *EIF2AK3* puncta number in
 *SNAP25*^+^ neurons and *AIF1*^+^ microglia in control and MDD samples.
 Sheet Fig. 1F: quantification of *Eif2ak3* puncta number in
 *S100β*^+^ astrocytes of WT mice treated with vehicle or corticosterone
 (CORT). Sheet Fig. 1G: immunoblot analysis of PERK protein levels (fold
 change) in astrocytes isolated from the mouse mPFC after chronic CORT
 treatment or chronic restraint stress (CRS). Sheet Fig. 1H_EPM: Pearson
 correlation between astrocytic PERK protein levels (%) and the animals’
 anxiety-like behavior, as measured by time (seconds) spent in the open
 arms of the elevated plus maze (EPM). Sheet Fig. 1H_TST: Pearson
 correlation between astrocytic PERK protein levels (%) and the animals’
 depressive-like behavior, as measured by immobility time (seconds) in the
 tail suspension test (TST). Sheet Fig. 1H_FST: Pearson correlation between
 astrocytic PERK protein levels (%) and the animals’ depressive-like
 behavior, as measured by immobility time (seconds) in the forced swim test
 (FST). Sheet Fig. 1J: immunoblot analysis of total eIF2α and Nrf2 levels
 (fold change) in mPFC astrocytes of mice treated with vehicle or CORT.
 Sheet Fig. 1K: immunofluorescent detection of puromycin incorporation
 (fold change) in mPFC astrocytes as a readout of de novo protein
 synthesis. **File:** SourceData_Fig_2.xlsx **Description:** Source data
 for Figure 2, panels B, C, D, and F. Sheet Fig. 2B: quantification of
 *Eif2ak3* puncta number per *Aldh1l1*^+^ astrocyte in WT and astrocytic
 PERK cKO mice (n = 5 mice per group). Sheet Fig. 2C: immunoblot analysis
 of Nrf2 protein levels (fold change) in astrocytes isolated from the mPFC
 of WT and PERK cKO mice (n = 5 mice per group). Sheet Fig. 2D: behavioral
 performance of WT (n = 12) and PERK cKO (n = 14) mice in the open field
 (total distance traveled, meter; center time, second), elevated plus maze
 (open-arm time, second), tail suspension (immobility, second), forced swim
 (immobility, second), and sucrose preference (%) tests. Sheet Fig. 2F:
 behavioral outcomes after PL-mPFC-targeted astrocytic PERK deletion (n = 8
 mice per group) in the elevated plus maze, tail suspension, forced swim,
 and sucrose preference tests. **File:** SourceData_Fig_3.xlsx
 **Description:** Source data for Figure 3, panels B–D, F, and G. Sheet
 Fig. 3B: quantification of baseline endoplasmic reticulum (ER) Ca^2+^
 levels (arbitrary units) in astrocytes with and without PERK cKO. Sheet
 Fig. 3C: quantification of ER Ca^2+^ levels in astrocytes from mice
 exposed to vehicle or chronic CORT, as measured by G-CEPIA1er fluorescence
 (arbitrary units). Sheet Fig. 3D: quantification of peak ER Ca^2+^
 response amplitude (Δ*F*/*F*₀) in WT and PERK cKO mice. Sheet Fig. 3F:
 frequency (per minute) and amplitude (peak Δ*F*/*F*₀) of astrocytic
 cytosolic Ca^2+^ events in WT and PERK cKO mice at rest. Sheet Fig. 3G:
 quantification of amplitude (peak Δ*F*/*F*₀) and area under the curve
 (AUC) for air-puff-evoked astrocytic cytosolic Ca^2+^ responses in WT and
 PERK cKO mice. **File:** SourceData_Fig_4.xlsx **Description:** Source
 data for Figure 4, panels C, E, and I–L. Sheet Fig. 4C: quantification of
 perisynaptic astrocyte Ca^2+^ activity (2-min AUC, %) within 2.5 µm of
 dendritic spine heads. Sheet Fig. 4E: normalized changes in spine-head
 size during a 60-min imaging session. Sheet Fig. 4I: dendritic spine
 elimination and formation (%) during P40–P50, P50–P60, and P60–P70 (n = 5
 mice per group). Sheet Fig. 4J: net dendritic spine loss (%) across
 P40–P70. Sheet Fig. 4K: quantification of PSD95 puncta (fold change) in
 the mPFC of WT and PERK cKO mice. Sheet Fig. 4L: quantification of Homer1
 puncta (fold change) in the mPFC of WT and PERK cKO mice. **File:**
 SourceData_Fig_5.xlsx **Description:** Source data for Figure 5, panels D,
 G, H, and J. Sheet Fig. 5D: quantification of dendritic Ca^2+^ transient
 frequency (per min) in WT and PERK cKO mice. Sheet Fig. 5G: quantification
 of somatic Ca^2+^ transient frequency (per min) in WT and PERK cKO mice.
 Sheet Fig. 5H: mean pairwise correlation coefficients of somatic Ca2+
 transients in WT and PERK cKO mice. Sheet Fig. 5J: peak amplitude (ΔF/F₀)
 and AUC (4-s window) of population neuronal Ca2+ responses at struggle
 onset in WT and PERK cKO mice. **File:** SourceData_Fig_6.xlsx
 **Description:** Source data for Figure 6, panels B–E and G–J. Sheet Fig.
 6_BulkRNAseq: bulk RNA-seq raw data collected from the mPFC of WT and
 astrocytic PERK cKO mice. Sheet Fig. 6G: RT-qPCR analysis of Thbs1 mRNA
 expression in WT and PERK-deficient astrocytes. Sheet Fig. 6H:
 quantification of Thbs1 puncta number per S100β⁺ astrocyte in the mPFC of
 WT and PERK cKO mice. Sheet Fig. 6I: quantification of THBS1 puncta number
 per GFAP⁺ astrocyte in the anterior PFC of control and MDD subjects. Sheet
 Fig. 6J: quantification of Thbs1 puncta number per S100β⁺ astrocyte in the
 mPFC of WT mice exposed to vehicle or CORT. **File:**
 SourceData_Fig_7.xlsx **Description:** Source data for Figure 7, panels B,
 C, and F–H. Sheet Fig. 7B: quantification of astrocytic TSP1 expression
 (fold change) in the mPFC of AAV5-Gfap-TSP1-treated mice versus EGFP
 controls. Sheet Fig. 7C: behavioral outcomes in the open field, elevated
 plus maze, tail suspension, and forced swim tests. Sheet Fig. 7F:
 quantification of neuronal Ca^2+^ transient frequency (per min) across
 groups. Sheet Fig. 7G: quantification of mean pairwise correlation
 coefficients of neuronal Ca^2+^ transients across groups. Sheet Fig. 7H:
 behavioral outcomes in the open field, elevated plus maze, tail
 suspension, and forced swim tests. **File:** SourceData_Fig_8.xlsx
 **Description:** Source data for Figure 8, panels A–C. Sheet Fig. 8A:
 behavioral performance of CORT-treated mice receiving AAV-PHP.eB-Gfap-TSP1
 or EGFP before CORT administration in the open field, elevated plus maze,
 tail suspension, and forced swim tests. Sheet Fig. 8B: behavioral
 performance of CRS-exposed mice receiving AAV-PHP.eB-Gfap-TSP1 or EGFP
 before CRS in the open field, elevated plus maze, tail suspension, and
 forced swim tests. Sheet Fig. 8C: behavioral performance of CORT-treated
 mice receiving AAV-PHP.eB-Gfap-TSP1 or EGFP after CORT administration in
 the open field, elevated plus maze, tail suspension, and forced swim
 tests.