PATJ regulates cell stress responses and vascular remodeling post-stroke

Abstract

PALS1-associated tight junction (PATJ) protein is linked to metabolic disease and stroke in human genetic studies. Despite the recognized role of PATJ in cell polarization, its specific functions in metabolic disease and ischemic stroke recovery remain largely unexplored. We explored the functions of PATJ in an in vitro model and in vivo in C. elegans and mice. Using a mouse model of stroke, we found post-ischemic stroke duration-dependent increase of PATJ abundance in endothelial cells. PATJ knock-out (KO) HEK293 cells generated by CRISPR-Cas9 suggest roles for PATJ in cell proliferation, migration, mitochondrial stress response, and interactions with the Yes-associated protein (YAP)-1 signaling pathway. Notably, PATJ deletion altered YAP1 nuclear translocation. PATJ KO cells demonstrated transcriptional reprogramming based on RNA sequencing analysis, and identified dysregulation in genes central to vascular development, stress response, and metabolism, including RUNX1, HEY1, NUPR1, and HK2. Furthermore, we found that mpz-1, the homolog of PATJ, was significantly upregulated under hypoxic conditions in C. elegans. Knockdown of mpz-1 resulted in abnormal neuronal morphology and increased mortality, both of which were exacerbated by hypoxia exposure, indicating a critical protective role of PATJ/MPZ-1 in maintaining neuronal integrity and survival, particularly during oxygen deprivation stress relevant to ischemic stroke. These insights offer a new understanding of PATJ's regulatory functions within cellular and vascular physiology and help lay the groundwork for therapeutic strategies targeting PATJ-mediated pathways for stroke rehabilitation and neurovascular repair.

Keywords: Cellular stress; Ischemic stroke; PATJ; Vascular remodeling; YAP1.

Graphical abstract

Fig. 1

PATJ expression across development, cell types and endothelial hypoxia. (A) Dotplot demonstrating expression of PATJ and other key regulators of neurogenesis and vasculogenesis in the cortex. PATJ expression is highest during fetal development and infancy and decreases in adolescence and adulthood. Data from Zhu et al., 2023 (GSE204684) and accessed at singlecell.broadinstitute.org. (B) Human and (C) Mouse single cell RNA sequencing brain atlas UMAP data demonstrating ubiquitous expression of PATJ in adult brain cell subtypes, with inset highlighting vascular cell subtypes. Data from Siletti et al., 2023 and Yao et al., 2023, respectively and accessed at knowledge.brain-map.org. (D) Boxplot showing significantly increased PATJ expression in hypoxic versus normoxic HUVECs. ∗∗∗∗ p < 0.0001. (E) Gene-set enrichment analysis showing top pathways significantly associated with PATJ expression in HUVECs.

Fig. 2

Generation and metabolic profiling of PATJ knock-out (KO) HEK293 cells. (A) Schematic representation of CRISPR-Cas9 strategy targeting exon 3 of the PATJ gene.) CRISPR-Cas9 introduced an insertion (KO1) or deletion (KO2) leading to a frameshift and premature stop codon (red underline). Lowercase sequence base pairs denote intronic sequence. Ex: exon. (B) RNA-seq gene counts demonstrate the absence of PATJ transcripts in KO versus WT cells, (n = 3 for each group). (C) Western blot verifying the absence of PATJ protein expression in knockout cell lines. (D) Growth comparison of PATJ knockout (KO) and wild-type (WT) HEK293 cells (n = 3 independent experiments with 3 technical replicates for each group). (E) Oxygen consumption rates of PATJ KO and WT cells following treatment with oligomycin (i), FCCP (ii), and antimycin A/rotenone (iii; n = 4–6 replicates for each group). (F) PATJ-KO cells demonstrate higher cell death as measured by propidium iodide staining than WT after 2 μM rotenone for 24 h (n = 3 independent experiments with 3 technical replicates for each group). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Fig. 3

Comparative analysis of cell migration in PATJ-modified HEK293 Cells. (A) Scratch assay time-lapse images at 0, 24, and 48 h post-wounding display cell migration for various HEK293 cell lines: wild-type (WT), PATJ knock-out (KO), PATJ overexpression (Ov-PATJ), and KO with PATJ overexpression (KO + Ov-PATJ). Dotted lines indicate scratch edges. (B) Quantitative analysis of cell migration at 24 h post-scratch. (C) Quantitative analysis of cell migration at 48 h post-scratch. n = 4 independent experiments with 3 technical replicates. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 4

PATJ modulates stress responses in C. elegans (A) Schematic diagram of C. elegans anatomy showing MPZ-1GFP is expressed in head neurons. (B) Representative confocal images of endogenous MPZ-1GFP expression in C. elegans under hypoxia (0.1 % O2) or normoxia for 72 h post L4 stages. Scale bars: 10 μm. (C) Quantification of MPZ-1GFP fluorescence intensity under hypoxic and normoxic conditions. n > 20 animals per condition. (D) Representative confocal images of MPZ-1GFP expression in C. elegans under heat stress at 20 °C, 25 °C, or 28 °C for 72 h post L4 stages. Scale bars: 10 μm. (E) Quantification of the percentage of worms showing enhanced MPZ-1GFP fluorescence intensity under different temperature conditions. Data points represent three independent plates with >20 animals assessed per condition. (F) Representative confocal images of individual neurons in otIs181[Pdat-1mCherry; Pttx-3mCherry] worms following control RNAi or mpz-1 RNAi treatment under normoxic and hypoxic conditions. Scale bars: 2 μm. (G) Quantification of the percentage of abnormal worms with abnormal neurons under different conditions. Data points represent three independent experiments with >20 animals assessed per condition. (H) Death rates of L4440 control and mpz-1 RNAi worms subjected to hypoxia for 72 h ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Fig. 5

YAP1 expression and localization in PATJ-modified HEK293 cells and ischemic stroke.(A) Western blot analysis of YAP1 protein levels in WT and PATJ KO cells, with quantification showing a significant increase in YAP1 protein levels in KO cells (n = 4 for each group). (B) Immunofluorescence staining of YAP1 (green) DAPI staining of nuclei (blue). Yellow arrows indicate cells with predominantly cytoplasmic YAP1 localization, red arrowheads indicate predominantly nuclear YAP1 localization, and white arrowheads indicate cells with YAP1 distributed in both compartments. Right panels show quantitative analysis of YAP1 subcellular distribution patterns, presented as the percentage of cells with YAP1 localization predominantly in the nucleus, cytoplasm, or both compartments (n = 4 independent experiments with >50 cells/group). (C) Confocal microscopy with 3D reconstruction of YAP1 subcellular distribution in WT and PATJ KO cells. Right panel shows quantification of the YAP1 nuclear-to-cytoplasmic intensity ratio (n = 4 independent experiments with 50 cells/group). (D) Scratch-wound assay of WT and KO monolayers treated with verteporfin, captured at 0, 24, and 48 h post-scratch. Dashed lines denote wound edge (n = 4 independent experiments with 3 technical replicates/group). (E) Cell survival following 48 h exposure to rotenone with or without verteporfin (n = 3 independent experiments with 3 technical replicates/group). (F) Representative images of endothelial cell marker CD31 (red) and YAP1 (green) in brain tissue from sham and 28 days post-middle cerebral artery occlusion (MCAO) mice, with DAPI staining of nuclei (blue) and merged images highlighting co-localization. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Fig. 6

Temporal expression of PATJ in endothelial cells post-tMCAO and colocalization with YAP1. (A)Schematic representation of the peri-ischemic region observed in the tMCAO mouse model. (B) Representative immunofluorescence images showing double staining of Patj (red) and CD31 (green) in brain sections from sham and tMCAO-treated mice at days 1, 3, 7, 14, and 28 post-stroke. (C) Quantitative analysis of Patj/CD31 double positive cells per mm² in the peri-ischemic region over time (D) Quantitative analysis of Patj positive cells in the peri-ischemic area over time. (E) Quantitative analysis of CD31 positive signal dynamics in the peri-ischemic zone. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (F) Triple immunofluorescence staining showing CD31 (red), YAP1 (green), and PATJ (blue) in brain sections from sham and 7 days post-tMCAO mice. The merged images demonstrate colocalization of all three markers, with white arrowheads indicating triple-positive regions in the vascular structures. (G) Quantification shows the percentage of CD31+/YAP1+/PATJ + triple-positive area in sham versus tMCAO 7d tissues. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗∗∗∗p < 0.0001.

Fig. 7

Temporal shifts in Patj expression in neurons after tMCAO. (A) Representative immunofluorescence images showing double staining of Patj (red) and the neuronal marker NeuN (green) in brain sections from sham and tMCAO mice at 1, 3, 7, 14, and 28 days post-stroke. (B) Quantitative analysis of Patj/NeuN double-positive cells per mm² in the peri-ischemic region across the specified time points. (C) Quantitative analysis of Patj positive signal in the peri-ischemic region over time (D) Quantitative analysis of NeuN positive cells in the peri-ischemic zone at each time point post-tMCAO. n = 4 mice per group; 3 non-consecutive sections per mouse with 5 randomly selected peri-infarct fields analyzed per section. ∗p < 0.05, ∗∗p < 0.01, or ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 8

Transcriptomic landscape and pathway analysis in PATJ-modulated HEK293 cells. (A,B,C) Volcano plots detailing significant gene expression changes for KO + Ov vs. KO, KO vs. WT, and KO + Ov vs. WT, with upregulated genes in red and downregulated genes in blue. (D) Heatmaps of gene expression contrasts for KO + Ov vs. KO vs. WT, with color intensity denoting gene expression levels. (E) Gene Ontology (GO) enrichment analysis bubble chart for the KO + Ov vs. KO comparison. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis bubble chart for the KO + Ov vs. KO comparison.