Neuronal BAG3 attenuates tau hyperphosphorylation, synaptic dysfunction, and cognitive deficits induced by traumatic brain injury via the regulation of autophagy-lysosome pathway

Abstract

Growing evidence supports that early- or middle-life traumatic brain injury (TBI) is a risk factor for developing Alzheimer's disease (AD) and AD-related dementia (ADRD). Nevertheless, the molecular mechanisms underlying TBI-induced AD-like pathology and cognitive deficits remain unclear. In this study, we found that a single TBI (induced by controlled cortical impact) reduced the expression of BCL2-associated athanogene 3 (BAG3) in neurons and oligodendrocytes, which is associated with decreased proteins related to the autophagy-lysosome pathway (ALP) and increased hyperphosphorylated tau (ptau) accumulation in excitatory neurons and oligodendrocytes, gliosis, synaptic dysfunction, and cognitive deficits in wild-type (WT) and human tau knock-in (hTKI) mice. These pathological changes were also found in human cases with a TBI history and exaggerated in human AD cases with TBI. The knockdown of BAG3 significantly inhibited autophagic flux, while overexpression of BAG3 significantly increased it in vitro. Specific overexpression of neuronal BAG3 in the hippocampus attenuated AD-like pathology and cognitive deficits induced by TBI in hTKI mice, which is associated with increased ALP-related proteins. Our data suggest that targeting neuronal BAG3 may be a therapeutic strategy for preventing or reducing AD-like pathology and cognitive deficits induced by TBI.

Keywords: Alzheimer’s disease; Autophagy-lysosome pathway; BAG3; Gliosis; Memory; Synaptic dysfunction; Tau; Traumatic brain injury.

Fig. 1

Single TBI increases tau hyperphosphorylation and gliosis in C57BL6/J (WT) and hTKI mice. a Experimental timeline of CCI TBI surgery, followed by behavioral testing and analysis (figure made with BioRender). b, c Left panel: Representative immunofluorescence (IF) images of PHF1 + (red) staining (b) in the retrosplenial cortex (RSC) and (c) the hippocampus (HC) region of WT Sham and WT TBI mice. Scale bar, 100 μm. Right Panel: The number of PHF1 + cells/µm² was quantitated (b) in the RSC and (c) in the HC of WT Sham and WT TBI mice (*P < 0.05, **P < 0.01; Mann–Whitney test, n = 6 mice/group). d, e Left panel: Representative IF images of PHF1 + staining (d) in the RSC and (e) the HC region of hTKI Sham and hTKI TBI. Scale bar, 100 μm. Right Panel: The number of PHF1 + cells/µm² was quantitated (d) in the RSC and (e) in the HC of hTKI Sham and hTKI TBI (*P < 0.05, **P < 0.01; Mann–Whitney test, n = 6 mice/group). f Representative IF images of GFAP (green) and IBA-1 (red) staining in the corpus callosum (CC) region of WT Sham and WT TBI mice. Scale bar, 100 μm. g (Left) Quantification of the % Area of GFAP and (Right) the mean intensity of GFAP in the CC region of WT Sham and WT TBI mice (**P < 0.01; Mann–Whitney test, n = 6 mice/group). h (Left) Quantification of the % Area of IBA-1 and (Right) the mean intensity of IBA-1 in the CC region of WT Sham and WT TBI mice (**P < 0.01; Mann–Whitney test, n = 6 mice/group). i Representative IF images of GFAP (green) and IBA-1 (red) in the CC region of hTKI Sham and hTKI TBI mice. Scale bar, 100 μm. j (Left) Quantification of the % Area of GFAP and (Right) the mean intensity of GFAP in the CC region of hTKI Sham and hTKI TBI mice (**P < 0.01; Mann–Whitney test, n = 6 mice/group). k (Left) Quantification of the % Area of IBA-1 and (Right) the mean intensity of IBA-1 in the CC region of hTKI Sham and hTKI TBI (*P < 0.05; Mann–Whitney test, n = 6 mice/group). Nuclei for all images were counterstained by Hoechst 33342

Fig. 2

TBI reduces postsynaptic density protein 95 (PSD95) and induces memory deficits in WT and hTKI mice. a, b Left Panel: Representative IF images of PSD95 (red) immunoreactivity in (a) the RSC and (b) the HC of WT Sham and WT TBI mice. Scale bar, 100 μm. Right Panel: Quantification of the mean intensity of PSD95 in (a) the RSC and (b) the HC of WT Sham and WT TBI mice (*P < 0.05 and **P < 0.01; Mann–Whitney test, n = 7 mice/group). c, d Left Panel: Representative IF images of PSD95 (red) immunoreactivity in (c) the RSC and (d) the HC of hTKI Sham and hTKI TBI mice. Scale bar, 100 μm. Right Panel: Quantification of the mean intensity of PSD95 in (c) the RSC and (d) in the HC of hTKI Sham and hTKI TBI mice (c **P < 0.01 and d P = 0.1450; Mann–Whitney test, n = 6 mice/group). Nuclei for all images were counterstained by Hoechst 33342. e, g Left Panel: The % of spontaneous alternations (actual alternations/possible alternations (total arm entries—2) × 100) in the Y-maze was used to assess spatial working memory performance 1 month following TBI in (e) WT Sham vs WT TBI mice and (g) hTKI Sham vs hTKI TBI mice (**P < 0.01; Mann–Whitney test, n = 8–10 mice/group). Right Panel: The number of total arm entries was used to confirm a similar amount of movement in the Y-maze in (e) WT Sham vs WT TBI mice and (g) hTKI Sham vs hTKI TBI mice (Mann–Whitney test, n = 8–10 mice/group). f, h Left Panel: The total time spent resting was measured to evaluate the exploratory and locomotor activity in (f) WT Sham vs WT TBI mice and (h) hTKI Sham vs hTKI TBI mice (**P < 0.01; Mann–Whitney test, n = 8–10 mice/group). Right Panel: The total time spent in the center was used as a measure of the exploratory and locomotor activity in (f) WT Sham vs WT TBI mice and (h) hTKI Sham vs hTKI TBI mice (Mann-Whiney test, n = 8–10 mice/group). ns non-significant

Fig. 3

TBI and AD increase tau hyperphosphorylation and gliosis in the inferior parietal lobe (IPL) of human brains. a Representative IF images of GFAP (green), IBA-1 (red), and AT8 (white) immunoreactivity in the IPL of post-mortem human brain tissue (CT, TBI, AD with TBI history (ADwTBI), and AD without TBI history (AD)). Scale bar, 100 μm. b, c Quantification of the mean intensity of (b) GFAP and (c) IBA-1 in the IPL (*P < 0.05, **P < 0.01, ****P < 0.0001 vs CT; unpaired t-test, n = 4–5 cases/group). d Quantification of the mean intensity of AT8 in the IPL (*P < 0.05, **P < 0.01 vs CT; unpaired t-test, n = 4–5 cases/group). Nuclei for all images were counterstained by Hoechst 33342. e The total protein lysates from the IPL of CT, TBI, ADwTBI, and AD cases were subjected to Western blot (WB) assay with specific antibodies against mouse anti-PHF1 (top), rabbit anti-TauC (middle), and rabbit anti-GAPDH (bottom, loading control). TL: recombinant tau ladder with 6 isoforms of tau protein. The isoforms are in order: 2N4R, 2N3R, 1N4R, 1N3R, 0N4R, 0N3R. f Quantification of the ratio of PHF1/TauC (*P < 0.05 TBI vs CT, ADwTBI vs CT, AD vs CT, and AD vs TBI; Mann–Whitney test, n = 4 cases/group). Full-length western blots can be visualized in Supplementary Fig. 10

Fig. 4

TBI increases BAG3 immunoreactivity in astrocytes in WT and hTKI mice while BAG3 immunoreactivity decreases in neurons and OLG with ptau accumulation. a, c Representative epifluorescence IF images of GFAP (green) and BAG3 (red) immunoreactivity in the CA1 region of (a) WT Sham and WT TBI, (c) hTKI Sham and hTKI TBI mice. Scale bar, 20 μm. b, d Left Panel: Quantification of the mean intensity of BAG3 in GFAP + astrocytes in (b) WT Sham vs WT TBI mice and (d) hTKI Sham vs hTKI TBI mice (*P < 0.05, ***P < 0.001; Mann–Whitney test, n = 6–7 mice/group, average of 20–23 cells/mouse). Right Panel: Correlation of log₁₀(GFAP Mean Intensity) and log₁₀(BAG3 Mean Intensity) in (b) WT Sham and WT TBI mice and in (d) hTKI Sham and hTKI TBI mice (P < 0.0001; Spearman Correlation: b r = 0.4263, n = 244, d r = 0.5820, n = 291). e, g Representative confocal IF images of BAG3 (green), PHF1 (red), and OLIG2 (white) immunoreactivity in the cortex of hTKI TBI mice. Yellow arrows indicate (e) PHF1 + neurons (PHF1 + /OLIG2-) and (g) PHF1 + oligodendrocytes (PHF1 + /OLIG2 +). White arrows indicate (e) PHF1- neurons (PHF1-/OLIG2-) and (g) PHF1- oligodendrocytes (PHF1-/OLIG2 +). Scale bar, 20 μm. f, h Quantification of the mean intensity of BAG3 in (f) PHF1 + vs PHF1- neurons of hTKI TBI mice and in (h) PHF1 + and PHF1- oligodendrocytes in hTKI TBI mice (**P < 0.01, ***P < 0.001; Mann–Whitney test, n = 7 mice/group, average of 10 cells/mouse). Nuclei for all images were counterstained by Hoechst 33342

Fig. 5

TBI and AD increases BAG3 immunoreactivity in astrocytes while BAG3 immunoreactivity decreases in neurons and OLG with ptau accumulation in the IPL of human postmortem brains. a Representative IF images of GFAP (green) and BAG3 (red) immunoreactivity in the IPL of CT, TBI, ADwTBI, and AD. Scale bar, 20 μm. b Quantification of the mean intensity of BAG3 in GFAP + cells (*P < 0.05 vs CT; Mann–Whitney test, n = 4–5 cases/group, average of 20–21 cells/case). c Correlation of log₁₀(GFAP Mean Intensity) and log₁₀(BAG3 Mean Intensity) with combined human cases (P < 0.0001; Spearman Correlation: r = 0.3671, n = 342). d, e Representative IF images of BAG3 (green), PHF1 (red), and OLIG2 (white) immunoreactivity in the IPL in (d) neurons and (e) OLG across the human conditions. Scale bar, 20 μm. f, g Quantification of the mean intensity of (f) PHF1 + neurons and (g) PHF1 + OLG in TBI, ADwTBI, and AD compared to (f) PHF1- neurons and (g) PHF1- OLG in the respective disease (*P < 0.05, **P < 0.01; Mann–Whitney test, n = 4–5 cases/group, average of 7–10 cells/case). Nuclei for all images were counterstained by Hoechst 33342

Fig. 6

Overexpression of neuronal BAG3 reduces tau hyperphosphorylation, synaptic dysfunction, and cognitive deficits in hTKI mice induced by TBI. a Experimental timeline of AAV9-BAG3 or AAV9-GFP (CT) injection followed by CCI TBI (figure made with BioRender). b Representative IF images of BAG3 overexpression (Magenta) after AAV9-BAG3 injection compared to AAV9-GFP control injection in hTKI mice. Scale bar, 4 mm. c Left Panel: hTKI mouse lysate from the hippocampus and cortex regions were collected and subjected to WB assay with a rabbit anti-BAG3 and mouse anti-GAPDH specific antibody. Right Panel: Quantification from WB assay to confirm BAG3 overexpression in AAV-BAG3-injected mice compared to AAV-CT (****P < 0.0001; unpaired t-test, n = 4 mice/group). d Left Panel: Representative IF staining of PHF1 (red) from the intra-CA2–CA3 region of HC. Scale bar, 100 μm. Right Panel: Quantification of the number of PHF1 + cells/µm² in the CA2–CA3 region of HC (*P < 0.05; unpaired t-test, n = 4 mice/group). e, f Left Panel: Mouse lysate from the hippocampus and cortex regions were collected and subjected to WB assay with (e) mouse anti-PHF1 and rabbit anti-GAPDH specific antibodies or (f) rabbit anti-TauC and mouse anti-GAPDH specific antibodies. Right Panel: Quantification from WB assay to determine the ratio of (e) PHF1/TauC or (f) TauC/GAPDH in AAV-CT vs AAV-BAG3-injected mice (*P < 0.05; unpaired t-test, n = 4 mice/group). g Left Panel: Representative IF images of PSD95 (red) in the HC of AAV-CT and AAV-BAG3 mice. Scale bar, 50 μm. Right Panel: Quantification of PSD95 mean intensity from the HC of AAV-CT vs AAV-BAG3 mice (*P < 0.05; Mann–Whitney test, n = 4 mice/group, 2 image/mouse). h, i Morris Water Maze behavioral test to assess the (h) short-term (2 h) and (i) long-term (24 h) spatial learning and memory in AAV-CT and AAV-BAG3-injected mice subjected to TBI. (Left) Measurement of the distance and (Right) time the mice remained in the target quadrant (P = 0.0578, P = 0.0770, *P < 0.05, and **P < 0.01; unpaired t-test, n = 7 mice/group). j Y-maze behavioral test to evaluate spatial working memory performance in AAV-BAG3 compared to AAV-CT injected mice after TBI. (Left) The percentage of spontaneous alternations was calculated by (actual alternations/possible alternations (total arm entries – 2) × 100)). (Right) The total number of arm entries was used to confirm similar levels of movement between groups in the Y-Maze (*P < 0.05; unpaired t-test, n = 7 mice/group). Nuclei for IF images were counterstained by Hoechst 33342. ns; non-significant. Full-length western blots can be visualized in Supplementary Fig. 10

Fig. 7

Overexpression of BAG3 promotes autophagy flux, while knockdown of BAG3 inhibits it. a The experimental timeline of FUW mCherry-GFP-LC3 virus treatment in BAG3 modulated HEK293 cells. b Left Panel: Representative IF images of HEK293 cells after 48 h of treatment with various BAG3 lentiviruses (scr-BAG3, sh-res-BAG3, sh-BAG3-BFP, and sh-BAG3) using a rabbit anti-BAG3 specific antibody. Scale bar, 50 μm (low-magnification images), 10 μm (inset images). Nuclei for IF images were counterstained by Hoechst 33342. Right Panel: Quantification of BAG3 mean intensity after lentivirus treatment (P = 0.0916, *P < 0.05 vs scr-BAG3; unpaired t-test, n = 4 replicates/group). c Protein lysate was extracted from HEK293 cells and subjected to WB assay with (top) rabbit anti-BAG3, (middle) mouse anti-p62, and (bottom) rabbit anti-GAPDH. d Quantification of the ratio of BAG3/GAPDH to confirm knock-down or overexpression of BAG3 after lentivirus treatment (**P < 0.01, ****P < 0.0001 vs scr-BAG3; unpaired t-test, n = 3 replicates/group). e Quantification of the ratio of p62/GAPDH to assess autophagic flux after lentivirus treatment (*P < 0.05, ***P < 0.001 vs scr-BAG3; P = 0.0569 sh-BAG3-BFP vs res-BAG3; **P < 0.01 sh-BAG3 vs res-BAG3; unpaired t-test, n = 3 replicates/group). f Left Panel: Representative images of live HEK293 cells after treatment with BAG3 and FUW lentiviruses. Yellow puncta (green and red double positive) indicate phagosomes/autophagosomes, and single red puncta indicates autolysosome formation after the GFP signal is quenched by the acidic lysosome. Scale bars, 10 μm. Right Panel: Quantification of the number of single red puncta per cell after modulating BAG3 (****P < 0.0001 Scr vs BFP; **P < 0.01 Res vs Scr; ****P < 0.0001 BFP vs Res; Mann–Whitney test, n = 3 replicates/group, 20–46 cells/replicate). g Left Panel: Representative images of live HEK293 cells after treatment with BafA1, an autophagy flux inhibitor. Yellow puncta indicate phagosome/autophagosomes. Scale bars, 10 μm. Right Panel: Quantification of the number of yellow (green and red double positive) puncta per cell after modulating BAG3 and treating with BafA1 (**P < 0.01 Scr vs BFP; ****P < 0.0001 BFP vs Res; Mann–Whitney test, n = 3 replicates/group, 18–43 cells/replicate). ns non-significant. Full-length western blots can be visualized in Supplementary Fig. 10

Fig. 8

TBI reduces ALP-associated genes (CTSD and LAMP1) and increases p62 puncta formation in mouse models, consistent with human TBI and AD cases. Overexpression of BAG3 in neurons in hTKI mice prevents these changes. a, b Representative images of IF staining of CTSD (green) and LAMP1 (red) in the CA1 region of the hippocampus in (a) WT Sham and WT TBI, (b) hTKI Sham and hTKI TBI mice. Scale bar, 200 μm. c Quantification of the % Area of CTSD in (Left) WT Sham vs WT TBI mice and (Right) hTKI Sham vs hTKI TBI mice in the CA1 region of the hippocampus (**P < 0.01; unpaired t-test, n = 5–6 mice/group). d Quantification of the % Area of LAMP1 in (Left) WT Sham vs WT TBI mice and (Right) hTKI Sham vs hTKI TBI mice in the CA1 region of the hippocampus (*P < 0.05, **P < 0.01; unpaired t-test, n = 5–6 mice/group). e, f Representative images of IF staining of p62 (green) in the RSC of (e) WT Sham and WT TBI, (f) hTKI Sham and hTKI TBI mice. Scale bar, 100 μm (low-magnification images), 10 μm (inset images). g Quantification of the number of cells with aggregated p62 puncta/mm² in (Left) WT Sham vs WT TBI mice and (Right) hTKI Sham vs hTKI TBI mice ((Left) **P < 0.01; Mann–Whitney test, n = 5 mice/group, (Right) P = 0.0584; unpaired t-test, n = 6 mice/group). h Representative images of IF staining of CTSD (green), LAMP1 (red), and p62 (white) in the IPL of post-mortem human brain tissue. Scale bar, 100 μm. i Quantification of the mean intensity of (Left) CTSD, (Middle) LAMP1, and (Right) p62 across different disease conditions (*P < 0.05; unpaired t-test, n = 3 cases/group). j Left Panel: Representative images of IF staining of CTSD (green) and LAMP1 (red) in AAV-CT and AAV-BAG3-injected mice in the CA2–CA3 region of the hippocampus. Scale bar, 200 μm. Right Panel: Quantification of the mean intensity of CTSD and LAMP1 in AAV-CT vs AAV-BAG3-injected mice (*P < 0.05 and **P < 0.01; Mann–Whitney test, n = 3–5 mice/group, 3 image/mouse). k Left Panel: Representative images of IF staining of p62 (green) in the CA1 region of the hippocampus in AAV-CT and AAV-BAG3-injected mice. Scale bar, 50 μm (low-magnification images), 10 μm (inset images). Right Panel: Quantification of the number of cells with aggregated p62 puncta in AAV-CT and AAV-BAG3-injected mice (*P < 0.05; Mann–Whitney test, n = 3–5 mice/group, 3 image/mouse). Nuclei for IF images were counterstained by Hoechst 33342