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Connexin43 Contributes to Alzheimer's Disease by Promoting the Mitochondria-Associated Membrane-Related Autophagy Inhibition - PubMed

Connexin43 Contributes to Alzheimer's Disease by Promoting the Mitochondria-Associated Membrane-Related Autophagy Inhibition

Weiwei Yu et al. Mol Neurobiol. 2025 Apr.

Abstract

The perturbed structure and function of mitochondria-associated membranes (MAM), instead of the amyloid cascade, have been gradually proposed to play a basic role in the pathogenesis of Alzheimer's disease (AD). Notably, autophagy inhibition is one of the main mechanisms of MAM dysfunction and plays an important role in neuronal injury. However, the upstream molecular mechanism underlying the MAM dysfunctions remains elusive. Here, we defined an unexpected and critical role of connexin43 (Cx43) in regulating the MAM structure. The expression levels of Cx43 and mitofusin-2 (MFN2, the MAM biomarker) increase significantly in 9-month-old APP_swe/PS1_dE9 double-transgenic AD model mice, and there is an obvious colocalization relationship. Moreover, both AD mice and cells lacking Cx43 exhibit an evident reduction in the MAM contact sites, which subsequently promotes the conversion from microtubule-associated protein 1 light-chain 3B I (LC3B-I) to LC3B-II via inhibition mTOR-dependent pathway and then initiates the generation of autophagosomes. Autophagosome formation ultimately promotes β-amyloid (Aβ) clearance and attenuates Aβ-associated pathological changes in AD, mainly including astrogliosis and neuronal apoptosis. Our findings not only reveal a previously unrecognized effect of Cx43 on MAM upregulation but also highlight the major player of MAM-induced autophagy inhibition in Cx43-facilitated AD pathogenesis, providing a novel insight into the alternative therapeutic strategies for the early treatment of AD.

Keywords: Alzheimer’s disease; Autophagy; Connexin43; Mitochondria-associated membranes; β-Amyloid.

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Conflict of interest statement

Declarations. Ethics Approval and Consent to Participate: All experiments with animals were approved by Peking University Shenzhen Hospital and complied with the ethics approval obtained from Peking University Shenzhen Hospital (the specific ethics batch number: 2023–271) as well as the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Consent for Publication: Yes, all the authors have given their consent for the publication of this manuscript in its present form. Competing Interests: The authors declare no competing interests.

Figures

**Fig. 1**
Cx43 is colocalized with the MAM structural marker MFN2 in APP/PS1 mice. A Double immunostaining for Cx43 (green) and Aβ42 (red) in APP/PS1 mice at different months of age (3 m, 6 m, 9 m, 12 m) showing that the immunoactivity of Cx43 around Aβ42 plaques increased significantly with the increase of the age of APP/PS1 mice. Scale bar, 100 µm. B Western blot assay to detect the Aβ42 and Cx43 expression levels in APP/PS1 mice at different months of age (3 m, 6 m, 9 m, 12 m). C Quantitative analysis of protein levels relative to GAPDH. D Double immunostaining for Cx43 (green) and MFN2 (red) in 9-month-old WT and APP/PS1 mice showing an obvious colocalization between Cx43 and MFN2, a MAM structural marker. Scale bar, 100 µm. E Scatter J is used for colocalization relationship analysis, and Pearson’s coefficient (r = 0.63334) further demonstrates a colocalization relationship between Cx43 and MFN2 in 9-month-old APP/PS1 mice. ****P < 0.0001, ***P < 0.001, **P < 0.01 versus WT mice, n > 6 mice for each group

**Fig. 2**
Cx43-mediated MAM increase promotes Aβ deposition in APP/PS1 mice. A Representative TEM images of the mitochondria membranes in contact with ER in four groups of mice reveal a significant increase of mitochondrial-ER coupling (at the top) in APP/PS1 mice compared with the other three groups, which can be more clearly observed in the enlarged images and its corresponding pattern diagrams (below). Scale bars, 1 µm (at top) and 500 nm (below); green polygons mark ER-mitochondria contact sites. B Quantitative analysis of the percentage of mitochondria membranes in contact with ER by ImageJ. C Representative western blot images of MFN2 and ACAT1 were used to evaluate the MAM structure and activation. D Quantitative analysis of optical densities of the bands relative to GAPDH. E Representative immunostaining images for Aβ42 in four groups of mice showing an obvious decrease in fluorescence intensity of Aβ42 in APP/PS1Cx43^± mice compared with APP/PS1 mice. Scale bar, 100 µm. F Representative images of Aβ42 measured by western blot in four groups of mice. G Quantitative analysis of optical densities of Aβ42 relative to GAPDH. ****P < 0.0001, **P < 0.01 versus WT mice, n > 6 mice for each group

**Fig. 3**
Cx43-mediated MAM formation further accelerates WML and neuronal injury in APP/PS1 mice. A HE staining was used to investigate the pathological changes of white matter in CC (critical areas indicated between two yellow dotted lines) and hippocampal neurons (white arrows) in four groups of mice. Scale bar, 1 mm. B Representative TEM images for observing the ultrastructural alterations of myelin sheath of four group mice indicating a significant decrease of myelinated lamina (red arrow) and segmental demyelination (red star). Scale bar, 500 nm. C Representative immunostaining images for MAG showing an obvious decrease of MAG staining in APP/PS1 mice. Scale bar, 50 µm. D Representative images depict immunofluorescent labeling of GFAP in four groups of mice to indicate the astrogliosis exhibiting an evident activation of astroglia in APP/PS1 mice. Scale bar, 100 µm. E Western blot analysis for GFAP and MAG expressions with GAPDH used as an internal control. F Quantitative analysis of GFAP and MAG proteins relative to GAPDH. ****P < 0.0001 versus WT mice, n > 6 mice for each group

**Fig. 4**
Cx43-mediated MAM increase impairs autophagy by activating mTOR-dependent pathway. A Representative images of double immunostaining for LC3B (green) and P62 (red) in four groups of mice showing a significant autophagy deficiency in APP/PS1 mice, which can be rescued by Cx43 knockout. Scale bar, 50 µm. B Western blot analysis for LC3B and P62 expression with GAPDH used as an internal control to determine autophagic flux. C Quantitative analysis of LC3B and P62 proteins relative to GAPDH. D Western blot was used to evaluate the expression levels of proteins associated with the mTOR-dependent autophagy signaling pathway. E Quantitative analysis of optical densities of bands in Fig. 4D relative to GAPDH. ****P < 0.0001, ***P < 0.001 versus WT mice, n > 6 mice for each group

**Fig. 5**
Cx43 contributes to the MAM formation in AD cells. A Western blot analysis for Aβ42 to identify the transfection efficiency of JLV-APP. B Quantitative analysis of Aβ42 relative to GAPDH. C Western blot analysis for Cx43 to identify the transfection efficiency of Cx43 siRNA. D Quantitative analysis of Cx43 relative to GAPDH. E Representative TEM images for observing the percentage of mitochondria membrane in contact with ER in cells showing an obvious increase of mitochondrial-ER coupling in JLV-APP-treated cells compared with the other three groups. Scale bar, 500 nm; green polygons mark ER-mitochondria contact sites. F ImageJ was used to quantitatively analyze the percentage of mitochondria membrane in contact with ER in four group cells. G Immunostaining for MFN2 in four group cells indicating an increased MFN2 fluorescence in JLV-APP-treated cells compared with the other three groups. Scale bar, 100 µm. H Western blot of MFN2 and ACAT1 to evaluate the MAM structure and activation in four group cells. I Quantitative analysis of optical densities of MFN2 and ACAT1 relative to GAPDH. ****P < 0.0001, ***P < 0.001 versus NC cells. All studies were performed independently at least three times

**Fig. 6**
Cx43-mediated MAM formation in AD cells promotes Aβ42 deposition and neuronal apoptosis. A Representative images depicting immunofluorescent labeling Aβ42 in the four cell groups. Scale bar, 100 µm. B Western blot analysis for Aβ42 expression of samples from the four cell groups. C Quantitative analysis of Aβ42 relative to GAPDH was performed. D Aβ42 concentrations in the culture medium of four cell groups were analyzed using an Aβ42 ELISA kit. E TUNEL staining was performed to evaluate the neuronal apoptosis in four cell groups. Scale bar, 100 µm. F An apoptosis assay was performed via flow cytometry after Annexin V-FITC/PI double staining. Viable cells appeared in the lower left quadrant, early apoptotic cells were presented in the upper left quadrant, and late apoptotic cells were shown in the upper right quadrant. G Quantitative analysis of the percentage of viable cells, early apoptotic cells, and late apoptotic cells. ****P < 0.0001, ***P < 0.001, **P < 0.01 versus NC cells. All studies were performed independently at least three times

**Fig. 7**
Cx43-mediated MAM generation negatively regulates autophagy through activating mTOR-dependent pathway in AD cells. A Representative electron micrographs of autophagosome structures (black arrows) in four cell groups. Scale bar, 500 nm. B Representative images of double immunostaining for LC3B (green) and P62 (red) in four cell groups. Scale bar, 50 µm. C Western blot analysis for LC3B and P62 expressions to evaluate autophagic flux in four cell groups, and GAPDH was an internal control. D Quantitative analysis of LC3B and P62 relative to GAPDH. E Representative images of a western blot for proteins related to mTOR-dependent signaling pathway in four cell groups. F Quantitative analysis of the band intensities of proteins related to mTOR-dependent signaling pathway. ****P < 0.0001, ***P < 0.001, **P < 0.01 versus NC cells. All studies were performed independently at least three times

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Connexin43 Contributes to Alzheimer's Disease by Promoting the Mitochondria-Associated Membrane-Related Autophagy Inhibition - PubMed