Bone marrow mesenchymal stem cells alleviate neurological dysfunction by reducing autophagy damage via downregulation of SYNPO2 in neonatal hypoxic-ischemic encephalopathy rats

Abstract

Neonatal hypoxic-ischemic encephalopathy (HIE) is worsened by autophagy-induced neuronal damage, with SYNPO2 playing a key role in this process. This study investigates the involvement of SYNPO2 in neuronal autophagy and explores the potential of bone marrow mesenchymal stem cells (BMSCs) to alleviate HIE-induced dysfunction by inhibiting SYNPO2-mediated autophagy. Using in vitro and in vivo neonatal HIE models, we observed an upregulation of SYNPO2 expression, accompanied by increased neuronal injury and aggregation of autophagy-related proteins. Intervention with BMSCs effectively reduced SYNPO2 expression, and SYNPO2 depression mitigated neuroautophagic damage and improved neurological dysfunctions. Moreover, SYNPO2 overexpression exacerbated neuroautophagy despite BMSC treatment, while SYNPO2 depletion notably reduced neuroautophagic damage and alleviated cognitive impairments, retaining the neuroprotective efficacy of BMSC treatment. These findings confirm the role of BMSCs in attenuating HIE injury by suppressing neuroautophagy and provide insights into the mechanistic involvement of SYNPO2. Ultimately, this study identifies SYNPO2 as a novel therapeutic target for neonatal HIE and supports the clinical potential of BMSCs in HIE management.

Fig. 1. CM suppressed neuronal autophagic injury in OGD neurons.

A Immunofluorescence double staining of cortical neurons with Tuj1 (green) and TUNEL (red) in the Normal, OGD, OGD + CM groups. Scale bar = 100 μm. Blue, DAPI-stained nuclei. a Magnified view of neuronal axons indicated by white arrows. b Magnified view of apoptotic neurons indicated by white arrows. Scale bar = 25 μm (a, b). B Quantitative bar graph of apoptotic rate and neuronal axon length in the Normal, OGD, OGD + CM groups. C Quantitative bar graph showing cell viability in neurons from Normal, OGD, OGD + CM groups. D RT-qPCR detected relative expression of SYNPO2 in neurons 24 hours after OGD. E. WB detected expression of SYNPO2 in neurons 24 hours after OGD. F. Expression of autophagy-related proteins, P62, AIFM1, Beclin-1, LC3B, and ATG12 in the Normal, OGD, and OGD + CM groups. Bar graphs display mean ± SD of independent biological replicates. n = 3–6 wells, two-sided unpaired one-way ANOVA. All experiments were repeated three times independently, with similar results. OGD oxygen-glucose deprivation; CM BMSC-conditioned medium.

Fig. 2. BMSCs alleviated brain autophagic injury and neurological dysfunction in HIE rats.

A Immunofluorescent double-labeling staining of brain tissues with NEUN (red) and autophagic markers (green) in HIE rats. Scale bar = 50 μm, 25 μm (magnified view). Blue, DAPI-stained nuclei. B TEM images showing autophagy in the brain tissue of HIE rats, with red arrows indicating autophagic vesicles. Scale bar = 500 nm. Nissl staining (C) and HE staining (D) of coronal brain sections from HIE rats in the Sham, HIE + PBS, HIE+BMSCs groups. Scale bar = 2 mm (panoramic view), 500 μm (magnified view). Forelimb grip test (E), rotarod test (F), righting test (G), geotaxis test (H), and climbing test (I) were used to evaluate the muscle strength and coordination in HIE rats. J Spatial exploration and autonomous behavior of HIE rats were detected by the Y Maze. K The number of entries into the center of the open field (left) and grooming time (right). L The latency to target and the number of platform crossings in the Sham, HIE, HIE+BMSCs groups. M RT-qPCR verified the expression of SYNPO2 in the Sham, HIE + PBS, HIE+BMSCs groups. Bar graphs display mean ± SD of independent biological replicates. n = 4-8 rats, two-sided unpaired one-way ANOVA. All experiments were repeated three times independently, with similar results. HIE + PBS, rats were given PBS before HIE modeling as control; HIE+BMSC, rats were infused with BMSCs before HIE modeling; TEM transmission electron microscope.

Fig. 3. Changes of autophagy-related proteins in cortical neurons infected with shSYNPO2 lentivirus.

A Immunofluorescent staining of cortical neurons with Tuj1 (green)/LC3B (red) and Tuj1 (green)/P62 (red) in OGD+shNC and OGD+shSYNPO2 groups. Scale bar = 25 μm. B Quantification of LC3B and P62-positive neurons from immunofluorescent staining. C Cell viability of neurons in OGD+shNC and OGD+shSYNPO2 groups. D WB detection of SYNPO2 and autophagy-related protein expression (P62, Beclin1, ATG12, AIFM1 and LC3B II/I) in the OGD+shNC and OGD+shSYNPO2 groups. Bar graphs display mean ± SD of independent biological replicates. n = 3–6 wells, two-sided unpaired independent sample t-test. All experiments were repeated three times independently, with similar results. OGD+shNC, cortical neurons infected with control lentivirus overexpressing SYNPO2 before OGD; OGD+shSYNPO2, cortical neurons infected with shSYNPO2 lentivirus before OGD.

Fig. 4. Suppression of SYNPO2 in HIE rats alleviated neuroautophagic damage and neurological dysfunction.

A RT-qPCR results showing the expression levels of SYNPO2 in the cortex and hippocampus of HIE rats. B Immunofluorescent double-labeling staining of brain tissues of HIE rats with NEUN (red) and autophagic markers (green). Scale bar = 50 μm, 25 μm (magnified view). Blue, DAPI-stained nuclei. C TEM images showing autophagy in the brain tissue of HIE rats, with red arrows indicating autophagic vesicles. Scale bar = 500 nm. Nissl staining (D) and HE staining (E) of coronal brain sections from HIE rats in the HIE+shNC and HIE+shSYNPO2 groups. Scale bar = 2 mm (panoramic view), 500 μm (magnified view). Evaluation of muscle strength and coordination ability of rats by forelimb grip test (F), rotarod test (G), righting test (H), geotaxis test (I), and climbing test (J). The spatial exploration, autonomous behavior, and anxiety performance in HIE rats were detected by the Y maze (K) and open field test (L). M. The latency to target and the number of platform crossings in the HIE+shNC and HIE+shSYNPO2 groups. Bar graphs display mean ± SD of independent biological replicates. n = 4–8 rats, two-sided unpaired independent sample t-test. All experiments were repeated three times independently, with similar results. HIE+shNC, rats injected with control interfering lentivirus before HIE modeling; HIE+shSYNPO2, rats injected with SYNPO2 interfering lentivirus before HIE modeling; TEM, Transmission electron microscope.

Fig. 5. CM reduced OGD-induced neuronal damage and autophagy by downregulating SYNPO2 expression.

A. Immunofluorescence staining of cortical neurons with TUJ1 (green) and C-cas3 (red) in the CM + OE-NC, CM + OE and CM+dPDZ groups. Scale bar = 50 μm. White arrows indicate apoptotic neurons. Blue, DAPI-stained nuclei. B Quantification of C-cas3-positive neurons from CM + OE-NC, CM + OE and CM+dPDZ groups. C Cell viability of neurons in CM + OE-NC, CM + OE and CM+dPDZ groups. D WB detection of SYNPO2 expression in the OGD+shNC and OGD+shSYNPO2 groups. E WB detection of autophagy-related protein expression (ATG12, P62, Beclin1 and LC3B II/I) in the neurons from CM + OE-NC, CM + OE and CM+dPDZ groups. Bar graphs display mean ± SD of independent biological replicates. n = 3–5 wells, two-sided unpaired one-way ANOVA. All experiments were repeated three times independently, with similar results. dPDZ, cortical neurons infected with the SYNPO2-dPDZ deletion mutant lentivirus before OGD and treated with BMSC-conditioned medium; CM + OE-NC, cortical neurons infected with SYNPO2 overexpressing control lentivirus before OGD and subjected to BMSC treatment; CM + OE, cortical neurons infected with SYNPO2 overexpressing lentivirus before OGD and with BMSC infusion.

Fig. 6. SYNPO2 downregulation by BMSC treatment ameliorated autophagy and neurological dysfunction in HIE rats.

A Lentiviral vector constructs of SYNPO2-dPDZ used in this study. B RT-qPCR results showing the relative expression levels of SYNPO2 in the cortex and hippocampus of HIE rats. C Immunofluorescent double-labeling staining of brain tissues of HIE rats with NEUN (red) and autophagic markers (green) in HIE rats. Scale bar = 50 μm, 25 μm (magnified view). Blue, DAPI-stained nuclei. D TEM images showing autophagy in the brain tissue of HIE rats, with red arrows indicating autophagic vesicles. Scale bar = 500 nm. Nissl staining (E) and HE staining (F) of coronal brain sections from HIE rats in the BMSCs+OE-NC, BMSCs+OE, BMSCs+dPDZ groups. Scale bar = 2 mm (panoramic view), 500 μm (magnified view). The muscle strength and coordination ability of rats in the BMSCs+OE-NC, BMSCs+OE, BMSCs+dPDZ groups were tested by rotarod test (G), righting test (H), geotaxis test (I), and climbing test (J). K The latency to target and the number of platform crossings in water maze test. Bar graphs display mean ± SD of independent biological replicates. n = 4-6 rats, two-sided unpaired one-way ANOVA. All experiments were repeated three times independently, with similar results. BMSCs+OE-NC, rats injected with SYNPO2 overexpressing control lentivirus before HIE model establishment and BMSCs treatment; BMSCs+OE, rats injected with SYNPO2 overexpressing lentivirus before HIE model establishment and BMSCs treatment; BMSCs+dPDZ, rats injected with SYNPO2-dPDZ deletion mutant lentivirus before HIE model establishment and BMSCs treatment; TEM transmission electron microscope.