GDNF alleviates constipation, restores colonic 5-HT secretion, and improves depression-like behavior in 6-OHDA mouse model
A PD-like model was established by stereotaxic injection of 6-OHDA (2 μL, 4 μg/μL) into the brains of 6-8-week-old mice (Fig. 1A). Two weeks later, intraperitoneal injection of APO (0.1 mg/100 g) was used to induce mouse rotation, with PD model mice showing stable rotation toward the contralateral side at speeds greater than 5 r/min (Fig. 1B). Western blot analysis revealed a significant decrease in TH expression in the SNc brain region of the 6-OHDA model group compared to the Sham group. Immunofluorescence showed reduced distribution and expression of TH in the 6-OHDA group (Fig. 1C-F), confirming successful PD-like modeling.
On the basis of 6-OHDA stereotaxic injection, AAV-GDNF or AAV-NC was injected intraperitoneally (Fig. 1A). Western blot analysis showed a significant increase in GDNF expression in the colon tissue of the 6-OHDA + AAV-GDNF group compared to the 6-OHDA + AAV-NC group (Fig. 1G and H), indicating successful GDNF overexpression.
At weeks 1-5 following 6-OHDA brain stereotaxic injection, 24-h fecal weight and 2-h fecal water content were measured. The results showed that as the modeling time increased, the 24-h fecal weight and 2-h fecal water content in the 6-OHDA + AAV-NC group were reduced compared to the Sham group, while the 6-OHDA + AAV-GDNF group showed improvements (Fig. 1I and J), confirming that GDNF overexpression improved constipation-related indicators in 6-OHDA mice.
A 5% activated carbon gavage (0.6 mL/mouse) was used to assess intestinal motility. Propulsion ratio (%) was calculated as (the length from the pylorus to the red arrow (activated carbon)/the length from the pylorus to the anus)×100. The higher propulsion ratio indicates better gut motility and less severe constipation symptoms. The results showed a reduced intestinal propulsion rate in the 6-OHDA + AAV-NC group compared to the Sham group, while the 6-OHDA + AAV-GDNF group showed improvements (Fig. 1K and L), indicating that GDNF could improve the weakened intestinal motility in 6-OHDA mice.
The 5-HT content in the colon supernatant of each group was measured, showing that the 6-OHDA + AAV-NC group had reduced 5-HT secretion compared to the Sham group, while the 6-OHDA + AAV-GDNF group showed improvements (Fig. 1M), suggesting that GDNF could improve the 5-HT secretion deficit in the colons of 6-OHDA mice.
The tail suspension test was used to measure immobility time in mice. The 6-OHDA + AAV-NC group showed reduced immobility time compared to the Sham group, indicating depressive-like despair behavior, while the 6-OHDA + AAV-GDNF group had a significantly shorter immobility time compared to the 6-OHDA + AAV-NC group (Fig. 1N), suggesting that GDNF intervention could improve the motor depression-like behavior.
Additionally, the sugar water preference rate was significantly reduced in the 6-OHDA + AAV-NC group compared to the Sham group, indicating anhedonia. The 6-OHDA + AAV-GDNF group had a significantly higher sugar water preference rate than the 6-OHDA + AAV-NC group (Fig. 1O), suggesting that GDNF has a significant role in improving anhedonia
This study performed RNA sequencing (RNA-Seq) on colonic tissues from Sham and 6-OHDA-induced PD-like model mice to detect gene transcriptional differences. After log-transformation of the transcriptomic data, both groups followed a normal distribution, and quantile normalization was performed to make the data comparable between groups (Fig. S1A and B). Principal component analysis (PCA) showed significant differences between the groups under different interventions (Fig. S1C). Differential analysis revealed that in the 6-OHDA group, 473 genes were upregulated and 249 genes were downregulated in the colonic tissue (Fig. S1D). Among the 473 upregulated genes, protein expression of inflammation-related pathway genes was significantly elevated in 6-OHDA model mice (Fig. 2A). Enrichment analysis of the top 50 upregulated differential genes was performed using the Enrichr database to analyze their pathways and biological processes (Biological Process, BP). KEGG analysis showed that these upregulated genes were mainly enriched in inflammation and cell response regulation, Toll-like receptor signaling, Jak-STAT signaling, and interleukin signaling pathways (Fig. S1E and F). GO-BP analysis indicated that these genes were involved in the regulation of lipopolysaccharide and inflammatory responses, intracellular signal transduction, and cytokine-mediated responses (Fig. S1G and H). Further network analysis was performed on the 50 significantly upregulated differential expression genes (DEGs). Protein-protein interaction (PPI) networks were constructed using the STRING database (v11.5) and imported into Cytoscape (v3.9.1). Four topological algorithms (maximal clique centrality (MCC), maximum neighborhood component (MNC), degree, closeness) were applied to evaluate node importance, and hub genes were identified. The results indicated that TLR4 occupies a central position in the differential regulation network of 6-OHDA model mice and may participate in disease progression through downstream inflammatory pathways (Fig. S1I-L).
Western blot analysis showed that TLR4 protein expression in the colonic tissue of the 6-OHDA + AAV-NC group was upregulated compared to the Sham group, while the 6-OHDA + AAV-GDNF group showed improvement (Fig. 2B, C). Additionally, immunofluorescence staining of EGCs labeled with GFAP (green) and TLR4 (red) demonstrated upregulated TLR4 expression in the 6-OHDA + AAV-NC group compared to Sham, and improvements were observed in the 6-OHDA + AAV-GDNF group, with co-localization of GFAP and TLR4 also changing accordingly (Fig. 2D-F). This confirmed that GDNF improves the abnormal upregulation of TLR4 expression in the colonic EGCs of 6-OHDA mice.
To verify whether GDNF improves various indicators in the 6-OHDA model mice through the TLR4 signaling pathway, 6-8-week-old mice were stereotaxically injected with 6-OHDA and given an intraperitoneal injection of AAV-GDNF. Four weeks later, the TLR4 agonist RS09 (6 mg/kg) was administered intraperitoneally for 3 days, forming the 6-OHDA + AAV-GDNF + RS09 experimental group (Fig. 2G). Activated carbon gavage (5%, 0.6 mL/mouse) was used to assess intestinal motility, and the results showed that the 6-OHDA + AAV-GDNF group had improved motility compared to the 6-OHDA + AAV-NC group, but this improvement disappeared after treatment with the TLR4 agonist RS09 (Fig. 2H and I). Furthermore, 5-HT levels in the colonic supernatant were measured, and the results indicated that the 6-OHDA + AAV-GDNF group had improved colonic 5-HT secretion compared to the 6-OHDA + AAV-NC group, but this effect was abolished upon treatment with RS09 (Fig. 2J). This suggests that GDNF likely exerts its effects through the blockade of TLR4 signaling.
Western blot analysis of colonic inflammatory markers showed elevated inflammation indicators in the 6-OHDA + AAV-NC group compared to the Sham group, which were reduced in the 6-OHDA + AAV-GDNF group. These reductions were reversed after RS09 administration (Fig. 2K and L), confirming that GDNF improves colonic inflammation and constipation in 6-OHDA mice by inhibiting TLR4 pathway activation.
TLR4 is an essential component of the immune system, and tumor necrosis factor receptor-associated factor 6 (TRAF6), as a key downstream signaling molecule of TLR4, plays a central role in immune responses and inflammation. Therefore, we further examined the expression changes of TRAF6 in the EGCs of the mouse colon and in the EGC cell line (EGC CRL-2690). Immunofluorescence staining of EGCs labeled with GFAP (green) and TRAF6 (red) showed that TLR4 expression was upregulated in the 6-OHDA + AAV-NC group compared to Sham, with corresponding increases in co-localization between GFAP and TRAF6. These changes were reversed in the 6-OHDA + AAV-GDNF group, and the improvement disappeared after the addition of the TLR4 agonist RS09 (Fig. 2M and N). This confirmed that GDNF inhibits the abnormal activation of TLR4, further reducing the elevated expression of TRAF6 in the colonic EGCs of 6-OHDA mice.
EGCs were treated with RS09 (64 μmol/L) for 24 h to mimic PD colonic glial cells, and GDNF (0.1 ng/mL) was added for intervention for 24 h to simulate the GDNF administration group. Western blot analysis showed that TRAF6 expression was elevated in the RS09 group compared to Sham, but this elevation was abolished after GDNF treatment (Fig. 2O and P). Correspondingly, immunofluorescence staining also confirmed that TRAF6 expression was elevated in the RS09 group compared to Sham, but the increase disappeared after GDNF treatment (Fig. 2Q and R). This further supports that GDNF inhibits the abnormal activation of TLR4, leading to a reduction in the elevated expression of TRAF6 in EGCs.
This experiment further investigates the relationship between TRAF6 expression and the regulation of NEDD4 ubiquitination. The study found that TRAF6 can form a complex with NEDD4, participating in and catalyzing the ubiquitination degradation of NEDD4. GDNF's mechanism of action in vivo can be progressively achieved through the activation of GDNF-Ret-Src signaling. Therefore, we explored whether activated Src plays a role in TRAF6-mediated NEDD4 ubiquitination regulation.
In the Zdock docking simulation of Src binding with TRAF6, Src binds to an α-helix between positions 195-200 and an unstructured loop and β-sheet between positions 215-255 of TRAF6, primarily interacting non-covalently with tyrosine at position 198, cysteine at position 218, threonine at position 237, and glutamine at position 254 (Fig. 3A). In the docking model of NEDD4 and TRAF6, NEDD4 binds between the α-helices at positions 205-235 of TRAF6, interacting non-covalently with serine at position 207 and aspartic acid at position 232 (Fig. 3B). Based on these results, we hypothesized a competitive binding relationship between Src-TRAF6-NEDD4 and conducted corresponding validation experiments.
We constructed a TRAF6 overexpression plasmid (Fig. 3C). CO-IP was used to detect the binding between TRAF6 and Src in EGCs from different experimental groups. The results showed that in the RS09 group, TRAF6 binding to Src was reduced compared to the Sham group, and after GDNF intervention, this reduction disappeared. TRAF6 overexpression further increased the binding between TRAF6 and Src (Fig. 3D and E). Additionally, CO-IP was used to detect the interaction between TRAF6 and NEDD4. The results showed that in the RS09 group, TRAF6 binding to NEDD4 increased compared to the Sham group, and after GDNF intervention, this increase was reversed. TRAF6 overexpression also showed trends consistent with the RS09 group (Fig. 3F and G).
In vivo experiments confirmed these binding differences between TRAF6 and Src, and TRAF6 and NEDD4, with results consistent with the cell experiments (Fig. 3H-J). These findings suggest that GDNF likely competes with TRAF6 to bind to NEDD4 through activated Src.
Next, we used protein immunoblotting and in vivo immunofluorescence experiments to further validate the expression and ubiquitination modification differences of NEDD4. Immunofluorescence staining of EGCs, with GFAP (green) marking and NEDD4 (red) dual labeling, showed that in the 6-OHDA + AAV-NC group, NEDD4 expression was downregulated compared to Sham. This downregulation was improved in the 6-OHDA + AAV-GDNF group, but the improvement disappeared in the 6-OHDA + AAV-GDNF + RS09 group (Fig. 3K and L).
Subsequently, Western blot experiments at the cellular level showed that NEDD4 expression was downregulated in the RS09 group compared to Sham. GDNF intervention improved this downregulation, and further TRAF6 overexpression showed trends consistent with the RS09 group (Fig. 3M and N). These results suggest that GDNF can improve the downregulation of NEDD4 expression by inhibiting the abnormal elevation of TLR4-TRAF6 signaling.
Finally, CO-IP was used to detect the differences in NEDD4 ubiquitination modifications. The results showed that in the RS09 group, NEDD4 ubiquitination was increased compared to Sham. However, after GDNF intervention, the increased ubiquitination modification of NEDD4 disappeared, and TRAF6 overexpression showed a trend consistent with the RS09 group (Fig. 3O and P). These findings suggest that GDNF may block the TRAF6-mediated NEDD4 ubiquitination degradation by activating Src, thereby increasing NEDD4 expression in 6-OHDA mice.
Previous studies from our lab have shown that 6-OHDA mice exhibit gut inflammation accompanied by abnormal expression of cell junction proteins, particularly the abnormally high expression of CX43. To investigate whether NEDD4 is directly associated with the abnormal changes in CX43, we developed a 6-OHDA + AAV-GDNF model in GFAP-cre mice and intraperitoneally injected RNAi-NEDD4 virus. Western blotting confirmed the effectiveness of the viral infection, showing significant suppression of NEDD4 expression (Fig. 4A-C). Subsequently, we performed immunofluorescence staining, marking EGCs with GFAP, and co-labeled GFAP (green) with CX43 (red). The results indicated that CX43 expression was upregulated in the 6-OHDA + AAV-NC group compared to the Sham group. In the 6-OHDA + AAV-GDNF group, this upregulation was improved, and in the 6-OHDA + AAV-GDNF + NEDD4 KD group, the improvement was lost (Fig. 4D and E).
We then examined CX43 expression at the cellular level using Western blot, and found that CX43 expression was upregulated in the RS09 group compared to the Sham group. After GDNF intervention, the upregulation of CX43 expression was improved. However, after NEDD4 knockdown, CX43 upregulation became more pronounced (Fig. 4F and G). Immunofluorescence results corroborated the Western blot data (Fig. 4H and I). These findings suggest that CX43 expression is abnormally elevated in the colons of 6-OHDA mice, GDNF has some improvement effects, and NEDD4 expression directly influences CX43 expression. As NEDD4 is an E3 ubiquitin ligase, we hypothesized that NEDD4 might regulate CX43 expression changes by participating in its ubiquitination modification. In the Zdock docking simulation, we found that NEDD4 binds to CX43 between positions 100-105 in the α-helix, primarily interacting non-covalently with arginine at position 101 and glutamic acid at position 104 (Fig. 4J). CO-IP was then used to detect the binding between NEDD4 and CX43 in EGCs from different experimental groups. The results showed that in the RS09 group, NEDD4 binding to CX43 was reduced compared to the Sham group. After GDNF intervention, NEDD4 binding to CX43 increased again, and after NEDD4 knockdown, the binding almost disappeared (Fig. 4K and L). We further validated the interaction between NEDD4 and CX43 using proximity ligation assay (PLA-Duolink). The appearance of distinct red fluorescence dots indicates protein-protein interaction, with the intensity of red fluorescence corresponding to the strength or quantity of the interaction. PLA-Duolink results were consistent with the previous CO-IP findings (Fig. 4M and N).
Finally, we used CO-IP to detect the differences in CX43 ubiquitination modifications across the experimental groups. The results showed that in the RS09 group, CX43 ubiquitination was reduced compared to the Sham group. After GDNF intervention, CX43 ubiquitination increased again. However, after NEDD4 knockdown, the ubiquitination of CX43 nearly disappeared (Fig. 4O and P). Additionally, ubiquitination prediction indicated that CX43 (GJA1) ubiquitination is largely regulated by NEDD4 (Fig. 4Q).
Together, the experimental results from CX43 expression, NEDD4-CX43 binding, and CX43 ubiquitination modifications confirm that NEDD4 targets CX43 for ubiquitination and participates in its degradation. As shown in Fig. 3, GDNF can increase NEDD4 expression, suggesting that GDNF may promote NEDD4 expression to facilitate its targeting of CX43 for ubiquitination and degradation.
To investigate the effects of overexpression of CX43 on the EGCs membrane, we used the EGC CRL-2690 cell line to overexpress CX43 through plasmid transfection (Fig. 5A). Western blotting was used to verify the transfection efficiency and expression of CX43 in other groups, showing that CX43 expression in the CX43 OE group was significantly higher than in the other groups. In the RS09 group, CX43 expression was abnormally upregulated compared to the Sham group, and this upregulation was improved after GDNF intervention (Fig. 5B and C). Next, we collected the extracellular medium and measured various indicators. We found significant differences in the relative ATP content in the extracellular fluid. The RS09 group showed an increase in ATP relative content compared to the Sham group, which was restored to Sham levels after GDNF intervention. Notably, the CX43 OE group exhibited significantly higher ATP relative content than other groups (Fig. 5D), suggesting that abnormally activated CX43 releases excessive ATP, creating an extracellular hyperuricemic environment that may contribute to neuroinflammation and other pathological processes. We further explored the effects of extracellular hyperuricemia on physiological states using in vivo experiments in mice. We found that purinergic receptors, such as P2Y12 and P2Y1, were regulated: through immunofluorescence staining with CgA to mark enteroendocrine cells (ECs), we co-labeled CgA (green) with P2Y12 (red). The results showed that P2Y12 expression in ECs was upregulated in the 6-OHDA + AAV-NC group compared to the Sham group, and the upregulation was inhibited in the 6-OHDA + AAV-GDNF group (Fig. 5E and F). This indicates that overactivation of CX43 on EGCs in the colon of 6-OHDA mice leads to excessive ATP release, creating an extracellular hyperuricemic environment that regulates purinergic receptors like P2Y12 and P2Y1 on ECs.
To further investigate the effects of different ATP concentrations on P2Y12 and P2Y1 receptor expression, we treated QGP-1 cells with various concentrations of exogenous ATP (10, 100, 250 μM). Western blotting revealed that as extracellular ATP concentration increased, P2Y12 expression was upregulated, while P2Y1 and TPH expression was downregulated (Fig. 5G-J). This confirms that P2Y12 is positively regulated by ATP, while P2Y1 is negatively regulated by ATP. In parallel in vivo experiments, we observed that P2Y12 expression was significantly upregulated in the ATP 250 μM group compared to the Sham group (Fig. 5K, L), consistent with the cell Western blot data. Thus, we hypothesize that overexpression of CX43 on the membrane of EGCs in 6-OHDA mice creates a hyperuricemic environment, leading to increased P2Y12 expression on ECs. To explore the effects of P2Y12 and P2Y1 receptor expression changes on 6-OHDA mice, we administered P2Y1 receptor antagonist (MRS2179) to the 6-OHDA + AAV-GDNF group and P2Y12 receptor antagonist (PSB0739) to the 6-OHDA + AAV-NC group. We then measured the 24-h fecal weight and 2-hour fecal moisture content at weeks 1-5 after 6-OHDA brain stereotactic injection. The results showed that, over time, the 6-OHDA + PSB0739 group exhibited significant improvements in fecal weight and moisture content compared to the 6-OHDA group. These improvements were suppressed in the 6-OHDA + AAV-GDNF + MRS2179 group compared to the 6-OHDA + AAV-GDNF group (Fig. 5M, N). This confirms that inhibition of the P2Y1 receptor can reverse the improvements in the GDNF group, while inhibition of the P2Y12 receptor can reverse the abnormal changes in the 6-OHDA group. These findings suggest that the improvement in the GDNF group may be mediated by P2Y1 activation, whereas the abnormal changes in the 6-OHDA group may be driven by P2Y12 activation. We then verified this hypothesis by testing gut motility with 5% activated charcoal gavage (0.6 mL per mouse). The results showed that the 6-OHDA + PSB0739 group had significantly improved gut propulsion compared to the 6-OHDA group, while the 6-OHDA + AAV-GDNF + MRS2179 group showed suppressed improvement compared to the 6-OHDA + AAV-GDNF group (Fig. 5O, P). Moreover, measuring the 5-HT content in the colon supernatant of each group showed that the 6-OHDA + PSB0739 group had increased 5-HT secretion compared to the 6-OHDA group, while the improvement in the 6-OHDA + AAV-GDNF + MRS2179 group was suppressed (Fig. 5Q).
In summary, we hypothesize that overactivation of P2Y1 and P2Y12 receptors, respectively, positively and negatively regulate 5-HT synthesis and secretion, contributing to constipation in PD-like mice. In the tail suspension test, the 6-OHDA + PSB0739 group showed significantly reduced immobility time compared to the 6-OHDA group. In contrast, the 6-OHDA + AAV-GDNF + MRS2179 group had significantly increased immobility time compared to the 6-OHDA + AAV-GDNF group (Fig. 5R). These results suggest that activation of the P2Y1 receptor can reverse the improvement in motor depression-like behavior in the GDNF group, while inhibition of the P2Y12 receptor can alleviate depressive-like behavior in mice. Furthermore, the 6-OHDA + PSB0739 group showed significantly increased sugar water preference compared to the 6-OHDA group, while the 6-OHDA + AAV-GDNF + MRS2179 group showed decreased sugar water preference compared to the 6-OHDA + AAV-GDNF group (Fig. 5S), indicating that P2Y1 receptor inhibition can reverse the improvement in depressive-like behavior in the GDNF group.
Several studies have reported that probiotics can improve certain motor and NMS in PD patients. To further explore the effect of the gut microbiota Akk11 on the improvement of 6-OHDA-induced PD-like models in mice, we administered Akk11 by gavage. 6-OHDA models were established in 6-8-week-old mice by stereotactic injection of 6-OHDA (2 μL, 4 μg/μL) into the brain, followed by Akk11 gavage treatment once a week for 4 weeks, starting 4 days after the injection (Fig. 6A). Compared to the Sham group, GDNF protein expression in the colon tissue of 6-OHDA group mice was significantly reduced. However, after adding Akk11 gavage treatment to the 6-OHDA intervention, GDNF protein expression was significantly increased compared to the 6-OHDA group (Fig. 6B and C). In vitro, GDNF expression in EGC cells was also higher in the Sham and RS09+Akk11 groups compared to the RS09 group (Fig. 6D and E). These results suggest that Akk11 can partially reverse the decline of GDNF in PD-like models. Next, we assessed gut motility using 5% activated charcoal gavage (0.6 mL per mouse). The results showed that the 6-OHDA+Akk11 group had significantly improved gut propulsion compared to the 6-OHDA group (Fig. 6F). Akk11 not only altered GDNF protein expression in the colon tissue of 6-OHDA mice but also significantly improved impaired gut motility. Additionally, the tail suspension test revealed that the 6-OHDA+Akk11 group had significantly reduced immobility time compared to the 6-OHDA group (Fig. 6G), suggesting that Akk11 alleviates the despair-like state in mice. Furthermore, the 6-OHDA+Akk11 group showed a significant increase in sugar water preference compared to the 6-OHDA group (Fig. 6H), indicating that Akk11 also has an improving effect on depressive-like behavior in mice.