Connexin 30 deficiency attenuates A2 astrocyte responses and induces severe neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride Parkinson’s disease animal model

The experimental procedures were designed to minimise the number of animals used as well as animal suffering. All animal experiments were carried out according to the guidelines for the proper conduct of animal experiments published by the Science Council of Japan, and ethical approval for the study was granted by the Animal Care and Use Committee of Kyushu University (#No. A29-179). The Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal research were followed.

Male heterozygous Cx30 KO mice were obtained from the European Mouse Mutant Archive [17]. Their spermatozoa were used to fertilise C57BL/6 oocytes in vitro. Heterozygous mice were interbred to obtain homozygous Cx30 KO and wild-type (WT) mice. Homozygous non-mutant mice were used as WT controls to ensure the control of the genetic background. All mice were provided food and water ad libitum and kept under a 12-h light/dark cycle in a specific pathogen-free room at the Biomedical Research Laboratory Station for Collaborative Research I of Kyushu University.

Eight-week-old male WT and Cx30 KO mice were intraperitoneally injected with 20 mg/kg of free base MPTP-HCl (Tokyo Chemical Industry, Tokyo, Japan) four times at 2 h intervals, and were sacrificed on days 1 and 7 after the last injection.

Total RNA was isolated from the striatum of each animal on day 1 after the last MPTP injection using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) and purified using the SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA). Total RNA (50 ng) was amplified and labelled with amplification and labelling kits (Agilent Technologies, Santa Clara, CA, USA) and then hybridised to a 60K Agilent 60-mer oligomicroarray (Agilent Technologies). The hybridised microarray slides were scanned using an Agilent Scanner, and the relative hybridisation intensities and background hybridisation values were calculated using Agilent Feature Extraction software (9.5.1.1). Gene set enrichment analysis (GSEA) (www.​broadinstitute.​org/​gsea) was performed to determine the enrichment score (ES), which indicates the degree to which each gene set is overrepresented at the top or bottom of a ranked list of genes. The false discovery rate (FDR) is the estimated probability of an ES representing a false-positive finding. An ES with a normalised p value less than 0.05 by an empirical phenotype-based permutation test and an FDR less than 0.25 was considered to be significant. We also calculated Z-scores and ratios from the normalised signal intensities of each probe for comparison. Z-scores are the number of standard deviations from the mean of log-scaled signal intensities. Ratios are non-log-scaled fold changes in signal intensities. We established criteria of Z-score ≥ 2.0 and ratio ≥ 1.5 for upregulated genes and Z-score ≤ − 2.0 and ratio ≤ 0.66 for downregulated genes. To determine significantly overrepresented categories of KEGG pathways, we used the tools and datasets provided at the Database for Annotation, Visualisation and Integrated Discovery (DAVID) (http://​david.​abcc.​ncifcrf.​gov/​home.​jsp). The raw data from this study have been submitted to the Gene Expression Omnibus (accession number: GSE113693).

Mice were euthanised and transcardially perfused with 4% paraformaldehyde. The brains were removed, fixed in 4% paraformaldehyde overnight, cryopreserved in 30% sucrose in phosphate-buffered saline (PBS), and stored at − 80 °C until analysis. For fluorescent immunostaining, 40-μm-thick sections were cut and incubated overnight at 4 °C with the primary antibodies against Cx30, Cx43, glial fibrillary acidic protein (GFAP), and ionised calcium-binding adapter molecule 1 (Iba1) (Additional file 1: Table S1) and then with an Alexa Fluor 488- or 594-conjugated secondary antibody for 1 h. The sections were mounted in mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) and visualised by confocal laser microscopy (Nikon A1, Nikon, Tokyo, Japan). For colorimetric immunostaining, the sections were incubated with primary antibodies against tyrosine hydroxylase (TH), dopamine transporter (DAT), GFAP, and Iba1 (Additional file 1: Table S1) followed by a rabbit or mouse Vectastain Elite ABC HRP kit (Vector Laboratories) and ImmPACT DAB Peroxidase Substrate (Vector Laboratories). We used Nikon NIS-Elements software (Nikon) to calculate the number of Iba1-positive cells, Cx30 dots, and Cx43 dots in the unilateral striata according to the previously reported procedures [18, 19]. The values from the three sections for each animal were averaged.

Mice were euthanised and perfused as described above. The brains were removed, fixed with G-Fix (Genostaff, Tokyo, Japan), and embedded in paraffin on a CT-Pro20 system (Genostaff) using G-Nox (Genostaff), which is a less toxic organic solvent than xylene. The brains were cut into 8-μm-thick sections and stained as follows with an in situ hybridisation (ISH) Reagent Kit (Genostaff) according to the manufacturer’s instructions. The tissue sections were deparaffinised with G-Nox and rehydrated through a graded ethanol series to PBS. The sections were fixed with 10% formalin in PBS for 30 min at 37 °C, washed with distilled water, placed in 0.2 N HCl for 10 min at 37 °C, washed in PBS, treated with 4 μg/ml proteinase K (Wako Pure Chemical Industries, Osaka, Japan) in PBS for 10 min at 37 °C, and washed again in PBS. The sections were then placed in a Coplin jar containing 1× G-Wash (Genostaff; equivalent to 1× saline sodium citrate buffer). Hybridisation was performed by incubation with probes for S100a10 and Gdnf (Additional file 1: Table S2) at 250 ng/ml in G-Hybo-L (Genostaff) for 16 h at 60 °C. The sections were then washed in 1× G-Wash for 10 min at 60 °C and incubated in 50% formamide in 1× G-Wash for 10 min at 60 °C. The sections were washed twice in 1× G-Wash for 10 min at 60 °C, twice in 0.1× G-Wash for 10 min at 60 °C, and twice in 0.1% Tween-20 in Tris-buffered saline (TBST) at room temperature. The sections were then incubated with 1× G-Block (Genostaff) for 15 min at room temperature and with alkaline phosphatase-coupled anti-digoxigenin (Roche Diagnostics, Mannheim, Germany) diluted 1:2000 with 1× 50 G-Block (Genostaff) in TBST for 1 h at room temperature. The sections were washed twice in TBST and then incubated in 100 mM NaCl, 50 mM MgCl₂, 0.1% Tween-20, 100 mM Tris-HCl, and pH 9.5. Colour development was performed by incubation with nitro blue tetrazolium and 5-bromo-4-chloro-3′-indolyphosphate solution (Sigma-Aldrich, St. Louis, MO, USA) overnight followed by washing in PBS.

Following ISH, S100β protein was detected by IHC. The sections were incubated with 0.3% H₂O₂ in PBS for 30 min to block endogenous peroxidase and then incubated with G-Block (Genostaff) and Avidin/Biotin Blocking reagent (Vector Laboratories). The sections were then incubated successively with anti-S100β antibody (Abcam) at 4 °C overnight, biotin-conjugated goat anti-rabbit antibody (Dako, Santa Clara, CA, USA) for 30 min at room temperature, and peroxidase-conjugated streptavidin (Nichirei, Tokyo, Japan) for 5 min. Peroxidase activity was visualised by incubation with diaminobenzidine, and the sections were mounted with G-Mount (Genostaff). Images of the bilateral striata were captured using a Leica DM2500 microscope with a × 40 objective (Leica Microsystems, Wetzlar, Germany). Finally, the number of cells double positive for S100β protein and either Gdnf or S100a10 mRNA was counted. The primer sequences specific for Gdnf and S100a10 are listed in Additional file 1: Table S2.

The total numbers of TH-, GFAP-, and Iba1-positive cells in unilateral SNcs were measured stereologically using an optical fractionator method as previously described [20‐22]. Every fourth section through the SNc was analysed using Stereo Investigator software (Stereo Investigator 10.0; MicroBrightField, Williston, VT, USA). Immunolabelled cells were counted by the optical fractionator method (× 40 objective; counting frame, 100 × 100 μm; sampling grid, 200 × 200 μm; counting frame thickness, 10 μm). The coefficient of error (Gundersen, m = 1) for cell count estimation was less than 0.15 for each animal.

The density of TH- and DAT-positive fibres in the striatum was assessed in the sections between Bregma + 0.62 and − 0.10 mm, as previously reported [23]. Every fourth section was immunolabelled, to give a total of six sections analysed per animal. Four to six images per section were captured using an Olympus B51 microscope with a × 100 objective (Olympus Corp., Tokyo, Japan). The optical density was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA), followed by background subtraction of the corpus callosum.

Total RNA was isolated from the striata using an RNA purification kit (Qiagen, Hilden, Germany), and cDNA was synthesised using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). Quantitative PCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific) with TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) and TaqMan Gene Expression Assays (Cx30, Mm00433661_s1; Cx43, Mm01179639_s1; S100a10, Mm00501457_m1; Gdnf, Mm01285715_m1; Thermo Fisher Scientific). Glyceraldehyde 3-phosphate dehydrogenase (Gapdh, Mm99999915_g1) was amplified as an internal control. The ∆∆CT efficiency correction method was used to calculate relative mRNA levels.

We first examined the changes in Cx30 expression in the sections of the striatum and SNc from WT mice on treated with normal saline (NS, control) or MPTP for 1 or 7 days. Immunofluorescence staining revealed Cx30 expression as small foci or dots (Fig. 1). The number of Cx30 dots was significantly increased between days 1 and 7 in MPTP-treated, but not NS-treated, mice (p < 0.0001, Fig. 2a), suggesting that MPTP treatment increased the expression of Cx30. This finding was supported by qRT-PCR and Western blot analysis which showed a significant increase in Cx30 mRNA levels (p = 0.0044, Fig. 2b) and Cx30 protein levels (p = 0.0269, Fig. 2c) in the striata of WT mice between days 1 and 7 after MPTP treatment (Additional file 1: Figures S1 and S2).

MPTP had no effects on the abundance of immunoreactive Cx30 dot counts or levels of Cx30 mRNA and protein in the striatum compared with NS on 1 day after treatment. However, on day 7, the striata from MPTP-treated mice contained significantly more Cx30 dots (p < 0.0001, Fig. 2a) and significantly higher Cx30 mRNA levels (p = 0.0277, Fig. 2b) than did the striata from NS-treated mice. In contrast, Cx30 protein levels were insignificantly higher in the MPTP-treated mice than NS-treated mice on day 7 post-treatment (p = 0.3302, Fig. 2c).

By immunofluorescence microscopy, small immunoreactive Cx30 dots were detectable along the striatal blood vessels of NS-treated mice, and their density in hypertrophic astrocytes was markedly increased 7 days after MPTP administration (Fig. 1a, b, upper panels). These findings are consistent with a previous report showing that Cx30 localisation is strongly increased around the striatal vessels following treatment with 6-OHDA [14]. Cx30 dots were also observed outside the perivascular areas of MPTP-treated mice, predominantly along astrocyte processes and occasionally in astrocyte cell bodies (Fig. 1a, b, lower panels). In the SNc, Cx30 expression was strongly upregulated by MPTP treatment and was present in both astrocyte processes and cell bodies (Additional file 1: Figure S3). To confirm the specificity of this analysis, we performed anti-Cx30 immunostaining of NS- and MPTP-treated striata from Cx30 KO mice, and we detected no Cx30 immunoreactivity (Additional file 1: Figure S4).

To determine whether Cx30 expression influences the expression of a second astrocyte gap junction protein, Cx43, we performed a similar analysis of Cx43 expression in the striatum and SNc of WT and Cx30 KO mice. As was observed for Cx30, Cx43 was detectable as fluorescent dots by IHC (Additional file 1: Figure S5). In both WT and Cx30 KO mice, the number of Cx43 dot counts did not change between days 1 and 7 in NS-treated mice but increased significantly between days 1 and 7 after MPTP treatment (p < 0.0001 for WT and Cx30 KO mice, Additional file 1: Figure S6a, b). As a result, Cx43 dot counts were comparable in NS- and MPTP-treated WT mice on day 1, but they were significantly higher in MPTP-treated than NS-treated WT mice on day 7 (p < 0.0001, Additional file 1: Figure S6a, b). The same trend was observed for Cx30 KO mice (p < 0.0001, Additional file 1: Figure S6a, b). Moreover, deletion of Cx30 caused a significant increase in the number of Cx43 dots in the striatum on day 1 after NS and MPTP treatment (p = 0.0310 and p = 0.0007, respectively, Fig. 3a) but not on day 7 (Fig. 3b).

RT-PCR analysis showed that Cx43 mRNA levels in the striatum were unchanged by NS treatment but were significantly increased by MPTP treatment between days 1 and 7 in both WT and Cx30 KO mice (p = 0.0322 and p = 0.0038, respectively, Additional file 1: Figure S6c, d). The levels of Cx43 mRNA did not differ significantly between NS- and MPTP-treated mice on day 1 after treatment, but they were significantly different after 7 days of treatment, with MPTP treatment inducing a marked increase in both WT and Cx30 KO mice (p = 0.0304 and p = 0.0044, respectively, Additional file 1: Figure S6c, d). Moreover, deletion of Cx30 caused no significant change in Cx43 mRNA levels in the striatum on days 1 and 7 after NS and MPTP treatment (Fig. 3c, d).

Cx43 protein levels did not change between days 1 and 7 after NS treatment in WT or Cx30 KO mice (Additional file 1: Figures S6e, S6f, S7, S8), whereas MPTP treatment significantly increased Cx43 protein expression between day 1 in Cx30 KO mice (p < 0.0001) but not in WT mice (p = 0.2795; Additional file 1: Figures S6e, f, S7, S8). As a result, Cx43 protein levels were comparable in NS- and MPTP-treated WT mice on day 1, but they were significantly higher in MPTP-treated than NS-treated WT mice on day 7 (p = 0.043, Additional file 1: Figure S6e, f). The same trend was observed for Cx30 KO mice (p < 0.0001, Additional file 1: Figure S6e, f). Moreover, deletion of Cx30 caused no significant change in Cx43 protein levels in the striatum on days 1 and 7 after NS and MPTP treatment (Fig. 3e, f).

On day 1 after NS or MPTP treatment, Cx43 dots were predominantly observed in astrocyte processes along the blood vessels in the striatum of WT and Cx30 KO mice (Fig. 4a); in contrast, Cx43 dots were also present beyond the perivascular areas by day 7 (Fig. 4b), which was similar to the pattern of Cx30 dots 7 days after MPTP treatment. In the SNc, the pattern of Cx43 staining was similar to that of Cx30. Cx43 dots were mainly detected in astrocyte processes along the vessels on day 1 after NS and MPTP treatment of both WT and Cx30 KO mice, and this was upregulated on day 7 after MPTP treatment (Additional file 1: Figure S5). These results suggest that Cx30 deficiency modestly increases the basal Cx43 expression and MPTP-upregulated Cx43 expression on day 1 after administration. This may reflect a partial compensation for the loss of Cx30, as observed in Cx43-deficient mice, which showed a twofold increase in Cx30 expression [24]. However, Cx30 deficiency did not alter either the levels or distribution of Cx43 on day 7 after MPTP treatment.

MPTP is converted by astrocytes to the neurotoxic molecule MPP⁺, which is then taken up into DA neurons via DAT. We used LC-MS/MS to measure MPP⁺ concentrations in the striatum of WT and Cx30 KO mice at 2 h after a single MPTP injection (peak MPP⁺ brain concentrations are observed at 1 h after injection [25]). We found no significant difference in the concentration of MPP⁺ in WT and Cx30 KO mice (Additional file 1: Figure S9), suggesting that Cx30 deficiency does not affect astrocyte MPTP metabolism. In addition, there was no significant difference between WT and Cx30 KO mice in DAT intensity (Additional file 1: Figure S10). Taken together, these results suggest that MPP⁺ uptake into DA neurons is comparable in WT and Cx30 KO mice.

Next, we examined the susceptibility of WT and Cx30 KO mice to MPTP neurotoxicity. MPTP caused severe acute toxicity, including immobility and, occasionally, death, in both the WT and Cx30 KO mice, as previously reported [26, 27]. The mortality rates on day 1 after MPTP treatment were higher in Cx30 KO mice than in WT mice, albeit not significantly (38% (19/50) vs. 20% (10/49), p = 0.0545). To evaluate DA neuron damage, we quantified TH-positive cells and dopamine levels at 7 days after MPTP injection. This time point was selected because TH expression and dopamine levels in the striatum of MPTP-treated mice have been reported to gradually decrease after 7 days, and the number of TH-positive cells in the SNc reaches a nadir 2 days after MPTP injection and then stabilises for at least 7 days thereafter [28]. We found that TH-positive fibre densities in the striatum and TH-positive cell numbers in the SNc were significantly decreased in Cx30 KO mice compared with WT mice after MPTP treatment (p = 0.0018 and p = 0.0246, respectively; Fig. 5a–d). Similarly, striatal levels of dopamine and its metabolites DOPAC and HVA were significantly lower in Cx30 KO mice than in WT mice after MPTP treatment (p = 0.0372, p < 0.0001, and p = 0.0007, respectively; Fig. 5e). Taken together, these findings indicate that Cx30 KO mice display hypersensitivity to MPTP.

Because Cx30 deficiency in astrocytes may alter their response to brain insults, we analysed the changes in gene expression associated with A1, A2, and pan-reactive astrocyte responses in the striatum on day 1 after NS or MPTP injection. Comparative microarray analyses showed that MPTP-induced gene expression changes in A1 astrocytes were similar in WT and Cx30 KO mice, whereas MPTP-induced upregulation of A2 and pan-reactive gene expression observed in WT mice was markedly attenuated in Cx30 KO mice (Fig. 6a; Additional file 1: Figure S11 and Table S3). Consistent with this, GSEA showed that the A2 and pan-reactive gene sets were significantly enriched in WT compared with Cx30 KO mice (A1: ES = 0.6435, normalised p = 0.1523, FDR = 0.1836; A2: ES = 0.8335, normalised p < 0.0001, FDR = 0.0076; pan-reactive: ES = 0.8886, normalised p < 0.0001, FDR < 0.0001; Fig. 6b). These data indicate that Cx30 is required for optimal induction of neuroprotective A2, as well as pan-reactive astrocyte responses against MPTP toxicity.

One of the A2 astrocyte genes that showed a reduced response to MPTP in Cx30 KO mice compared with WT mice was S100a10 (Z-score = − 3.9153, ratio = 0.4566). We selected this for further analysis because S100A10 plays important roles in intracellular trafficking and cell migration, thereby contributing to neuroprotection [29, 30]. RT-PCR analyses of striatal tissues 1 day after treatment demonstrated that MPTP significantly increased S100a10 mRNA expression compared with NS treatment in both WT and Cx30 KO mice (p < 0.0001 and p = 0.0063, respectively), but the upregulation in Cx30 KO mice was significantly less than that in WT mice (p = 0.0303; Fig. 7a), in agreement with the microarray results. However, these differences were not observed on day 7 after treatment (Fig. 7b).

The microarray analyses revealed that the expression of Gdnf, a well-known neurotrophic factor constitutively expressed in the striatum [31], was decreased in Cx30 KO mice compared with WT mice after MPTP treatment (Z-score = − 2.3141 and ratio = 0.5661). Using RT-PCR, we confirmed that striatal Gdnf expression was significantly decreased in Cx30 KO mice compared with WT mice 1 day after MPTP treatment (p = 0.0139), and its expression in Cx30 KO mice was significantly lower on day 1 after MPTP treatment compared with NS treatment (p = 0.0275; Fig. 7c). Similar to S100a10, Gdnf mRNA levels were not significantly different between WT and Cx30 KO mice at 7 days after treatment with NS or MPTP (Fig. 7d). ELISA analysis of GDNF protein in the striatum 1 day after MPTP treatment showed that levels were significantly lower in Cx30 KO mice compared with WT mice (p = 0.0192; Fig. 7e), consistent with the RT-PCR results. Collectively, these findings suggest that expression of both S100a10, a representative MPTP-upregulated A2 astrocyte response gene, and Gdnf, a constitutively expressed neuroprotective factor, is lower in Cx30 KO mice compared with WT mice at 1 day, but not 7 days, after MPTP administration.

We performed ISH combined with IHC on the striatal sections to detect cells in which Gdnf or S100a10 mRNA were colocalised with S100β protein. We detected Gdnf or S100a10 mRNA and S100β-positive striatal astrocytes and other cell types that were Gdnf or S100a10 mRNA-positive S100β-negative in both WT and Cx30 KO mice after treatment with NS or MPTP (Fig. 8a, b). MPTP had no effect on the number of Gdnf and S100β double-positive cells in WT mice, while it significantly decreased the number in Cx30 KO mice (p = 0.0221, Fig. 8a). As a result, the number of Gdnf and S100β double-positive cells was significantly lower in Cx30 KO mice compared with WT mice at 1 day after MPTP treatment (p = 0.0011, Fig. 8a). The number of S100a10 and S100β double-positive cells was significantly increased after MPTP treatment in WT mice (p = 0.0010, Fig. 8b) but was unchanged in Cx30 KO mice (Fig. 8b). As a result, the number of S100a10 and S100β double-positive cells was significantly lower in Cx30 KO mice compared with WT mice at 1 day after MPTP treatment (p = 0.0121, Fig. 8b).

In the microarray analysis of pan-reactive astrocyte genes, Gfap expression was upregulated by MPTP to a smaller extent in Cx30 KO mice than in WT mice (Z-score = − 6.9796 and ratio = 0.2812). To explore this further, we analysed GFAP protein expression levels in the striatum and SNc by IHC and Western blotting. We detected no effects of MPTP on striatal GFAP expression on day 1 after MPTP treatment in either mouse strain (Fig. 9a). However, on day 7, a robust MPTP-induced increase in GFAP levels was observed in WT and Cx30 KO mice, although the change in Cx30 KO mice was less marked (Fig. 9b), as confirmed by Western blotting (p = 0.0346; Fig. 9c and Additional file 1: Figure S12). Stereological counting of GFAP-positive cells in the SNc also revealed significantly fewer cells in the SNc of Cx30 KO mice compared with WT mice on day 7 after MPTP treatment (p = 0.0008; Fig. 9d, e). Taken together with the microarray results, these findings indicate that Cx30 deficiency reduces the MPTP-induced upregulation of Gfap, a pan-reactive astrocyte gene, at both the mRNA and protein levels.

Microglia play a critical role in neuroinflammation in PD; therefore, we examined microglial activation in our PD mouse model [32]. Previous work has shown that microglia are activated to a greater extent on day 1 after MPTP treatment compared with day 7 [33]. Therefore, we analysed microglial activation in the striatum and SNc at this time point. No significant differences between WT and Cx30 KO mice were observed in Iba1-positive cell numbers in either the striatum or SNc after NS or MPTP treatment (Fig. 10a, b), although MPTP caused vigorous activation of microglia in both locations on day 1 (Fig. 10c). Microglial activation was diminished by day 7 after MPTP administration to similar extents in WT and Cx30 KO mice (Fig. 10d).

Depending on the predominance of secreted factors, microglia can be classified into classical (M1: proinflammatory) or alternative (M2: anti-inflammatory) activation phenotypes. Thus, we used gene expression microarrays to examine M1-related proinflammatory, M2-related anti-inflammatory, and mixed M1/M2-related chemokine/cytokine expression in the striatum on day 1 after NS or MPTP treatment [34]. However, we found no difference between WT and Cx30 KO mice in the expression of any chemokines/cytokines after MPTP injection (Fig. 11a and Additional file 1: Figure S13 and Table S4). GSEA also revealed no significant differences in M1, M2, or M1/M2 gene expression between WT and Cx30 KO mice (M1: ES = − 0.3246, normalised p = 0.7926, FDR = 0.7410; M2: ES = 0.4118, normalised p = 0.7222, FDR = 0.7258; M1/M2 mixed: ES = 0.7616, normalised p = 0.0755, FDR = 0.1043; Fig. 11b).

From our gene expression analyses, we extracted genes that met the following conditions: (i) differentially expressed in the striatum of MPTP-treated Cx30 KO mice compared with NS-treated CX30 KO mice and (ii) not differentially expressed in the striatum of MPTP-treated WT mice compared with NS-treated WT mice on day 1. The resulting gene set was evaluated by DAVID functional annotation and pathway enrichment analysis, which revealed a total of 11 KEGG pathways that were significantly enriched in Cx30 KO compared with WT mice after MPTP treatment (Additional file 1: Table S5). The most highly enriched pathway was the axon guidance pathway (p = 0.00033), which includes netrins, ephrins, semaphorins, and their receptors (Additional file 1: Figure S14). Within this pathway, we found that ten genes were downregulated and four genes were upregulated, suggesting an overall downregulation of this pathway in the striatum of Cx30 KO mice compared with WT mice after MPTP treatment (Additional file 1: Table S6). This is consistent with the results shown in the scatter plots and heat map of differentially expressed genes detected by microarray analysis (Additional file 1: Figure S15). These results therefore suggest that loss of Cx30 KO may reduce the potential for axon regeneration and repair in the striatum after MPTP treatment.