Cx32 facilitates the uptake of oα-syn assemblies in vitro
To determine whether connexins play a role in the uptake of different α-syn assemblies, we first generated recombinant α-syn monomers, oligomers (oα-syn), and fibrillar assemblies fluorescently tagged with ATTO-550 . We then characterized soluble protein assemblies by size exclusion chromatography (SEC) as previously reported (Fig. 1a, b) . As expected, we observed prolonged retention values for oα-syn assemblies compared to α-syn monomers. The absence of free dye within our preparations was corroborated by measuring the absorbance of ATTO-550 (550 nm) using SEC (Fig. 1c). We next characterized all protein assemblies by transmission electron microscopy (TEM) and concomitant to the prolonged SEC retention values observed, the characteristic donut-like and filament ultrastructures typical of oα-syn and fibrillar assemblies, respectively, were identified (Fig. 1d–f and Suppl. Figure S1a, b, Online Resource 4) . We next analyzed the fibril length by TEM and observed an average length of 133.74 nm ± 3.53 nm, a result consistent with the length of non-sonicated α-syn fibrils (Suppl. Figure S1c, Online Resource 4) . Following protein characterization, we transiently transfected human embryonic kidney cells (HEK-293) with increasing concentrations of the neuronal, non-canonically expressed Cx32 or the non-neuronally associated Cx43 (fused to mCherry and GFP, respectively), to exclude a general Cx effect. As expected, increased concentration of plasmid DNA transfection (15 µg; low and 50 µg; high) enhanced Cx expression in HEK-293 cells as shown by the increased intensity of the fluorescent tags observed under confocal microscopy and densitometry analysis (Fig. 1g and Suppl. Figure S1d, Online Resource 4). Consistent with the previous observations , the addition of fluorescent tags had no effect on Cx activity, gap junction formation or membrane compartmentalization, as shown by the subcellular localization of Cx32-mCherry or Cx43-GFP at the cellular membrane (Suppl. Figure S1e, f, Online Resource 4). Interestingly, treatment of Cx-expressing cells with α-syn monomers, oligomers, or fibrillar assemblies demonstrated a selective increase in the uptake of oα-syn that was Cx32-dependent (Fig. 1h, i). In contrast, HEK-293 cells expressing Cx43 showed no differences in protein uptake regardless of the assembly state of the α-syn molecule (Fig. 1h, i). Moreover, the increased levels of Cx32 in high-expression cells further amplified oα-syn uptake, suggesting a correlation between Cx32 expression and oα-syn uptake (Suppl. Figure S1d, g, Online Resource 4).
To verify that Cx32-mediated oα-syn uptake was not due to differential expression in HEK-293 cells but a Cx32-dependent mechanism, we generated multiple clonal SH-SY5Y cell lines expressing Cx32 (Cx32-mCherry) or its heteromeric gap junction binding partner Cx26 (Cx26-mCherry) . We also generated clonal cell lines expressing Cx43 (Cx43-HA) as well as untagged mCherry proteins alone to use as controls. The contribution of Cx32 to oα-syn uptake was further tested by deletion of the Cx32 gene (GJB1) using CRISPR/Cas9 (Cx32-KO). Following SH-SY5Y differentiation to generate a neuron-like phenotype with morphological and biochemical characteristics of mature neurons [2, 73], we observed Cx32 expression localized to the cellular membrane. We next incubated SH-SY5Y cells with α-syn monomers, oligomers or fibrillar assemblies for 24 h. Consistent with the HEK-293 results (Fig. 1h, i), expression of Cx32 in differentiated neuronal SH-SY5Y cells selectively increased the uptake of oα-syn as analyzed by Western blot and immunocytochemistry (Fig. 1j–l) when compared to α-syn monomers (Suppl. Figure S1 h, Online Resource 4) and fibrillar assemblies (Fig. 1m, n). Consistent with the involvement of Cx32 in oα-syn uptake, CRISPR/Cas9-based deletion of the Cx32-mCherry construct significantly blocked oα-syn uptake, validating the involvement of Cx32 in the uptake of oα-syn assemblies (Suppl. Figure S1i, Online Resource 4). As expected, overexpression of Cx26 or Cx43 in differentiated SH-SY5Y cells showed no differences in the uptake of any α-syn assemblies, corroborating a Cx32-dependent mechanism in the selective uptake of oα-syn assemblies (Fig. 1j–n).
The mitogen-activated protein kinase (MAPK) pathway modulates oα-syn uptake via Cx32
To further demonstrate a direct link between Cx32 expression and oα-syn uptake in differentiated human neuronal SH-SY5Y cells, we investigated the impact of inhibiting the p38 MAPK pathway, which negatively regulates Cx32 protein turnover, on oα-syn uptake . We incubated differentiated SH-SY5Y cells with the potent p38 MAPK inhibitor SB203580 (10 and 25 μM), together with oα-syn for 24 h. As expected, we observed a concentration-dependent increase in Cx32 protein expression and this effect led to a decrease in Cx32 mRNA (Fig. 2a, b). Correspondingly, the increase in Cx32 protein expression induced by SB203580 treatment correlated with a significant increase in oα-syn uptake that was concentration dependent (Fig. 2c, d). In contrast, cells exposed to the p38 MAPK activator anisomycin (1, 3, and 5 µM), which promotes Cx32 degradation , showed a significant decrease in Cx32 protein expression and promoted a significant upregulation of Cx32 mRNA (Fig. 2e, f). Concomitant with the results above, the observed reduction in Cx32 expression correlated with a significant decrease in oα-syn uptake, emphasizing a direct link between Cx32 expression and oα-syn uptake (Fig. 2g, h).
Cx32-mediated protein uptake is energy dependent and preferential for oα-syn assemblies
Next, we investigated whether Cx32-mediated oα-syn uptake is mediated by an energy-dependent mechanism rather than via channel diffusion. We incubated Cx32-mCherry and mCherry control cells with oα-syn assemblies at 4 °C or 37 °C for 6 h. As expected, we observed a greater uptake of oα-syn in cells expressing Cx32-mCherry than its wild-type counterpart (mCherry) under normal culture conditions (37 °C) (Fig. 2i). However, reduced temperatures (4 °C) significantly reduced oα-syn uptake in both cell types, indicating an energy-dependent mechanism required for oα-syn uptake (Fig. 2i, j). Given the accumulation of protein oligomers in other neurodegenerative conditions including AD , we next assessed whether Cx32 mediates the uptake of oligomers composed of Aβ, one of the pathological hallmarks of AD . Similar to our oα-syn uptake conditions, we incubated differentiated SH-SY5Y cells expressing Cx32 or mCherry proteins alone for 24 h then assessed oAβ uptake by immunocytochemistry and Western blot analysis. We observed a significant increase in the uptake of oAβ in cells expressing Cx32 compared to controls by both Western blot analysis and immunocytochemistry (Fig. 2k–n). However, the differential uptake of oAβ via Cx32 compared to controls was not as effective as that for oα-syn assemblies (compare Figs. 1j and 2k), suggesting that, in addition to its oligomeric state, the underlying protein sequence may play a role in this process.
Cx32 expression increases oα-syn cell-to-cell transfer
In a previous study, we generated a co-culture model system under proliferating conditions to monitor and quantify the transfer of human α-syn-GFP from donor to recipient cells that was suitable for flow cytometry and high content screening . Using a similar approach (Fig. 3a), we assessed whether proliferating human SH-SY5Y cells overexpressing Cx32-mCherry (as recipient cells) efficiently internalized α-syn-GFP derived from donor cells compared to cells expressing untagged mCherry proteins. Following 5 days of co-culture, we quantified a higher percentage of Cx32-mCherry cells containing α-syn-GFP puncta compared to control cells expressing untagged mCherry proteins (Fig. 3b). We next co-cultured donor and recipient cells under differentiation conditions to promote a mature neuron-like phenotype and enhance neuronal connectivity  (Suppl. Figure S2a, b, Online Resource 5). Consistent with our results above, we observed a higher percentage of Cx32-mCherry cells containing α-syn-GFP puncta compared to control cells expressing mCherry proteins alone under differentiating conditions (Fig. 3c). Although a higher trend for α-syn-GFP transfer in cells under differentiation was observed compared to proliferating conditions, this difference was not statistically significant. To further assess the role of neuronal connectivity in the transfer of α-syn, we co-cultured donor and recipient cells in separate compartments to prevent cellular connectivity but allowing co-cultured cells to share the same medium (Suppl. Figure S2c, Online Resource 5). Consistent with the results above, neuronal connectivity increased the transfer of α-syn-GFP to cells expressing Cx32-mCherry compared to mCherry controls. However, blocking neuronal connectivity prevented α-syn transfer even though the presence of oα-syn within the media could be demonstrated . Collectively, our results indicate that neuronal connectivity plays a major role in the transfer of oα-syn, suggesting that Cx32 expression increases oα-syn uptake in a manner enhanced by neuronal connectivity.
oα-Syn binds and colocalizes with Cx32 during cellular uptake
To validate oα-syn transfer to recipient cells during our co-culture conditions, we sorted double-labeled cells expressing either Cx32-mCherry or mCherry containing α-syn-GFP puncta using fluorescence-activated cell sorting (FACS). Using confocal microscopy, we visualized the presence of α-syn-GFP puncta within the cell soma and around the cell membrane of both Cx32-mCherry and mCherry recipient cells (Fig. 3d, Suppl. Figure S2d, Online Resource 5). We then evaluated the extent of colocalization between α-syn-GFP and Cx32-mCherry fluorophores or α-syn-GFP and mCherry by calculating the Pearson correlation coefficient (P). Our results point to a direct protein–protein interaction between oα-syn and Cx32 during cellular uptake that is not evident in cells expressing mCherry proteins alone (Fig. 3d, Suppl. Figure S2d, Online Resource 5). To further assess a direct interaction between Cx32 and oα-syn, we incubated Cx32-mCherry or mCherry cells with oα-syn for 24 h followed by immunoprecipitation (IP) of oα-syn assemblies using a pan-specific antibody that does not interfere with the N-terminal region of α-syn required for membrane interaction  (epitope 117-125). Given the observed interaction between Cx32 and oα-syn by confocal microscopy, we identified the presence of Cx32-mCherry but not of untagged mCherry proteins following IP of oα-syn (Fig. 3e). Moreover, the presence of beads with IgG in our IP conditions failed to pull down α-syn or Cx32 within the samples tested, confirming a direct interaction between Cx32 and oα-syn during cellular uptake.
Cx32 interacts with oα-syn and facilitates uptake in oligodendrocytes
We next determined whether Cx32 also plays a role in the uptake of oα-syn in oligodendrocytes, the cell type associated with canonical Cx32 expression in the central nervous system . Using the rat oligodendrocyte cell line OLN-93 , we generated Cx32-KO cells using CRISPR/Cas9 or overexpressed Cx32-mCherry. We then differentiated these cells by serum deprivation as previously reported , followed by incubation for 24 h with α-syn monomers, oligomers or fibrillar assemblies. Consistent with our Cx32 neuronal model system, we observed a significant increase in the uptake of oα-syn assemblies compared to monomers and fibrillar assemblies in Cx32-mCherry OLN-93 cells (Fig. 3f, g). Moreover, OLN-93 cells lacking Cx32 (Cx32-KO) showed a decrease in oα-syn uptake compared to control conditions and although this was not statistically significant, it validates the involvement of other cellular mechanisms in this process (Fig. 3f, g). It is worth noting that whereas Cx32 in SH-SY5Y cells was observed at the cellular membrane (Fig. 1l), Cx32-mCherry in OLN-93 oligodendrocytes localized primarily to the cell soma (Fig. 3h, asterisk). However, in some instances, we found that Cx32 was properly localized to the cellular membrane, forming the typical Cx32 plaques (Fig. 3h, arrowheads).
To further validate the involvement of Cx32 in the uptake of oα-syn in oligodendrocytes, we transfected human oligodendrocyte precursor cells (OPCs) derived from induced pluripotent stem cells (iPS) with Cx32-mCherry or untagged mCherry proteins. We observed Cx32-mCherry in differentiated human OPCs (2′,3′-Cyclic-nucleotide 3′-phosphodiesterase, CNPase+) localized to the cellular membrane, where it remained throughout the differentiation process (Fig. 3i). In contrast, untagged mCherry expression was observed throughout the entire cytoplasm (data not shown). We next differentiated OPCs for 30 days and used the expression of the myelin basic protein (MBP) by immunocytochemistry as a marker for cell maturity (Suppl. Figure S3a, b, Online Resource 6). Subsequently, we incubated differentiated OPCs with α-syn monomers, oligomers or fibrillar assemblies for 24 h. Using Western blot analysis, we found that expression of Cx32-mCherry in differentiated oligodendrocytes (MBP+) significantly increased the uptake of oα-syn assemblies compared to monomeric or fibrillar assemblies (Fig. 3j, k). To validate a direct interaction between oα-syn and Cx32 in human oligodendrocytes, we next treated differentiated OPCs (CNPase+) overexpressing Cx32-mCherry with ATTO-488-labeled oα-syn for 24 h. Similar to the results in our neuronal culture model, we observed a remarkable colocalization between Cx32 and oα-syn-ATT0-488 proteins in human oligodendrocytes (Suppl. Figure S3c. Online Resource 6). We next treated OPCs expressing Cx32-mCherry cells with oα-syn followed by IP of oα-syn as described above, and identified the presence of Cx32 within the immunoprecipitated samples by Western blot analysis (Suppl. Figure S3d, Online Resource 6). Collectively, our results confirm that Cx32 interacts with oα-syn and facilitates its internalization in neurons and oligodendrocytes.
Cx32 peptide mimetic inhibitors reduce the uptake of oα-syn assemblies
We next explored whether pharmacological strategies targeting Cx32 activity inhibit oα-syn uptake. We exposed differentiated wild-type SH-SY5Y cells to oα-syn or fibrillar assemblies tagged with ATTO-550 , in the presence of specific Cx32 peptide mimetic sequences (Gap3211 and Gap2409) known to bind and block Cx32 hemichannel activity [9, 13]. Gap3211 binds to the first extracellular loop [amino acid (AA) 52–63], whereas Gap2409 binds to the inner cytoplasmic transmembrane loop (AA100–110) of Cx32. Treatment of differentiated SH-SY5Y cells with oα-syn assemblies in the presence of Gap3211 (Fig. 4a, b) or Gap2409 (Suppl. Figure S4a, c, Online Resource 7) for 24 h significantly reduced oα-syn uptake. This treatment, however, had a limited effect on fibrillar α-syn uptake (Fig. 4a, Suppl. Figure S4a, e, Online Resource 7). Given the high sequence similarity of Cx32 between humans and rodents, we next exposed primary rat cortical neurons (DIV 10) to oα-syn assemblies in the presence of Gap3211 or Gap2409. Consistent with our differentiated human neuronal SH-SY5Y results, we observed a significant reduction in oα-syn uptake following Gap3211 or Gap2409 peptide mimetic treatment on primary neurons (Fig. 4c, d). Similarly, OLN-93 oligodendrocytes exposed to oα-syn or fibrillar assemblies in the presence of Gap3211 or Gap2409 showed a marked decrease in oα-syn uptake (Fig. 4e, f, Suppl. Figure S4b, d); however, this effect did not translate to fibrillar assemblies (Fig. 4e, f Suppl. Figure S4b, f, Online Resource 7).
To verify that inhibition of oα-syn is dependent on the sequence of Cx32, we next treated differentiated human neuronal SH-SY5Y cells with Cx32 scrambled mimetic sequences (SC) of both GAP3211-SC (Suppl. Figure S5a, Online Resource 8) and 2409-SC (data not shown). In contrast to functional Cx32 sequences, we observed no inhibition of oα-syn or fibrillar uptake using Cx32-SC (Suppl. Figure S5a, Online Resource 8). We next assessed whether functional mimetic sequences targeting Cx43 blocked oα-syn or fibrillar uptake. We treated cells with peptides targeting the first extracellular loop of Cx43 (Gap2605, AA64–76), but no effect on oligomeric or fibrillar α-syn assemblies could be detected (Suppl. Figure S5b, Online Resource 8). These results indicate that blockade of Cx32 using peptide mimetic sequences partially impedes the uptake of oα-syn, but this treatment has limited effect on fibrillar α-syn assemblies.
Pharmacological gap junction inhibitors reduce α-syn uptake
To further assess the potential of Cx32 as a suitable target for pharmacological intervention to block α-syn uptake, we next tested the widely used pan-connexin gap junction inhibitor carbenoxolone (CBX) . Treatment of differentiated neuronal SH-SY5Y cells with increasing concentrations of CBX (50 and 100 µM) significantly blocked oα-syn and fibrillar uptake in a concentration-dependent manner (Suppl. Figure S6a, b, Online Resource 9). Similarly, treatment of oligodendrocytes (OLN-93) with CBX led to a significant decrease in oα-syn and fibrillar uptake that was concentration dependent (Suppl. Figure S6c, d, Online Resource 9). Given the non-specific inhibitory profile of CBX to different Cx proteins, we next assessed the effect of mefloquine (MQ), a selective inhibitor that targets gap junctions composed primarily of Cx26, Cx32 and/or Cx43 . In our culture conditions, MQ (25 and 50 µM) significantly blocked the uptake of oα-syn and fibrillar assemblies in human differentiated SH-SY5Y neuronal cells, primary cortical neurons (DIV 10) and differentiated OLN-93 oligodendrocytes in a concentration-dependent manner (Suppl. Figure S7a–f, Online Resource 10). Finally, we tested the inhibitory effect of 2-aminoethoxydiphenyl borate (2-APB), a highly selective gap junction inhibitor known to primarily target Cx26 and Cx32 . Treatment of cells with 2-APB (100 and 200 µM) significantly blocked the uptake of oα-syn and fibrillar assemblies in human SH-SY5Y neuronal cells (Fig. 5a, b), primary cortical neurons (DIV 10) (Fig. 5c, d) and OLN-93 oligodendrocytes in a concentration-dependent manner (Fig. 5e, f).
α-Syn expression differentially regulates Cx32 in neurons and oligodendrocytes
Having established that Cx32 expression preferentially modulates oα-syn uptake in neurons and oligodendrocytes, we next assessed the effect of oα-syn uptake in differentiated SH-SY5Y cells overexpressing human α-syn-GFP, a useful cellular system to model PD in vitro . We treated differentiated SH-SY5Y cells overexpressing human α-syn-GFP with exogenously generated oα-syn assemblies for 24 h, then measured protein uptake by Western blot analysis. We observed a significant increase in the uptake of oα-syn in cells expressing α-syn-GFP compared to controls (Fig. 6a). Importantly, this effect was not due to the proteolytic degradation of the endogenous α-syn-GFP construct, which remained fully intact (~ 40 kDa) throughout the experiment (Suppl. Figure S8a, Online Resource 11). Prompted by the observed increase in oα-syn uptake in our in vitro cellular model of PD, we next examined the levels of Cx32 protein and mRNA before and after oα-syn treatment. We found that Cx32 protein levels increased upon expression of α-syn-GFP compared to controls (Fig. 6b), with no further detectable increase following exogenous oα-syn treatment. However, an increase in Cx32 mRNA following exogenous oα-syn treatment compared to that of the untreated α-syn-GFP cells was observed, an effect that was more pronounced in the treated α-syn-GFP expressing cells than in wild-type-treated controls (Fig. 6c).
Next, we investigated whether Cx32 upregulation correlates with the levels of α-syn expression. We, therefore, separated SH-SY5Y cells expressing low and high levels of α-syn-GFP using FACS (Suppl. Figure S8b, Online Resource 11). We then induced neuronal differentiation as previously reported , and assessed the levels of Cx32 expression by Western blot and qRT-PCR. Interestingly, we identified an increase in Cx32 protein upregulation that associated with the levels of α-syn-GFP expression, both at the protein and mRNA level (Suppl. Figure S8c, d, Online Resource 11), demonstrating a direct correlation between Cx32 upregulation and α-syn expression, (Suppl. Figure S8e, Online Resource 11). Collectively, our results suggest that expression of human α-syn or exposure to oα-syn assemblies promotes Cx32 upregulation in differentiated human neuronal SH-SY5Y cells.
We next investigated whether exogenous oα-syn treatment or expression of α-syn-GFP in human oligodendrocytes would increase Cx32 expression. We exposed differentiated wild-type OPCs (CNPase +) to oα-syn assemblies for 24 h then measured Cx32 protein expression. As expected, we observed a significant increase in Cx32 upregulation, albeit to a lesser extent than that observed in neuronal SH-SY5Y cells (Fig. 6d). However, in contrast to the neuronal SH-SY5Y model, we identified no differences in Cx32 protein expression between wild-type OPCs and OPCs overexpressing human α-syn-GFP (Fig. 6d). In fact, relative to wild-type OPC-treated cells, OPC-α-syn-GFP cells exposed to exogenous oα-syn assemblies showed a significant decrease in Cx32 expression. However, although a trend for a reduction in Cx32 expression was observed following incubation of OPC’s expressing α-syn-GFP with oα-syn assemblies compared to untreated α-syn-GFP cells, this effect was not statistically significant (Fig. 6d). We then asked whether OPCs overexpressing Cx32 would show a decrease in Cx32 following oα-syn treatment. We incubated OPCs overexpressing Cx32-mCherry with oα-syn proteins for 24 h then measured Cx32 protein levels by Western blot analysis. We observed a significant decrease in endogenous Cx32 protein expression in cells incubated with oα-syn compared to untreated OPC-Cx32 cells (Fig. 6e). These results suggest that, compared to neuronal cells, human oligodendrocytes differentially regulate Cx32 expression following α-syn expression or exposure to oα-syn assemblies.
α-Syn accumulation increases Cx32 in Tg models of PD and MSA
To assess a possible connection between α-syn and Cx32 in vivo, we analyzed adult and aged cortical brain homogenate samples from Tg mice overexpressing human wild-type α-syn (Line 61, 6 months) or mutant α-syn harboring the familial A30P mutation (18 months), both driven by the Thy-1 promoter (Suppl. Table S2 [23, 46], Online Resource 2). Compared to respective non-transgenic (non-Tg) age-matched controls, we observed a significant upregulation in Cx32 protein expression in L61 (Fig. 6f) and A30P Tg mice (Fig. 6g). The upregulation of monomeric Cx32 was corroborated using a different immunological probe targeting the C-terminal region of the Cx32 molecule (Suppl. Figure S8f, g, Online Resource 11). We then analyzed the levels of Cx32 mRNA in respective age-matched control mice and L61 and A30P cohorts. While we observed no statistically significant differences between non-Tg and Tg models (L61 and A30P), a trend of increased Cx32 mRNA levels in both PD models was noted (Suppl. Figure S8 h, Online Resource 11). To further assess whether Cx32 colocalizes with α-syn in situ, we performed immunohistochemistry on tissue sections from the A30P mice (Fig. 6h). We observed a clear colocalization between Cx32 and human A30P-α-syn that, in some instances, appeared within the membrane of cells of the oligodendrocyte lineage, as indicated by the morphological pattern of Cx32 labeling within oligodendrocytes (Fig. 6h, I–III). To further validate the presence of α-syn within cells of the oligodendrocyte lineage, we performed immunohistochemistry on adjacent tissue sections using the oligodendrocyte-specific marker CNPase and observed the presence of α-syn within CNPase+ cells [Suppl. Figure S9a (I–IV), Online Resource 12]. Moreover, we observed a clear colocalization between Cx32 and the neuronal-specific protein β3-tubulin, confirming the presence of Cx32 proteins within neuronal cell types (Fig. 6h, IV–VI). Finally, we immunolabeled tissue sections with a highly specific α-syn antibody that labels α-syn phosphorylated at serine 129 (pS129) and does not cross-react with a similar epitope in the neurofilament light chain protein (NFL ). Our qualitative observations revealed a clear colocalization between Cx32 and pS129 in neuronal cells (Fig. 6h, VII–XII) and oligodendrocytes co-labeled with CNPase and pS129 [Suppl. Figure S9a (V–VIII), Online Resource 12], corroborating the direct interaction between Cx32 and α-syn in neuronal and oligodendrocyte cell types in the Tg A30P mouse model of PD.
We then investigated the relationship between Cx32 and α-syn in the MBP29 mouse model of MSA, which overexpresses human wild-type α-syn in oligodendrocytes driven by the MBP promoter . We prepared brain homogenate samples from the cortex (ctx) and corpus callosum (cc) of young (3 weeks) and adult cohorts (3 months) followed by Western blot analysis. We observed no statistically significant differences in the Cx32 profile between young age-matched controls and Tg MBP29 mice (Fig. 7a, b). Importantly, and consistent with the accumulation of α-syn deposits within these mice over time, we observed a significant age-dependent increase in Cx32 protein levels in Tg cohorts compared to age-matched controls (3 months, Fig. 7c, d) as well as young MBP29 Tg mice (Suppl. Figure S9b, Online Resource 12). These results are similar to those in the mouse PD cohorts; however, a significant decrease in Cx32 mRNA in both young and adult Tg MBP29 cohorts compared to non-Tg age-matched controls within the cortex and corpus callosum was clearly observed (Fig. 7e, f). These results are consistent with our in vitro oligodendrocyte results pointing to an overall attempt to downregulate Cx32 in oligodendrocytes following oα-syn expression, indicating that Cx32 expression in oligodendrocytes is differentially regulated compared to its neuronal counterpart.
Cx32 interacts with α-syn in PD brains
To further assess the relationship between Cx32 and α-syn in human PD brains, we analyzed the putamen and substantia nigra pars compacta (SNpc) from four neuropathologically diagnosed PD cases and four age-matched controls (Suppl. Table S3, Online Resource 3). While we observed no statistically significant differences in Cx32 expression between PD and control cases within the putamen (Fig. 7g), the SNpc, a region highly vulnerable to PD pathogenesis, showed a marked decrease in Cx32 expression (Fig. 7h). Interestingly and consistent with the decrease in Cx32 within the substantia nigra, we observed no significant differences in the levels of α-syn from control and PD cases from the same region (Suppl. Figure S9c, Online Resource 12). We then assessed the levels of Cx32 in an area outside the nigrostriatal pathway, the Brodmann area 9 (BR9) of the dorsal lateral pre-frontal cortex, a region relatively unaffected by α-syn pathology in PD during the early stages of the disease process. In contrast to the substantia nigra, we observed no significant differences in Cx32 protein levels between controls (n = 15) and PD cases (n = 15) (Fig. 7i, j), suggesting that the reduction of Cx32 levels within the nigral region may serve as a protective mechanism to reduce α-syn uptake and accumulation. To further validate the interaction between Cx32 and α-syn in human PD brains, we next immunoprecipitated α-syn from the putamen, the region containing higher levels of Cx32 relative to the SNpc. Cx32 co-immunoprecipitated with α-syn in two out of four PD cases but this interaction was not observed in any of the four age-matched control cases tested (Fig. 7k, Suppl. Figure S9d, Online Resource 12). We next analyzed Cx32 levels from the putamen region of control and pathologically diagnosed MSA cases (Suppl. Table S3, Online Resource 3), a region highly vulnerable to α-syn accumulation in oligodendrocytes of MSA patients [54, 64]. Similar to the substantia nigra in PD, we identified a significant decrease in Cx32 levels in MSA cases compared to age-matched controls (Fig. 7l), further validating a potential relationship between human α-syn and Cx32 in the pathophysiology of PD and MSA.