Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons

Abstract

Small-conductance Ca(2+)-activated K(+) channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson's disease, where dopaminergic cell loss is associated with progression of the disease.

Figure 1

SK channels are expressed in LUHMES neurons. Human LUHMES dopaminergic neurons were differentiated for 6 days. The expression of SK channel subtypes was analyzed by (a) qPCR, (b) RT-PCR (SK1 channels depicted as KCNN1, SK2 channels depicted as KCNN2 and SK3 channels depicted as KCNN3 for mRNA levels) and (c) western blotting. As loading controls, GAPDH, actin (depicted as ACTB for mRNA levels) and Cyclophilin B (depicted as PPIB for mRNA levels) are shown

Figure 2

Dopaminergic neurons undergo cell death upon rotenone challenge. (a) Cell viability of LUHMES dopaminergic neurons treated with rotenone (0.1–2 μM) for 24 h (white bars) or 48 h (black bars) was investigated by an MTT assay. (b) ATP production was analyzed following rotenone challenge for 24 h (white bars) or 48 h (black bars). (c) LUHMES dopaminergic cells were treated with NS309 (1–100 μM) for 24 h and analyzed by an MTT cell viability assay. (*P<0.05, ^#P<0.05, ***P<0.001 versus non-treated dopaminergic cells, ANOVA Scheffé's test, n=6 wells per group, the experiment was repeated three times)

Figure 3

Activation of SK channels prevents rotenone-induced neuronal cell death and network disintegration. Dopaminergic cells were treated with rotenone (0.5 μM) in the presence or absence of NS309 (25 μM). (a) Representative microscopic pictures and DAPI stainings depicted for dopaminergic cells untreated or treated for 24 h with rotenone (0.5 μM) and NS309 (25 μM). (b) The neuronal network was visualized by DAT immunostaining. The degree of neuronal network disintegration (depicted as Neuronal network) was analyzed using the Image J software, with the Neurite Tracer plug-in (bar graph: 20 μm). (c) The analysis of neuronal network disintegration (depicted as Neuronal network) was assessed in relation to the total area covered by DAT immunostaining and DAPI-stained nuclei. The bar graph shows the analysis of neurite tracing for n=9–10 fields per condition, n=3, **P<0.01 versus non-treated dopaminergic neurons, ^#P<0.05 versus rotenone-treated dopaminergic neurons, ANOVA Scheffé's test). (d) Nuclear damage was assessed by counting the total numbers of DAPI-positive nuclei and fragmented or pyknotic DAPI-positive nuclei. The bar graph shows the percentage of fragmented DAPI versus total DAPI staining (**P<0.001 versus non-treated dopaminergic neurons, ^###P<0.001 versus rotenone-treated dopaminergic neurons, ANOVA Scheffé's test, n=500 cells per condition, n=4). (e and f) Dopaminergic cells were challenged with rotenone (0.5 μM) in the presence and absence of NS309 (25 μM) and apamin (10–20 μM). NS309 mediated neuroprotection against rotenone toxicity as analyzed by (e) an MTT assay and (f) an ATP assay (non-significant data are shown as NS; **P<0.01, ****P<0.0001 versus rotenone-treated dopaminergic neurons, ANOVA Scheffé's test, n=6 wells per group, the experiment was repeated three times with similar results)

Figure 4

Rotenone alters the expression of SK channels. Dopaminergic cells challenged with rotenone (0.5 μM) in the presence and absence of NS309 (25 μM) for 24 h were investigated for SK channel expression. The expression of SK channel subtypes was analyzed by (a–c) qPCR (SK1 channels depicted as KCNN1, SK2 channels depicted as KCNN2 and SK3 channels depicted as KCNN3 for mRNA levels) and (d–f) western blotting. Immunodetection of whole-protein lysates probed with antibodies to SK1 (d) SK2 (e) and SK3 (f) channels and actin, as loading control. Upper panels show SK1 (d), SK2 (e) or SK3 (f) channel expression, and the lower panels show the corresponding actin expression. Densiometric analysis of western blotting bands of SK channels (depicted as ‘Integrated optical densities' in percentage of non-treated control cells) shows that rotenone treatment induces a downregulation of SK2 channels and an upregulation of SK3 channels (non-significant data are shown as NS, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus non-treated dopaminergic neurons, ^#P<0.05, ^##P<0.01, ^####P<0.0001 versus rotenone-treated dopaminergic neurons, ANOVA Scheffé's test, n=3)

Figure 5

SK2 channels are present in mitochondria. (a) Immunoblot analysis of whole-cell extract (depicted as ‘Cell'), cytosol supernatant (depicted as ‘Cyto') or crude mitochondrial pellets (depicted as ‘Mito'), using antibodies against (a) SK1, (b) SK2 and (c) SK3 channels and control antibodies for protein location (COX IV—mitochondrial matrix, Tubulin—cytosol). (d) Confocal images of SK1, SK2 and SK3 channels in human dopaminergic cells. Mitochondrial staining was performed using a specific mitochondrial marker, MitoTracker Deep Red (bar graph: 5 μm). (e) Quantitative colocalization analysis of dopaminergic cells with SK (SK1/SK2/SK3) channels and MitoTracker Deep Red. (f) Image-generated scatter plots of acquired images for colocalization analysis processed by the LAS AF software (Leica). Mean percentages of colocalization rate were calculated at ROIs placed at cell body. Mean of colocalization rate: MitoTracker/SK1: 34.1%, MitoTracker/SK2: 86.1%, MitoTracker/SK3: 26.7%. Colocalization rate: This value indicates the extent of colocalization in percentage. It is calculated from the ratio of area of colocalizing fluorescence signals and the area of the image foreground (Obs. ROI: region of interest)

Figure 6

Activation of SK channels prevents calcium dysregulation. (a) Intracellular calcium was investigated in dopaminergic cells challenged with toxic concentrations of rotenone in the presence or absence of SK channel opener, NS309 (*P<0.05, versus rotenone and NS309-treated dopaminergic neurons were considered to be significant, ANOVA Scheffé's test, n=6). (b) Changes of the MMP (ΔΨ_m) were detected by DIOC6(3) fluorescence-based assay. Isolated 25–50 μg mitochondria were incubated with 20 nM DIOC6(3) dye. As a positive control for a complete loss of ΔΨ_m, CCCP (50 μM) protonophore was applied on intact mitochondria. ΔΨ_m was analyzed by a FLUOstar Optima fluorescence plate reader using 485/520-nm filters for excitation/emission

Figure 7

ΔΨ_m is altered by SK channel opening. Isolated mitochondria from (a, c, e) the mouse brain and synaptosomes (b, d, f) were loaded with fluorescent dye Rhodamine 123 (depicted as Rh123), and the ΔΨ_m was measured. Representative Rh123 fluorescent traces of NS309 effect (25 μM) on isolated mitochondria (a and b) in the absence of extracellular calcium, (c and d) in the presence of 10 μM Ca²⁺ and (eand f) of 40 μM Ca²⁺. As a positive control for a complete loss of ΔΨ_m, FCCP (50 μM) protonophore was applied on intact mitochondria