PDYN R212W cerebella show alterations in GABAergic connectivity
Mice expressing human PDYN containing the p.R212W SCA23 mutation (PDYNR212W mice) suffer from loss of motor function and balance, coinciding with loss of CF height compared to control mice, as well as to mice expressing wild type human PDYN (PDYNWT mice) (13). This pattern of findings for SCA23 is reminiscent of that seen for SCA1 (26, 28). Therefore, we hypothesized that SCA23 may also mirror other aspects of SCA1 pathology. Edamakanti et al. found alterations in GABAergic signalling in SCA1 mice (30), leading us to investigate GABAergic signalling in PDYNR212W mice. We examined the inhibitory synapses from basket cells (BCs) on PC soma in the vermal lobules at 2, 3, 4, and 8 weeks of age, using the vesicular inhibitory amino acid transporter (vGAT) as a marker for inhibitory synapses and Calbindin as a marker for PCs (Fig. 1A). PDYNR212W mice showed a reduced number of somatic inhibitory synapses at 2 weeks of age in lobules II, III, IV/V, IX, and X as compared to PDYNWT and control mice, as well as in lobule I as compared to PDYNWT mice (Fig. 1B). At 3 weeks of age, PDYNR212W lobule IV/V showed fewer synapses as compared to both PDYNWT and control, as well as PDYNR212W lobules VI and X as compared to control mice (Fig. 1B). Lobules II, IV/V, and X showed a decreased number of inhibitory synapses in PDYNR212W mice as compared to PDYNWT and control mice, as well as lobule I as compared to PDYNWT mice at 4 weeks of age (Fig. 1C). At 8 weeks of age, PDYNR212W mice showed fewer somatic synapses in lobules IV/V and VI as compared to both PDYNWT and control mice, in lobule II as compared to control mice, and in lobules I and IX as compared to PDYNWT mice (Fig. 1C). Taken together, these data indicate that SCA23 shows an opposite pathology to SCA1, as inhibitory synapses are lost on PDYNR212W PC soma.
CF development is disrupted in PDYNR212W mice
As inhibitory connectivity plays a role in the early phase of CF synapse elimination by BC collaterals taking over somatic spines from weak CFs on the PCs, we next investigated CF-PC somatic synapses using vGlut2 as a marker for the CF-PC synapse and Calbindin as a marker for PCs (Fig. 2A). Control and PDYNWT mice demonstrated normal early phase CF synapse elimination as demonstrated by significant reduction of vGlut2+ somatic puncta over time from 2 to 4 weeks of age (Fig. 2B and C). PDYNR212W mice, however, do not show a significant reduction of these puncta between 2 and 3 weeks of age in lobules II, III, IV/V, VI, and IX (Fig. 2D). Additionally, in PDYNR212W mice, lobule VI does not show significantly fewer vGlut2+ synapses from 3 weeks of age on and lobule X does not demonstrate any loss of CF somatic synapses (Fig. 2D). While these data suggest that PDYNR212W mice do not start out with fewer somatic CF synapses, they point towards a disruption of early phase CF synapse elimination.
As CF development is a finely tuned process, these findings suggested that further CF development could also be affected. Using the same markers, we analysed the reach of CFs by examining the CF-PC synapses along the PC dendrites (Fig. 3A). The PC dendrites of PDYNR212W mice showed a significantly reduced CF reach in vermal lobules I, II, III, VI/V, VI, and IX at 2 weeks of age in comparison to those of PDYNWT and control mice (Fig. 3B). The deficit persisted in these lobules and included lobule X at 3, 4, and 8 weeks of age (Fig. 3B and C). At 8 weeks of age, significance was lost in lobule IX (Fig. 3C). CF reach did not decline between 2 and 8 weeks of age, but does eventually decrease by 12 months of age, as observed previously (Smeets et al., 2015). Altogether, these data indicate disruption of normal CF development in PDYNR212W mice, specifically a delay in early phase CF synapse elimination and discontinued CF translocation, leading to a loss of the CF monopoly of the PC proximal dendrites.
PDYN R212W mice display increased PF-PC connectivity
On the distal PC dendritic tree, CFs and PFs are under intense competition for PC dendritic territory, and loss of CF synapses allows for an increase in PF synapses. This process, known as heterosynaptic competition, is employed during development and synaptic plasticity (22, 23). Given the striking loss of CF-PC synapses, we hypothesized that the number of PF-PC synapses may have increased. However, there is currently no suitable antibody available for a detailed histological quantification of vGlut1, the marker for PF-PC synapses. Therefore, we compared the protein levels of vGlut1 in the vermis of PDYNR212W mice to that of PDYNWT and control mice. Since vGlut1 and − 2 are highly expressed in the cerebellar granule layer, we first ascertained whether we could see a change in vGlut2 protein levels representing the loss of CF-PC synapses by determining vGlut2 protein levels in whole vermis protein lysate. At 8 weeks of age, we observed the expected reduction of overall vGlut2 levels in PDYNR212W cerebella (Fig. 4A). Although this reduction was only significant at 8 weeks of age, we were able to observe changes in overall vGlut2 protein level, indicating that changes in vGlut1 protein levels point to changes in PF-PC synapse count. The vGlut1 protein levels in PDYNR212W vermis were significantly increased at 2, 3, and 4 weeks of age compared to control vermis, and at 8 weeks of age compared to PDYNWT vermis (Fig. 4B). The elevated levels of vGlut1 suggest that PFs have increased their synapse numbers in the vermis of PDYNR212W mice, as CFs cannot reach terminal height to populate their natural PC dendritic territory.
Reduced GAD67 expression could indicate internal changes in PDYNR212W PCs
As both GABAergic and glutamatergic inputs are altered in PDYNR212W mice, we hypothesized that perhaps PCs adapted internal changes to counteract these alterations. Therefore, we determined the expression of glutamate decarboxylase 67 (GAD67), the main enzyme used by PCs to convert glutamate to GABA. Using GAD67 immunostaining, we determined expression levels by measuring fluorescence intensity in the PC layer of control, PDYNWT, and PDYNR212W mice (Fig. 5A). We observed a loss of GAD67 in PDYNR212W PCs at 2, 3, and 4 weeks of age as compared to control mice, and at 4 weeks as compared to PDYNWT mice (Fig. 5B). These data suggest that PDYNR212W PCs produce less GABA, which could be a reaction to the lost input from basket cells and/or CFs, possibly to normalise their impact on cerebellar nuclei neurons.
Changes in NMDA receptor subunits suggest altered Ca2+ signalling
Loss of vGlut2 has been shown to impair glutamatergic transmission (31), and we have previously demonstrated changes in NMDA receptor Grin2a subunit expression in PDYNR212W mice at 3 months of age (13). Therefore, we determined the mRNA expression levels of the Grin2 NMDA receptor subunits in the vermis of 2-, 3-, 4-, and 8-week-old PDYNWT, PDYNR212W and control mice. The expression levels of these subunits were relatively low and no significant alterations were detected in PDYNR212W mice (Additional File 2A). We also determined the mRNA expression levels of the remaining NMDA receptors subunits Grin1, Grin3a and -b. No alterations in Grin1, Grin3a and –b subunit expression were observed in 2-, 3-, and 8-week-old PDYNR212W mice (Fig. 6A). However, the expression of Grin1 was significantly increased at 4 weeks of age in PDYNR212W mice as compared to control and PDYNWT mice (Fig. 6A), while the inhibitory subunits Grin3a and –b displayed significantly decreased expression (Fig. 6A). Since Grin1 is the essential subunit for surface expression of NMDA receptors (Low and Wee, 2010), it is possible that at 4 weeks of age, PDYNR212W mice have increased surface expression of NMDA receptors. Taken together, in 4-week-old PDYNR212W mice, when NMDA receptors are expressed at PF- and CF-PC synapses in wild type mice (Watanabe and Kano, 2011), the expression of crucial NMDA receptor subunits is altered, potentially causing altered Ca2+ signalling.
VGCC dysregulation may be evidence of a compensatory mechanism for PDYN-R212W expression
As PDYNR212W mice display alterations in the development of crucial PC inputs and NMDA receptor subunit expression is affected, we hypothesized that the expression of voltage-gated Ca2+ channels (VGCCs) may also be affected. Cav2.1, a VGCC encoded by Cacna1a, the SCA6 disease gene (32), is crucial for proper CF maturation and regulates the expression of several genes involved in PC development (22, 33–35). Based on these prior findings, dysregulation of Cacna1a could potentially underlie the observed CF and PC deficits (24, 25), and ultimately ataxia (24, 36–39). To investigate whether PDYNR212W mice exhibit altered Ca2+ signalling via VGCCs, we assessed the mRNA expression levels of the cerebellar VGCC subunits Cacna1a and –c, Cacna2d2 and − 3, Cacnb2 and − 4, and Cacng2 and − 7 in the vermis of PDYNWT, PDYNR212W, and control mice. The mRNA level of Cacna1a was significantly increased at 3, 4, and 8 weeks of age in PDYNR212W mice (Fig. 6B). A similar effect was observed for Cacna1c; its expression was increased at 2, 4, and 8 weeks of age in PDYNR212W mice (Fig. 6B). At 8 weeks of age, the expression of both Cacna2d2 and − 3 was upregulated in PDYNR212W vermis (Fig. 6B), which was also observed for Cacnb2 and − 4 (Fig. 6C). Additionally, Cacnb2 expression was also increased at 2 weeks of age, and Cacnb4 at 4 weeks of age (Fig. 6C). As these last four subunits are auxiliary subunits, regulating the function of Cacna1a and Cacna1c, the observed increases could be a response to the increased Cacna1a and Cacna1c mRNA levels. Cacng2 and Cacng7 mRNA expression was increased at 8 and 4 weeks of age, respectively (Fig. 6C). Since γ2 and γ7 primarily regulate trafficking, localization and biophysical properties of AMPA receptors (Buraei and Yang, 2010; Yamazaki et al., 2015), we also studied the mRNA levels of Gria1-4. However, we found no correlation with the expression levels of Cacng2 and Cacng7 (Additional file 2B). These data demonstrate that Cav2.1, a key player in CF maturation, is markedly upregulated in PDYNR212W vermis around the time of CF maturation. We therefore suggest that dysregulated expression of crucial VGCCs and their auxiliary subunits contributes to the CF maturation deficits and loss of CF-PC connectivity in PDYNR212W cerebella.