DOI: https://doi.org/10.21203/rs.3.rs-26966/v1
Spinocerebellar ataxia type 23 (SCA23) is a late-onset neurodegenerative disorder characterized by slowly progressive gait and limb ataxia, for which there is no therapy available. It is caused by mutations in PDYN, which encodes the opioid precursor protein prodynorphin (PDYN). PDYN is processed into the opioid peptides α-neoendorphin, and dynorphins (Dyn) A and B; inhibitory neurotransmitters that function in pain signalling, stress-induced responses, and addiction. Mutations causing SCA23 mostly affect Dyn A, leading to loss of secondary structure and increased peptide stability. PDYNR212W mice express human PDYN containing the SCA23 p.R212W mutation. These mice show gait deficits and progressive loss of motor function from 3 months of age. The cerebella of PDYNR212W mice show climbing fibre (CF) deficits from 3 months of age and Purkinje cell (PC) loss from 12 months of age. A mouse model for SCA1 showed similar CF deficits, and a recent study found additional developmental abnormalities, namely hyperproliferation of stem cells leading to increased GABAergic interneuron connectivity and non-cell autonomous disruption of PC function. As SCA23 mice show a similar pathology to SCA1 mice in adulthood, we hypothesized that SCA23 may also follow SCA1 pathology during development.
In the present study, we examined the cerebella of PDYNR212W mice during cerebellar development, from 2 to 8 weeks of age, using immunohistochemistry, protein, and RNA analysis.
We uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma in PDYNR212W mice, possibly leading to the observed delay in early phase CF elimination between 2 and 3 weeks of age. Furthermore, CFs did not reach terminal height leaving proximal PC dendrites open to be occupied by parallel fibres (PFs). The observed increase in vGlut1 protein -a marker for PF-PC synapses- indicates that PFs indeed take over CF territory and have increased connectivity with PCs. Additionally, we detected altered expression of several critical Ca2+ channel subunits, potentially contributing to altered Ca2+ transients in PDYNR212W cerebella.
These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.
Spinocerebellar ataxia type 23 (SCA23) is a late-onset, slowly progressive neurodegenerative disorder for which there is no available therapy. It is caused by missense mutations in PDYN and characterized by loss of neurons in the Purkinje cell (PC) layer, dentate nuclei, and inferior olivary nuclei (1, 2). Patients suffer from gait ataxia, dysarthria, slowed saccades, ocular dysmetria, Babinski’s sign, and hyperreflexia. PDYN encodes the opioid precursor protein prodynorphin (PDYN), which is processed into the opioid peptides α-neoendorphin and dynorphins (Dyn) A and B. These peptides normally function as inhibitory neurotransmitters in pain processing, stress-induced responses, and addiction (3–6). Dyn A is also known to elicit non-opioid-mediated neurotoxic effects including allodynia, neuronal loss and paralysis (3, 7, 8). It can cause cell death via the NMDA (N-methyl-D-aspartate) receptor (9), and elicit neurotoxic effects via the AMPA (α-amino-hydroxy-5-methylisoxazole-4-propionate) receptor and acid-sensing ion 1a channels (10, 11). Additionally, we have shown that mutations causing SCA23 affect the secondary structure of Dyn A, reducing its affinity with its natural κ-opioid receptor and peptide stability, leading to peptide aggregation (12).
We previously generated PDYNR212W mice, expressing human PDYN containing the p.R212W mutation, and showed that they recapitulate features of SCA23 pathology, showing progressive gait deficits from 3 months of age and loss of motor coordination and balance at 12 months of age (13). Examination of the cerebella of these mice revealed a loss in climbing fibre (CF) height from 3 months of age, PC loss at 12 months of age, and pathologically elevated levels of Dyn A peptide (13).
CFs form a crucial component in synaptic plasticity, a process of great importance to the functioning of the cerebellum; it has long been thought to be the molecular mechanism underlying motor functioning and learning (14–17). CFs and parallel fibres (PFs) are the excitatory inputs of the cerebellum, and synapse upon the singular output of the cerebellum, the PC (18, 19). CFs form a crucial part of the cerebellar machinery, as they exert enormous control over the synaptic plasticity of the PFs (14, 15, 20, 21). In order to maintain the delicate balance of synaptic plasticity, both CFs and PFs have their own PC dendrite territories. CFs populate the proximal PC dendritic tree, whereas the PFs synapse upon the distal PC dendritic tree (18, 19). CFs and PFs compete for PC territory throughout life, and loss of one of these types of fibres leads to an increase in the other (22, 23). Alterations in components of this process lead to synaptic deficits in several mouse models suffering from ataxia and absence seizures (24, 25). The observed CF deficits in SCA23 are particularly interesting, as similar effects have been observed in other SCA types, including SCA1 (26–29).
A recent publication demonstrated that in SCA1, stem cells hyperproliferate and preferably differentiate into GABAergic interneurons leading to increased inhibitory connections with PCs and non-cell autonomous PC dysfunction (30). As the CF deficits of SCA23 match those of SCA1, we hypothesized that SCA23 may also follow this newly identified SCA1 pathology. Therefore, we studied PDYNR212W cerebella around the time of cerebellar circuit maturation, from 2 to 8 weeks of age. Interestingly, we found that in SCA23, GABAergic innervation shows reduced synapse connectivity. Here, we report the molecular changes observed in the developing PDYNR212W cerebellum, and propose an alternative disease model for SCA23.
All animal experiments were performed according to the ethical guidelines of the Animal Welfare Committee of the University of Groningen, the Netherlands. The experimental protocols were approved by the Animal Welfare Committee of the University of Groningen. All efforts were made to reduce the number of animals and minimize their suffering. Transgenic mice were bred and genotyped as previously described (13), and housed with same-sex litter mates under standard conditions including environmental enrichment, with ad libitum access to food and water. Twelve animals were assigned to each group based on genotype, aged to 2, 3, 4, or 8 weeks of age and sacrificed humanely. Cerebellar vermes were dissected and snap-frozen in liquid nitrogen. In preparation for immunohistochemical stainings, mice were perfused with 4% PFA, post-fixed for up to 24 h in PFA, cryopreserved in 20% and 30% sucrose solutions until saturated, and then frozen on dry ice. Researchers were blind to genotypes during collection of tissue and data collection.
Sectioning and staining were performed as described previously (13). The primary antibodies used were vesicular glutamate transporter 2 (vGlut2, rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), vesicular GABA transporter (vGAT, rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), glutamate decarboxylase 67 (GAD67, rabbit, 1:1000, Abcam, Cambridge, UK), and Calbindin (mouse, 1:500, Abcam, Cambridge, UK). The secondary anti-rabbit antibody was conjugated with Alexa Fluor 488, the anti-mouse antibody with Cy3 (both: donkey, 1:250, Jackson ImmunoResearch Laboratories, Suffolk, UK). Sections were imaged using an AxioObserver Z1 fluorescence microscope (Zeiss, Oberkochen, Germany), an AxioScan Z1 scanning microscope (Zeiss, Oberkochen, Germany), and a TCS SP8 confocal microscope (Leica, Wetzlar, Germany), and the images were analyzed using Fiji software (National Institutes of Health, http://fiji.sc/). vGAT and vGlut2 vesicles on PC soma were quantified using a custom-made pipeline. First, PC soma were identified by creating masks using Calbindin stained images. These masks were subsequently used in the vGAT or vGlut2 stained images to count puncta by determining local maxima in each PC soma. Per lobule, 4–7 images were collected and at least 5 PC soma were analysed per image. CF height was analysed by measuring both Calbindin and vGlut2 staining height from the tip of the PC soma, and calculating the ratio. Per lobule 4–7 images were collected and analysed. GAD67 stainings were performed without Calbindin co-staining. Here, fluorescence intensity was measured in the PC layer. Per lobule, 4–7 images were collected and analysed.
Reverse transcription PCR and quantitative real-time PCR were performed as described previously (13). A full list of primers can be found in the Additional File 1.
Proteins were isolated from snap-frozen mouse vermis. Organs were homogenized in ice-cold Ripa buffer supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland) and PMSF (Sigma-Aldrich, Saint Louis, MO, USA). Samples were centrifuged for 15 min at 10,000 rpm, and protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal amounts were loaded onto SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes (Life Technologies, Carlsbad, CA, USA) and blocked with 5% non-fat milk in Tris-buffered saline (TBS)-Tween. Blots were probed with primary antibodies against vGlut1 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), vGlut2 (rabbit, 1:1000, Synaptic Systems, Göttingen, Germany), and actin (mouse, 1:5000, MP Biomedicals, Irvine, CA, USA). Secondary antibodies were conjugated with horseradish peroxidase (goat, 1:10 000, Jackson ImmunoResearch Laboratories, Suffolk, UK). Densitometric analysis was performed using ImageLab software (BioRad Laboratories, Hercules, CA, USA).
All data were normalized against 3-week-old control data and expressed as means ± SEM. Two-way ANOVA was used to determine the significance of the observed differences between the genotypes, and over time (p < 0.05 was considered statistically significant).
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.
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.
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.
Our data are the first to demonstrate a neurodevelopmental role for PDYN, as we observed cerebellar developmental deficits in PDYNR212W mice that include loss of GABAergic connectivity, disrupted CF development, increased PF-PC connectivity, and dysregulation of key VGCC subunits that are involved in CF maturation between 2 and 8 weeks of age. Moreover, the loss of CF-PC synapses persisted up to 12 months of age, and likely contributes to PC degeneration (13). The alterations in the maturation and number of CF-PC synapses, the number of BC-PC synapses, and the expression of vGlut2 and vGlut1 support our hypothesis that developmental deficits in synaptic wiring contribute to motor dysfunction and ataxia. While one study found development of the brain was not affected in Pdyn knockdown mice, the cerebellum was not studied in detail (40). Our evidence leads us to propose that PDYN has different functions in the cerebrum versus the cerebellum, and that, in the cerebellum, it plays key roles in development.
BCs innervate the PC soma and form the pinceau on the PC initial axonal segment, inhibiting PC firing. A recent publication demonstrated that the loss of stellate and basket cell GABAergic transmission does not affect PC dendritic tree development and maintenance (41). However, Brown et al. also demonstrated that stellate and basket cells cooperate to establish the correct rate and pattern of simple and complex spike firing of PCs in vivo, and that loss of BC inhibition increased PC simple spike firing, while it decreased the PC complex firing rate (41). The loss of vGAT+ somatic synapses in PDYNR212W vermis suggests that SCA23 PCs suffer from reduced inhibition leading to changes in simple and complex spike firing rates as well as reduced synchronous firing of PC zones, altering cerebellar output and leading to motor function and ataxia (42–44). The observed loss of GAD67 likely contributes to this altered cerebellar output. Notably, reduced levels of GAD67 mRNA in PCs have been observed in the cerebella of people with autism (45). Additionally, BC-PC synapse formation is critical for early phase CF synapse elimination, as BC collaterals take over PC somatic spines from CFs (35). Therefore, the absence of BC-PC synapses may underlie the observed delay in early CF synapse elimination. Nakayama et al. have shown that diazepam, a GABAA receptor sensitizer, can restore impaired CF maturation due to altered GABAergic transmission (46). Hence, we put forward the malformation of GABAergic innervation as a possible therapeutic target for SCA23.
Loss of vGlut2 in the vermis of PDYNR212W mice supports a developmental deficit in the cerebellum, in line with findings showing developmental roles for vGlut2 in the hippocampus (31). A vGlut2 conditional knockout mouse displayed increased open-field exploratory behavior and impaired spatial learning and memory, a phenotype similar to that of NMDA-R knockdown mice (31, 47). Deficiency of vGlut2 led to reductions of evoked glutamate transmission, neurotransmitter release probability, and long-term depression at hippocampal CA3-CA1 synapses during postnatal development. This led to a loss of arborization of the dendritic tree and reductions in the number of dendritic spines in adult mice, suggesting widespread alterations in synaptic connectivity (31). We hypothesize that vGlut2 serves a similar purpose in the cerebellum, with deficiency leading to reduced glutamatergic transmission and, consequently, disturbed Ca2+ signaling and malformation of PC dendrites. This hypothesis is strengthened by our previous observation that cultured neurons of PDYNR212W cerebella show reduced neuronal excitability (13), which could be caused by the loss of vGlut2 expression.
PDYN R212W mice displayed elevated vGlut1 levels at 2, 3, and 4 weeks of age, which suggests that PFs in these animals extended their innervation territory on PCs to include more proximal dendrites. The timing of increased vGlut1 expression coincides with late phase CF synapse elimination, a process critically dependent on normal PF-PC synapse formation (35), suggesting that aberrant PF-PC synapse formation supports the malformation of CFs. A cause of aberrant PF-PC synapse formation could be the increased expression levels of Cacna1a, which encodes Cav2.1, at 3, 4, and 8 weeks of age. Increased expression of this subunit may affect heterosynaptic competition between PFs and CFs and distal extension of CFs (22). However, as Cacna1a is elevated from 3 weeks of age, the increase in vGlut1 at 2 weeks of age could be caused by the loss of vGlut2 at that time. Cacna1a dysregulation may also disrupt CF maturation, distal CF extension, and Ca2+ signaling, as Cav2.1 plays crucial roles in these processes (22, 48, 49). Moreover, the C-terminal tail of the channel functions as a transcription factor, coordinating the expression of genes involved in PC development (34). PC loss has not been observed until 12 months of age (13), therefore altered cerebellar development may lead to PC dysfunction before 12 months of age. Increased expression of Cacna1c, encoding Cav1.2, very likely contributes to the SCA23 pathology, as it plays significant roles in neuronal activity and survival, dendritic development, synaptic plasticity, memory formation, and learning (50–54). Additionally, an intronic variant in CACNA1C was recently proposed to be disease-causing in a Chinese family with autosomal dominant cerebellar ataxia (55), suggesting a role for Cacna1c in the pathology of ataxias. Furthermore, as a loss of vGlut2 could lead to reduced glutamatergic transmission, and consequently, disturbed Ca2+ signaling, the increases in expression of these VGCCs and their auxiliary subunits could be a compensatory mechanism for these changes. Altered expression of other Ca2+ channel subunits including Cacng2, Cacng7, Grin1, Grin3a, and Grin3b further hint towards dysregulation of Ca2+ signalling, however, due to the absence of consistent changes, we cannot draw strong conclusions from these data.
It has long been established that the loss of CF input causes PCs to malfunction, as CFs play a dual role in cerebellar functioning; 1) control of synaptic plasticity at dendritic PC synapses, and 2) generating the distinct complex spike output in the PC axon, and motor learning and performance crucially depend on them (Smeets and Verbeek, 2016). Additionally, CF deficits have been observed in other SCA types as well, including SCA1 (26, 28, 29, 56). The arrested development of CFs in PDYNR212W mice therefore likely underlies the ataxic phenotype (13). Interestingly, while the SCA23 pathology may mirror that of SCA1 in adulthood, the underlying developmental deficits oppose each other. In SCA1, Edamakanti et al. found hyperproliferation of stem cells that preferentially differentiated into GABAergic interneuons, leading to increased GABAergic interneuron connectivity and non-cell autonomous disruption of PC function (30). As discussed above, there could be several explanations as to why CFs develop abnormally in SCA23. We believe that the most likely culprit is the loss of GABAergic connectivity that should be provided to PCs by the BCs, as this is the earliest disruption in normal cerebellar development that we have detected. How expression of PDYN-R212W leads to dysfunction of BCs should be studied further.
In conclusion, the early loss of BC-PC and, consequently, CF-PC synapses plays a crucial role in the neuropathology of SCA23. We previously hypothesized that an increase in intracellular Ca2+ underlies the SCA23 pathology (13); with the evidence presented here, it now appears more likely that the disturbance in Ca2+ signalling lies on the other end of the scale, with decreased intracellular Ca2+ disrupting normal cellular functioning. This fits the SCA23 pathology more closely, as increased intracellular Ca2+ would lead to PC loss more quickly than previously observed (13). We demonstrate a developmental role for PDYN in the cerebellum and show that developmental abnormalities in neuronal wiring and disturbance in the PC simple/complex spike balance underlie SCA23. In addition, we propose that diazepam should be explored as a potential therapy, as it could sensitize remaining GABAA receptors and thereby alleviate SCA23 symptoms.
AMPA = α-amino-hydroxy-5-methylisoxazole-4-propionate, BC = basket cell, CF = climbing fibre, Dyn = Dynorphin, GAD67 = glutamate decarboxylase 67, NMDA = N-methyl-D-aspartate, PC = Purkinje cell, PDYN = prodynorphin, PF = parallel fibre, SCA = spinocerebellar ataxia, vGAT = vesicular GABA transporter, VGCC = voltage-gated calcium channel, vGlut = vesicular glutamate transporter
Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.
Competing interests
The authors declare that they have no competing interests.
Data availability
The data from this study will be made available upon request.
Funding
This work was supported by a Rosalind Franklin Fellowship from the University of Groningen, the Jan Kornelis de Cock-Stichting, the U4 PhD program of the Behavioral and Cognitive Neuroscience graduate school of the University of Groningen, and the Max Planck Society. Part of this work was performed at the University Medical Centre Groningen Microscopy and Imaging Centre, which is sponsored by the Netherlands Organization for Scientific Research (NWO grants 40-00506-98-9021 and 175-010-2009-023). None of the funding bodies were involved in the collection, analysis, and interpretation of data, nor in the writing of the manuscript.
Authors’ contributions
CJLMS and DSV designed the project. CJLMS collected samples and performed immunohistochemistry, Western blotting, and qPCR experiments and analysis, and wrote the manuscript. KYM performed Western blotting experiments and analysis and immunohistochemistry analysis. CJLMS, KYM, SEF, and DSV interpreted data and revised the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We would like to thank Harm H Kampinga for critically reading this manuscript, and Jackie Senior and Kate Mc Intyre for editing this manuscript.
Additional files
The additional files includes one figure and one table that can be found online.