Cerebellar developmental deficits underlie neurodegenerative disorder spinocerebellar ataxia type 23

Abstract

Background

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. PDYN^R212W 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 PDYN^R212W 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.

Methods

In the present study, we examined the cerebella of PDYN^R212W mice during cerebellar development, from 2 to 8 weeks of age, using immunohistochemistry, protein, and RNA analysis.

Results

We uncovered developmental deficits from 2 weeks of age, namely a reduced number of GABAergic synapses on PC soma in PDYN^R212W 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 PDYN^R212W cerebella.

Conclusions

These findings indicate that developmental abnormalities contribute to the SCA23 pathology and uncover a developmental role for PDYN in the cerebellum.

Background

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 PDYN^R212W 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 PDYN^R212W 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 PDYN^R212W cerebellum, and propose an alternative disease model for SCA23.

Materials And Methods

Results

PDYN ^R212W cerebella show alterations in GABAergic connectivity

Mice expressing human PDYN containing the p.R212W SCA23 mutation (PDYN^R212W 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 (PDYN^WT 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 PDYN^R212W 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). PDYN^R212W 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 PDYN^WT and control mice, as well as in lobule I as compared to PDYN^WT mice (Fig. 1B). At 3 weeks of age, PDYN^R212W lobule IV/V showed fewer synapses as compared to both PDYN^WT and control, as well as PDYN^R212W 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 PDYN^R212W mice as compared to PDYN^WT and control mice, as well as lobule I as compared to PDYN^WT mice at 4 weeks of age (Fig. 1C). At 8 weeks of age, PDYN^R212W mice showed fewer somatic synapses in lobules IV/V and VI as compared to both PDYN^WT and control mice, in lobule II as compared to control mice, and in lobules I and IX as compared to PDYN^WT mice (Fig. 1C). Taken together, these data indicate that SCA23 shows an opposite pathology to SCA1, as inhibitory synapses are lost on PDYN^R212W PC soma.

CF development is disrupted in PDYN^R212W 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 PDYN^WT 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). PDYN^R212W 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 PDYN^R212W 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 PDYN^R212W 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 PDYN^R212W 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 PDYN^WT 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 PDYN^R212W 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 PDYN^R212W mice to that of PDYN^WT 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 PDYN^R212W 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 PDYN^R212W vermis were significantly increased at 2, 3, and 4 weeks of age compared to control vermis, and at 8 weeks of age compared to PDYN^WT vermis (Fig. 4B). The elevated levels of vGlut1 suggest that PFs have increased their synapse numbers in the vermis of PDYN^R212W mice, as CFs cannot reach terminal height to populate their natural PC dendritic territory.

Reduced GAD67 expression could indicate internal changes in PDYN^R212W PCs

As both GABAergic and glutamatergic inputs are altered in PDYN^R212W 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, PDYN^WT, and PDYN^R212W mice (Fig. 5A). We observed a loss of GAD67 in PDYN^R212W PCs at 2, 3, and 4 weeks of age as compared to control mice, and at 4 weeks as compared to PDYN^WT mice (Fig. 5B). These data suggest that PDYN^R212W 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.

Conclusions

Our data are the first to demonstrate a neurodevelopmental role for PDYN, as we observed cerebellar developmental deficits in PDYN^R212W 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 PDYN^R212W 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 GABA_A 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 PDYN^R212W 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 PDYN^R212W 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 PDYN^R212W 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 GABA_A receptors and thereby alleviate SCA23 symptoms.

Abbreviations

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

Declarations

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.

References

1. Verbeek DS, van de Warrenburg BP, Wesseling P, Pearson PL, Kremer HP, Sinke RJ. Mapping of the SCA23 locus involved in autosomal dominant cerebellar ataxia to chromosome region 20p13-12.3. Brain: a journal of neurology. 2004 Nov;127(Pt 11):2551–7.
2. Bakalkin G, Watanabe H, Jezierska J, Depoorter C, Verschuuren-Bemelmans C, Bazov I, et al. Prodynorphin mutations cause the neurodegenerative disorder spinocerebellar ataxia type 23. American journal of human genetics. 2010 Nov 12;87(5):593–603.
3. Hauser KF, Aldrich JV, Anderson KJ, Bakalkin G, Christie MJ, Hall ED, et al. Pathobiology of dynorphins in trauma and disease. Frontiers in bioscience: a journal and virtual library. Jan. 2005;1:10:216–35.
4. Altier C, Zamponi GW. Opioid, cheating on its receptors, exacerbates pain. Nature neuroscience. 2006 Dec;9(12):1465–7.
5. Schwarzer C. 30 Years of Dynorphins–New Insights on Their Functions in Neuropsychiatric Diseases. Pharmacology & therapeutics. 2009 Sep;123(3):353–70.
6. Chavkin C. Dynorphin–still an extraordinarily potent opioid peptide. Mol Pharmacol. 2013 Apr;83(4):729–36.
7. Herman BH, Leslie F, Goldstein A. Behavioral effects and in vivo degradation of intraventricularly administered dynorphin-(1–13) and D-Ala2-dynorphin-(1–11) in rats. Life Sci. 1980 Sep;27(10):883–92.
8. Walker JM, Moises HC, Coy DH, Baldrighi G, Akil H. Nonopiate effects of dynorphin and des-Tyr-dynorphin. Science (New York). 1982 Dec 10;218(4577):pp. 1136–8.
9. Tan-No K, Cebers G, Yakovleva T, Hoon Goh B, Gileva I, Reznikov K, et al. Cytotoxic effects of dynorphins through nonopioid intracellular mechanisms. Experimental cell research. 2001 Sep 10;269(1):54–63.
10. Singh IN, Goody RJ, Goebel SM, Martin KM, Knapp PE, Marinova Z, et al. Dynorphin A (1–17) induces apoptosis in striatal neurons in vitro through α-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome C release and caspase-3 activation. Neuroscience. 2003 Jan;122(4):1013–23.
11. Sherwood TW, Askwith CC. Dynorphin opioid peptides enhance acid-sensing ion channel 1a activity and acidosis-induced neuronal death. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2009 Nov;11(45):14371–80. 29(.
12. Smeets CJLM, Zmorzyńska J, Melo MN, Stargardt A, Dooley C, Bakalkin G, et al. Altered secondary structure of Dynorphin A associates with loss of opioid signalling and NMDA-mediated excitotoxicity in SCA23. Human Molecular Genetics. 2016 Jun 3;6(13):ddw130.
13. Smeets CJLM, Jezierska J, Watanabe H, Duarri A, Fokkens MR, Meijer M, et al. Elevated mutant dynorphin A causes Purkinje cell loss and motor dysfunction in spinocerebellar ataxia type 23. Brain. 2015 Sep;138(9):2537–52.
14. Ito M. Cerebellar control of the vestibulo-ocular reflex–around the flocculus hypothesis. Annu Rev Neurosci. 1982 Jan;5:275–96.
15. Hansel C, Linden DJ, D’Angelo E. Beyond parallel fiber LTD: the diversity of synaptic and non-synaptic plasticity in the cerebellum. Nature neuroscience. 2001;4(5):467–75.
16. Hirano T. Long-term depression and other synaptic plasticity in the cerebellum. Proceedings of the Japan Academy, Series B. 2013;89(5):183–95.
17. Wang W, Nakadate K, Masugi-Tokita M, Shutoh F, Aziz W, Tarusawa E, et al. Distinct cerebellar engrams in short-term and long-term motor learning. Proceedings of the National Academy of Sciences of the United States of America. 2013;1–6.
18. 10.1007/978-3-662-13147-3
    Eccles JC, Ito M, Szentágothai J. The Cerebellum as a Neuronal Machine [Internet]. Berlin, Heidelberg: Springer Berlin Heidelberg; 1967 [cited 2015 Jun 11]. Available from: http://link.springer.com/10.1007/978-3-662-13147-3.
19. Ito M. Cerebellar circuitry as a neuronal machine. Progress in neurobiology. 2006 Jan;78(3–5):272–303.
20. Wang SS, Denk W, Häusser M. Coincidence detection in single dendritic spines mediated by calcium release. Nature neuroscience. 2000 Dec;3(12):1266–73.
21. Linden DJ. Neuroscience. From molecules to memory in the cerebellum. Science (New York. 2003 Sep 19;301(5640):pp. 1682–5.
22. Miyazaki T, Hashimoto K, Shin H-S, Kano M, Watanabe M. P/Q-type Ca2 + channel alpha1A regulates synaptic competition on developing cerebellar Purkinje cells. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2004 Mar;18(7):1734–43. 24(.
23. Miyazaki T, Yamasaki M, Takeuchi T, Sakimura K, Mishina M, Watanabe M. Ablation of glutamate receptor GluRδ2 in adult Purkinje cells causes multiple innervation of climbing fibers by inducing aberrant invasion to parallel fiber innervation territory. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2010 Nov 10;30(45):15196–209.
24. Leitch B, Shevtsova O, Guévremont D, Williams J. Loss of calcium channels in the cerebellum of the ataxic and epileptic stargazer mutant mouse. Brain research. 2009 Jul 7;1279:156–67.
25. Oda S-I, Lee KJ, Arii T, Imoto K, Hyun B-H, Park IS, et al. Differential regulation of Purkinje cell dendritic spines in rolling mouse Nagoya (tg/tg), P/Q type calcium channel (α1(A)/Ca(v)2.1) mutant. Anatomy & cell biology. 2010 Sep;43(3):211–7.
26. Duvick L, Barnes J, Ebner B, Agrawal S, Andresen M, Lim J, et al. SCA1-like disease in mice expressing wild-type ataxin-1 with a serine to aspartic acid replacement at residue 776. Neuron. 2010 Sep 23;67(6):929–35.
27. Perkins EM, Clarkson YL, Sabatier N, Longhurst DM, Millward CP, Jack J, et al. Loss of beta-III spectrin leads to Purkinje cell dysfunction recapitulating the behavior and neuropathology of spinocerebellar ataxia type 5 in humans. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2010 Apr 7;30(14):4857–67.
28. Ebner B, Ingram M, Barnes J, Duvick L, Frisch JL, Clark HB, et al. Purkinje cell ataxin-1 modulates climbing fiber synaptic input in developing and adult mouse cerebellum. Journal of Neuroscience. 2013 Mar 27;33(13):5806–20.
29. Furrer SA, Waldherr SM, Mohanachandran MS, Baughn TD, Nguyen K-T, Sopher BL, et al. Reduction of mutant ataxin-7 expression restores motor function and prevents cerebellar synaptic reorganization in a conditional mouse model of SCA7. Human molecular genetics. 2013 Mar 1;22(5):890–903.
30. Edamakanti CR, Do J, Didonna A, Martina M, Opal P. Mutant ataxin1 disrupts cerebellar development in spinocerebellar ataxia type 1. J Clin Invest. 2018 Jun 1;128(6):2252–65.
31. He H, Mahnke AH, Doyle S, Fan N, Wang C-C, Hall BJ, et al. Neurodevelopmental role for VGLUT2 in pyramidal neuron plasticity, dendritic refinement, and in spatial learning. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2012 Nov 7;32(45):15886–901.
32. Zhuchenko O, Bailey J, Bonnen P, Ashizawa T, Stockton DW, Amos C, et al. Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet. 1997 Jan;15(1):62–9.
33. Nakayama H, Miyazaki T, Kitamura K, Hashimoto K, Yanagawa Y, Obata K, et al. GABAergic Inhibition Regulates Developmental Synapse Elimination in the Cerebellum. Neuron. 2012 Apr 26;74(2):384–96.
34. Du X, Wang J, Zhu H, Rinaldo L, Lamar K-M, Palmenberg AC, et al. Second cistron in CACNA1A gene encodes a transcription factor mediating cerebellar development and SCA6. Cell. 2013 Jul;3(1):118–33. 154(.
35. Kano M, Watanabe T, Uesaka N, Watanabe M. Multiple Phases of Climbing Fiber Synapse Elimination in the Developing Cerebellum. Cerebellum. 2018 Jul 16;1–13.
36. Fletcher CF, Lutz CM, O’Sullivan TN, Shaughnessy JD, Hawkes R, Frankel WN, et al. Absence Epilepsy in Tottering Mutant Mice Is Associated with Calcium Channel Defects. Cell. 1996 Nov 15;87(4):607–17.
37. Burgess DL, Jones JM, Meisler MH, Noebels JL. Mutation of the Ca2 + Channel β Subunit Gene Cchb4 Is Associated with Ataxia and Seizures in the Lethargic (lh) Mouse. Cell. 1997 Feb 7;88(3):385–92.
38. Letts VA, Felix R, Biddlecome GH, Arikkath J, Mahaffey CL, Valenzuela A, et al. The mouse stargazer gene encodes a neuronal Ca2+-channel gamma subunit. Nat Genet. 1998 Aug;19(4):340–7.
39. Barclay J, Balaguero N, Mione M, Ackerman SL, Letts VA, Brodbeck J, et al. Ducky Mouse Phenotype of Epilepsy and Ataxia Is Associated with Mutations in the Cacna2d2 Gene and Decreased Calcium Channel Current in Cerebellar Purkinje Cells. J Neurosci. 2001 Aug;15(16):6095–104. 21(.
40. Ménard C, Tse YC, Cavanagh C, Chabot J-G, Herzog H, Schwarzer C, et al. Knockdown of prodynorphin gene prevents cognitive decline, reduces anxiety, and rescues loss of group 1 metabotropic glutamate receptor function in aging. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2013 Jul;31(31):12792–804. 33(.
41. Brown AM, Arancillo M, Lin T, Catt DR, Zhou J, Lackey EP, et al. Molecular layer interneurons shape the spike activity of cerebellar Purkinje cells. Sci Rep [Internet]. 2019 Feb 11 [cited 2020 Mar 17];9. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370775/.
42. Heiney SA, Kim J, Augustine GJ, Medina JF. Precise Control of Movement Kinematics by Optogenetic Inhibition of Purkinje Cell Activity. J Neurosci. 2014 Feb;5(6):2321–30. 34(.
43. Lee KH, Mathews PJ, Reeves AMB, Choe KY, Jami SA, Serrano RE, et al. Circuit Mechanisms Underlying Motor Memory Formation in the Cerebellum. Neuron. 2015 Apr 22;86(2):529–40.
44. Ramirez JE, Stell BM. Calcium Imaging Reveals Coordinated Simple Spike Pauses in Populations of Cerebellar Purkinje Cells. Cell Reports. 2016 Dec;17(12):3125–32.
45. Yip J, Soghomonian J-J, Blatt GJ. Decreased GAD67 mRNA levels in cerebellar Purkinje cells in autism: pathophysiological implications. Acta Neuropathol. 2007 May;113(5):559–68.
46. Nakayama H, Abe M, Morimoto C, Iida T, Okabe S, Sakimura K, et al. Microglia permit climbing fiber elimination by promoting GABAergic inhibition in the developing cerebellum. Nature Communications [Internet]. 2018 Dec [cited 2018 Jul 24];9(1). Available from: http://www.nature.com/articles/s41467-018-05100-z.
47. Sakimura K, Kutsuwada T, Ito I, Manabe T, Takayama C, Kushiya E, et al. Reduced hippocampal LTP and spatial learning in mice lacking NMDA receptor epsilon 1 subunit. Nature. 1995 Jan 12;373(6510):151–5.
48. Catterall WA. Structure and regulation of voltage-gated Ca2 + channels. Annual review of cell and developmental biology. 2000 Jan 28;16:521–55.
49. Watanabe M, Kano M. Climbing fiber synapse elimination in cerebellar Purkinje cells. Eur J Neurosci. 2011 Nov;34(10):1697–710.
50. Moosmang S, Lenhardt P, Haider N, Hofmann F, Wegener JW. Mouse models to study L-type calcium channel function. Pharmacology therapeutics. 2005 Jun;106(3):347–55.
51. Moosmang S, Haider N, Klugbauer N, Adelsberger H, Langwieser N, Müller J, et al. Role of hippocampal Cav1.2 Ca2 + channels in NMDA receptor-independent synaptic plasticity and spatial memory. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2005 Oct;26(43):9883–92. 25(.
52. Striessnig J, Koschak A, Sinnegger-Brauns MJ, Hetzenauer A, Nguyen NK, Busquet P, et al. Role of voltage-gated L-type Ca2 + channel isoforms for brain function. Biochemical Society transactions. 2006 Nov 1;34(Pt 5):903–9.
53. White JA, McKinney BC, John MC, Powers PA, Kamp TJ, Murphy GG. Conditional forebrain deletion of the L-type calcium channel Ca V 1.2 disrupts remote spatial memories in mice. Learning & memory (Cold Spring Harbor, NY). 2008 Jan 3;15(1):1–5.
54. Bhat S, Dao DT, Terrillion CE, Arad M, Smith RJ, Soldatov NM, et al. CACNA1C (Cav1.2) in the pathophysiology of psychiatric disease. Progress in Neurobiology. 2012 Oct;99(1):1–14.
55. Chen J, Sun Y, Liu X, Li J. Identification of a novel mutation in the CACNA1C gene in a Chinese family with autosomal dominant cerebellar ataxia. BMC Neurology. 2019 Jul;10(1):157. 19(.
56. Shuvaev AN, Horiuchi H, Seki T, Goenawan H, Irie T, Iizuka A, et al. Mutant PKCγ in spinocerebellar ataxia type 14 disrupts synapse elimination and long-term depression in Purkinje cells in vivo. The Journal of neuroscience: the official journal of the Society for Neuroscience. 2011 Oct 5;31(40):14324–34. Altier C, Zamponi GW. Opioid, cheating on its receptors, exacerbates pain. Nature neuroscience 2006; 9: 1465–7.

Supplementary File Caption