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. 2018 Apr 15;27(8):1396-1410.
doi: 10.1093/hmg/ddy050.

Protein Kinase C Activity Is a Protective Modifier of Purkinje Neuron Degeneration in Cerebellar Ataxia

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Free PMC article

Protein Kinase C Activity Is a Protective Modifier of Purkinje Neuron Degeneration in Cerebellar Ataxia

Ravi Chopra et al. Hum Mol Genet. .
Free PMC article

Abstract

Among the many types of neurons expressing protein kinase C (PKC) enzymes, cerebellar Purkinje neurons are particularly reliant on appropriate PKC activity for maintaining homeostasis. The importance of PKC enzymes in Purkinje neuron health is apparent as mutations in PRKCG (encoding PKCγ) cause cerebellar ataxia. PRKCG has also been identified as an important node in ataxia gene networks more broadly, but the functional role of PKC in other forms of ataxia remains unexplored, and the mechanisms by which PKC isozymes regulate Purkinje neuron health are not well understood. Here, we investigated how PKC activity influences neurodegeneration in inherited ataxia. Using mouse models of spinocerebellar ataxia type 1 (SCA1) and 2 (SCA2) we identify an increase in PKC-mediated substrate phosphorylation in two different forms of inherited cerebellar ataxia. Normalizing PKC substrate phosphorylation in SCA1 and SCA2 mice accelerates degeneration, suggesting that the increased activity observed in these models is neuroprotective. We also find that increased phosphorylation of PKC targets limits Purkinje neuron membrane excitability, suggesting that PKC activity may support Purkinje neuron health by moderating excitability. These data suggest a functional role for PKC enzymes in ataxia gene networks, and demonstrate that increased PKC activity is a protective modifier of degeneration in inherited cerebellar ataxia.

Figures

Figure 1.
Figure 1.
Increased cerebellar PKC substrate phosphorylation is observed in SCA1. (A) Representative western blot for phosphorylated PKC substrates at 5 weeks of age showing increased PKC substrate phosphorylation in cerebella from ATXN1[82Q]tg/tg mice, summarized in (B). Tubulin was used as a loading control. (C) Representative western blot for phosphorylated PKC substrates at 15 weeks of age show increased PKC substrate phosphorylation in cerebella from ATXN1[82Q]tg/tg mice, summarized in (D). Tubulin was used as a loading control. (E) Representative immunohistochemistry experiment showing PKC substrate phosphorylation (green) in Purkinje neurons (Calbindin: red) from the anterior cerebellar vermis in 15-week-old wild-type and ATXN1[82Q]tg/tg mice. Scale bars, 50 µm. (F) Western blot demonstrating increased phosphorylation of Ser880 of GluA2 in ATXN1[82Q]tg/tg mice at 15 weeks of age, summarized in (G). Total GluA2 was used as a loading control. (H) Representative images of phosphorylated PKC substrate staining (green) in calbindin-stained (red) Purkinje neurons from an SCA1 patient sample and healthy control sample. Scale bars, 50 µm. (I) Summarized data show an increase in phosphorylated PKC substrate-to-calbindin staining ratio in the somatic compartment of Purkinje neurons from SCA1 patient samples and age-matched healthy control samples. Each data point represents an individual patient, with error bars representing SEM for measurements from multiple Purkinje neurons in that individual. (J) Phosphorylated PKC substrate staining intensity in the somatic compartment shows a significant increase in SCA1 patient samples relative to age-matched healthy control samples. Each data point represents an individual patient, with error bars representing S.E.M. for measurements from multiple Purkinje neurons in that individual. Throughout, data are represented as means with error bars representing S.E.M. * P<0.05, ** <0.01; *** <0.001. Statistical significance derived by unpaired two-tailed Student’s t-test (B, D, G) or two-way ANOVA (I, J).
Figure 2.
Figure 2.
Increased PKC substrate phosphorylation is mediated by conventional PKC isozymes in SCA1. (A) Representative western blot for phosphorylated PKC substrates in organotypic cerebellar slice cultures treated with 5 mm EGTA from 15-week-old mice. Calcium chelation with EGTA reduced PKC substrate phosphorylation in ATXN1[82Q]tg/tg slice cultures but not wild-type slice cultures, summarized in (B). Tubulin was used as a loading control. (C) Representative western blot for PKCα and PKCγ in 15-week-old mice. Unchanged expression of PKCα in ATXN1[82Q]tg/tg cerebella at 15 weeks of age is summarized in (D), while reduced expression of PKCγ in ATXN1[82Q]tg/tg cerebella at 15 weeks of age is summarized in (E). Tubulin was used as a loading control. (F) Dagla transcripts are reduced at 5 and 15 weeks in ATXN1[82Q]tg/tg cerebella as assessed using quantitative RT-PCR. Number of animals from each group indicated within bars. Throughout, data are represented as means with error bars representing S.E.M. NS = not significant; * <0.05; ** <0.01; *** <0.001. Statistical significance derived by two-way ANOVA (B) or unpaired two-tailed Student’s t-test (D, E, F).
Figure 3.
Figure 3.
Increased PKC substrate phosphorylation is a protective modifier of neurodegeneration in SCA1 mice. (A) Representative western blot for PKC substrate phosphorylation in wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- mice at 20 weeks of age demonstrating reduction of PKC substrate phosphorylation in ATXN1[82Q]tg/-; PKCitg/- mice. Tubulin was used as a loading control. (B) Reduced molecular layer thickness in lobule V of ATXN1[82Q]tg/-; PKCitg/- mice at 20 weeks of age. (C) Whole-cell capacitance measurements in ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons from the anterior cerebellar vermis show a greater reduction in cell size relative to ATXN1[82Q]tg/- Purkinje neurons, which are atrophied relative to wild-type and PKCitg/- Purkinje neurons at 20 weeks of age. (D) Representative images at the cerebellar primary fissure in wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- mice stained with calbindin at 20 weeks of age. Scale bar, 100 µm. Throughout, data are represented as means with error bars representing S.E.M. * P<0.05. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test (B, C).
Figure 4.
Figure 4.
Increased PKC substrate phosphorylation is a shared protective pathway in SCA2 mice. (A) Representative western blot for PKC substrate phosphorylation in wild-type and ATXN2[127Q]tg/- cerebella at 12 weeks of age demonstrating increased PKC substrate phosphorylation, summarized in (B). Tubulin was used as a loading control. (C) Expression of Dagla is reduced at 12 weeks in ATXN2[127Q]tg/- cerebella as assessed using quantitative RT-PCR. (D) Representative western blot for PKC substrate phosphorylation in wild-type, PKCitg/-, ATXN2[127Q]tg/- and ATXN2[127Q]tg/-; PKCitg/- mice at 25 weeks of age demonstrating reduction of PKC substrate phosphorylation in ATXN2[127Q]tg/-; PKCitg/- mice. Tubulin was used as a loading control. (E) Representative images at the cerebellar primary fissure in sections from wild-type, PKCitg/-, ATXN2[127Q]tg/- and ATXN2[127Q]tg/-; PKCitg/- mice stained with calbindin at 25 weeks of age. Scale bar, 100 µm. (F) Reduced molecular layer thickness in lobule VI of ATXN2[127Q]tg/-; PKCitg/- mice at 25 weeks of age. Throughout, data are represented as means with error bars representing S.E.M. NS = not significant; * <0.05; ** <0.01. Statistical significance derived by unpaired two-tailed Student’s t-test (B, C) or one-way ANOVA with Holm-Sidak multiple comparison test (F).
Figure 5.
Figure 5.
The increase in PKC activity in SCA1 mice is not associated with alterations in Purkinje neuron spiking. (A) Representative firing from wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons. ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- mice show irregular spiking, with abnormal pauses marked. (B) ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons show reduced firing frequency. (C) ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons show more irregular firing, indicated by a higher CV. (D) Representative spontaneous action potentials from wild-type, PKCitg/-, ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons. ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons have an AHP which does not achieve the same degree of hyperpolarization as wild-type and PKCi Purkinje neurons, summarized in (E). Throughout, data are represented as means with error bars representing S.E.M. NS = not significant; * <0.05. Statistical significance derived by one-way ANOVA with Holm-Sidak multiple comparison test (B, C, E).
Figure 6.
Figure 6.
Increased PKC activity reduces membrane excitability in SCA1 and SCA2 Purkinje neurons. (A) Representative traces from ATXN1[82Q]tg/- and ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons injected with +700 pA of current. ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons undergo depolarization block of repetitive spiking at lower levels of injected current than ATXN1[82Q]tg/- Purkinje neurons, summarized in (B). (C) Representative traces where dendritic calcium spikes were evoked with somatic current injection (injected current amount indicated on the trace) from ATXN1[82Q]tg/- (left) and ATXN1[82Q]tg/-; PKCitg/- (right) Purkinje neurons treated with 1 µm TTX. ATXN1[82Q]tg/-; PKCitg/- Purkinje neurons require less injected current to elicit a dendritic calcium spike than ATXN1[82Q]tg/- Purkinje neurons, summarized in (D). (E) ATXN1[82Q]tg/- Purkinje neurons require less injected current to elicit a dendritic calcium spike in the presence of 1 µM TTX when PKC activity is inhibited with 1 µM Go6983. All slices were pre-incubated with dimethyl sulfoxide (DMSO) or Go6983 for 40 min before recording. (F) ATXN2[127Q]tg/-; PKCitg/- Purkinje neurons require less injected current to elicit a dendritic calcium spike in the presence of 1 µM TTX than ATXN2[127Q]tg/- Purkinje neurons. All data from experiments using ATXN1[82Q]tg/-; PKCitg/- were performed at 20 weeks of age, when dendritic degeneration is detected in these mice. All data from experiments using ATXN2[127Q]tg/-; PKCitg/- were performed at 12 weeks of age, when dendritic degeneration is first detected in ATXN2[127Q]tg/- mice (38). Throughout, data are represented as means with error bars representing S.E.M. * <0.05. Statistical significance derived by unpaired two-tailed Student’s t-test (B, D, E, F).

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