Protein kinase N1 critically regulates cerebellar development and long-term function
Stephanie zur Nedden, … , Gottfried Baier, Gabriele Baier-Bitterlich
Stephanie zur Nedden, … , Gottfried Baier, Gabriele Baier-Bitterlich
Published March 1, 2018
Citation Information: J Clin Invest. 2018;128(5):2076-2088. https://doi.org/10.1172/JCI96165.
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Protein kinase N1 critically regulates cerebellar development and long-term function

Abstract

Increasing evidence suggests that synapse dysfunctions are a major determinant of several neurodevelopmental and neurodegenerative diseases. Here we identify protein kinase N1 (PKN1) as a novel key player in fine-tuning the balance between axonal outgrowth and presynaptic differentiation in the parallel fiber–forming (PF-forming) cerebellar granule cells (Cgcs). Postnatal Pkn1–/– animals showed a defective PF–Purkinje cell (PF-PC) synapse formation. In vitro, Pkn1–/– Cgcs exhibited deregulated axonal outgrowth, elevated AKT phosphorylation, and higher levels of neuronal differentiation-2 (NeuroD2), a transcription factor preventing presynaptic maturation. Concomitantly, Pkn1–/– Cgcs had a reduced density of presynaptic sites. By inhibiting AKT with MK-2206 and siRNA-mediated knockdown, we found that AKT hyperactivation is responsible for the elongated axons, higher NeuroD2 levels, and reduced density of presynaptic specifications in Pkn1–/– Cgcs. In line with our in vitro data, Pkn1–/– mice showed AKT hyperactivation, elevated NeuroD2 levels, and reduced expression of PF-PC synaptic markers during stages of PF maturation in vivo. The long-term effect of Pkn1 knockout was further seen in cerebellar atrophy and mild ataxia. In summary, our results demonstrate that PKN1 functions as a developmentally active gatekeeper of AKT activity, thereby fine-tuning axonal outgrowth and presynaptic differentiation of Cgcs and subsequently the correct PF-PC synapse formation.

Authors

Stephanie zur Nedden, Rafaela Eith, Christoph Schwarzer, Lucia Zanetti, Hartwig Seitter, Friedrich Fresser, Alexandra Koschak, Angus J.M. Cameron, Peter J. Parker, Gottfried Baier, Gabriele Baier-Bitterlich

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Figure 6

Adult Pkn1–/– mice show cerebellar shrinkage and late-onset PC degeneration.

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Adult Pkn1–/– mice show cerebellar shrinkage and late-onset PC degenerat...
(A) Size differences of adult WT and Pkn1–/– cerebella (3–9 months), as seen in Hoechst-stained sagittal vermis sections and calbindin-stained ML pictures (representative images of 8–11 separate experiments). (B–D) Analysis of the cerebellar vermis area (B) [2-tailed unpaired t test, t(15) = 2.510, *P = 0.0236, n = 9 WT, 8 Pkn1–/– animals from 4–6 litters per group], the IGL thickness (C) [2-tailed unpaired t test with Welch’s correction, t(12) = 2.772, *P = 0.0169, n = 11 WT, 8 Pkn1–/– animals from 4–5 litters per group], and the ML thickness (D) [2-tailed unpaired t test, t(17) = 3.210, **P = 0.0051, n = 11 WT, 8 Pkn1–/– animals from 4–5 litters per group] in 3- to 9-month-old animals. (E) PC number in 3- to 9-month-old and 15- to 22-month-old animals [1-way ANOVA with Newman-Keuls multiple-comparisons test, F(3,38) = 23.12, P < 0.0001, post-test **P < 0.01, ***P < 0.001; n = 5–15 WT, 7–15 Pkn1–/– animals]. (F) Cerebellar protein extracts from 3- to 9-month-old animals were analyzed for the VGlut2/VGlut1 ratio [2-tailed unpaired t test, t(7) = 4.138, **P = 0.0044, n = 4 WT, 5 Pkn1–/– animals from 4 litters per group]. (G) Calbindin- and VGlut2-stained cerebellar sections of 3- to 9-month-old animals (representative images of 11–12 experiments; see Supplemental Figure 6C for analysis). All data are presented as individual n values with mean ± SEM. Scale bars: A, Hoechst, 500 μm; calbindin, 50 μm; E and G, 100 μm. All analyses were performed by experimenters blinded to the genotype, except F.