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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Research Article Neuroscience

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 1

Pkn1–/– mice show a defective PF-PC synapse formation during development.

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Pkn1–/– mice show a defective PF-PC synapse formation during developmen...
(A) Cerebellar vermis sections of P8–P15 animals (n = 4–12). Arrows mark distal and perisomatic varicosities of VGlut2-stained CFs. Scale bar: 50 μm. (B) The ratio of the VGlut2-stained CF innervation depth (μm) to the ML thickness (μm) was analyzed [1-way ANOVA with Newman-Keuls multiple-comparisons test, F(3,27) = 16.7, P < 0.0001, post-test *P < 0.05, **P < 0.01, ***P < 0.001; n = 4 WT, 4 Pkn1–/– animals for P8, n = 12 WT, 11 Pkn1–/– animals for P15 from 5–8 litters per group]. (C) The score of PC perisomatic VGlut2 staining in P15 animals [χ2 test = 4.286, P = 0.0384, n = 5 WT, 5 Pkn1–/– animals from 5 litters per group]. (D) CF-induced ePSCs were recorded from PCs in acute slices. With increasing stimulation strength, ePSCs were obtained in an all-or-none fashion (single CF) or in 2 or more discrete steps (multiple CFs) [χ2 test = 9.68, P = 0.0019, n = 23 WT, 23 Pkn1–/– cells from 7 P15–P17 animals per group]. (E) Spontaneous PC ePSC frequencies [2-tailed unpaired t test with Welch’s correction, t(5) = 2.865, *P = 0.0352, n = 6 WT, 4 Pkn1–/– cells from 3–5 P13–P15 animals per group]. (F) Western blot analysis of Cbln1 and GluD2 levels (n = 3–4). (G and H) Analysis of the Cbln1/tubulin ratio (G) [2-tailed unpaired t test, t(5) = 3.365, *P = 0.0200, n = 3 WT, 4 Pkn1–/– extracts from 3–4 animals per group] and GluD2/tubulin ratio (H) [2-tailed unpaired t test, t(5) = 1.016, P = 0.3561, n = 3 WT, 4 Pkn1–/– extracts from 3–4 animals per group] in P15 animals. Data are presented as individual n values with mean ± SEM. All analyses/experiments except F–H were performed in a blinded manner.