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.
View: Text | PDF
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

×

Figure 4

siRNA-mediated Akt knockdown reduces axonal length and NeuroD2 protein levels and increases the density of presynaptic sites in Pkn1–/– Cgcs.

Options: View larger image (or click on image) Download as PowerPoint
siRNA-mediated Akt knockdown reduces axonal length and NeuroD2 protein l...
(A) siRNAs targeting Akt123 reduce pan-AKT expression after DIV1. Pictures are representative of 5 separate experiments. For analysis at DIV1 and DIV4 in WT and Pkn1–/– Cgcs, see Supplemental Figure 4, A and B. (B) siRNAs targeting Akt123 significantly reduce axonal length of Pkn1–/– Cgcs at DIV1 [1-way ANOVA with Newman-Keuls multiple-comparisons test, F(3,16) = 20.78, P < 0.0001, post-test ***P < 0.001, n = 5 WT, 5 Pkn1–/– Cgc preparations from 3–5 litters per group]. Axons were stained with TAU. Representative WIS-NeuroMath–analyzed output images are shown. (C) siRNAs targeting Akt123 significantly reduce NeuroD2 intensity in WT and Pkn1–/– Cgcs at DIV4 [1-way ANOVA with Newman-Keuls multiple-comparisons test, F(3,16) = 18.73, P < 0.0001, post-test *P < 0.05, ***P < 0.001; n = 5 WT, 5 Pkn1–/– Cgc preparations from 3–5 litters per group]. (D) Presynaptic sites (varicosities) were stained with TAU and synapsin I. Insets represent higher-magnification single- and double-labeled examples of axonal varicosities (indicated by arrows). White varicosities in double-labeled insets demonstrate TAU and synapsin I colocalization. siRNAs targeting Akt123 significantly increase the density of presynaptic sites in Pkn1–/– Cgcs at DIV4 [1-way ANOVA with Newman-Keuls multiple-comparisons test, F(3,16) = 16.62, P < 0.0001, post-test ***P < 0.001, n = 5 WT, 5 Pkn1–/– Cgc preparations from 3–5 litters per group]. All data are presented as individual n values with mean ± SEM. Scale bars: 50 μm, except in inset in D: 10 μm. Experimenters were not blinded to the genotype or treatment.