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 Article has an altmetric score of 1

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 2

Pkn1–/– Cgcs have a reduced density of presynaptic sites and enhanced axonal outgrowth.

Options: View larger image (or click on image) Download as PowerPoint
Pkn1–/– Cgcs have a reduced density of presynaptic sites and enhanced a...
(A) In WT Cgcs, TAU-stained axonal en passant swellings colocalized with the presynaptic marker synapsin I (images were taken at DIV4 and are representative of at least 3 separate experiments in WT and Pkn1–/– Cgcs). (B) The number of en passant swellings (varicosities) per axonal section was analyzed in WT and Pkn1–/– Cgcs at DIV7 [2-tailed unpaired t test, t(6) = 4.413, **P = 0.0045, n = 4 WT, 4 Pkn1–/– Cgc preparations from 4 litters per group]. (C) Pkn1–/– Cgcs were transfected with GFP together with a control HA-plasmid (Co-HA) or human HA-tagged PKN1 (hPKN1), and varicosities per axonal section were analyzed at DIV7 in GFP-expressing/TAU-stained Cgcs [2-tailed unpaired t test, t(4) = 7.147, **P = 0.002, n = 3 from 3 litters]. (D) Axonal length after DIV1 [2-tailed unpaired t test, t(5) = 4.431, **P = 0.0068, n = 4 WT, 3 Pkn1–/– Cgc preparations from 3–4 litters per group] and DIV7 [2-tailed unpaired t test, t(6) = 2.692, *P = 0.0360, n = 4 WT, 4 Pkn1–/– Cgc preparations from 4 litters per group] in TAU-stained Cgcs. (E) Pkn1–/– Cgcs were transfected with GFP together with Co-HA or hPKN1, and axonal length at DIV7 was analyzed in GFP-expressing/TAU-stained Cgcs [2-tailed unpaired t test, t(4) = 4.752, **P = 0.0090, n = 3 from 3 litters]. GFP-expressing cells also expressed hPKN1, as seen in overlapping GFP/HA staining (image is representative of 3 separate experiments). Data are presented as individual n values with mean ± SEM. Cgcs were grown on laminin-coated coverslips, and representative WIS-NeuroMath–analyzed output images are shown in D and E. All scale bars: 50 μm. Experimenters were not blinded to the genotype or treatment.