PKN1 Is a Novel Regulator of Hippocampal GluA1 Levels

Abstract

Alterations in the processes that control α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) expression, assembly and trafficking are closely linked to psychiatric and neurodegenerative disorders. We have recently shown that the serine/threonine kinase Protein kinase N1 (PKN1) is a developmentally active regulator of cerebellar synaptic maturation by inhibiting AKT and the neurogenic transcription factor neurogenic differentiation factor-2 (NeuroD2). NeuroD2 is involved in glutamatergic synaptic maturation by regulating expression levels of various synaptic proteins. Here we aimed to study the effect of Pkn1 knockout on AKT phosphorylation and NeuroD2 levels in the hippocampus and the subsequent expression levels of the NeuroD2 targets and AMPAR subunits: glutamate receptor 1 (GluA1) and GluA2/3. We show that PKN1 is expressed throughout the hippocampus. Interestingly, not only postnatal but also adult hippocampal phospho-AKT and NeuroD2 levels were significantly elevated upon Pkn1 knockout. Postnatal and adult Pkn1 ^-/- hippocampi showed enhanced expression of the AMPAR subunit GluA1, particularly in area CA1. Surprisingly, GluA2/3 levels were not different between both genotypes. In addition to higher protein levels, we also found an enhanced GluA1 content in the membrane fraction of postnatal and adult Pkn1 ^-/- animals, while GluA2/3 levels remained unchanged. This points toward a very specific regulation of GluA1 expression and/or trafficking by the novel PKN1-AKT-NeuroD2 axis. Considering the important role of GluA1 in hippocampal development as well as the pathophysiology of several disorders, ranging from Alzheimer's, to depression and schizophrenia, our results validate PKN1 for future studies into neurological disorders related to altered AMPAR subunit expression in the hippocampus.

Keywords: AMPA receptor; GluA1; NeuroD2; PKN1; hippocampus.

FIGURE 1

PKN1 regulates AKT phosphorylation and NeuroD2 levels in postnatal and adult hippocampi. (A) P10 and adult hippocampal sections from WT animals were tested for PKN1 expression by in situ hybridization. Pictures are representative of 2–3 separate WT animals. Scale bar refers to 100 μm. (B) PKN1 expression levels from P1 to P15 old WT hippocampi were assessed by western blotting (**P < 0.01, one way ANOVA with Tukey’s multiple comparisons test). (C) NeuroD2 expression in hippocampal whole cell protein extracts of P8 and P15 old WT and Pkn1^–/– animals was assessed by western blotting (two way ANOVA: Interaction: P = 0.0745, Age: P = 0.0747, Genotype: P < 0.0001, Sidak’s multiple comparisons test: P8: P < 0.0001, P15: P = 0.0025). (D) NeuroD2 expression in adult WT and Pkn1^–/– hippocampal whole cells extracts (*P = 0.0496, unpaired t-test). (E) Hippocampi from P12 old WT and Pkn1^–/– animals were separated into cytosolic and membrane fractions. Membrane extracts were probed for phosphorylated AKT[T308] (pAKT[T308]) and total AKT and the ratio was calculated (*P = 0.0104, unpaired t-test). (F) Membrane fractions of adult WT and Pkn1^–/– animals were probed for pAKT[T308] and AKT (*P = 0.045, unpaired t-test). The markers in the representative blots in (E,F) are shown in separate lanes as samples were not directly next to the markers in the blots. Data is presented as individual n-values with mean ± S.E.M.

FIGURE 2

Adult Pkn1^–/– animals have enhanced GluA1 protein levels (A) Whole cell protein extracts from adult WT and Pkn1^–/– hippocampi were prepared, probed for GluA1 and the ratio to the loading control actin was calculated (*P = 0.0321, unpaired t-test). The marker in the representative blot is shown in a separate lane as samples were not directly next to the marker in the blot. (B) Hippocampal GluA1 levels were further assessed by immunofluorescence staining. Pictures are representative of 3 animals/genotype. Scale bar in overview images refers to 500 μm and scale bar in high resolution inserts refers to 20 μm. (C) The mean intensity of hippocampal GluA1 levels from (B) was quantified in Fiji. CA1 sr refers to CA1 stratum radiatum, CA1 so refers to CA1 stratum oriens. (CA1 sr *P = 0.0156, unpaired t-test, CA1 so *P = 0.0233, unpaired t-test). All data is presented as individual n-values with mean ± S.E.M.

FIGURE 3

Postnatal and adult Pkn1^–/– animals show enhanced membrane-association of GluA1. (A) The detergent-soluble cytosolic fraction of extracts prepared from P12 old WT and Pkn1^–/– hippocampi was probed for the loading control Tubulin and GluA1 (P = 0.393, unpaired t-test). (B) The detergent-insoluble membrane fraction of extracts prepared from P12 old WT and Pkn1^–/– hippocampi was probed for the loading control Na⁺/K⁺-ATPase and GluA1 (*P = 0.0416, unpaired t-test). (C) The detergent-soluble cytosolic fraction of extracts prepared from adult WT and Pkn1^–/– hippocampi was probed for the loading control GAPDH and GluA1 (**P = 0.0075, unpaired t-test). (D) The detergent-insoluble membrane fraction of extracts prepared from adult WT and Pkn1^–/– hippocampi was probed for the loading control Na⁺/K⁺-ATPase and GluA1 (*P = 0.0430, unpaired t-test). All data is presented as individual n-values with mean ± S.E.M. Images of PKN1 in (A,B) were derived from a second blot, loaded in the same order. All markers in the representative blots are shown in separate lanes as samples were not next to the markers in the blots.