Cellular and subcellular localization of PKMζ

Abstract

In contrast to protein kinases that participate in long-term potentiation (LTP) induction and memory consolidation, the autonomously active atypical protein kinase C isoform, protein kinase Mzeta (PKMζ), functions in the core molecular mechanism of LTP maintenance and long-term memory storage. Here, using multiple complementary techniques for light and electron microscopic immunolocalization, we present the first detailed characterization of the cellular and subcellular distribution of PKMζ in rat hippocampus and neocortex. We find that PKMζ is widely expressed in forebrain with prominent immunostaining in hippocampal and neocortical grey matter, and weak label in white matter. In hippocampal and cortical pyramidal cells, PKMζ expression is predominantly somatodendritic, and electron microscopy highlights the kinase at postsynaptic densities and in clusters within spines. In addition, nuclear label and striking punctate immunopositive structures in a paranuclear and dendritic distribution are seen by confocal microscopy, occasionally at dendritic bifurcations. PKMζ immunoreactive granules are observed by electron microscopy in cell bodies and dendrites, including endoplasmic reticulum. The widespread distribution of PKMζ in nuclei, nucleoli and endoplasmic reticulum suggests potential roles of this kinase in cell-wide mechanisms involving gene expression, biogenesis of ribosomes and new protein synthesis. The localization of PKMζ within postsynaptic densities and spines suggests sites where the kinase stores information during LTP maintenance and long-term memory.

Multicolor Electron Microscopy for Simultaneous Visualization of Multiple Molecular Species

“…To confirm that the apparent signal was representative of newly produced PKMζprotein, we used EELS to visualize Ce at the same synapse. We found that the Ce signal was enhanced at the postsynaptic membrane (Figure 5C and D at lower magnification, E and F at higher magnification) confirming that new copies of PKMζpreferentially localize to the post-synaptic membrane, consistent with previous reports for PKMζlocalization (Hernández, et al, 2014). Unstimulated neurons that were treated with BILN-2061 and analyzed similarly (Figure 5G–I) showed only a minimal signal for Ce by EELS in synapses (Figure 5J) despite comparable post-synaptic density staining in conventional EM.…”

Persistent Increases of PKMζ in Sensorimotor Cortex Maintain Procedural Long-Term Memory Storage

“…Confocal microscopy revealed PKMζ in the sensorimotor cortex is compartmentalized in small puncta (Figure 3), similar to its distribution in the hippocampus (Hernández et al., 2014). The puncta number and size were quantified in each hemisphere, and the interhemispheric ratios (trained/untrained hemispheres) were used to compare the amounts of PKMζ in each group with those in controls (naive rats) (Figures 4 and S5 and Transparent Methods).…”

Persistent increased PKMζ in long-term and remote spatial memory

“…Similar to LTP (Kelly et al, 2007), PKMζ protein increases in long-term memory without changes in mRNA levels. Therefore, we speculate that spatial conditioning causes PKMζ increases through new synthesis from pre-existing dendritic PKMζ mRNA (Muslimov et al, 2004) located at or near the specific synapses that are strongly activated during conditioning (Hernandez, Oxberry, Crary, Mirra, & Sacktor, 2014; Hernandez et al, 2003). The persistent local increases in the autonomously active kinase then maintain increased postsynaptic AMPAR-mediated synaptic transmission at these specific synapses by, for example, increasing the trafficking of GluA2 to postsynaptic sites through the action of N-ethylmaleimide sensitive factor (NSF) (Yao et al, 2008; Migues et al, 2010), so as to persistently modify the networks of neurons activated during learning to facilitate memory storage (Sacktor, 2011).…”