Dendritic transport and localization of protein kinase Mzeta mRNA: implications for molecular memory consolidation

Abstract

Protein kinase Mzeta (PKMzeta) is an atypical protein kinase C isoform that has been implicated in the protein synthesis-dependent maintenance of long term potentiation and memory storage in the brain. Synapse-associated kinases are uniquely positioned to promote enduring consolidation of structural and functional modifications at the synapse, provided that kinase mRNA is available on site for local input-specific translation. We now report that the mRNA encoding PKMzeta is rapidly transported and specifically localized to synaptodendritic neuronal domains. Transport of PKMzeta mRNA is specified by two cis-acting dendritic targeting elements (Mzeta DTEs). Mzeta DTE1, located at the interface of the 5'-untranslated region and the open reading frame, directs somato-dendritic export of the mRNA. Mzeta DTE2, in contrast, is located in the 3'-untranslated region and is required for delivery of the mRNA to distal dendritic segments. Colocalization with translational repressor BC1 RNA in hippocampal dendrites suggests that PKMzeta mRNA may be subject to translational control in local domains. Dendritic localization of PKMzeta mRNA provides a molecular basis for the functional integration of synaptic signal transduction and translational control pathways.

Fig. 1

Dendritic localization of PKMζ mRNA in hippocampal neurons in primary culture. A–F, substantial labeling signal is observed throughout the dendritic extent. In dark field photomicrographs (A–C), the signal appears as white silver grains over a dark blue background. Corresponding phase contrast photomicrographs are shown in D–F. The signal often appears clustered at branch points and intersections (arrows, B and F). No specific labeling is detectable along axonal shafts (arrowheads in D). G shows a sense strand control. Scale bar, 15 μm (B–D and F) and 40 μm (all others).

Fig. 2

Dendritic localization of PKMζ mRNA in sympathetic neurons in primary culture. Labeling signal is evident throughout the dendritic extent. Overall signal levels are about 10 times lower than in hippocampal neurons, partially compensated for by longer exposure times. The signal is robust and clustered in dendrites but is absent from axonal processes. C is a sense strand control. A–C, dark field photomicrographs. D–F, corresponding phase contrast photomicrographs. Scale bar, 15 μm (B and E) and 45 μm (all others).

Fig. 3

Expression of PKMζ mRNA, BC1 RNA, and NSE mRNA in forebrain. PKMζ mRNA (A) and BC1 RNA (B), but not NSE mRNA (C), extend into dendritic layers of CA3-CA1. Note that PKMζ mRNA and BC1 RNA, but not NSE mRNA, are low or absent in dentate gyrus granule cells. Conversely, PKMζ mRNA and BC1 RNA, but not NSE mRNA, are detectable at substantial levels in the hilus. Coexpression of PKMζ mRNA and BC1 RNA was also observed in several other brain areas but not universally in all (a notable exception being the cerebellum). A slight but consistent lateral hemispherical asymmetry is apparent with PKMζ mRNA and BC1 RNA, but to a much lower degree, if at all, with NSE mRNA (for discussion of left-right asymmetry in brain, see Refs. and 51). CA, cornu ammonis; DG, dentate gyrus. Scale bars, 1.2 mm.

Fig. 4

Localization profiles of PKMζ mRNA, BC1 RNA, MAP2 mRNA, and NSE mRNA in the CA3 field of hippocampus. PKMζ mRNA (A), BC1 RNA (B), and MAP2 mRNA (C), but not NSE mRNA (D) are expressed at substantial levels in dendritic layers. PKMζ mRNA and BC1 RNA are expressed at equally high levels in stratum radiatum and in stratum lacunosum moleculare but at lower relative levels in stratum lucidum. In contrast, relative MAP2 mRNA expression levels are highest in stratum radiatum but lower in stratum lacunosum moleculare. The results with all four RNAs were imaged and quantified (E). For each RNA analyzed, relative signal intensities in stratum pyramidale were set at 100%. Or, stratum oriens; Py, stratum pyramidale; Luc, stratum lucidum; Rad, stratum radiatum; LMol, stratum lacunosum moleculare. Scale bar, 250 μm.

Fig. 5

Design of PKMζ mRNA segments that were used to identify cis-acting DTEs. PKMζ mRNA segments were generated 1) by successive trimming in the 3′ to 5′ direction (Segments 1–4), 2) by trimming in the 5′ to 3′ direction (Segments 5 and 6), and 3) by deleting a 42-nt element in the 3′-UTR (Segment Δ).

Fig. 6

Dendritic transport competence of PKMζ mRNA Segment 1. Segment 1, representing nt 48–1982, is delivered to distal dendritic tips (indicated by arrows) in sympathetic neurons in culture. A–C, dark field photomicrographs. D–F, corresponding phase contrast photomicrographs. Scale bar, 50 μm.

Fig. 7

Dendritic transport competence of PKMζ mRNA Segments 2–4. Segment 2 (nt 48–1898; A and D) and Segment 3 (nt 48–906; B and E) are delivered to proximal but not to distal dendritic domains. Maximal extent of signal in proximal dendrites is indicated by arrows. In contrast, PKMζ mRNA Segment 4 (nt 48–347; C and F) fails to exit the soma to any appreciable extent. A–C, dark field photomicrographs. D–F, Corresponding phase contrast photomicrographs. Scale bar, 50 μm.

Fig. 8

Dendritic transport competence of PKMζ mRNA Segments 5, 6, and Δ. Segment 5 (1635 nt, lacking 5′ nt 1–347; A and D) is delivered to distal dendrites (arrows) in a manner indistinguishable from Segment 1. In contrast, Segment 6 (1136 nt, lacking 5′ nt 1–846; B and E) fails to enter even proximal dendritic domains. Segment Δ (1893 nt, lacking nt 1905–1946; C and F) is delivered to proximal, but not to distal, dendritic domains (extent of significant labeling indicated by arrows). A–C, dark field photomicrographs. D–F, corresponding phase contrast photomicrographs. Scale bar, 50 μm.

Fig. 9

Dendritic transport competence of PKMζ mRNA: quantitative analysis. PKMζ mRNA segments used in this analysis are schematically shown in the inset (see also Fig. 5). Relative dendritic signal intensities were established for each PKMζ mRNA segment at various distances along the dendritic extent. In the bar diagram, PKMζ mRNA segments are grouped as follows. Segments 1 and 5 are shown on the left; these segments were transported to the distal-most dendritic domains at substantial levels. Grouped on the right are Segments 2, 3, and Δ; these segments were delivered to dendrites but fail to reach distal domains (>200 μm). Segments 4 and 6 were not exported from the soma (>40 μm) at significant levels and therefore do not appear in the diagram.

Fig. 10

Secondary structure analysis of putative Mζ DTE2. A 44-nt stable stem-loop structure is predicted for nt 1905–1948 of PKMζ mRNA. Two A-residues (red) are unpaired. The asymmetric A/G bulge (blue ring) and the unpaired A-residues are likely to result in a distinct tertiary structure conformation.