Identification of process-localized mRNAs from cultured rodent hippocampal neurons

Abstract

The regulated translation of localized mRNAs in neurons provides a mechanism for spatially restricting gene expression in a synapse-specific manner. To identify the population of mRNAs present in distal neuronal processes of rodent hippocampal neurons, we grew neurons on polycarbonate filters etched with 3 microm pores. Although the neuronal cell bodies remained on the top surface of the filters, dendrites, axons, and glial processes penetrated through the pores to grow along the bottom surface of the membrane where they could be mechanically separated from cell bodies. Quantitative PCR and immunochemical analyses of the process preparation revealed that it was remarkably free of somatic contamination. Microarray analysis of RNA isolated from the processes identified over 100 potentially localized mRNAs. In situ hybridization studies of 19 of these transcripts confirmed that all 19 were present in dendrites, validating the utility of this approach for identifying dendritically localized transcripts. Many of the identified mRNAs encoded components of the translational machinery and several were associated with the RNA-binding protein Staufen. These findings indicate that there is a rich repertoire of mRNAs whose translation can be locally regulated and support the emerging idea that local protein synthesis serves to boost the translational capacity of synapses.

Figure 1.

Mechanical separation of neuronal processes from cell bodies. Dissociated hippocampal neurons were grown on filter membranes etched with 3 μm pores and imaged at 14 d in vitro. Removal of cells from the top surface with a cell scraper revealed that a pure preparation of dendrites, axons, and glial processes was present on the bottom surface. A, The cultures consisted of MAP2-immunopositive neurons (green) and GFAP-immunopositive astrocytes. B, When the top surface of the filter was removed with a cell scraper, MAP2-immunopositive dendrites and some GFAP-immunopositive glial processes remained on the bottom surface. F, After removal of the top surface, the bottom surface also contained tau-immunopositive axons (red) in addition to MAP2-immunopositive dendrites (green). Staining of intact filters with propidium iodide (PI) for nuclei (red) and MAP2 for neuronal somata and dendrites (green) in intact filters (C) and in filters after removal of the top surface (D) revealed that the bottom surface was devoid of nuclei. E, MAP2-stained cultures were imaged by confocal microscopy (E), and the reconstructed image was visualized in the z-plane (E, inset), showing that cell bodies (white vertical arrows) were restricted to the top of the filter (bottom left inset, green bar), and that MAP2-immunopositive dendrites penetrated through the 10-μm-thick filter (bottom left inset, light blue bar) to grow along the bottom surface of the filter (bottom left inset, red bar). G, Immunoblot analysis of the top (cell soma, S) and bottom (process, P) preparations revealed that the bottom lacked histone H3, a nuclear marker. Scale bars: (in A) A–E, 10 μm; F, 10 μm.

Figure 2.

Unsupervised hierarchical clustering of gene expression demonstrates reproducibility and reveals distinct patterns of gene expression between the arrays hybridized with either whole-cell sample or sample from cell processes. Transcripts that were analyzed were from the p < 0.01 without FDR. Similar results were also obtained using p < 0.05 with FDR threshold. Array hybridizations are designated X-Y, where X denotes which harvest the RNA was collected from and Y denotes the replicate number.

Figure 3.

Functional categories present in the process mRNA sample.

Figure 4.

mRNAs encoding translation factors are present in dendrites of cultured rat hippocampal neurons (14 div) as detected by double-label ISH and MAP2 immunocytochemistry. Left to right, ISH with antisense riboprobe (green); 4× zoom of a linearized region of dendrite >20 μm from soma; double-label MAP2 immunoreactivity (red) in the antisense sample; ISH with sense control riboprobe (green); and double-label MAP2 immunoreactivity in the sense sample (red). eIFγ2, Eukaryotic initiation factor γ2; eIF3s9, eukaryotic initiation factor 3, subunit 9. Scale bar, 10 μm.

Figure 5.

mRNAs encoding a variety of proteins are present in dendrites of cultured rat hippocampal neurons (14 div) as detected by double-label ISH and MAP2 immunocytochemistry. Left to right, ISH with antisense riboprobe (green); 4× zoom of a linearized region of dendrite >20 μm from soma; double-label MAP2 immunoreactivity (red) in the antisense sample; ISH with sense control riboprobe (green); and double-label MAP2 immunoreactivity in the sense sample (red). PPP1cc, Protein phosphatase 1, catalytic subunit, γ-isoform; PPP2cb, protein phosphatase 2, catalytic subunit, β-isoform. Scale bar, 10 μm.

Figure 6.

Staufen protein associates with dendritically localized mRNAs encoding translation factors. Hippocampal lysates were immunoprecipitated with Staufen antibody (Stau) or were incubated with anti-rabbit IgG beads in the absence of primary antibody (mock) followed by reverse transcription-PCR with gene-specific primers. A, Left, The immunoblot on the right shows that anti-Staufen antibody recognizes a single band of the appropriate molecular weight in hippocampal lysates. Middle, Top to bottom, Immunoblot of Staufen immunoprecipitation with anti-Staufen antibody showing that the anti-Staufen antibody immunoprecipitates Staufen protein, followed by PCR analysis of mRNAs coimmunoprecipitated with Staufen [CaMKIIα (25 cycles), Histone 1 (30 cycles), and importin β1 (32 cycles)]. Right, Top to bottom, EF1α (25 cycles), EF2 (28 cycles), eIF4γ2 (28 cycles), and PABPc1 (28 cycles). CaMKIIα, EF1α, EF2, eIF4γ2, and PABPc1 were all enriched in the Staufen immunoprecipitates, whereas neither histone 1 nor importin β1 were. B, Transfection of dissociated hippocampal neurons (8 div) with Staufen 1-EGFP construct (Stau-EGFP) and co-ISH with either EF1α (top row) or EF2 (middle row) antisense riboprobes. EF1α ISH was performed with an EGFP control plasmid (bottom row). Scale bar, 10 μm; inset, 20 μm.