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. 2014 Nov;20(11):1789-802.
doi: 10.1261/rna.047373.114. Epub 2014 Sep 22.

Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation

Lena Saal  1 , Michael Briese  1 , Susanne Kneitz  2 , Michael Glinka  1 , Michael Sendtner  3
Affiliations

Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation

Lena Saal et al. RNA. 2014 Nov.

Abstract

Neuronal function critically depends on coordinated subcellular distribution of mRNAs. Disturbed mRNA processing and axonal transport has been found in spinal muscular atrophy and could be causative for dysfunction and degeneration of motoneurons. Despite the advances made in characterizing the transport mechanisms of several axonal mRNAs, an unbiased approach to identify the axonal repertoire of mRNAs in healthy and degenerating motoneurons has been lacking. Here we used compartmentalized microfluidic chambers to investigate the somatodendritic and axonal mRNA content of cultured motoneurons by microarray analysis. In axons, transcripts related to protein synthesis and energy production were enriched relative to the somatodendritic compartment. Knockdown of Smn, the protein deficient in spinal muscular atrophy, produced a large number of transcript alterations in both compartments. Transcripts related to immune functions, including MHC class I genes, and with roles in RNA splicing were up-regulated in the somatodendritic compartment. On the axonal side, transcripts associated with axon growth and synaptic activity were down-regulated. These alterations provide evidence that subcellular localization of transcripts with axonal functions as well as regulation of specific transcripts with nonautonomous functions is disturbed in Smn-deficient motoneurons, most likely contributing to the pathophysiology of spinal muscular atrophy.

Keywords: axon; compartment; motoneuron; spinal muscular atrophy.

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Figures

FIGURE 1.
FIGURE 1.
Compartmentalized motoneuron cultures for microarray analysis of somatodendritic and axonal RNA. (A) Schematic of a microfluidic chamber in which primary motoneurons (E12.5) cultured on the left (somatodendritic) side extend axons through 150-μm-long microgrooves to the right (axonal) side. (B) Immunofluorescence staining for Tau (green) showing a dense network of axonal processes on the right side of the microfluidic chamber. Nuclear staining with DAPI (blue) and immunofluorescence staining for GFAP (red) shows the complete absence of somata on the axonal side. Scale bar: 150 μm. (C) Reproducibility of microarray data.
FIGURE 2.
FIGURE 2.
GO term analysis of genes expressed in the somatodendritic and axonal compartment of cultured wild-type motoneurons. (A) Correlation of axonal with somatodendritic microarray expression values for the three replicate wild-type motoneuron compartmentalized chambers. (B) GO terms enriched among the top 5000 expressed and bottom 5000 expressed somatodendritic and axonal transcripts. (C) Scatter plots depicting the enrichment of GO terms common to the top 5000 expressed somatodendritic and axonal transcripts for the different ontologies.
FIGURE 3.
FIGURE 3.
Transcript level changes in compartmentalized cultures of Smn-deficient motoneurons. (A) Smn transcript levels in wild-type and Smn knockdown motoneurons measured by quantitative PCR (qPCR). RNA was linearly amplified for one round prior to qPCR analysis. (B) MA plots of Smn knockdown microarray data from the somatodendritic compartment showing the relationship between the change in gene expression (log2 ratio) upon Smn knockdown relative to wild-type and GFP-expressing controls and the average level of gene expression (mean intensity) for each microarray probeset. Significantly up-regulated (black) and down-regulated (dark gray) transcripts are indicated. (C) MA plots of microarray data from axonal compartments. For axonal transcript changes an additional cutoff of |log2 ratio| > 2 was applied. (D) Volcano plot of Smn knockdown microarray data for somatodendritic compartments indicating the significance of change in gene expression [−log10(P)] for each probeset relative to the magnitude of change (log2 ratio). The top 50 most significantly deregulated genes are marked by filled circles. The magnified boxed area is shown on the right. (E) Volcano plot for axonal microarray data. In D and E, up-regulated genes are shown in black and down-regulated genes are shown in dark gray.
FIGURE 4.
FIGURE 4.
GO term analysis of transcripts deregulated in the somatodendritic compartment of Smn knockdown motoneurons. (A) The top 10 GO categories for “biological process,” “cellular component,” and “molecular function” for transcripts down-regulated or up-regulated upon Smn knockdown relative to controls (P < 0.05). (B) Transcripts in the GO category “RNA splicing” and “antigen processing and presentation of peptide antigen via MHC class I” up-regulated as a result of Smn knockdown.
FIGURE 5.
FIGURE 5.
Analysis of transcript level changes of axonal compartments of Smn knockdown motoneuron cultures. (A) GO term analysis of transcripts down-regulated or up-regulated in response to Smn knockdown (P < 0.05 and |log2 ratio| > 2). (B) Volcano plot of 195 microarray probesets with an average expression >10 in wild-type and control axons and which are deregulated upon Smn knockdown (P < 0.05 and |log2 ratio| > 2). (C) Transcript level changes of the 195 transcripts in B in the axonal and somatodendritic compartments of Smn-deficient motoneurons. Data points marked in black indicate a P-value <0.05 in the somatodendritic compartment. (D) GO term analysis of the 195 genes described in B. The table lists individual probesets changed in axons upon Smn knockdown that are contained in the GO term “neuron projection.”
FIGURE 6.
FIGURE 6.
Validation of microarray predictions by quantitative PCR for (A) somatodendritic and (B) axonal transcripts. Axonal genes shown in light gray in B have average crossing points >30 in the wild-type samples.
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