A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons

Abstract

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function.

Keywords: Campenot cell culture chambers; Microarray analyses; Subcellular localization; Superior cervical ganglion; mRNAs.

Fig. 1

RNA prepared from the distal axons of SCG neurons is free of neuronal cell body contamination. RT-PCR analysis was conducted on RNA obtained from the lateral and central compartments of the Campenot cell culture chambers using gene-specific primer sets for Scn3b and Glrb. PCR products were fractionated on 2.5% agarose gels and amplicons visualized by ethidium bromide staining. PCR reactions lacking cDNA served as negative (H₂O) controls.

Fig. 2

The presence of nuclear-encoded mitochondrial mRNAs can be visualized in the somata and axons of primary SCG neurons. Visualization of nuclear-encoded mitochondrial mRNAs was achieved using RNAscope in situ hybridization. No signal was detected in axons hybridized with the DAPb control riboprobe. In axons hybridized with a COXIV riboprobe, mRNA appears as puncta of varying dimensions (arrows). In contrast, barely detectable fluorescent signals were observed using AADAT riboprobes.

Fig. 3

Subcellular distributions of nuclear-encoded mitochondrial mRNAs in the axons and cell body of SCG neurons as assessed by microarray analysis. (A) A scatter plot depicting the relative abundance of nuclear-encoded mitochondrial mRNA in the axons and cell bodies of SCG neurons. The distribution of the mRNAs levels in the axons and cell bodies is highly correlated (Pearson correlation coefficient of 0.967). (B) Comparison of axonal and cell body abundance ratios (fold-change) as a function of relative expression (intensity) for nuclear-encoded mitochondrial mRNAs suggests that Prss35, Cryab and AT5i are highly enriched in SG axons.

Fig. 4

qRT-PCR based quantification of Prss35, Cryab, ATP5i, Ndufa1 and Lyrm2 expression levels in the cell body and axons of SCG neurons. Data are shown as mean +/– SEM; p-values are determined by Student's t-test. *p < 0.05, **p < 0.001.