Evidence for developmentally regulated local translation of odorant receptor mRNAs in the axons of olfactory sensory neurons

Abstract

Odorant receptor mRNAs are transported within axons of olfactory sensory neurons that project into the olfactory bulb. Odorant receptor proteins have been identified along the distal part of these axons, which raises the possibility of their local synthesis in axons. We took advantage of the anatomical separation between the olfactory mucosa (which contains the sensory neuron cell bodies) and the bulb (which contains sensory axons but no sensory neuron cell bodies) to address this issue using a quantitative biochemical approach. Combining a method that separates polysome-associated mRNAs from untranslated mRNAs with a reverse transcription-quantitative PCR approach, we demonstrate that significant amounts of odorant receptor mRNAs are associated with polysomes in the sensory axons of the adult mouse bulb. We thus provide the first evidence for local synthesis of odorant receptor proteins in these axons. Interestingly, the rate of odorant receptor mRNA translation in axons is significantly greater during periods when the proportion of immature axons is higher (i.e., at postnatal day 4 or on regeneration after chemical lesion in adults). In contrast, the olfactory marker protein mRNA, which is restricted to mature axons, is translated at a low and constant level. Overall, we demonstrate that translation levels of odorant receptor mRNAs in axons are developmentally regulated, and positively correlated to the stage of axonal growth into the bulb. Given the established function of odorant receptors in the axonal wiring of sensory projections, we propose that this regulated axonal translation may play a role in the development and maintenance of the glomerular array.

Figure 1.

Assessment of axonal odorant receptor mRNAs in adult mice olfactory bulb during reversible sensory deafferentation. A, Protocol of chemical lesion of olfactory sensory neurons: intraperitoneal injections of dichlobenil were performed at days 0, 2, and 4 in adult mice; mice were killed at days 7, 14, or 28. B, Immunolocalization of OMP (a specific marker for mature olfactory sensory neurons; red label) and βIII-Tubulin (a marker for immature neurons—TUJ1 antibody; green label) in the olfactory epithelium after dichlobenil treatment. Blue label (DAPI) stains cell nuclei. At day 7, few OMP-positive neurons remain in the lesioned olfactory epithelium characterized by a dramatically reduced thickness. However, the lesion triggers the regeneration of new sensory neurons, which were initially TUJ1-immunoreactive before they progressively mature to become OMP-expressing neurons. C, The ratio between GAP43 mRNA (a marker for immature neurons) and OMP mRNA is transiently increased in the olfactory epithelium, further demonstrating the strong regeneration of sensory neurons 1–2 weeks after the onset of the lesion. D, E, RT-qPCR assessment of OMP (D) and odorant receptors (E) mRNAs in olfactory bulb (OB) and olfactory epithelium (OE) on dichlobenil treatment. mRNA levels represent the percentage of measured mRNAs compared with that in untreated mice. D, Dichlobenil treatment induces a dramatic decrease in OMP mRNA contents in the OE and in the OB at days 7 and 14, which subsequently increases as the epithelium regenerates, indicating that OMP mRNA molecules measured in the bulb originated in the axons. E, Similarly, dichlobenil treatment induces a dramatic decrease in odorant receptor mRNA contents in the olfactory epithelium and bulb at day 7, which subsequently increases, indicating that odorant receptor mRNAs measured in the bulb likewise originated in the axons. Data are expressed as mean ± SEM (n = 3–5). RT-qPCR data in D and E were normalized using α1b-Tubulin mRNA as an internal standard.

Figure 2.

Developmentally regulated transport of odorant receptor mRNAs in axons of olfactory sensory neurons. A, RT-qPCR quantification of MOR253-5, MOR83, and MOR10 mRNAs from adult and P4 mice olfactory epithelium. B, RT-qPCR quantification of MOR253-5, MOR83, and MOR10 mRNAs from adult or P4 mice olfactory bulb. C, Relative olfactory bulb/olfactory epithelium (OB/OE) transport index for odorant receptor mRNAs, representing the percentage of odorant receptor mRNAs quantified in the olfactory bulb over the percentage quantified in the olfactory epithelium. These indices are higher in P4 mice than in adult mice, indicating a greater axonal transport in sensory axons at P4 than in adults. Data are expressed as mean ± SEM (n = 4–5). *Mann–Whitney U test: p = 0.016 for MOR10, p = 0.029 for MOR253-5 and MOR83. All RT-qPCR data were normalized using α1b-Tubulin mRNA as an internal standard.

Figure 3.

Developmentally regulated translation of odorant receptor mRNAs in bulbar sensory axons. A, Typical polysomal profile obtained in a sucrose gradient fractionation of olfactory bulb. B, C, Odorant receptor mRNAs were quantified in both mRNP and polysome fractions from olfactory bulb, and the PMP was calculated for each of them. Data are expressed as mean ± SEM. B, MOR253-5, MOR83, and MOR10 PMPs in olfactory bulb from adult and P4 mice. PMPs for odorant receptor mRNA in the bulb are higher in P4 than in adult mice. *Mann–Whitney U test: p = 0.052 for MOR10 (n = 5–6), p = 0.057 for MOR253-5 and MOR83 (n = 3–4). C, MOR253-5 and MOR10 PMPs in olfactory bulb from adult mice after dichlobenil treatment (refer to Fig. 1 A for the dichlobenil experiment protocol). A transitory increase in MOR253-5 and MOR10 PMPs is observed at days 7 and 9, respectively, before PMPs return to lower values by day 28. *Mann–Whitney U test: p = 0.057 for MOR253-5 (n = 3–4), p = 0.038 for MOR10 (n = 4–6).

Figure 4.

OMP mRNA local translation in bulbar sensory axons is not higher in P4 mice than in adult mice, and it does not increase in the regeneration process after dichlobenil lesion. A, OMP and α1b-Tubulin PMPs in bulbs from adult and P4 mice. B, OMP and α1b-Tubulin PMPs in olfactory bulbs from adult mice on dichlobenil treatment. PMPs for OMP mRNA are equally low (∼10%) in all tested conditions. OMP and α1b-Tubulin mRNAs were quantified in both mRNP and polysome fractions from olfactory bulbs, and the PMP was calculated for each of them. Data are expressed as mean ± SEM (n = 4).

Figure 5.

Odorant receptor and OMP mRNAs local translation in bulbar sensory axons are differentially regulated. Correlation between sensory neuron maturation indices (calculated from OMP/GAP43 mRNAs ratio in the olfactory epithelium; the value for untreated adult mice is 100%) and PMPs for the MOR253-5 (A), MOR10 (B), OMP (C), and α1b-Tubulin (D) mRNAs in the olfactory bulb in various physiological or experimental conditions: P4 (purple); untreated adults (blue); adults after dichlobenil treatment (7 d after the onset of dichlobenil treatment in red, 9 d in orange, 14 d in brown, 28 d in green). Linear trend lines are shown for each mRNA (slope: MOR253-5, −29.6; MOR10, −32.3; OMP, −0.6; α1b-Tubulin, 7.2).