Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains

Abstract

Local mRNA translation in growing axons allows for rapid and precise regulation of protein expression in response to extrinsic stimuli. However, the role of local translation in mature CNS axons is unknown. Such a mechanism requires the presence of translational machinery and associated mRNAs in circuit-integrated brain axons. Here we use a combination of genetic, quantitative imaging and super-resolution microscopy approaches to show that mature axons in the mammalian brain contain ribosomes, the translational regulator FMRP and a subset of FMRP mRNA targets. This axonal translational machinery is associated with Fragile X granules (FXGs), which are restricted to axons in a stereotyped subset of brain circuits. FXGs and associated axonal translational machinery are present in hippocampus in humans as old as 57 years. This FXG-associated axonal translational machinery is present in adult rats, even when adult neurogenesis is blocked. In contrast, in mouse this machinery is only observed in juvenile hippocampal axons. This differential developmental expression was specific to the hippocampus, as both mice and rats exhibit FXGs in mature axons in the adult olfactory system. Experiments in Fmr1 null mice show that FMRP regulates axonal protein expression but is not required for axonal transport of ribosomes or its target mRNAs. Axonal translational machinery is thus a feature of adult CNS neurons. Regulation of this machinery by FMRP could support complex behaviours in humans throughout life.

Figure 1.

FXG identification in olfactory sensory neurons. (A) Cre-dependent tdTomato reporter expression in an olfactory bulb from a #123-Cre;R26^tdT/WT mouse expressing Cre under control of the olfactory sensory neuron-specific #123 promoter. tdTomato expression (red) was observed in olfactory sensory neuron axons but not in cells resident within the olfactory bulb (DAPI; blue). (B,C) Olfactory glomerulus (representative of boxed region in A) from a #123-Cre;R26^tdT/WT mouse. tdTomato (red) in olfactory sensory neuron axons colocalizes with FXR2P (green) in FXGs (arrows) but not with FXR2P in cell bodies of resident olfactory bulb neurons (arrowheads). (D–M) Analysis of FMRP signal in FXGs and resident olfactory bulb neurons in wild type (#123-Cre;Fmr1^wt/y) and cKO (#123-Cre;Fmr1^flox/y) mice in which a floxed Fmr1 allele was selectively ablated in olfactory sensory neurons. (D,G) Schematic of FMRP and FXG expression in experiments depicted in E-F, H-M. Images were collected in olfactory bulb glomeruli. Image processing of the FXR2P signal was used to identify FXGs in these images. (E,F,H,I) Representative images of data analysed in J-M. Image processing of FXR2P immunostaining (red) identifies FXGs (blue). (E,F) In wild type brains, FMRP (green) is present in FXGs in OSN axons as well as in cell bodies of olfactory bulb neurons. (H-I) In cKO brains, FMRP is selectively ablated from FXGs in OSN axons but retained in cell bodies of olfactory bulb neurons. (J) Quantitation of FMRP levels in FXGs. FMRP intensity was quantified for each FXG. FMRP intensity in olfactory FXGs was significantly different between cKO (0.93 ± 0.012) and wild type brains (2.84 ± 0.022; P = 0.003661 by ANCOVA). Background fluorescence: 1.00 ± 0.0078. (K) Cumulative frequency distribution of average FMRP fluorescence intensity in olfactory FXGs for each genotype. Dashed line indicates the median signal in wild type FXGs. (L) Quantitation of FMRP levels in olfactory bulb somata. For each image, the average somatic signal in cells was calculated. There was no change in FMRP intensity in these cells between wild type (2.47 ± 0.027) and cKO (2.35 ± 0.019) animals (P = 0.7437 by ANCOVA). Background fluorescence: 0.95 ± 0.011. (M) Cumulative frequency distribution of average FMRP fluorescence intensity in olfactory bulb neurons. Dashed line indicates the median signal for wild type somata. For J-M, FMRP fluorescence in wild type and cKO regions of interest was compared using background fluorescence as a covariate. Background is depicted separately to provide context for interpreting FMRP signal. For all panels, arrows: FXGs; arrowheads: cell bodies. OE: olfactory epithelium; OB: olfactory bulb; tdT: tdTomato; OSN: olfactory sensory neuron. Scale bar = 500 μm in A; 20 μm in B; 10 μm in C; 15 μm in E,G; 5 μm in F,H. Wild type: n = 2720 FXGs and 325 cell bodies; 9 images; 3 animals. cKO: n = 2146 FXGs and 461 cell bodies; 9 images; 2 animals.

Figure 2.

FXGs associate with ribosomes in the juvenile mouse. (A) Schematic indicating location of depicted FXGs. Confocal micrographs from P15 mouse brains show that FXGs associate with: (B-E) 5S/5.8S rRNA including in neurofilament-expressing axons; (F) 18S and 28S rRNA; and (G) ribosomal protein S6. Quantification of this colocalization is in Table 2. For all panels, arrows: FXGs; arrowheads: cell bodies. SL: stratum lucidum; SP: stratum pyramidale. Scale bar = 10 μm in B; 5 μm in C, E-G; 450 nm in D.

Figure 3.

FXGs associate with mRNA in the juvenile mouse. (A) Schematic indicating location of depicted FXGs. Confocal micrographs of P15 mouse brains show that FXGs (identified by FXR2P) associate with (B) polyA⁺RNA; (C-D) β-catenin mRNA, which colocalizes with rRNA; and (E) OMP mRNA. Quantification of this colocalization is in Table 2. For all panels, arrows: colocalizing FXGs; arrowheads: non-colocalizing FXGs; double arrowheads: non-FXG granules. Scale bar = 5 μm.

Figure 4.

FXGs exhibit species-dependent expression in the adult brain. (A) FXG abundance in selected rat and mouse brain regions across postnatal development; n = 5 for each age/species. FXG density in frontal cortex declined with age in both species (2-way ANOVA; species P = 0.9952, age P < 0.0001, interaction P = 0.0013). In mouse frontal cortex, FXG densities were: P16, 0.124 ± 0.017 FXGs/100 μm²; P70, 0.012 ± 0.003; P150, 0.015 ± 0.008. For rats, the FXGs densities were: P16, 0.077 ± 0.006; P70, 0.037 ± 0.005; P150, 0.038 ± 0.011. Olfactory sensory neuron FXG expression varied between species but remained high in adults of both species (2-way ANOVA; species P = 0.0052, age P = 0.0168, interaction P = 0.0109). For mouse olfactory bulb, FXG densities were: P16, 1.963 ± 0.146; P70, 1.484 ± 0.143; P150, 1.181 ± 0.218. For rat olfactory bulb, FXG densities were: P16, 1.799 ± 0.261; P70, 2.832 ± 0.210; P150, 1.720 ± 0.335. Hippocampal mossy fibre FXG density declined with age in mice, but remained high in rats (2-way ANOVA; species P < 0.0001, age P = 0.0693, interaction P < 0.0001). For mouse hippocampus, FXG densities were: P16, 0.385 ± 0.038; P70, 0.039 ± 0.008; P150, 0.041 ± 0.009. In rats, hippocampal FXG densities were: P16, 0.273 ± 0.029; P70, 0.562 ± 0.072; P150, 0.434 ± 0.035. (B-G) FXGs decline with age in (B-D) the mouse hippocampus, but remain abundant across the lifespan in (E-G) the rat hippocampus as visualized in mossy fibres (MF). (H-J) FXGs in adult rat hippocampal mossy fibres (MF; H,I) and CA3 associational fibres (AF; J) contain ribosomes (H,J) and RNA (I,J) including the Ctnnb1 mRNA (J). For all panels, arrows: FXGs. Scale bar = 15 μm in B-G, 10 μm in H-J.

Figure 5.

Ribosomes and mRNA are expressed in axons of mature, circuit-integrated neurons. (A) Schematic indicating location of depicted FXGs. (B-C) FXGs were not detected in immature (GAP43-expressing) olfactory sensory neuron axons in adult mice in olfactory nerve layer tracts (ONL; B) or in glomerular neuropil (GL; C). (D-G) Ablation of neurogenesis in GFAP-TK transgenic adult rats by valganciclovir administration for 10 weeks had no effect on FXG density (control: 0.401 ± 0.033 FXGs/100 μm²; neurogenesis-deficient: 0.444 ± 0.040; unpaired t-test: P = 0.474; n = 5 rats for each condition). For all panels, arrows: colocalizing FXGs; arrowheads: non-colocalizing FXGs. Scale bar = 5 μm in A-B; 10 μm in E-F.

Figure 6.

FMRP regulates axonal protein expression of FXG targets independent of RNA expression or localization. (A) Schematic indicating location of images. In brains from Fmr1 null mice, FXGs associate with: (B-C) ribosomes; (D) polyA⁺RNA; (E)Ctnnb1 mRNA; and (F)Omp mRNA. Quantification of the colocalization is in Table 2. (G)Omp mRNA expression and axonal localization in Fmr1 null mice are comparable to those in controls. For somatic expression in olfactory epithelium, qPCR values were 1.451 ± 0.621 in wild type and 1.610 ± 0.798 in Fmr1 null mice. For axonal localization in olfactory bulb, values were 1.292 ± 0.444 in wild type and 1.122 ± 0.431 in Fmr1 nulls. n = 5 for each genotype. (H)Fmr1 null mice exhibit increased axonal expression of OMP protein (wild type: 0.870 ± 0.109; Fmr1 null: 1.617 ± 0.238) but not GAP43 protein (wild type: 1.474 ± 0.403; Fmr1 null: 1.072 ± 0.199). (I-J) Representative images of olfactory glomeruli depicting OMP and GAP43 proteins filling olfactory sensory neuron axons as they innervate the olfactory bulb from wild type and Fmr1 null mice. n = 5 for each genotype. For all panels, arrows: colocalizing FXGs; arrowheads: non-colocalizing FXGs; double arrowheads: non-FXG granules. Scale bar = 5 μm for B,D-F; 450 nm for C; 35 μm for I-J.

Figure 7.

Axonal mRNA and ribosomes in FXGs of the adult human brain. Confocal micrographs of hippocampal sections from a 44-year-old individual. In these sections, FMRP and FXR2P localize to both FXGs (arrows) and neuronal cell bodies (arrowheads). (A-D) FXGs are found in adult human mossy fibre axons where they contain FXR2P as well as FMRP (A-B), ribosomes (C), and polyA+ RNA (D). (E-H) FXGs in CA3 associational fibres contain FXR2P as well as FMRP (E,F), ribosomes (G) and polyA⁺RNA (H). Scale bar = 10 μm in A,E; 5 μm in B-D,F-H.

Figure 8.

A model for FMRP-regulated axonal translation. Axons in the central nervous system contain mRNA and ribosomes that associate with FXGs. In FXG-containing neurons, local translation contributes to the axonal proteome. In the absence of FMRP, these neurons contain FXGs that associate with ribosomes and mRNA but exhibit dysregulated axonal translation. This dysregulation results in a cell type-specific increase in the dosage of select presynaptic proteins that mediate axonal plasticity.