Systematic mapping of fragile X granules in the mouse brain reveals a potential role for presynaptic FMRP in sensorimotor functions

Abstract

Loss of Fragile X mental retardation protein (FMRP) leads to Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism. Although the functions of FMRP and its homologues FXR1P and FXR2P are well studied in the somatodendritic domain, recent evidence suggests that this family of RNA binding proteins also plays a role in the axonal and presynaptic compartments. Fragile X granules (FXGs) are morphologically- and genetically-defined structures containing Fragile X proteins that are expressed axonally and presynaptically in a subset of circuits. To further understand the role of presynaptic Fragile X proteins in the brain we have systematically mapped the FXG distribution in the mouse central nervous system. This analysis revealed both the circuits and the neuronal types that express FXGs. FXGs are enriched in circuits that mediate sensory processing and motor planning - functions that are particularly perturbed in FXS patients. Analysis of FXG expression in the hippocampus suggests that CA3 pyramidal neurons utilize presynaptic Fragile X proteins to modulate recurrent but not feedforward processing. Neuron-specific FMRP mutants revealed a requirement for neuronal FMRP in the regulation of FXGs. Finally, conditional FMRP ablation demonstrated that FXGs are expressed in axons of thalamic relay nuclei that innervate cortex, but not in axons of thalamic reticular nuclei, striatal nuclei, or cortical neurons that innervate thalamus. Together, these findings support the proposal that dysregulation of axonal and presynaptic Fragile X proteins contribute to the neurological symptoms of FXS.

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

“…(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 work, we confirmed the restricted expression of this transgene by crossing these mice to conditional FMRP knockout mice and showing that Cre-mediated recombination occurs in neurons in thalamic relay nuclei including VAL but not in neurons in the TRN (14). Supplementary Material, Fig.…”

“…In previous work we took advantage of these traits to develop standardized image analyses for identifying FXGs. FXGs identified using these approaches always colocalized with axonal markers in the brain and were not observed to colocalize with markers of neuronal cell bodies or dendrites (14,15). Combining these approaches with genetic and ablation experiments established that FXGs are restricted to axons and are not present in glia or in neuronal somata or dendrites (14,15).…”

“…We first tested this approach in the olfactory system, where FXGs are found in olfactory sensory neuron axons innervating olfactory glomeruli in the olfactory bulb (14,15). For these experiments, we used mice in which the #123 promoter drives Cre recombinase in olfactory sensory neurons but not in the resident cells of the olfactory bulb (16)(17)(18).…”

“…We also tested this approach for selectively detecting FXGassociated proteins in the thalamus, where FXGs are found in axons of ventral anterior and ventrolateral (VAL) thalamic nuclei that project to the thalamic reticular nucleus (TRN) (14). For these experiments, we used mice in which a Syn1-Cre promoter drives Cre in restricted neuronal populations (21).…”

“…In mouse brain, FXGs show striking temporal regulation that correlates with epochs of robust synaptic plasticity (14,15). Several lines of evidence have established that FXGs are exclusively axonal: 1) colocalization with axonal but not somatodendritic markers; 2) ultrastructural demonstration of FMRP localization in axons and presynaptic boutons; 3) identification of FXG-containing axonal tracts within specific circuits in the intact brain; 4) loss of FXGs in axonal tracts and target neuropil following chemical ablation of projection neurons; and 5) selective depletion of FMRP from FXGs following circuitspecific genetic ablation of FMRP in projection neurons (14,15). As an endogenous, readily identifiable and uniquely axonal structure containing RNA binding proteins, the FXG is an attractive portal for investigating translational machinery in axons in the brain.…”