The most striking features of FXGs are their localization to axonal and presynaptic compartments, restriction to a subset of brain circuits during defined developmental periods, absolute dependence on FXR2P and regulation by FMRP. All of these attributes distinguish FXGs from the ubiquitous pool of Fragile X proteins investing the somatodendritic domain. Here we will discuss the evidence supporting the assignment of FXGs as a novel context for Fragile X proteins and the implications of these findings for FXS and autism.
Several lines of evidence support the conclusion that FXGs are predominantly localized to the axonal and presynaptic compartments of selected neurons. 1) FXGs are most abundant in neuropil – e.g., strata oriens and lucidum in hippocampal area CA3 as well as the glomeruli of the olfactory bulb (-). 2) FXGs are also observed in the axons in the hilus of the hippocampus and in the olfactory nerve layer (, S1
). 3) FXGs are enriched in the axonal/presynaptic compartments as compared to dendritic domains of the olfactory glomerulus (). 4) The number of FXGs in the olfactory glomerulus drops precipitously following ablation of olfactory sensory neurons (). 5) Immunoelectron microscopy shows that FMRP is localized presynaptically in olfactory glomeruli () and in hippocampal area CA3 (). Importantly, we could detect postsynaptic FMRP by immunoelectron microscopy in many brain regions including olfactory glomeruli (), hippocampal areas CA3 () and CA1 (data not shown). However, we only observed presynaptic FMRP in those areas where FXGs were observed (e.g., olfactory glomeruli and area CA3). An earlier ultrastructural study (Feng et al., 1997
) also reported postsynaptic and “rare” presynaptic FMRP in cerebral cortex, which is consistent with our immunohistochemical observations in this brain region (; supplemental Fig. S3b-d
). Thus, FXGs correlate with presynaptic and axonal localization of the Fragile X proteins.
Several studies have provided evidence that local protein synthesis in growth cones can play a role in axonal growth and guidance (Cox et al., 2008
; Lin and Holt, 2008
; Willis et al., 2007
). Moreover, FMRP has been localized to growth cones in cultured neurons (Antar et al., 2006
). However, we saw little evidence for FXG localization in growth cones in the systems examined here. For example, FXG number in regenerating olfactory circuits peaked at the time of synapse formation and refinement, rather than during the earlier phase characterized by axonal growth (). Moreover, we observed few FXGs in area CA3 at P7, a time when growth cones are abundant. It is important to stress that these observations in no way preclude a role for non-FXG-associated Fragile X proteins in growth cones and axonal guidance. Indeed, there is little doubt that the Fragile X proteins are components of a variety of complexes (Jin et al., 2004
; Zalfa et al., 2006
), each of which is likely to carry out distinct functions within the cell.
The functions of FXGs at the presynaptic apparatus are not known, but one attractive idea is that they are involved in translation-dependent synaptic plasticity. Such presynaptic plasticity has been observed in cultured neurons for BDNF-mediated potentiation and for serotonin-induced long term facilitation (Lyles et al., 2006
; Zhang and Poo, 2002
). The molecular mechanisms acting in these cases are not known. Mutations of dfmr1
, the sole homolog of the Fragile X gene family in Drosophila, lead to abnormal structure and function of the larval neuromuscular junction. Genetic experiments indicate at least some of this role is in the presynaptic cell, potentially through interaction with MAP1b message (Zhang et al., 2001
). Notably, several of the known mRNA targets of mammalian FMRP encode proteins active in the presynaptic apparatus including Munc13, semaphorin 3F, NAP-22, SEC-7, RAB-5, ID3 and Cadherin 11 (Brown et al., 2001
; Miyashiro et al., 2003
The developmental regulation of FXGs may reflect a linkage between neurogenesis and the expression of these granules. In hippocampal area CA3 as well as in cerebral cortex FXG expression peaks at approximately P15 and then falls off rapidly, with almost no FXGs detected after P60 (). Dentate granule neurons, which form the FXG-rich mossy fiber terminals in area CA3, show peak neurogenesis around P7 (Bayer and Altman, 1974
; Schlessinger et al., 1975
). In the developing cerebellar cortex, FXGs are only detected during the developmental time window when granule cells are born, migrate and make initial synaptic contacts with Purkinje cells (P6-P20; Supp. Fig. S5
) (Altman, 1969
; Miale and Sidman, 1961
). Finally, the time course of FXG expression during regeneration of OSN afferents showed a similar discrete window of peak expression following neurogenesis () (Schwob et al., 1995
). Taken together, these results argue that FXGs are expressed as newly generated neurons are making their initial synaptic contacts.
It should be noted that most neuronal types express few if any FXGs, regardless of developmental stage. For example, olfactory granule cells are born throughout life, but we did not detect FXGs in the external plexiform layer, where these cells form synapses (). Further, FXGs were not detected in hippocampal area CA1 at P7 or P15. Taken together, these results suggest that FXG expression is a function of both neuron age and type.
Our morphological and genetic experiments provide insights into the mechanisms of FXG formation and regulation. All FXGs express FXR2P (,). Further, FXR2P is necessary for FXG formation in all brain regions examined (). In contrast, FMRP is not required for FXG formation, but serves as a negative regulator of their expression (). Interestingly, while FMRP regulates the number and timing of FXG expression, its loss has no effect on the neuronal cell-type specificity of FXG expression. We were unable to test the role of FXR1P in depth, since mutants in this gene die at birth (Mientjes et al., 2004
) and this protein is present in multiple types of granules (). Nonetheless, the absence of FXR1P in most hippocampal and olfactory bulb FXGs of either wild type or fmr1 mutants suggests that this member of the Fragile X family may play a less prominent role in these structures. Finally, the levels of FMRP and FXR2P in the somatodendritic compartment were unaffected in the fxr2 and fmr1 mutants, respectively (; see also (Bakker et al., 2000
). Thus, the mechanisms underlying Fragile X protein expression in FXGs are distinct from those in the somatodendritic compartment.
Fmr1 mutations cause FXS and are the most common single gene basis for autism. A host of recent studies support the view that both FXS and autism spectrum disorders are synaptic diseases (Chao et al., 2007
; Huber et al., 2002
; Moretti et al., 2006
; Morrow et al., 2008
; Zoghbi, 2003
). Although the nature of the synaptic abnormalities are likely to be diverse, it seems likely that many of these defects will involve experience-dependent plasticity in selected neural circuits during development. For example, over 50% of autistic patients show regression, where they are apparently normal until approximately two years of age. Moreover, the best-characterized physical sign of autism spectrum disorder is a transiently enlarged brain during early childhood, which might reflect a deficit in axonal pruning (Aylward et al., 1999
; Courchesne et al., 2003
). Pruning defects – which must involve presynaptic elements – are also characteristic of FXS (Irwin et al., 2001
). We suggest that presynaptic defects, at least in part involving the FXG pathway, could be relevant to the etiology and treatment of FXS and autism.