Is metabotropic glutamate receptor 5 upregulated in prefrontal cortex in fragile X syndrome?

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is associated with multiple cognitive, behavioral, and neuropsychiatric abnormalities [1]. The disorder is caused by expansion of the CGG trinucleotide repeat in the 5′ untranslated region of the fragile X mental retardation 1 (FMR1) gene located at the distal end of the long arm of the X chromosome. The length of repeat expansion determines the expression of FXS disorder: alleles containing ≤44 repeats are considered ‘normal’, those containing between 44 and 54 repeats form a ‘gray zone’, those containing between 55 and 200 repeats are ‘premutation carriers’, and alleles with ≥200 repeats are ‘full mutations’ [2]. Only the full mutation leads to hypermethylation of the FMR1 promoter and consequent transcriptional silencing of the FMR1 gene that encodes fragile X mental retardation protein (FMRP) [3]. FMRP is a cytoplasmic RNA-binding protein known to repress the translation of specific messenger RNAs at neuronal synapses, thereby regulating expression of various synaptic proteins [4, 5]. FMRP thus plays a major role in synaptic signal transduction, and its loss dysregulates neuronal signaling pathways. One of the key signaling pathways believed to be dysregulated in FXS as the result of a loss of FMRP is mediated by metabotropic glutamate receptors (mGluRs), particularly subtype 5 (mGluR5). Studies have demonstrated that dysregulated mGluR5 signaling results in uncontrolled activation of synaptic protein synthesis, owing to the loss of normal repressor action of FMRP on protein synthesis [6, 7].

Recent studies using various animal models of FXS have suggested that many features of the FXS phenotype, including behavioral abnormalities, cognitive deficits, and altered dendritic spines, may be attributable to exaggerated mGluR5 signaling. Some studies that sought to suppress mGluR5 signaling by genetic knockdown or by using negative allosteric modulators of mGluR5, such as 2-methyl-6-(phenylethynyl)-pyridine (MPEP), corrected most of these phenotypic features in mouse models of FXS [8, 9]. Although evidence from rodent studies suggests that mGluR5 signaling is increased in FXS, mGluR5 density has yet to be examined in the postmortem brain tissue of individuals with FXS. Answering this question is important because measuring mGluR5 levels could allow categorization of FXS patient subgroups based on receptor density and, relatedly, help identify those FXS patients who might be promising candidates for treatment with mGluR5 allosteric modulators.

To determine whether mGluR5 levels are altered in FXS, we measured mGluR5 receptor density via radioligand binding assays, and expression levels via Western blotting. These measures were ascertained in the postmortem brain tissue of FXS patients and premutation carriers, and compared with measures ascertained in age- and sex-matched healthy controls.

Using in-vitro radioligand binding assays and Western blotting, we found a marginally significant increase in mGluR5 receptor density and a statistically significant increase in mGluR5 expression from the postmortem prefrontal cortex of FXS patients or carriers, compared with age- and sex-matched controls without neurological disorders. Highly significant differences in mGluR5 density could not be measured, mainly because of the limited availability of postmortem human brain tissues from FXS patients; nevertheless, the results raise the important question of whether mGluR5 density is indeed increased in the brains of individuals with FXS. While numerous preclinical studies have linked increased mGluR5 receptor signaling to various syndromic features of FXS [6, 7], this study is the first to measure mGluR5 receptor density or expression levels directly in the human brain of individuals with FXS or carriers. It is important to note, however, that several reports have described changes in mGluR5 expression in patients with autism or in FXS model mice [13‐15].

Notably, FXS is the most common single-gene cause of autism, accounting for 2 to 6% of all cases of autism, and numerous studies suggest that similar sets of proteins are dysregulated in both autism and FXS, including mGluR5 [14, 15]. For instance, Fatemi and colleagues [13] recently reported a statistically significant increase in mGluR5 protein levels in postmortem tissue samples from the superior frontal cortex of autistic children, a finding that was not significant in autistic adults. Interestingly, the same study also found significantly lower levels of FMRP in autistic adults, despite the fact that no records indicated that any of the individuals were tested for or diagnosed with FXS. Considering that autism and FXS are intertwined at the molecular level [15], the findings of the autism study are in agreement with the significantly increased mGluR5 expression observed in the present study in human prefrontal cortex of individuals with FXS.

Reports of mGluR5 expression levels in brain samples from animal models of FXS are limited. Giuffrida and colleagues [16] recently found reduced mGluR5 expression in the detergent insoluble fraction of synaptic plasma membranes isolated from forebrains of Fmr1 knockout mice compared with wild-type mice; however, the mRNA and total mGluR5 protein expression in total membrane protein preparation were comparable between the two mouse groups. The study attributed the altered mGluR5 distribution to a complex interaction with Homer scaffolding proteins. Another study found that mGluR5 levels were significantly reduced in the lateral geniculate nucleus of Fmr1 KO mice [17]. In the Fmr1 KO hippocampus, however, Western blot analysis revealed no difference in mGluR5 protein expression [8]. In contrast to these reports, our results indicated significantly elevated expression of mGluR5 in human prefrontal cortex samples of FXS patients or carriers compared with healthy controls. This discrepancy might be due to several key factors, including differences between species, between the brain regions studied, and between experimental procedures.

Presently, we can only speculate about the mechanisms that might explain the increased mGluR5 levels observed in prefrontal cortex of FXS patients or carriers. The absence of FMRP in FXS has been implicated for both decreases and increases of different mRNA pools, owing to translational repression or activation of diverse mRNAs [5]. Although FMRP is not believed to be directly associated with Grm5 mRNA, its association with other mRNAs may indirectly upregulate mGluR5 levels [18]. However, overexpression of mGluR5 secondary to absence of FMRP may lead to reduced 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid/N-methyl-D-aspartate receptors as well as alterations in several other signaling molecules; thus, FXS is currently considered a disease of dysregulated neuronal signaling [5].

Preclinical studies in FXS model mice suggest that synaptic protein synthesis is dysregulated by hypersensitivity to mGluR5 signaling in FXS. Indeed, reducing mGluR5 signaling by genetic knockdown or by administering mGluR5 negative allosteric modulators, such as MPEP, improved cognitive and behavioral measures in a mouse model of FXS [8, 9]. Negative allosteric modulators of mGluR5 are presently being considered as potential treatments for FXS. In a pilot, open-label, single-dose, Phase I study with fenobam, a negative allosteric modulator of mGluR5 comparable to MPEP, 9 of 12 subjects exhibited calmed behavior within one hour of dosing, and 6 of 12 subjects had improved prepulse inhibition [19]. Another trial administered AFQ056, an mGluR5 negative allosteric modulator, for several weeks. Although the overall therapeutic efficacy in FXS was negative, a post-hoc analysis found significant therapeutic efficacy in 7 patients who had full FMR1 promoter methylation, but no response in 18 patients with partial promoter methylation [5, 20]. Full methylation would be expected to cause complete silencing of the FMR1 gene with minimal expression of FMRP (comparable to the mouse KO model), whereas partial methylation would be associated with variable FMRP levels; this could perhaps affect the degree of mGluR5 hyperactivation and, thus, treatment response. Future treatment trials are likely to prospectively separate patients with and without hypermethylation of the FMR1 gene.

This study was associated with several limitations. First, the increased mGluR5 binding and expression observed in human prefrontal cortex samples from individuals with FXS patients or carriers might not reflect actual disease status; instead, it could result from treatment with antipsychotic medications. FXS patients are commonly treated with antipsychotic drugs in childhood and early adulthood, though usually not in late adulthood [21]. Medication status, which might have helped elucidate this issue, was not available for most of these tissue samples. Nevertheless, one study of rats chronically treated with the antipsychotic drugs haloperidol or risperidone found that mGluR5 expression in the frontal cortex was not altered, although significant changes were observed in subcortical regions, such as the caudate nucleus [22]. Second, tissue samples were only obtained from white male subjects. Thus, the presence of any potential sex-related or ethnic variation in mGluR5 binding or expression cannot be ruled out. The mutation responsible for FXS is known to affect men and women at equal rates, but owing to lyonization or X chromosome inactivation, FMRP levels are higher in women, with commensurately fewer and less severe physical, cognitive, and behavioral phenotypes [1]. Third, most tissue samples were from full mutation cases of FXS; only four samples were from FXS carriers. When FXS carrier samples were excluded from the analysis, both mGluR5 density and expression were higher, but only mGluR5 density was marginally significant between FXS and control samples. Unlike FXS patients with full mutation, premutation carriers are known to express low levels of functional FMRP. However, the carriers are known to have increased transcription of FMR1 mRNA, causing RNA toxicity by sequestering several proteins to form intracellular inclusion bodies [2]. These inclusion bodies have been found to contain several RNA-binding proteins, such as hnRNPA2/B1, CUGBP1 and Sam68 [23, 24]. Currently, it is not known whether RNA toxicity in carriers is associated with sequestration of receptor protein, such as mGluR5. Thus, the current finding of mGluR5 density or expression warrants further study in a greater number of subjects who are only FXS full mutation or FXS carriers. Furthermore, for most samples, neither information on the number of CGG repeats nor the methylation status in the FMR1 gene was available. The availability of this information would have allowed us to assess the correlation between methylation or CGG repeats and mGluR5 binding or expression, given that epigenetic modification of the FMR1 gene has been implicated in differential therapeutic response to mGluR5 antagonists in individuals with FXS [20]. Finally, owing to the limited availability of tissue samples, we were only able to evaluate mGluR5 measures in the prefrontal cortex of humans; other subcortical regions known to contribute to the various cognitive and psychomotor effects observed in FXS (for example, hippocampus, amygdala, and cerebellum) might have yielded different mGluR5 measures.

Despite these limitations, the marginally increased mGluR5 receptor density and expression observed in this study in individuals with FXS provide a strong rationale for measuring mGluR5 in living patients using positron emission tomography (PET) [25, 26]. Such studies could address many questions raised in the postmortem study. First, PET could determine whether increased mGluR5 binding is present in live subjects and in all brain areas, including the prefrontal cortex. However, PET typically does not separately measure receptor density (B_max) and affinity (1/K_D). Instead, PET uses low tracer doses of the radioligand, which measures the product of these two variables, called the binding potential (BP = B_max/K_D). In our postmortem study, both BP and B_max were increased (16% and 17%, respectively) with marginal statistical significance (P = 0.058 and 0.092, respectively) for FXS patients compared with controls (Table 2). Even after excluding four FXS carriers, both BP and B_max were increased (17% and 19%, respectively) with marginal statistical significance (P = 0.071 and 0.094, respectively) for FXS patients compared with controls. Because PET can only measure BP, based on 80% power and two-tailed P ≤ 0.05, the required sample size for PET to detect a significant elevation in BP in the prefrontal cortex of FXS patients would be approximately 45 subjects. Second, the records from the postmortem samples contained inadequate information about prior drug treatment, which may have upregulated mGluR5. By selecting live, unmedicated subjects, PET imaging can more directly assess the possible confound of concomitant medications. Third, baseline mGluR5 levels could be correlated to any response to treatment with mGluR5 negative allosteric modulators. For example, would those subjects with the highest elevation of mGluR5 have the greatest response to this antagonist treatment?