The quest for fragile X biomarkers

A biomarker is a measurable and quantifiable biological characteristic that can serve as an indicator of healthy or pathological processes. For example, HDL and LDL are biomarkers for cardiovascular health and autoantibodies are biomarkers for autoimmune disease. Biomarkers are extremely useful in evaluating the clinical benefit of pharmaceutical interventions. A good biomarker assay will be sensitive, specific, rapid, simple to perform, inexpensive and applicable to easily obtained sample material. There is an urgent need to develop such biomarkers for fragile X syndrome (FXS).

FXS is the most common form of inherited mental retardation with a frequency of 1 in 2,500 births [1]. FXS results from a mutation in the fragile X mental retardation-1 (FMR1) gene, which was discovered by Drs. Ben Oostra, David Nelson and Stephen Warren in 1991. The FMR1 gene codes for fragile X mental retardation protein (FMRP), an RNA binding protein that plays a critical role in dendrite development. Thus, the absence of FMRP in FXS has a profound effect on synaptic plasticity. The clinical symptoms of FXS include intellectual disability, attention deficit and hyperactivity, anxiety, autistic behaviors, sensory integration problems, speech delay, seizures, hyperextensible joints, hypotonia, postpubescent macroorchidism, flat feet and vertical maxillary excess with protruding ears [2]. There is currently no cure or approved medication for FXS although various drugs target specific disease symptoms and a large number of therapeutics are in various stages of clinical development.

ERK is a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. ERK signaling can be activated by either protein tyrosine-linked receptors or by G protein-coupled receptors with signaling propagated through a series of phosphorylation reactions. Although there are contradictory results regarding basal phospho-ERK levels and mGluR-induced phosphorylation of ERK in FXS models, a specific inhibitor of the upstream mitogen-activated protein kinase kinases 1 and 2 (MEK1/2) eliminated audiogenic seizure activity in Fmr1 ^KO mice [7]. Findings from the Greenough laboratory suggest that the early phase kinetics of ERK activation in lymphocytes is delayed in FXS subjects and could serve as a disease biomarker [8]. ERK activation rates normalize in response to lithium and riluzole treatment [9],[10].

PI3Ks are a family of enzymes involved in cell growth, proliferation, differentiation, motility, survival and intracellular trafficking. The catalytic p110β subunit of class I PI3Ks activates protein kinase B (PKB, aka Akt), which is part of the mammalian target of rapamycin (mTOR) signaling pathway. The Zukin laboratory has shown that p110β as well as mTOR phosphorylation and activity are elevated in juvenile Fmr1 ^KO mice [11]. Gross and Bassell demonstrated that FMRP regulates the synthesis of p110β and that peripheral lymphocytes from FXS patients exhibit excessive PI3K activity as well as protein synthesis levels [12]. They utilized an ELISA-based colorimetric assay to detect PI3K activity and a fluorescent metabolic labeling assay to detect protein synthesis in FXS lymphocytes. Both methods could be adapted for clinical evaluation of PI3K activity and protein synthesis levels in FXS lymphocytes in response to drug treatment.

MMPs are involved in the breakdown of the extracellular matrix during processes such as embryonic development, wound healing and learning and memory. MMP-9 is involved in activity-dependent reorganization of dendritic spine architecture. MMP-9 mRNA is part of the FMRP complex, and translation of MMP-9 is increased at Fmr1 ^KO synapses [13]. The Ethell laboratory found that the antibiotic minocycline decreases MMP-9 in the hippocampus of Fmr1 ^KO mice while promoting dendritic spine maturation and improving anxiety and strategic exploratory behavior [14]. Minocycline also prevents all neuroanatomical defects in FXS flies and improves language and social communication skills, anxiety, attention, irritability, stereotypy, hyperactivity and inappropriate speech in humans [15]–[17]. The pro- and active-forms of plasma MMP-9 are substantially elevated in FXS individuals compared to typically developing age-matched controls although a significant overall correlation between reduced MMP-9 activity and observed improvement in the Clinical Global Impression-Improvement (CGI-I) was not observed [18]. The preliminary analysis consisted of plasma samples from ten subjects who received minocycline for 3 months with blood samples collected at baseline and after treatment. Six of the ten subjects exhibited some decrease in MMP-9 activity after minocycline treatment and the remaining four showed no change. Five of six patients with decreased MMP-9 activity exhibited improvement in the CGI-I. Thus, a larger study is required to determine if MMP-9 is a viable blood-based biomarker to monitor minocycline efficacy in FXS.

APP and metabolites including amyloid-beta (Aβ) are potential FXS biomarkers. We found that FMRP binds to and regulates the translation of App mRNA [19]. In the absence of FMRP (Fmr1 ^KO mice), APP and Aβ are overexpressed. Genetic knockout of one App allele in the Fmr1 ^KO mice rescues many disease phenotypes including seizures, anxiety, the ratio of mature versus immature dendritic spines and mGluR-LTD [20]. In human blood plasma, the level of Aβ_1-42 was significantly reduced in full-mutation FXS adult males compared to age-matched controls while APP and Aβ_1-40 levels were not altered. These data suggest that Aβ_1-42 may be a plausible blood-based biomarker for FXS. APP and Aβ are currently under evaluation as blood-based biomarkers in a prospective open-label trial of acamprosate in FXS youth and preliminary results indicate that APP levels are normalized in response to drug treatment [4]. Accumulating evidence from the Lahiri laboratory shows that Aβ_1-40, Aβ_1-42 and sAPPβ levels are decreased in plasma of youth with severe autism compared to controls whereas sAPPα levels are elevated [21]. In total, these data suggest that there may be age-dependent differences in APP expression and processing and that these proteins may be valuable biomarkers for both FXS and autism. An important caveat in developing Aβ as a blood-based biomarker is that anticoagulants appear to alter APP processing; thus, blood collection procedures need to be standardized [22]. The advantage of APP and Aβ as blood-based biomarkers for FXS, as opposed to measuring the activity of the aforementioned signaling molecules, is the stability of the proteins in serum.

In addition to the biomarkers discussed above, other potential candidates include brain-derived neurotrophic factor (BDNF), p70 ribosomal subunit 6 kinase 1 (S6K1), and cytokine and chemokine profiles. Erickson and colleagues observed increased BDNF levels in a pilot FXS trial testing acamprosate [23]. Hoeffer and colleagues found increased phosphorylation of S6K1 in FXS lymphocytes [24]. Ashwood and colleagues found altered plasma cytokine and chemokine levels in FXS subjects; specifically, they observed elevated interleukin-1 alpha (IL-1α), regulated on activation normal T-cell expressed and secreted protein (RANTES) and 10 kDa interferon gamma-induced protein (IP-10) compared to typically developing controls [25].

In summary, there are numerous drugs exhibiting promise in preclinical testing for FXS but no validated blood-based biomarkers or behavioral outcome measures for drug efficacy testing. This mini-review highlights research findings demonstrating that ERK activation rates, PI3K and MMP-9 activity levels, and APP and metabolite levels are potential blood-based biomarkers for FXS. These candidate biomarkers are at early stages of validation and deserve further investigation.