Excess Protein Synthesis in FXS Patient Lymphoblastoid Cells Can Be Rescued with a p110β-Selective Inhibitor
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The fragile X mental retardation protein (FMRP) plays a key role for neurotransmitter-mediated signaling upstream of neuronal protein synthesis. Functional loss of FMRP causes the inherited intellectual disability fragile X syndrome (FXS), and leads to increased and stimulus-insensitive neuronal protein synthesis in FXS animal models. Previous studies suggested that excess protein synthesis mediated by dysregulated signal transduction contributes to the majority of neurological defects in FXS, and might be a promising target for therapeutic strategies in patients. However, possible impairments in receptor-dependent protein synthesis have not been evaluated in patient cells so far. Using quantitative fluorescent metabolic labeling, we demonstrate that protein synthesis is exaggerated and cannot be further increased by cytokine stimulation in human fragile X lymphoblastoid cells. Our previous work suggested that loss of FMRP-mediated regulation of protein expression and enzymatic function of the PI3K catalytic subunit p110β contributes to dysregulated protein synthesis in a mouse model of FXS. Here, we demonstrate that these molecular mechanisms are recapitulated in FXS patient cells. Furthermore, we show that treatment with a p110β-selective antagonist rescues excess protein synthesis in synaptoneurosomes from an FXS mouse model and in patient cells. Our work suggests that dysregulated protein synthesis and PI3K activity in patient cells might be suitable biomarkers to quantify the efficacy of drugs to ameliorate molecular mechanisms underlying FXS, and could be used for drug screens to refine treatment strategies for individual patients. Moreover, we provide rationale to pursue p110β-targeting treatments as potential therapy in FXS, and possibly other autism spectrum disorders.
Fragile X syndrome (FXS), the most common inherited intellectual disability, is caused by loss of function of the fragile X mental retardation protein (FMRP). The analysis of animal models has shown that absence of FMRP causes pathological changes in the regulation of basal and stimulus-induced protein synthesis in the brain (1, 2, 3, 4). These changes in neuronal protein expression are believed to underlie or contribute to most of the neuronal dysfunctions observed in FXS (5). FMRP is an mRNA binding protein shown to regulate translation, localization and stability of many target mRNAs (6). FMRP influences the expression of members of several different protein families, such as scaffolding proteins or proteins involved in receptor trafficking. However, evidence is emerging that FMRP has a major function in regulating neurotransmitter-induced signal transduction upstream of protein synthesis, which might cause the aberrant protein synthesis observed in the absence of FMRP (1,3,4,7, 8, 9). Pharmacological inhibition or genetic reduction of a few signal transduction pathways regulating protein synthesis, such as group 1 metabotropic glutamate receptors (mGlu1/5), glycogen synthase kinase 3 (GSK3), extracellular signal regulated kinase 1/2 (ERK1/2) and phosphoinositide-3 kinase (PI3K), were shown to rescue aberrant protein synthesis and several protein synthesis-dependent phenotypes in FXS mice [reviewed in (10,11)]. Of note, treatment with two different protein synthesis inhibitors rescued cognitive impairments in a Drosophila model for FXS (12). Taken together, these studies suggest that correcting dysregulated protein synthesis or defective signaling pathways regulating protein synthesis might be a promising therapeutic strategy for patients with FXS, and provided rationale for the initiation of several clinical trials [reviewed in (10)].
A major challenge of current FXS research is to refine and improve treatment strategies by identification of more specific and effective drugs that target the underlying pathomechanisms. Basic research in FXS animal models that further elucidates the molecular mechanisms regulated by FMRP could help to identify more potent drugs. In addition, drug screens in easily obtainable peripheral patient cells measuring FXS-specific bio-markers would accelerate the identification of even more efficient therapies, which might differ for individual patients (13). An assay quantifying ERK1/2 activation kinetics has been suggested and used as a biomarker in FXS clinical trials, however, the underlying mechanisms of the detected ERK1/2 dysfunctions are not fully understood (14, 15, 16). Studies in Fmr1 knockout (KO) mice suggest that the ERK1/2 pathway is hypersensitive to receptor activation, but the precise mechanisms remain obscure (1).
So far, there are no biochemical cell-based assays available that quantify a molecular function shown to be directly regulated by FMRP, and thus could be used in drug screens. Such assays would also be crucial to evaluate whether a drug used in a clinical trial for FXS ameliorates underlying molecular defects.
We have shown recently that FMRP regulates the mRNA translation and protein expression of the PI3K catalytic subunit p110β, leading to excess PI3K activity, downstream signaling and protein synthesis in Fmr1 KO mice (3). Excess p110β expression and activity could also be detected in cultured nonneuronal cells treated with siRNA to knockdown FMRP, implying that the molecular pathomechanism is not neuron specific. A broad spectrum PI3K inhibitor rescued several phenotypes in the mouse model (3). Based on these previous observations, we hypothesized that reduction of p110β subunit-specific PI3K activity might be an efficient therapeutic strategy in FXS and that the underlying molecular mechanism might be detectable in peripheral cells, such as lymphoblastoid cell lines from humans with FXS.
Here, using a quantitative and scalable fluorescent metabolic labeling assay, we show that protein synthesis rates are increased and dysregulated in FXS patient lymphoblastoid cells. We provide evidence suggesting that the underlying mechanisms observed in neurons, that is, increased p110β protein expression, excess p110β-specific PI3K activity and downstream signaling are recapitulated in patient nonneuronal cells. Furthermore, we show that a p110β-selective antagonist rescues excess protein synthesis in synaptic fractions from Fmr1 KO mice and in FXS patient lymphoblastoid cells, providing rationale for p110β-selective inhibition as potential novel therapeutic strategy for FXS. Moreover, our results suggest that, in the future, similar assays quantifying excessive protein synthesis might be suitable to screen for drugs targeting FXS-underlying pathomechanisms.
Materials and Methods
Drugs and Antibodies
TGX-221 (Selleck Chemicals, Boston, MA, USA) was dissolved in dimethyl sulfoxide (DMSO) (5 mmol/L). Human interleukin (IL)-2 (PeproTech, Rocky Hill, NJ, USA) was dissolved in 0.02 N HCl (106 units/mL). Anisomycin (Sigma- Aldrich, St. Louis, MO, USA) was dissolved in DMSO (25 mmol/L). phospho-Akt, Akt, phosphoS6 and S6 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA), p110β antibody for Western blotting was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), p110β antibody for immunoprecipitation was purchased from Millipore (Billerica, MA, USA). Tubulin antibody for Western blotting was purchased from Sigma-Aldrich, the β-tubulin antibody for immunocyto-chemistry was purchased from Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA, USA). The Cy2-coupled anti-mouse secondary antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).
Mice and Tissue Preparation
Male Fmr1 KO mice in C57BL/6J background and male wild-type (WT) littermates (The Jackson Laboratory, Bar Harbor, MA, USA) were used at postnatal d 17–20. Synaptoneurosomes (SNS) were prepared as described previously (4). The animal protocol was approved by the Institutional Animal Care and Use Committee, Emory University, and complied with the Guide for the Care and Use of Laboratory Animals (17).
Lymphoblastoid Cell Lines and Cell Culture
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) from healthy controls (GM10851: male, Caucasian, 52 years, unaffected [Coriell Institute, Camden, NJ, USA], called “Ctr”; J1: male, unaffected , called “Ctr-b”), and FXS (GM03200: male, Caucasian, 34 years, hypermethylated CGG repeat expansion, affected [Coriell Institute], called “FXS”; DM316: male, Caucasian, 3 years, nucleotide deletion within the FMR1 gene, affected [19,20], called “Fdel”) patients were cultured in RPMI supplemented with 10% fetal bovine serum and antibiotics at 37°C and 5% CO2 (cell density ∼500,000 cells/mL).
Radioactive PI3K Assays
Synaptoneurosomes (SNS) from one mouse cortex or 1–2 million LCLs, respectively, were used per experiment. LCLs were washed once with ice-cold PBS, and lysed in ice-cold PI3K assay lysis buffer (50 mmol/L Tris-HCl, pH 7.4, 40 mmol/L NaCl, 1 mmol/L EDTA, 0.5% Triton X-100, 1.5 mmol/L Na3VO4, 50 mmol/L NaF, 10 mmol/L sodium pyrophosphate, and 10 mmol/L sodiumglycerol phosphate, supplemented with proteinase inhibitors). SNS were lysed as described previously (3). 100 µg protein was used for subsequent immunoprecipitation with a p110β-specific antibody. PI3K activity assays and thin-layer chromatography were performed as described previously (3,21).
ELISA PI3K Assays
PI3K activity enzyme-linked immunosorbent assays (PI3-Kinase Activity ELISA: Pico, Echelon Biosciences, Inc, Salt Lake City, UT, USA) were performed with p110β protein immunoprecipitated from SNS or LCLs (as above), following the manufacturer’s instructions, with these modifications: kinase reactions were conducted in 60 µL volume with 75 µmol/L adenosine triphosphate (ATP), 1 mmol/L dithiothreitol (DTT) and 15 µmol/L phosphatidylinositol (3,4) biphosphate diC8 for 3 h. Reactions were stopped with 2.4 mmol/L ethylenediaminetetraacetic acid (EDTA). A standard curve was used to quantify the amount of phosphatidylinositol (3,4,5) triphosphate present after the reaction.
PhosphoS6 and S6-Specific ELISAs
Equal amounts of protein were analyzed for phosphoS6 and S6 protein levels using PathScan® phosphoS6 or total S6 ribosomal protein sandwich ELISA antibody pairs, respectively, according to the manufacturer’s protocol (Cell Signaling Technology). Ratios of phosphoS6 and S6 in the same samples were compared and quantified.
Metabolic Labeling in SNS
Metabolic labeling in SNS was performed as described previously (3,4). Where indicated, SNS were incubated for 10 min with 1 µmol/L TGX-221 or an equal amount of dimethyl sulfoxide (DMSO) before labeling.
Bioorthogonal Labeling and Click-iT® Chemistry
Bioorthogonal labeling in LCLs and subsequent Click-iT chemistry (Life Technologies Invitrogen, Carlsbad, CA, USA) were performed according to the manufacturer’s protocol. Briefly, LCLs were kept for 1 h in methionine-free media. Where indicated, TGX-221 or anisomycin was added to the methionine-free medium after 30 min, or IL-2 was added after 45 min. Cells were pulsed with 50 µmol/L L-azidohomoalanine for a total time of 1 h. After 45 min, cells were plated on poly-lysine-coated coverslips, washed once with PBS 15 min after plating, and fixed with 4% paraformaldehyde. Click-iT reaction was performed as described in the manual with 5 µmol/L tetramethylrhodamine (TAMRA) alkyne. Cells were further processed for immunocytochemistry with a β-tubulin antibody and a Cy2-labeled secondary antibody.
Western Blot Analysis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and densitometric analysis using ImageJ software (NIH) were performed as described previously (3,4). Signals for phospho antibodies were normalized to the signal of the respective total antibody and tubulin on the same blot, p110β- p110α-, p110δ- and FMRP-specific signals to tubulin-specific signal on the blots.
Image Acquisition and Analysis
Images were acquired with a wide-field fluorescent Nikon Eclipse inverted microscope equipped with a cooled chargecoupled device (CCD) camera and built-in Z-drives. Z-Stacks were deconvolved using AutoQuant X (Media Cybernetics, Bethesda, MD, USA). Fluorescent signal intensities were quantified with Imaris Software (Bitplane, Zurich, Switzerland). Total fluorescent intensities for both channels were background subtracted and measured for the entire stack. Background was determined as fluorescent intensity in an area of the image that did not contain cells. Approximately 15 images per condition and experiment were acquired and analyzed. Fluorescent signal intensities of newly incorporated amino acids were normalized to tubulin signal.
Dysregulated Protein Synthesis in FXS Patient Lymphoblastoid Cells
A prominent phenotype of the FXS mouse model is increased and stimulusinsensitive protein synthesis, which leads to impairments of several protein synthesis-dependent forms of synaptic plasticity (5,6). To test whether dysregulated protein synthesis in the absence of FMRP can be detected in nonneuronal, peripheral cells from human patients with FXS, we quantified protein synthesis rates in lymphoblastoid cell lines (LCLs) from a healthy control (called “Ctr” in figures and legends) and a patient with FXS that carried the full mutation, that is, completely methylated trinucleotide expansion in the FMR1 gene (subsequently called FXS cells). We chose LCLs, because they are a virtually unlimited source of patient material, and do not require an invasive biopsy. Furthermore, lymphocytes have been recognized as a valid model for cell signal transduction, because of the variety of different signaling pathways present in these cells (22). This suggests that they are especially suitable to analyze diseases like FXS that are characterized by dysregulated signal transduction. To quantify newly synthesized proteins in LCLs, we used a fluorescent metabolic labeling method (Click-iT technology, Invitrogen), which employs bioorthogonal amino acids that can be labeled with fluorescent molecules via alkyne-azide-based Click-iT chemistry (23). Here, we used a fluorophore-coupled alkyne and the bioorthogonal amino acid azidohomoalanine to visualize and quantify newly synthesized proteins. Either pretreatment with the protein synthesis inhibitor anisomycin, or omitting the bioorthogonal amino acid in the reaction, significantly reduced the fluorescent intensity, indicating that the fluorescent signal indeed represented newly synthesized proteins, and was dependent on the presence of azidohomoalanine (Figure 1A, n = 5, *P = 0.034, #P = 0.028, 1-way analysis of variance (ANOVA) with Games-Howell post hoc analyses). In contrast, tubulin counterstaining was unaffected by these treatments. Using this method, we detected increased basal protein synthesis in LCLs from a patient with FXS (Figure 1B). Furthermore, stimulation with IL-2 (100 U/mL, 15 min prior labeling), which significantly increased protein synthesis rates in control cells, did not induce protein synthesis in FXS cells, but led to significantly reduced translation rates [Figure 1B, n (Ctr untreated) = 60, n (Ctr IL-2) = 58, n (FXS untreated) = 57, n (FXS IL-2) = 58; 4 independent experiments, 2-way ANOVA shows significant interaction of treatment and genotype, Bonferroni post hoc analyses, *P = 0.005, #P = 0.002, ≠P = 0.001]. These results suggest that FMRP regulates cell surface receptor-mediated protein synthesis in peripheral lymphocytes, leading to dysregulated protein synthesis in LCLs from FXS patients, similarly to what has been observed in neurons from Fmr1 KO mice (1,3,4,9).
Increased PI3K Activity and Downstream Signaling in FXS Patient Lymphoblastoid Cells
Increased Protein Expression of the PI3K Catalytic Subunit p110β in Fragile X Patient LCLs
A p110β-Selective Inhibitor Rescues Excess Protein Synthesis in Fmr1 KO Mouse SNS and in FXS Patient LCLs
A major challenge for the development of disease-targeted therapeutic strategies for FXS and other cognitive and autism spectrum disorders is to provide a reliable biomarker assay that quantifies improvements in the underlying pathological mechanisms in easily accessible patient cells as an additional outcome measure for use in human clinical trials. While behavioral and cognitive tests are important to evaluate the overall benefits of the therapeutic strategy, biomarker assays targeted at the underlying molecular defects and applicable to accessible peripheral cells will help to optimize and refine drug therapies.
Our data suggest that excess protein synthesis and PI3K activity in LCLs from patients with FXS might be potential biomarkers that quantify molecular defects directly caused by the absence of FMRP. This assumption is corroborated by our observation that the underlying pathomechanisms occurring in neurons are recapitulated in peripheral lymphoblastoid cells. We could detect increased and dysregulated protein synthesis in FXS patient lymphoblastoid cells, similar to what we and others have observed in neuronal synaptic fractions and brain slices from Fmr1 KO mice (1, 2, 3, 4,25). Furthermore, we show that PI3K activity and downstream signaling is upregulated in these cells, likewise resembling observations in Fmr1 KO mice (3,26). We have shown previously that FMRP controls PI3K activity by regulating at least two of its target mRNAs, namely p110β and PIKE-L (3). FMRP limits the expression of these proteins (3,26), leading to increased and stimulus-insensitive PI3K activity, which might underlie dysregulated synaptic protein synthesis in the absence of FMRP (3). Here, we demonstrate that p110β levels are also increased in LCLs from two different patients with FXS, suggesting that the pathological molecular mechanisms that occur in neurons are recapitulated in peripheral lymphocytes.
A previous study has shown that polysomal profiles of several potential FMRP target mRNAs are abnormal in LCLs from patients with FXS, suggesting their dysregulated translation (18). However, to our knowledge, no study has reported dysregulated basal and stimulus-induced general protein synthesis in cells from human patients with FXS so far. The absence of IL-2-induced stimulation of protein synthesis in LCLs from a patient with FXS suggests that an intracellular signaling pathway downstream of IL-2, which regulates protein synthesis, is over-active and insensitive to stimulation in these cells. Interestingly, contrary to the effect in healthy control cells, we observed a significant reduction of protein synthesis rates in patient LCLs upon IL-2 stimulation. At present, we cannot explain this observation, but speculate that it might be caused by a negative feedback mechanism, which is still functional in the patient cells. Cytokines such as IL-2, play an important role for cell proliferation and survival. The lack of a proper response to these prosurvival stimuli in the absence of FMRP might therefore contribute to the reduced cancer rates that have been reported for patients with FXS (27).
We show that a p110β-selective antagonist can rescue increased protein synthesis in patient LCLs to control levels, reporting a significant interaction between genotype and treatment, which suggests that increased p110β-mediated PI3K activity and downstream signaling underlies the dysregulated protein synthesis in these cells. Furthermore, together with our observation of excess p110β-associated PI3K activity, this suggests that targeting the p110β subunit might be a potential therapeutic strategy for FXS. Of note, our data show that in contrast to p110β, the two other class 1A PI3K catalytic subunits, p110α and δ, are unchanged in LCLs from FXS patients, thereby corroborating the potential benefits of a p110β-targeted therapy in FXS. As mentioned above, previous work has suggested that FMRP also regulates another modulator of PI3K activity, the PI3K enhancer PIKE (3,26). Future studies could analyze whether PIKE is also dysregulated in cells from patients with FXS and thus might serve as valuable therapeutic target. However, in contrast to p110β, no specific antagonists of PIKE are currently available for the use in humans, suggesting that pursuing p110β-specific therapeutic strategies might have a more immediate beneficial effect for patients with FXS.
Previous studies have corroborated the importance of PI3K function for IL-2-induced protein synthesis in T-lymphocytes by showing that a broad-spectrum PI3K antagonist leads to a strong reduction of protein synthesis rates in T-cells cultured in the presence of IL-2 (28). Here, we show that even a high dose of the p110β-subunit selective antagonist TGX-221 (10 µmol/L, 1000× over IC50) does not significantly affect basal protein synthesis rates in healthy control LCLs. The lack of effect on control cells suggests that under healthy conditions, p110β (or general PI3K) activity might only be required for stimulus-induced protein synthesis. In the future, it will be interesting to examine the effect of p110β-selective antagonists on IL-2-induced protein synthesis in LCLs from healthy controls and FXS patients. An alternative explanation for the absence of an effect of the p110β antagonist on protein synthesis in healthy control cells might be that other class 1 p110 catalytic subunits can compensate for the loss of p110β activity to sustain protein synthesis in normal LCLs. It was shown previously that genetic or pharmacological inhibition of one or more class 1 PI3K catalytic subunits in immortalized leukocytes and fibroblasts leads to functional compensation by the other subunits to preserve cell survival and proliferation (29). In line with this observation, results from cancer research suggest that a monotherapy using single subunit-selective PI3K antagonists might not be efficient to stop tumor growth due to functional compensation by other catalytic subunits (30). This further supports the applicability of therapies targeting p110β in patients with FXS, because it suggests that basic cell functions, such as mitosis and cell survival might not be affected by p110β-selective antagonists.
Our observation that neuronal disease mechanisms of FXS, such as dysregulated PI3K-mediated protein synthesis, are detectable in nonneuronal, peripheral cell lines from human patients has two important implications: firstly, lymphoblastoid cells and peripheral blood lymphocytes might be suitable as a biomarker tool for FXS that detects underlying pathomechanisms, and secondly, they may thus be used for drug screens to identify disease-targeted therapeutics. This hypothesis is corroborated by our study showing that a p110β-selective antagonist, TGX-221, can rescue excess protein synthesis not only in synaptic fractions from the FXS mouse model, but also in patient lymphoblastoid cells. Lymphoblastoid cell lines are a valuable and efficient tool for personalized drug screens to identify therapeutics in specific patients. They are easy to obtain and are a virtually unlimited source of patient material. Of note, two of the here described assays, the PI3K activity ELISA and the Click-iT protein synthesis assay, are colorimetric or fluorescent, respectively, and thus suitable for larger-scale applications needed to perform drug screens.
Quantification of PI3K signaling and protein synthesis rates in peripheral lymphocytes might also have a broader applicability as a biomarker beyond FXS, for example, for autism research. Autism spectrum disorders (ASDs) are a highly variable group of disorders. The majority is idiopathic, and only a few monogenic defects have been shown to lead to autism (31). FXS is the most frequent monogenic ASD, but of note, it is not the only one that is characterized by dysregulated signaling through PI3K/mTOR. Another example is tuberous sclerosis, which is caused by mutations or deletions in the tuberous sclerosis complex 1/2 (TSC1/2). TSC1/2 regulates mTOR signaling, and approximately 25–50% of patients with tuberous sclerosis develop ASD (32,33). Furthermore, mutations in phosphatase and tensin homologue on chromosome 10 (PTEN) have been associated with several forms of ASD (34). PTEN regulates the PI3K/mTOR pathway by dephosphorylating phosphoinositide-(3,4,5)-triphosphate (PIP3), the product of PI3K. Apart from these known monogenic causes for ASD, a considerable number of chromosomal copy number variations associated with ASDs were shown to affect genes within the PI3K/mTOR pathway (35). The high frequency of PI3K/mTOR defects in ASDs with known underlying gene defects suggests that dysregulated PI3K/mTOR signaling and/or protein synthesis might also be the cause of other, so far idiopathic, ASDs. Interestingly, a recent study showed that a mouse model for Rett syndrome, a rare form of autism caused by mutations in the gene encoding the epigenetic regulator methyl CpG-binding protein 2 (MeCP2), displays reduced PI3K/mTOR signaling and protein synthesis (36), suggesting that mutations or defects in other pathways might have an effect on PI3K/mTOR signaling. Moreover, aberrant neuronal protein synthesis was hypothesized to be a shared pathomechanism of several inherited ASDs (37). Taken together, this suggests that the here described assays detecting aberrant PI3K/mTOR signaling as well as dysregulated protein synthesis in peripheral patient cells might also be useful biomarker tools for other ASDs apart from FXS. In particular, such assays could be used for screens using lymphoblastoid cell lines from patients with idiopathic ASD to identify those who would qualify for a PI3K/mTOR-based therapy. Several collections of ASD lymphoblastoid cell lines are already available for researchers, for example, from the Simons Simplex Collection (SSC) or the Autism Genetic Research Exchange (AGRE).
Our results suggest that quantitative analysis of PI3K activity and protein synthesis rates in LCLs from patients with FXS may be a valuable tool for drug screens to identify more potent therapeutic strategies for FXS and other ASDs that directly target underlying mechanisms. Together with our previous work demonstrating that a broad spectrum PI3K inhibitor can rescue several phenotypes in Fmr1 KO mouse neurons (3), the current study also suggests that PI3K subunit-selective antagonists might be a valuable therapeutic treatment for FXS. Several different forms of cancers are caused by multiple mutations within the PI3K signaling pathway, and subunit-selective PI3K inhibitors have been developed and are currently being tested for the treatment of specific tumors (30). In the future, FXS and autism research could greatly benefit from these developments in the field of cancer research, where PI3K targeting drugs are already being tested for their safety and applicability in human patients.
The authors would like to thank M Kim and A Poopal for excellent technical assistance, and J Mowrey for valuable advice on LCL culturing. Lymphoblastoid cell lines from FXS patients and healthy controls were a kind gift from S Warren (Emory University). The authors thank S Warren for helpful discussions. The authors thank S Swanger for critically reading the manuscript, and all members of the Bassell lab for helpful discussions. This work was supported by a postdoctoral fellowship from FRAXA (to C Gross), the NIH Grant MH085617 (to GJ Bassell), the Emory/Baylor Fragile X Center Grant 3P30HD024064 (to GJ Bassell), and a Suzanne and Bob Wright Trailblazer Award form Autism Speaks (to GJ Bassell).
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