The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. Fragile X syndrome is the most prevalent inheritable mental retardation. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and is proposed to act as a translation regulator of specific mRNA. Despite extensive research, the mechanism by which FMRP loss leads to the fragile X syndrome remains unclear. Thus, there is high interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies of the protein function. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and dialysis of full-length FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing for the further study of the direct effects of these modifications individually and simultaneously. Additionally, FMRP has been shown to undergo alternative splicing, with one of the splicing sites in close proximity to the FMRP domain shown to be involved in binding G quadruplex mRNA with high affinity and specificity. We have analyzed how naturally occurring truncations in the FMRP sequence affect its RNA binding affinity, by applying the expression, purification and dialysis process to the second and third longest FMRP isoforms, followed by subsequent analysis of the G quadruplex mRNA binding properties by fluorescence spectroscopy. Our results show that as FMRP gets truncated by alternative splicing, its mRNA binding affinity increases. To test a model we proposed for FMRP translation regulation activity, we developed a luciferase reporter gene construct that contains the G quadruplex structure in the mRNA 5���-untranslated region. Using luminescence spectroscopy to analyze luciferase translation, we showed that low levels of full-length FMRP reduces luciferase translation, and as the concentration of full-length FMRP increases the luciferase translation increases. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry; / PhD; / Dissertation;

Fragile X Syndrome is the most common form of heritable cognitive disability. It is caused by a genetic mutation that leads to a lack of protein from the FMR1 gene. This protein (FMRP) is used to regulate the translation of many other proteins, thereby leading to a wide range of effects. Because the origin of this disease is based on the lack of a single protein, an animal model with construct validity can be used to investigate the potential effects leading to the symptoms of the disease. Many studies have investigated the synaptic plasticity differences of CA1 pyramidal neurons between a mouse model of fragile X syndrome (KO) and a wild type mouse (WT). This study investigates the differences in firing properties of a CA1 pyramidal neuron between the KO and WT. Specifically, contributions of two ion channels are investigated: the Ca2+ and voltage activated potassium channel (BK) and the potassium channel (M) inhibited by the muscarinic acetylcholine receptor. This study finds some differences that warrant further investigation, including differences in spike timing, spike width and the initial rate of rise of an action potential. However, several areas of investigation yield subtle or confounding results, which may indicate that the CA1 pyramidal neurons affected by the lack of FMRP may make up more than one population. / text

Faces provide important information necessary for social communication. The current study aimed to evaluate Event-Related Brain Potentials (ERPs) as a method of exploring face processing abilities in fragile X syndrome (FXS), a genetic disorder where social deficits lie at the core of the cognitive phenotype. Neural changes were investigated in three children with FXS across various conditions such as upright vs. inverted faces, intact faces vs. faces with no eyes as well as faces vs. cars. Relative to chronological age matched controls, children with FXS displayed greater N170 amplitudes and shorter latency peaks across conditions. In addition, the FXS group showed right hemispheric specialization for both face and non-face stimuli. Heightened electrophysiological responses in FXS are discussed in the context of reported hyper-sensitivity and arousal.

A series of empirical studies is presented that examine the contribution of Fragile X Mental Retardation 1 (FMR1) gene expression to the structure and function of the visual system. This contribution is documented using a histological approach in human and nonhuman primate tissue in conjunction with psychophysical testing of Fragile X Syndrome affected patients who are lacking FMR1 expression. / In the first set of experiments, immunohistological studies of unaffected human and primate brain tissue were carried out to reveal the staining pattern for Fragile X Mental Retardation Protein (FMRP), the protein product of the FMR1 gene, within the two main subcortical pathways at the level of the lateral geniculate nucleus (LGN). FMRP is expressed in significantly greater quantity within the magnocellular (M) neurons of the LGN when compared to levels obtained from the parvocellular (P) neurons. This finding suggests that M neurons depend on FMRP to greater extent than P neurons for determining their normal structure and function. A subsequent histological analysis of the LGN from a FXS affected individual revealed atypical LGN composed of small-sized neurons that were more P- than M-like. This result supports the notion that with the lack of FMR1 expression as occurs in FXS, the impact is greatest to M neuron morphology. / A second set of experiments explored the idea that the M neuron pathology in FXS results in a functional deficit for processing of visual information carried by this pathway. Detection thresholds for stimuli known to probe either M or P-pathway integrity were obtained from individuals affected by FXS as well as age- and developmental-matched control participants. In support of this hypothesis, FXS affected individuals displayed significantly elevated thresholds for M-but not P-specific achromatic visual stimuli. The selectivity of this deficit was verified in a consequent experiment that evaluated colour vision, a visual attribute known to be exclusively processed by the P-pathway. Affected individuals did not differ significantly from developmental-matched control participants in their ability to detect chromatic stimuli. Finally, the effect of the M pathway deficit on cortical visual function was assessed. Results of these experiments reveal that the thresholds for detection of coherent motion, but not form, are significantly elevated in the FXS group. This finding suggests that the parietal (dorsal) visual stream, the major cortical recipient of input from the M pathway, is detrimentally affected in FXS. / A third experiment examines the extent to which the M pathway deficit impacts on cortical visual functioning by employing stimuli of varying complexity that probe the parietal (dorsal) and temporal (ventral) visual streams separately. Results suggest that FXS affected individuals have a pervasive deficit in their ability to detect both simple and more complex forms of motion. In contrast, these same individuals have normal detection thresholds for simple form stimuli. However, with more complex form stimuli affected individuals have significant elevations in threshold. Taken together these results support the notion that the M pathway deficit is amplified at higher levels of visual processing and further, that FXS affected individuals have difficulties integrating all early visual information.

Fragile X syndrome (FXS) is the leading cause of inherited mental retardation and developmental delay. In the vast majority of cases, this X-linked disorder is due to a CGG expansion in the 5' untranslated region of the fmr-1 gene and the resulting decreased expression of its associated protein, FMRP (Fragile X associated Mental Retardation Protein). FXS is characterized by a number of cognitive, behavioural, anatomical, and biological abnormalities. This monogenic disorder provides a unique opportunity to study the consequence of a mutation in a single gene on the development and proper functioning of the CNS. / Histological work on FMRP expression in the monkey lateral geniculate nucleus (LGN) has revealed differential staining in the magnocellular and parvocellular layers, with increased expression in the magnocellular layers (Kogan, Boutet, Cornish, Zangenehpour, & Mullen, 2004). In individuals with fragile X, this differential expression pattern is correlated at the behavioural level with impairments in the M but not the P-visual pathway processing. These findings by Kogan and colleagues, led to the hypothesis that brain regions that express high levels of FMRP are particularly susceptible to its reduced expression, as occurs in FXS. It was therefore of interest to extend this work to determine the pattern of FMRP expression throughout the monkey brain, with the aim of identifying the brain structures most susceptible to reduced expression of the fmr-1 gene product. / The current focus on the role of FMRP in RNA translation and neuronal maturation makes it timely to assemble the extant information on how reduced expression of the fmr-1 gene leads to neuronal dysmorphology. The first section of this manuscript offers a summary of recent genetic, neuroanatomical, and behavioural studies of fragile X syndrome, and provides potential mechanisms to account for the pleiotropic phenotype of this disorder. The following section presents a detailed account of the FMRP expression profile in the monkey brain, and reveals the striking correlation between the expression of the protein and behavioural deficits associated with its reduced expression, as occurs in FXS. / The last chapter of this manuscript offers insight into future trends in FXS research. A number of electrophysiological and behavioural studies point to a particular involvement of the metabotropic glutamatergic system in FXS, with a preeminent role for metabotropic glutamate receptor type V (mGluR5). The involvement of this receptor in FXS and the potential therapeutic implications of pharmacological regulation of this receptor will be discussed. / There is a body of work pointing to the remarkable behavioural similarities between FXS and autism. While FXS is a single-gene disorder, autism is associated with a number of genes, which have not yet been precisely identified. The final section of this manuscript delineates the neuroanatomical, behavioural, and linguistic overlap, as well as the differences between the two conditions. Finally, this section affords some insight as to how FXS, a single-gene disorder, may assist us in our understanding of autism. / Keywords: Fragile X Syndrome (FXS), fmr-1 mutation, lateral geniculate nucleus (LGN), anterior cingulated cortex (ACC), dentate gyros (DG), superior frontal gyrus (SFG), cerebellum, metabotropic glutamate receptor type V (mGluR5), postsynaptic density protein 95 (PSD-95), dendritic spine, autism / Abbreviations: fragile X syndrome (FXS), long-term potentiation (LTP), long-term depression (LTD), RNA-binding protein (RBP), lateral geniculate nucleus (LGN), anterior cingulated cortex (ACC), dentate gyrus (DG), superior frontal gyrus (SFG), metabotropic glutamate receptor type V (mGluR5), postsynaptic density protein 95 (PSD-95), region of interest (ROI), deep cerebellar nucleus (DCN).

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