Our work this year addressed the following specific aims: 1) Develop improved numerical techniques for analyzing PET data to determine rCPS. 2) Determine rCPS in human subjects with FXS. 3) Examine social and nonsocial anxiety in a mouse model of fragile X syndrome. 4) Test the effects of chronic lithium treatment on rCPS in a mouse model of FXS. 5) Examine the response to chronic stress in a mouse model of FXS. 6) Determine rCPS in a mouse model of the fragile X premutation. 1) We have developed two new approaches for the analysis of L-1-11Cleucine PET data that are more robust to the effects of the limited spatial resolution of the PET camera and noise in the data. One is a basis function method applied to PET data at the voxel level (SNM Meeting Abstracts, Tomasi et al., 2009;BrainPET09 Meeting Abstracts, Tomasi et al., 2009). The second approach is spectral analysis with an iterative filter that is applied to regions of interest (BrainPET09 Meeting Abstracts, Veronese et al., 2009). Analysis of completed studies in healthy young male volunteers with the basis function method confirms that in human subjects low variance and highly reproducible measures of rCPS can be made with the L-1-11Cleucine PET method (Tomasi G, Bertoldo A, Bishu S, Unterman A, Smith CB, Schmidt KC, JCBF&M. 29:1317-1331, 2009). Last year we had shown in healthy young male volunteers (19-24 years of age) rCPS measured with the L-1-11Cleucine PET method is not significantly altered by anesthesia with propofol (Bishu S, Schmidt KC, Burlin T, Channing M, Horowitz L, Huang T, Liu Z-H, Qin M, Vuong B-K, Unterman A, Xia Z, Herscovitch P, Quezado Z, Smith CB. Propofol anesthesia does not alter regional rates of cerebral protein synthesis measured with L-1-11-Cleucine and PET in healthy male subjects. JCBF&M 29:1035-1047, 2009). Further analysis of these data with the voxel-based and spectral analysis methods confirms that rCPS is not affected by anesthesia with propofol;these data were presented at the 24th International Symposium on Cerebral Blood Flow &Metabolism and 9th International Conference on Quantification of Brain Function with PET (BrainPET09 Meeting Abstracts, Bishu et al., 2009) and at the 2009 Annual Meeting of the Society for Nuclear Medicine (SNM Meeting Abstracts, Bishu et al., 2009). 2) Currently we are applying these new approaches to the analysis of ongoing L-1-11Cleucine PET studies of subjects with fragile X syndrome. This year we have completed L-1-11Cleucine PET studies in six subjects with the full fragile X mutation. Subjects were between the ages of 18 and 23 and were studied under propofol anesthesia. Preliminary results suggest that in some brain regions rCPS are elevated in the fragile X subjects. 3) Anxiety is a common symptom in fragile X patients. However, an anxiety-prone phenotype in mouse models of FXS has not been clearly demonstrated. Abnormal social interactions suggestive of elevated social anxiety are reported in some studies, but decreased anxiety-like responses in exploratory-based tests are also found. In studies of social approach behavior and exploratory behavior in an elevated zero maze we found support for the hypothesis that social and nonsocial anxiety can be dissociated and that, in the fragile X mouse model, behavior consistent with hyper-social anxiety coexists with hypo-nonsocial anxiety (Liu Z-H, Smith CB. Dissociation of social and nonsocial anxiety in a mouse model of fragile X syndrome. Neurosci Lett 454:62-66, 2009). 4) Our studies of the effectiveness of chronic lithium carbonate (Li) treatment (0.24% added to normal mouse chow at weaning) have been carried out in the fragile X knockout (FXKO) mouse model. We tested for effects of treatment on dendritic spine morphology, behaviour, and rCPS, and we found that chronic Li treatment normalized spine morphology, partially reversed hyperactivity, alleviated effects on general anxiety, and ameliorated effects on social interactions and fear learning/memory (Liu Z-h, Chuang D-M, Smith CB, Lithium ameliorates behavioral deficits in mouse model of fragile X syndrome, Annual Meeting of the Society for Neuroscience, 2008). Preliminary results of studies of the effects of Li on rCPS in FXKO indicate that increases in rCPS are also reversed by Li. These results in mice suggest that chronic lithium treatment may have therapeutic value in FXS and coupled with the results from other laboratories studying FXS make a strong case for instituting a placebo-controlled trial of lithium in subjects with FXS. 5) Clinical reports suggest that FXS may involve a dysregulation of the hypothalamic-pituitary-adrenal axis. In a systematic investigation of the response to acute restraint stress or spatial novelty in FXKO mice we found that hormonal responses were similar to those of wild type (WT) mice (Qin M, Smith CB. Unaltered Hormonal Response to Stress in a Mouse Model of Fragile X Syndrome. Psychoneuroendocrinology 33:883-889, 2008). We have also examined the effects of chronic restraint stress in WT and FXKO mice. We found that mice of both genotypes underwent adrenal hypertrophy and had similar hormonal responses to a test of anxiety after chronic restraint. Only WT mice demonstrated the increased anxiety characteristic of rodents exposed to chronic stress and only WT mice had increased dendritic branching on pyramidal cells in basolateral amygdala. FXKO did not exhibit either the behavioural or morphological response. We posit that these differences in responses to chronic stress reflect a diminished adaptive response in FXKO mice. 6) A knockin (FXKI) mouse model of the fragile X premutation developed by Dr. K Usdin (Laboratory of Molecular &Cellular Biology, NIDDK) has many of the attributes found in human subjects with the fragile X premutation, i.e., intranuclear inclusions, increased levels of fmr1 mRNA, and a loss of Purkinje cells. In later life individuals with the fragile X premutaion sometimes develop the fragile X tremor and ataxia syndrome (FXTAS) a disease associated with the accumulations of neuronal inclusion bodies in some brain regions. It has also been reported that in boys with the fragile X premutation there is a higher prevalence of attention deficit hyperactivity disorder, learning disabilities, and autism spectrum disorders. In the FXKI mouse model, Dr Usdin has demonstrated that in some regions of the brain and, despite the increases in fmr1 mRNA, FMRP concentrations are reduced. Our results show that FXKI mice exhibit abnormalities in behavior, morphology, and rCPS similar to those found in the FXKO mice (Qin M, Entezam A, Usdin K, Smith CB, Hyperactivity and impaired social interaction in fragile X premutation mice, Annual Meeting of the Society for Neuroscience, 2008). It appears that in this novel mouse model, reductions in FMRP herald profound effects on brain structure and function. Studies described address the central question that symptoms of FXS are caused by the lack of regulation of translation that may occur in the absence of FMRP. Our focus is on FXS because the process of translation regulation is likely at the heart of the malady in this single gene disease. Understanding the essential neurobiological abnormalities in FXS is vital to rational development of novel therapies. Further, measurement of rCPS in FXS may provide an objective and quantitative means of evaluating new therapies. It is also likely that dysregulation of protein metabolism underlies other neurodevelopmental disorders.
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