Studies On Protein Synthesis And Long-Term Adaptive Responses in the CNS

在上一个资助期（2010年），我们致力实现以下具体目标：

During the last funding period (2010), we addressed the following specific aims: 1) Determine rCPS in human subjects with FXS 2) Refine numerical techniques for analysis of PET data to determine rCPS 3) Test the effects of chronic lithium treatment on rCPS in a mouse model of FXS 4) Determine rCPS in a mouse model of the fragile X premutation. 5) Examine the response to chronic stress in a mouse model of FXS 1) We are measuring rCPS with the L-1-C-11leucine PET method in young men with FXS (18-24 years of age) to determine if the effect of a lack of FMRP in human subjects has similar effects as those found in the mouse model. This year we have completed PET studies in seven subjects. Subjects were studied under deep propofol anesthesia. We have also studied 12 age-matched healthy volunteers under the same conditions. Preliminary results in ten fragile X subjects suggest that, in some brain regions, rCPS are elevated. Taking into account the effects of partial volume of PET measurements the magnitude of the effects is similar to what we had observed in studies on the fmr1 KO mouse model of FXS (Presented at the 2010 FRAXA Investigators Meeting in Durham, NH in May 2010). 2) We have developed two new approaches for the analysis of L-1-C-11leucine 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 (BFM) applied to PET data at the voxel level. We have adapted and validated this method and demonstrated that voxel-level estimates of rCPS averaged over a region of interest (ROI) are substantially less biased than estimates based on direct fitting of the ROI time-activity curve with a homogeneous tissue model (Tomasi et al, 2009). The second approach is spectral analysis with an iterative filter (SAIF) that is applied to ROI data. SAIF takes into account kinetic heterogeneity within the ROI and produces low bias, low variance estimates of rCPS (Veronese et al, 2010). Simulation of normal count-rate studies showed that SAIF applied to ROI time-activity curves performed comparably to BFM applied to voxel time-activity curves when voxel-wise estimates were averaged over all voxels in the ROI. At low count-rates, however, SAIF performed better. In PET data measured after injection of 20-30 mCi L-1-C-11leucine, we found good agreement between ROI-based SAIF estimates and average voxel-wise BFM estimates of rCPS. 3) We studied the effects of chronic dietary lithium treatment begun at the time of weaning on the fmr1 KO phenotype. We measured effects on robust behavioral and morphological phenotypes and rCPS. We found that lithium treatment reversed behavioral phenotypes of fmr1 KO mice including hyperactivity, reduced anxiety, reduced social interaction, and a deficit on the passive avoidance test. It also mitigated abnormal spine length and density in medial prefrontal cortex (Liu, Z-H, Chuang D-M, Smith CB. Lithium ameliorates phenotypic deficits in a mouse model of fragile X syndrome. Int J Neuropsychopharm doi:10.1017/S1461145710000520, Available on CJO 25 May 2010). Further studies of lithium treatment of fmr1 KO mice indicate that lithium also reverses increased rCPS found in these mice (presented at the 2010 FRAXA Investigators Meeting in Durham, NH in May 2010). Taken together our studies indicate that lithium treatment has a wide range of effects in fmr1 KO mice suggesting that lithium treatment may affect underlying chemical pathology in FXS. 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. 4) Silencing of FMR1 is due to the presence of an expanded CGG-repeat sequence (>200 repeats) in the 5-UTR. In the normal population, the repeat sequence on FMR1 is <54. Carriers of FMR1 premutation alleles have 55-200 CGG repeats. These sequences are unstable and can expand in the next generation to the full fragile X mutation. Premutation carriers are at risk for fragile X associated primary ovarian insufficiency (FXPOI) and, in late life, fragile X associated tremor and ataxia syndrome (FXTAS). Premutation carrier status can also be associated with autism spectrum disorder, attention deficit hyperactivity disorder, and some cognitive deficits. In premutation carriers, FMR1 mRNA levels are often higher than those with normal sized alleles, and it is thought that RNA toxicity contributes to the symptoms. We have characterized a knock-in (KI) mouse model of the fragile X premutation developed in the laboratory of Dr. K. Usdin (NIDDK). Our results show that young, adult, male, KI mice have a distinct behavioral phenotype which includes hyperactivity, some subtle social deficits, profound deficits on the passive avoidance test, reduced levels of general anxiety, and normal motor learning. Protein synthesis rates in brain were generally higher in KI mice compared with wild type (WT). We also found clear neuropathology in the KI mouse including hypotrophic dendritic arborization and increased length and density of dendritic spines. Consistent with findings in the human premutation, the level of fmr1 mRNA was increased throughout the brain. Despite the increased message, FMRP levels were profoundly reduced (10-20% of WT). Generally, it is thought that symptoms of patients with the fragile X premutation are due to the FMR1 mRNA toxicity in brain and the age-dependent increase in inclusion bodies. Results of our study highlight similarities in phenotype between KI and fmr1 KO mice and suggest that it is the decreased concentration of FMRP that contributes to the phenotype in young adult KI mice rather than the increased concentration of fmr1 mRNA. A manuscript reporting these results is currently under review. 5) Clinical reports suggest that FXS may involve a dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. In a systematic investigation of the response to acute restraint stress or spatial novelty in fmr1 KO mice we found that hormonal responses were similar to those of WT mice. We have extended these studies of the HPA axis by investigating the effects of chronic restraint stress in WT and fmr1 KO mice. We found that mice of both genotypes underwent adrenal hypertrophy and had similar hormonal responses, but only WT mice demonstrated the increased anxiety characteristic of rodents exposed to chronic stress. Moreover only WT mice had morphological changes in dendrites in basolateral amygdala in response to chronic stress. These differences in response to chronic stress may reflect a diminished adaptive response in fmr1 KO mice. 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.