Influence of the fragile X mental retardation (FMR1) gene on the brain and working memory in men with normal FMR1 alleles.

The fragile X mental retardation 1 (FMR1) gene plays an important role in the development and maintenance of neuronal circuits that are essential for cognitive functioning. We explored the functional linkage(s) among lymphocytic FMR1 gene expression, brain structure, and working memory in healthy adult males. We acquired T1-weighted and diffusion tensor imaging from 37 males (18-80 years, mean ± SD= 40.7 ± 17.3 years) with normal FMR1 alleles and performed genetic and working memory assessments. Brain measurements were obtained from fiber tracts important for working memory (i.e. the arcuate fasciculus, anterior cingulum bundle, inferior longitudinal fasciculus, and the genu and anterior body of the corpus callosum), individual voxels, and whole brain. Both FMR1 mRNA and protein (FMRP) levels exhibited significant associations with brain measurements, with FMRP correlating positively with gray matter volume and white matter structural organization, and FMR1 mRNA negatively with white matter structural organization. The correlation was widespread, impacting rostral white matter and 2 working-memory fiber tracts for FMRP, and all cerebral white matter areas except the fornix and cerebellar peduncles and all 4 fiber tracts for FMR1 mRNA. In addition, the levels of FMR1 mRNA as well as the fiber tracts demonstrated a significant correlation with working memory performance. While FMR1 mRNA exhibited a negative correlation with working memory, fiber tract structural organization showed a positive correlation. These findings suggest that the FMR1 gene is a genetic factor common for both working memory and brain structure, and has implications for our understanding of the transmission of intelligence and brain structure.

CGG-repeat length threshold for FMR1 RNA pathogenesis in a cellular model for FXTAS.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects carriers of premutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The presence of elevated levels of expanded mRNA found in premutation carriers is believed to be the basis for the pathogenesis in FXTAS, but the exact mechanisms by which the mRNA causes toxicity are not known. In particular, it is not clear whether there is a threshold for a CGG-repeat number below which no cellular dysregulation occurs, or whether toxicity depends on mRNA concentration. We have developed a doxycycline-inducible episomal system that allows us to study separately the effects of CGG-repeat number and mRNA concentration (at fixed CGG-repeat length) in neuroblastoma-derived SK cells. Our findings show that there is a CGG-repeat size threshold for toxicity that lies between 62 and 95 CGG repeats. Interestingly, for repeat sizes of 95 CGG and above, there is a clear negative correlation between mRNA concentration and cell viability. Taken together, our results provide evidence for an RNA-toxicity model with primary dependence on CGG-repeat size and secondary dependence on mRNA concentration, thus formally ruling out any simple titration model that operates in the absence of either protein-binding cooperativity or some form of length-dependent RNA structural transition.

Altered zinc transport disrupts mitochondrial protein processing/import in fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset neurodegenerative disorder that affects individuals who are carriers of small CGG premutation expansions in the fragile X mental retardation 1 (FMR1) gene. Mitochondrial dysfunction was observed as an incipient pathological process occurring in individuals who do not display overt features of FXTAS (1). Fibroblasts from premutation carriers had lower oxidative phosphorylation capacity (35% of controls) and Complex IV activity (45%), and higher precursor-to-mature ratios (P:M) of nDNA-encoded mitochondrial proteins (3.1-fold). However, fibroblasts from carriers with FXTAS symptoms presented higher FMR1 mRNA expression (3-fold) and lower Complex V (38%) and aconitase activities (43%). Higher P:M of ATPase ß-subunit (ATPB) and frataxin were also observed in cortex from patients that died with FXTAS symptoms. Biochemical findings observed in FXTAS cells (lower mature frataxin, lower Complex IV and aconitase activities) along with common phenotypic traits shared by Friedreich's ataxia and FXTAS carriers (e.g. gait ataxia, loss of coordination) are consistent with a defective iron homeostasis in both diseases. Higher P:M, and lower ZnT6 and mature frataxin protein expression suggested defective zinc and iron metabolism arising from altered ZnT protein expression, which in turn impairs the activity of mitochondrial Zn-dependent proteases, critical for the import and processing of cytosolic precursors, such as frataxin. In support of this hypothesis, Zn-treated fibroblasts showed a significant recovery of ATPB P:M, ATPase activity and doubling time, whereas Zn and desferrioxamine extended these recoveries and rescued Complex IV activity.

Fibroblast phenotype in male carriers of FMR1 premutation alleles.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder among carriers of premutation expansions (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. The clinical features of FXTAS, as well as various forms of clinical involvement in carriers without FXTAS, are thought to arise through a direct toxic gain of function of high levels of FMR1 mRNA containing the expanded CGG repeat. Here we report a cellular endophenotype involving increased stress response (HSP27, HSP70 and CRYAB) and altered lamin A/C expression/organization in cultured skin fibroblasts from 11 male carriers of premutation alleles of the FMR1 gene, including six patients with FXTAS and five premutation carriers with no clinical evidence of FXTAS, compared with six controls. A similar abnormal cellular phenotype was found in CNS tissue from 10 patients with FXTAS. Finally, there is an analogous abnormal cellular distribution of lamin A/C isoforms in knock-in mice bearing the expanded CGG repeat in the murine Fmr1 gene. These alterations are evident even in mouse embryonic fibroblasts, raising the possibility that, in humans, the expanded-repeat mRNA triggers pathogenic mechanisms early in development, thus providing a molecular basis for the neurodevelopmental abnormalities observed in some children and clinical symptoms in some adults who are carriers of premutation FMR1 alleles. Cellular dysregulation in fibroblasts represents a novel and highly advantageous model for investigating disease pathogenesis in premutation carriers and for quantifying and monitoring disease progression. Fibroblast studies may also prove useful in screening and testing the efficacy of therapeutic interventions.

Effects of Dexamethasone on DNA Synthesis in Lens Epithelial Cells Are Dependent on Cell Type and Growth Factor.

PURPOSE: To determine the factors that influence the ability of dexamethasone (dex) to inhibit or stimulate the growth of lens epithelial cells. METHOD: Different growth factors with or without dex (10-6 M) were added to quiescent cultures of two clones of Nakano mouse lens epithelial cells (NK11) in serum-free medium. DNA synthesis was then measured after 8-12 hours by the incorporation of tritiated thymidine. RESULTS: Dex was found to both stimulate and inhibit mitogen-induced 3H-thymidine incorporation into the DNA of cultured mouse lens epithelial cells. Enhancement or repression by dex was found to depend on the growth factor used to stimulate the quiescent cell. EGF and insulin were consistently inhibited with dex. Basic fibroblast growth factor (bFGF) and retinoblastoma-derived growth factor (RbDGF) were both enhanced and inhibited by dex, depending on the growth factor concentration and the cell clone used for the experiment. Additionally, RbDGF protects against the dex inhibition of insulin stimulation, but not the inhibition of EGF stimulation. Progesterone, an inhibitor of the activation of the glucocorticoid receptor, blocks the dex inhibitory effect on the EGF and insulin stimulation of DNA synthesis. The ability of progesterone to affect the dex inhibition is consistent with the dex receptor modulating DNA synthesis. The dex effect on DNA synthesis, either stimulatory or inhibitory, was still seen if dex was added as late as 10 hours after the growth factor. CONCLUSIONS: The study demonstrated that dex reduces the overall growth and activity of lens epithelial cells in vitro. This result provides insight into the risk of developing posterior subcapsular cataracts (PSC) in patients on oral glucocorticoid therapy. Understanding the basic mechanisms by which steroids mediate lens cell growth may provide the ability to more accurately predict who will develop PSC. The present studies show the difference in the effect of dex from lens cell to lens cell, but, more importantly, suggest a pattern of dependent variables that might prove useful in such predictions.

Evidence of mitochondrial dysfunction in fragile X-associated tremor/ataxia syndrome.

FXTAS (fragile X-associated tremor/ataxia syndrome) is a late-onset neurodegenerative disorder that affects individuals who are carriers of premutation expansions (55-200 CGG repeats) in the 5' untranslated region of the FMR1 (fragile X mental retardation 1) gene. The role of MD (mitochondrial dysfunction) in FXTAS was evaluated in fibroblasts and brain samples from premutation carriers with and without FXTAS symptoms, with a range of CGG repeats. This study resulted in several important conclusions: (i) decreased NAD- and FAD-linked oxygen uptake rates and uncoupling between electron transport and synthesis of ATP were observed in fibroblasts from premutation carriers; (ii) a lower expression of mitochondrial proteins preceded both in age and in CGG repeats the appearance of overt clinical involvement; (iii) the CGG repeat size required for altered mitochondrial protein expression was also smaller than that required to produce brain intranuclear inclusions from individuals with the premutation who died, suggesting that MD is an incipient pathological process occurring in individuals who do not display overt features of FXTAS; and (iv) on the basis of the CGG repeats, MD preceded the increase in oxidative/nitrative stress damage, indicating that the latter is a late event. MD in carriers of small CGG repeats, even when the allele size is not sufficient to produce FXTAS, may predispose them to other disorders (e.g. Parkinson's disease) that are likely to involve MD, and to environmental stressors, which may trigger the development of FXTAS symptoms. Detection of MD is of critical importance to the management of FXTAS, since it opens up additional treatment options for this disorder.

FMR1 RNA within the intranuclear inclusions of fragile X-associated tremor/ataxia syndrome (FXTAS).

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a recently identified neurodegenerative disorder affecting older adult males with premutation alleles of the fragile X mental retardation 1 (FMR1) gene. The principal clinical features of FXTAS include progressive intention tremor, gait ataxia, parkinsonism, and autonomic dysfunction. The disorder affects at least one-third of carrier males over 50 years of age and, with an estimated carrier frequency of approximately 1/800 males, is likely to be one of the most common heritable forms of tremor and ataxia among older adult males in the general population. Brains from all FXTAS cases examined to date (10/10) possess numerous ubiquitin-positive intranuclear inclusions in broad distribution throughout the cerebrum and brainstem. The absence of either the neurodegenerative disorder or inclusions among adults with fragile X syndrome (who lack the FMR1 protein), coupled with elevated FMR1 mRNA with expanded CGG repeats in premutation carriers, has led us to propose an RNA toxic gain-of-function model for FXTAS. Consistent with this model, we have now identified FMR1 mRNA within the intranuclear inclusions isolated from post-mortem (FXTAS) brain tissue.

High-throughput screening of FDA-approved drugs using oxygen biosensor plates reveals secondary mitofunctional effects.

Repurposing of FDA-approved drugs with effects on mitochondrial function might shorten the critical path to mitochondrial disease drug development. We improved a biosensor-based assay of mitochondrial O2 consumption, and identified mitofunctional defects in cell models of LHON and FXTAS. Using this platform, we screened a 1600-compound library of clinically used drugs. The assay identified drugs known to affect mitochondrial function, such as metformin and decoquinate. We also identified several drugs not previously known to affect mitochondrial respiration including acarbose, metaraminol, gallamine triethiodide, and acamprosate. These previously unknown 'mitoactives' represent novel links to targets for mitochondrial regulation and potentially therapy, for mitochondrial disease.

Decreased fragile X mental retardation protein expression underlies amygdala dysfunction in carriers of the fragile X premutation.

BACKGROUND: The fragile X premutation provides a unique opportunity for the study of genetic and brain mechanisms of behavior and cognition in the context of neurodevelopment and neurodegeneration. Although the neurodegenerative phenotype, fragile X-associated tremor/ataxia syndrome, is well described, evidence of a causal link between the premutation and psychiatric disorder earlier in life, clear delineation of a behavioral/cognitive phenotype, and characterization of the physiological basis of observed symptoms have been elusive. METHODS: We completed functional magnetic resonance imaging targeting the amygdala with an emotion-matching task and concurrent infrared eye tracking, FMR1 molecular genetic testing, and neuropsychological assessment in 23 men with the premutation (mean age = 32.9 years) and 25 male control subjects (mean age = 30.1 years). RESULTS: Premutation carriers had significantly smaller left and right amygdala volume and reduced right amygdala activation during the task relative to control subjects. Although both elevated FMR1 messenger RNA and reduced fragile X mental retardation protein (FMRP) were associated with the reduced activation, multiple regression analysis suggested that reduced FMRP is the primary factor. Premutation carriers also had higher ratings of autism spectrum symptoms than control subjects, which were associated with the reduced amygdala response. CONCLUSIONS: Although prior studies have emphasized a toxic gain-of-function effect of elevated messenger RNA associated with the premutation, the current results point to the role of reduced FMRP in alterations of brain activity and behavior.

A quantitative ELISA assay for the fragile x mental retardation 1 protein.

Non-coding (CGG-repeat) expansions in the fragile X mental retardation 1 (FMR1) gene result in a spectrum of disorders involving altered neurodevelopment (fragile X syndrome), neurodegeneration (late-onset fragile X-associated tremor/ataxia syndrome), or primary ovarian insufficiency. While reliable and quantitative assays for the number of CGG repeats and FMR1 mRNA levels are now available, there has been no scalable, quantitative assay for the FMR1 protein (FMRP) in non-transformed cells. Using a combination of avian and murine antibodies to FMRP, we developed a sensitive and highly specific sandwich enzyme-linked immunosorbent assay (ELISA) for FMRP in peripheral blood lymphocytes. This ELISA method is capable of quantifying FMRP levels throughout the biologically relevant range of protein concentrations and is specific for the intact FMRP protein. Moreover, the ELISA is well-suited for replicate protein determinations across serial dilutions in non-transformed cells and is readily scalable for large sample numbers. The FMRP ELISA is potentially a powerful tool in expanding our understanding of the relationship between FMRP levels and the various FMR1-associated clinical phenotypes.

Isolation of pathology-associated intranuclear inclusions.

An emerging theme in neurodegenerative diseases is the aggregation of proteins as inclusions in neural cells. Their presence is a useful tool in the differential diagnosis of the particular illness, although in no instance is the specific role of the inclusions in disease pathogenesis understood at present. However, apart from their role in the disease mechanism, the inclusions themselves may contain important molecular clues as to the mechanism(s) behind the specific inclusion-associated disease. Thus, isolation and analysis of the composition of the inclusions is likely to yield biochemical evidence of the cellular pathways that are involved in the disease process.

Induction of inclusion formation and disruption of lamin A/C structure by premutation CGG-repeat RNA in human cultured neural cells.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder that affects some adult carriers of pre-mutation alleles (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. FXTAS is thought to be caused by a toxic 'gain-of-function' of the expanded CGG-repeat FMR1 mRNA, which is found in the neuronal and astrocytic intranuclear inclusions associated with the disorder. Using a reporter construct with a FMR1 5' untranslated region harboring an expanded (premutation) CGG repeat, we have demonstrated that intranuclear inclusions can be formed in both primary neural progenitor cells and established neural cell lines. As with the inclusions found in post-mortem tissue, the inclusions induced by the expanded CGG repeat are alphaB-crystallin-positive; however, inclusions in culture are not associated with ubiquitin, indicating that incorporation of ubiquitinated proteins is a later event in the disease process. The absence of ubiquitinated proteins also argues against a model in which inclusion formation is due to a failure of the proteasomal degradative machinery. The presence of the expanded CGG repeat, as RNA, results in reduced cell viability as well as the disruption of the normal architecture of lamin A/C within the nucleus. This last observation, and the findings that lamin A/C is present in both the inclusions of FXTAS patients and the inclusions in cell culture, suggests that lamin A/C dysregulation may be a component of the pathogenesis of FXTAS; in particular, the Charcot-Marie-Tooth-type neuropathy associated with FXTAS may represent a functional laminopathy.