1. Specific Aims
The overall Specific Aim of this proposal is to hold a series of five annual interdisciplinary conferences on basic and clinical research relevant to fragile X syndrome (FXS), at the Banbury Conference Center, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. The initial and model conference of what we plan as a continuing series was held April 16-19, 2000, at the Banbury Center under the joint sponsorship of the FRAXA Research Foundation, NIMH and NICHD and concentrated upon molecular biological research relevant to FXS, but also included work on other related topics including behavior symptoms and phenotype, other characteristics of the knockout mouse including molecular phenotype and seizure symptoms, experimental therapeutic approaches, and synaptic function and plasticity (see Appendix for program). Ample time was allowed for discussion and interaction with regard to the topics presented, and a number of potential collaborations and novel approaches emerged from these sessions. (See “Feedback from the previous meeting,” Sec. 3.)
· A specific aim of the series of annual FXS conferences is to bring together a broad range of scientists working on various aspects of basic and clinical research appropriate to FXS and, in the context of a focal theme each year, to both exchange data regarding recent progress in the focal area and to educate individuals in other areas with regard to relevant details of the focal theme. An essential outcome of this process is keeping researchers in the focal area fully cognizant of the approaches and the resources currently available, everything from the latest technology to available antibodies.
· One of the principal strengths of these meetings is the second specific aim: to introduce scientists from other areas to the best work in a current focal area, while at the same time introducing those in the focal area to important work in other areas. Hence each conference will include a core of scientists working on that meeting’s topic area and a set of scientists working in other areas who can benefit from exposure to this knowledge. This approach was particularly popular among attendees of the first Banbury Conference. (See “Feedback from the previous meeting,” Sec. 3.)
· A third specific aim is to provide training and an introduction to the area to younger scientists. The first Banbury Conference invited and supported participation by young scientists, particularly postdoctoral researchers. We will select 10 young scientists (of 36 total participants) from those who respond to the meeting announcement for invitation to attend and participate in the meeting. This is essential to promote scientific networking, continuing exchange of knowledge and ideas, and collaboration, both among younger scientists and between younger and established scientists.
Examples of focal meeting topics appear in Sec. D. We believe that FXS is both a remarkably rapidly moving research area and remarkably broad in the number of approaches that will need to be integrated in the ultimate search for treatments. Hence an annual meeting with different focal topics is clearly warranted. Moreover, this meeting can serve as a model for approaches to other disorders such as autism, and mental disorders of unknown etiology, for which the genetic origins are less well understood.
2. BACKGROUND AND SIGNIFICANCE
2. What is known about FXS?
2.1. Fragile X Syndrome
Fragile X syndrome is the second leading genetic cause (and the leading inherited cause) of mental retardation in humans, surpassed only by Down syndrome; the incidence in males (as high as 1/2-3000) is more frequent than in females (Brown, 1996; Turner et al., 1996). It has no ethnic boundaries and is caused by a novel, poorly understood, mutational mechanism. Fragile X manifests with relatively subtle facial appearance symptoms, enlarged testicles and reduced IQ (Warren & Nelson, 1994; Davies, 1989; Hagerman & Cronister, 1996). Problems are first noticed when an afflicted child presents at age two or three as a temperamentally difficult child that is unable to speak in sentences. IQ deficits are, on average, greater in males. Wright-Talamante et al. (1996) reported a significant decline in IQ among fragile-X males which were administered IQ tests anywhere from 7 months to 13 years apart. Fisch et al. (1996) investigated cognitive ability (Stanford-Binet) and adaptive behavior (Vineland Adaptive Behavior Scales) in 24 male fragile-X patients three to fifteen years of age. They described declines in IQ scores in 75% of the subjects and in adaptive behavior scores in 92% of the subjects. These scores did not reflect a decline in intellectual or social skills with age, but rather a widening gap between these fragile-X and control patients. Fragile X retardation is often accompanied by symptoms similar to those of attentional deficit and hyperactivity disorder and of autism. Development of treatments for fragile X patients exhibiting these clinical symptoms might be facilitated if the normal functions of FMRP were understood.
2.1.1. Genetic and Molecular Basis of Fragile X Syndrome
Originally, fragile X was diagnosed by a karyotype in which a limb of the X chromosome exhibited breaks during replication in lymphocytes cultured in folate-deficient medium (Lubs, 1969). The genetic basis for the fragile X syndrome is an unstable region of trinucleotide repeats [(CGG)n] in the 5’ untranslated region which causes hypermethylation of a CpG island that spans the promotor and first exon (e.g., Warren & Nelson, 1994; Davies, 1989; Hagerman & Cronister, 1996; Stoger et al., 1997). This unstable repeat may exhibit a longer or shorter repeat length as cells replicate (Telenius et al., 1994). Fragile X mRNA expression is dramatically reduced in cases exhibiting the clinical fragile X syndrome (Pieretti et al., 1991), reflecting interference of the excessive repeat length and the inactivating effect of cytosine methylation on transcription of the gene. Recent work has shown that a repeat length beyond 200 also impedes the migration of the 40S ribosomal subunit along the 5' untranslated region, which may inhibit translation (Feng et al., 1995). (The fragile X gene is expressed in a number of other body tissues as well and may have multiple roles or processes in which it is involved; Hinds et al., 1993; Hanzlik et al., 1993 Coy et al., 1995.) Symptoms of fragile X syndrome can also result from mutations in the coding region of the gene: FMRP contains RNA-binding (KH) domains similar to those of some heterogeneous nuclear ribonucleoproteins and mutations to the KH domain, which impair binding of the protein to RNA (Siomi et al, 1994), result in fragile X syndrome (DeBoulle et al., 1993). Alternative splicing of the gene (Ashley et al, 1993a) is responsible for a number of isoforms, some with altered cellular localization. RNA binding proteins have a wide range of roles, including posttranscriptional regulation of gene expression (Dreyfuss et al., 1988). FMRP binds to about 4% of human fetal brain mRNAs, including its own (Ashley et al., 1993b), suggesting the capacity for selective regulatory effects. It has been proposed, based on the presence of nuclear export and nuclear localization signal sequences, that the protein may be involved in posttranscriptional alteration of RNA in the nucleolus (Eberhart et al., 1996). However, in neurons FMRP is seen in the cytoplasm and immunoreactivity appears only rarely in the nucleus (Devys et al., 1993; our unpublished immuno-EM observations). Other possible FMRP roles include mRNA processing as well as ribosomal complex targeting and docking of cytoplasmic RNPs (Dreyfuss et al., 1988; Bandziulis et al., 1989). FMRP has been shown to dimerize in vitro with two homologs, termed FXR1 and FXR2, which also have KH domains (Zhang et al, 1995). However, at least in mouse spermatozoa, expression of these proteins appears to be independently regulated on different time courses (Coy et al., 1996). Another neuron-specific RNA-binding protein, Nova-1, is rendered non-functional by autoantibody in a paraneoplastic motor disorder (Buckanovich et al, 1996; see Sec. 3 presentation by Darnell), emphasizing the importance of this class of proteins in neural function. Several CNS-specific paraneoplastic disorders involve RNA binding proteins, including Paraneoplastic Encephalomyetis (e.g., King et al., 1999), such that the outcomes of these conferences may well have implications for other disorders.
2.1.2 Knockout Mouse Model of FXS
A mouse model of Fragile-X Syndrome (KO mice; fmr1-knockout mice) was developed by the Dutch-Belgian Fragile-X Consortium (1994) who inserted a neo-cassette in exon 5, resulting in disrupted expresion of the FMR-1 gene. These mice initially exhibited macroorchidism, mild learning deficits on a reversal condition in the water-maze task, and hyperactivity. More extensive studies of the FraX mice were carried out by Kooy et al. (1996), which again demonstrated macroorchidism (caused by an increase in the rate of Sertoli cell proliferation from postnatal day 12 to 15 (Slegtenhorst-Eegdeman et al., 1998) and possible learning deficits in the water-maze; however, there was no indication of hyperactivity upon re-testing. No impairment of short- or long-term potentiation was found in hippocampal slices from these knockout mice (Godfraind et al., 1996). Furthermore, despite a possible role for FMRP in the transport of mRNA to the synapse, no disruptions in the dendritic localization of the mRNAs for MAP2, CaMKII, or dendrin or in the rapid dendritic transport of the mRNA for activity-regulated cytoskeletal protein (ARC) were found in FraX mice (Steward et al., 1998).
2.2 Brain Morphological Phenotype in FXS
While much of the molecular work on FXS has been done on other tissues, there is general consensus that the proximate cause of most of the behavioral aspects of the system probably involves some sort of pathology of the brain. While abnormalities in both gross and fine structure have been reported, they tend to be rather subtle. There was some controversy regarding brain phenotype at the recent Banbury Conference, as Nimchinsky and Svoboda reported that they were unable to repeat prior observations of longer and more immature spines in the knockout mice using their method, fluorescence visualization of neurons that had been virally-infected with green fluorescent protein. This report is already stimulating collaborative and individual attempts to optimally characterize the structure of synapses in the knockout mouse. The published data are reviewed here in the absence of any change in perspective that may result from this work.
A number of early reports, appearing before the delineation of the fragile X syndrome, showed that mental retardation was often associated with the presence of an immature spine morphology in the cerebral cortex (Marin-Padilla, 1972, 1974; Purpura, 1974). Recent studies of autopsy cases have reported an apparently very similar result for fragile X syndrome: cerebral cortical spines exhibit a thin, elongated morphology in Golgi preparations and a reduced synaptic contact size in electron microscopy (e.g., Rudelli et al., 1985; Hinton et al., 1991), both of which are also characteristic of the immature, or the experience-deprived synapse in the cerebral cortex. A similar morphology appears to be present, perhaps less dramatically, in fragile X knockout mice. While an initial report (Comery et al., 1997) was fatally flawed by the inclusion of an unknown number of subjects with a retinal degeneration syndrome, additional reports have shown a mild morphological phenotype in the KO mouse that parallels that seen in humans (Irwin et al., in prep.). Hinton et al. (1991) noted that neither gross pathology nor any indication of cell migratory failures or neuronal density changes is associated with fragile X syndrome, in contrast to a variety of other mental retardation syndromes (including Down syndrome). Thus while "dendritic spine dysgenesis" also occurs in other mental deficiency syndromes, it is the only cellular-level neuropathology thus far found in fragile X syndrome (Irwin et al., in press). There are also deficits in gross brain morphology. Reiss, et al. (e.g., 1994) has found some brain regions to be larger and others reduced in FraXS patients.
2.3. Current FXS Research Advances
One thing that was very clear from the Banbury Conference described in the following section is that a combination of increasingly widespread interest in FXS among scientists and some breakthrough findings have tremendously advanced this field in recent years. We find ourselves on an accelerating curve of knowledge regarding both the basic character of the disorder as it occurs in humans and in the biological substrates at the cellular and molecular levels. The early 1980s witnessed widespread recognition of the karyotype-based diagnosis first described by Lubs (1969). From 1985 to the present there have been increasingly detailed descriptions of the brain morphological phenotype including, most recently and controversially, that of the knockout mouse. In 1991 the FMR-1 gene was cloned (Pieretti et al., 1991). In 1994 the knockout mouse model for the disorder was produced, described, and began to be distributed to investigators worldwide (Consortium, 1994). In 1997 it was discovered that FMRP synthesis occurred at the synapse under the control of the neurotransmitter glutamate acting through a metabotropic receptor. As the Banbury Conference showed, the accumulation of discoveries is reaching a crescendo in the year 2000, with information beginning to accumulate to suggest specific roles of FMRP in nuclear and cellular trafficking of mRNA and regulation of protein synthesis.
Thus fragile X has become a fast moving field in which rapid changes across a broad range of basic and clinical research approaches justify holding a conference every year. We propose to hold an annual conference at Banbury Center following the general pattern of the meeting described herein, but changing to encompass relevant disciplines and adapting to important discoveries that are moving the field. Each year’s meeting agenda will be tailored to take a different focal area and bring together both researchers in that area and researchers from other relevant approaches to broadly educate those working in the field while ensuring optimal communication of the latest research results.
At present, there is no interdisciplinary research forum for fragile X syndrome-related research quite like that proposed here. The intense, small meeting format with detailed presentations and extensive interactive discussion both following each presentation and at the end of each session clearly leads to excellent communication. We observed much more learning and critical interactions than has been typical of other meetings at which FXS-related research is discussed.
We believe that this fragile X syndrome meeting can also be significant for other disorders in which interactions between basic and clinical researchers or molecular and behavioral researchers are of value. We believe that this conference can become a model for how to bring together basic and clinical researchers, as well as basic researchers in very broad ranges of subdisciplines in a maximally communicative and productive small group format.