A. SPECIFIC AIMS
The overall Specific Aim of this proposal is to continue a series of 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. A conference will be held in each of the years 2006 through
2010, modeled on the preceding series of conferences. (There have been 6 previous
meetings, beginning in 2000.) The principal investigators, representing basic research,
clinical research, and sophisticated parent-centered advocacy will select additional
scientists to participate in planning the meeting for successive years. The scientists
contributing to planning will come largely from those who have participated in previous
meetings, but advice will be sought from whomever is most appropriate to the topics at
hand. Selection of the subsequent year’s topic(s) has in previous years been based upon a
discussion held near the end of the meeting. The topics of the preceding 6 conferences
have ranged from fundamental molecular biological, morphological and behavioral
research on the mechanisms of the disorder to the development of treatments for the
disorder as it presents clinically. The conferences have a central theme, with most
sessions organized around it. The theme of the 2005 conference was defining a
molecular, anatomical, behavioral and clinical phenotype of FXS and its animal models
for use in developing and evaluating treatments, particularly drugs.
In all meetings to date, molecular biological and pharmacological research relevant to
FXS has been an important theme, but topics have also emphasized work on other FXSrelated areas including behavioral symptoms and phenotype, other characteristics of the
disorder in humans and the knockout mouse including molecular phenotype and seizure
symptoms, experimental therapeutic approaches, and synaptic function and plasticity.
Ample time is allowed for discussion and face to face interaction with regard to the
findings presented, and a substantial number of fruitful collaborations and novel
approaches have emerged from these meetings. (See Sec. C.4.)
 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 to the latest unpublished results.
 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 most recent Banbury Conference. (See Feedback from the previous meeting,
Sec. C.4.)
A third specific aim is to provide training and an introduction to the area to younger
scientists. Previous Banbury Conferences have invited and supported participation by
young scientists, particularly postdoctoral researchers and relatively young untenured
faculty. 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.
We believe that FXS is both a remarkably rapidly moving research area and remarkably
broad in the array of approaches that will need to be integrated in the ultimate search for
effective 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.
B. BACKGROUND AND SIGNIFICANCE
B.1. Some Background on FXS
This brief background is for general orientation only. Two review articles in the
Appendix further detail general and molecular aspects of 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 is
(consensus estimate) 1/4-6000 and it is less frequent in females (Brown, 1996; Turner et
al., 1996). It has no ethnic boundaries and is caused by a poorly understood mechanism.
Fragile X manifests with relatively subtle facial appearance symptoms, enlarged testicles
and reduced IQ (Warren & Nelson, 1994; Davies, 1989; Hagerman & Cronister, 1996
Best recent reference?). 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 normative subjects. 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.
b.1.1. Genetic and Molecular Basis of Fragile X Syndrome
The genetic basis for the fragile X syndrome is an unstable region of trinucleotide
repeats [(CGG)n] in the 5’ untranslated region which is associated with hypermethylation
of a CpG island that spans the promotor and first exon (e.g., Warren & Nelson, 1994;
Davies, 1989; Hagerman & Cronister, 1996; Stoeger et al., 1997; more recent
reference?). 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 more recent
reference?), 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). 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 particularly severe fragile X syndrome
(DeBoulle et al., 1993). Alternative splicing of the gene (Ashley et al, 1993a) is
responsible for a number of isoforms, which may cause altered cellular localization.
RNA binding proteins have a wide range of roles, including posttranscriptional regulation
of gene expression (Dreyfuss et al., 1988 more recent reference?). FMRP binds to about
4% of human fetal brain mRNAs, including its own (Ashley et al., 1993b), suggesting the
capacity for selective regulatory effects. In vitro, FMRP tends to have an inhibitory
effect on protein synthesis (Warren reference?), while in more intact tissues, it appears as
if activation of fragile X-associated or -dependent pathways may trigger selective
translation of mRNA with which it is associated (Weiler et al., 2004). It has been
proposed, based on the presence of nuclear export and nuclear localization signal
sequences, that the protein may bind specific species of mRNA in the nucleus,
accompany the export of the mRNA and association with actively transported granules
that, in neurons, may transport them to distal dendrites (Bassell reference; Ceman? more
recent reference?). FMRP has been shown to dimerize in vitro with two homologs,
termed FXR1 and FXR2, which also have KH domains (Zhang et al, 1995; more recent
reference?). However, in at least some tissues, expression of these proteins appears to be
independently regulated on different time courses (Coy et al., 1996).
C.2. Summaries of past Banbury Conferences (Katie)
This section summarizes all Banbury conferences to date. The most recent 5 Banbury
Fragile X Conferences, 2001-2005, have been supported by the current period of funding
through NIMH. Brief summaries of each meeting serve as illustrative Background with
respect to the scientific nature of this annual meeting. Some comments from participants
regarding the quality of some of the meeting and its impact on their research are also
included.
C.2.0. 2000 Banbury Conference
The initial session of the conference focused on the human phenotype. Following a
patient presentation by Katie Clapp, Randi Hagerman described the spectrum of
involvement in fragile X patients and in premutation carriers. Don Bailey compared the
phenotype in fragile X syndrome and autism, and Elizabeth Berry-Kravis presented
pharmacological approaches to treating seizures, behavior and cognitive symptoms in
Fragile X Syndrome.
The next addressed animal phenotypes. Bill Greenough discussed FMRP expression
and neural morphology in the knockout mouse. Ted Brown and Ben Oostra each
presented work on strain differences in the knockout mice and work toward developing
improved mouse models. Linda Crnic described evidence for abnormalities in the
auditory startle response in FMR1 targeted mutants on several genetic backgrounds and
Richard Paylor described behavioral phenotyping of several mouse models including an
FXR2/FMR1 double-knockout. Crnic and Paylor and several other labs now have data
indicating increased prepulse inhibition in the FMR1 knockout. Haruhiko Siomi
presented cell culture and whole animal approaches to understanding the function of the
FMR1 protein in Drosophila. James Eberwine discussed protein translation and a novel
approach to proteomic analysis of the neuronal dendrite. Finally, John Larson described
methods for assessing olfactory learning and memory deficits in FMR1 knockout mice.
The third session addressed FMR1 mRNA transport and regulation of FMRP
synthesis and interacting proteins. David Nelson described functional consequences of
mutations of the FMR1 and FXR genes in mouse and drosophila. Gary Bassell talked
about FMRP trafficking in cultured hippocampal neurons. Giovanni Neri and Pietro
Chiurazzi discussed pharmacological reactivation of the fully mutated FMR1 gene using
a combined demethylation and histone deacetylation approach, and Paul Hagerman
presented studies on the expression of the FMR1 gene in males with large premutations
and small full mutations. Kimberly Huber presented data supporting a role for dendritic
protein synthesis in hippocampal long-term depression. Kenneth Mack reported that
sensory experience increases, and seizures reduce, expression of FMRP in mice. Robert
Bauchwitz offered a molecular analysis of FMR1 transgenic and knockout mice, and
Frank Kooy described diffentially expressed sequences in the knockout.
The final sessions continued to focus on the cellular functions of FMRP and
interacting RNAs and proteins. Stephen Warren presented a DNA chip analysis of FMRP
binding mRNAs and identified several hundred genes that his group found to be
associated with FMRP by co-immune precipitation. Ivan Jeanne Weiler described a
deficit of synaptic protein translation in FMR1 knockout mice. Mario Rattazzi described
work aimed at gene therapy of Fragile X using non-viral vectors. Barbara Bardoni
characterized four novel FMRP-interacting proteins, and Herve Moine presented a
specific binding motif for FMRP on its own mRNA. Jennifer Darnell presented a new
method for identifying sequence-specific RNA targets for KH2 and newly characterized
high-affinity RNA binding domains of FMRP, which identified the same purine quartet
motif described by Moine). Finally, Andre Hoogeveen described FMRP and its related
proteins and Edouard Khandjian discussed the expanding family of Fragile X-related
proteins and their expression in non-neural tissues such as sperm.
INSERT 2001 conference
C. 2.1. 2001 Banbury Conference
C.2.2. 2002 Banbury Conference
The highly productive discussions focused attention on the role of FMRP as a mRNA
binding protein and upon the likelihood that the phenotype of FXS reflects where and if
the mRNAs that FMRP binds are translated within the neuron (or other cell). Of
particular interest were the first reports, independently discovered by Herve Moine and
Jennifer Darnell, of a motif in mRNA to which FMRP binds and the first report of a
number of mRNAs that appear to be associated with FMRP by co-precipitation. (All of
these findings have subsequently been published.) The conference focused interest on
the function of FMRP at a molecular level and at the level of the cell.
Robert Darnell’s introduction highlighted the uniqueness of fragile X. Loss of
function of one protein leads to an intriguing clinical syndrome, suggesting fragile X as
an important system for identifying RNA targets -- many of which may be candidate
genes for other neuropsychiatric disorders, understanding protein action on the targets,
and understanding RNA metabolism in neurons. The involvement of FMRP in regulation
activity-dependent protein synthesis strongly suggests a key role in learning and memory.
The initial session of the conference covered FMRP’s role in regulating RNA and
protein metabolism. Stephanie Ceman proposed a model of FMRP regulation in which
FMRP enters the nucleus, there binds to mRNAs, is then phosphorylated, stopping any
further RNA binding, and transports its bound mRNAs elsewhere in the cell. This model
helps to explain the regulation of the varied and sometimes contradictory functions which
have been proposed for FRMP; the study of alternative phosphorylation states of FMRP
is difficult, requiring specific antibodies which have only recently become available. Ben
Oostra described his model of FMRP transport using PC12 cells with an inducible Tet-on
system fused with FMR1 and GFP. He concluded that FMRP can be transported from
the perikaryon via neurites to growth cones; the movement is microtubule-dependent and
occurs at a fast transport rate, similar to RNA transport. Yue Feng showed that absence
of FMRP may cause developmental abnormalities by defective regulation of glial cell
proteins, including MBP, opening up the question of whether FMRP has distinct roles in
neurons vs. glial cells. Kirk Jensen discussed the roles of RNA binding proteins in
neurons, focusing on how Nova regulates alternative splicing of several neuronal
mRNAs; FMRP itself exists in several isoforms, with little evidence thus far of
differential functions of these isoforms. Harry Orr discussed spinocerebellar ataxia type
1 (SCA1), demonstrating that trinucleotide expansion in the ataxin 1 protein leads to
over/under expression of ten mRNAs and interferes with RNA transport. Doug Black
described neuronal regulation of pre-mRNA splicing, demonstrating that stimulation of
neurons can feed back to affect splicing of mRNAs; this may suggest a neuronal
mechanism for regulating expression of different isoforms of FMRP.
The second session focused on RNA targets of FMRP. Jennifer Darnell discussed her
methods for identifying specific targets of the RGG box and KH domains of FMRP,
including several which are phenotypically relevant to fragile X syndrome. She has
identified a 3 dimensional RNA structure called a G-quartet which appears to predict
binding to FMRP. William Greenough discussed possible phenotype contributions of
some FMRP-interacting mRNAs discovered by a technique in which an antibody to
FMRP was used to locate a degenerate primer for oligonucleotide sythesis
complementary to mRNAs in the vicinity of the antigen. One putative binding mRNA
discovered in ths manner is the glucocorticoid receptor. Herve Moine further described
the detailed physical interaction of FMRP with RNAs containing G-quartets, and Lynne
Regan presented data on the role of FMRP’s KH domains in recognizing RNAs. These
detailed descriptions of FMRP-mRNA interactions have shed new light on the
mechanism of the increased phenotypic severity of the I304 point mutation previously
described by the Belgian-Dutch Collaborative group.
The next session covered the involvement of FMRP in RNA trafficking and
translational control. Oswald Steward discussed targeting of mRNA to synaptic sites on
dendrites; Gary Bassell talked about regulation and function of FMRP and FMR1 mRNA
trafficking in developing neurons. Mathias Hentze described translational regulation by
mRNA binding proteins, many of which are now known to form protein complexes with
FMRP. Joel Richter described CPEB-mediated translational control and how this may
interact with the FMRP-RNP complex. Finally, Justin Fallon discussed regulation of
FMR1 mRNA translation in neurons.
In the fourth session, drosophila models of fragile X were described. Tom Jongens
analyzed behavior of dFXR mutant drosophila, demonstrating that the flies have
abnormal circadian rhythms and courtship rituals, and noted morphological abnormalities
in neurons. These are likely fly homologs of hyperarousal and social anxiety in humans
with fragile X, and may serve as a useful behavioral assay for potential therapeutic
interventions. Alexandre Costa presented studies of dFXR-Orb interactions and mRNA
localization/translation; the 2 proteins interact extensively during drosophila development
and appear to regulate transport and translation of a subset of mRNA’s. Joanella Morales
described how dFXR regulates brain morphology and function in the CNS; null mutant
flies displayed distinctly abnormal neuronal morphology which was somewhat different
from the abnormalities previously described in the neuromuscular junction. She
discussed the role of futsch as well, and how these findings could be reconciled with
those of Broadie et al. Heinrich Matthies (from the Broadie group) showed evidence that
dFXR acts as a translational suppressor of futsch to control microtubule dynamics
underlying synaptic structure and function. Kevin Moses presented a genetic screen for
dominant modifiers of DFXRP/ Jean-Louis Mandel presented data on FMRP interactors.
The final session of the meeting focused on physiology. Kimberly Huber demonstrated
a role for ERK in mGluR and protein-synthesis dependent LTD. Mark Bear presented
evidence that mGluR-mediated LTD is excessive in the FMR1 knockout mouse.
Roberto Malinow described AMPA receptor trafficking during synaptic plasticity. Karen
Zito described studies identifying genes differentially expressed in wildtype and FMR1
knockout mouse barrel cortex. Robert Darnell summed up the meeting and a general
discussion followed.
Comments from participants, 2002 Conference
Because this was the first conference supported under the new grant we solicited
written comments from participants.
Gary Bassell indicated that his collaborative work with Jennifer and Robert Darnell to
identify domains on FMRP necessary for activity-dependent regulation arose the from
Banbury meetings and that one of his former grad students is now a postdoctoral fellow
with Bob Darnell. Gary commented: “This remains my favorite meeting each year.
There are many groups of investigators from different disciplines i.e. Neuroscience,
Biochemistry, Cell Biology and Genetics- people that you don't see all year at specialized
society meetings. Another strength of the meeting is the collegial atmosphere with much
time for lively discussion about research in Fragile X.”
Laura Antar added “Once again, the Banbury has been the highlight of my year. I learn
more at the Banbury than at any other meeting-- partly because of the tight focus, partly
because of the high quality of investigators present, and partly because of the small
number of participants.”
Jennifer Darnell reported that “following the last meeting we began working with Jerry
Yin and started a new collaborative project with Steph Ceman in S. Warren's lab. So
these two collaborations definitely arose from the 2002 meeting. I find these small, very
specialized meetings to be the most valuable in terms of information exchange and
establishing or renewing collaborations. I also feel that they are a great venue for
exchanging data that's really fresh and novel.”
Steve Warren summarized “The Banbury meetings on fragile X syndrome have always
been the yearly highlight as meetings on this subject go. Every year it gets better and last
year was the best. The invited speakers are better picked from year to year with stronger
and stronger scientists attending. It is particularly good that the invited attendees are a
good mix of long standing fragile X investigators, those junior and new to the field and
leading investigators not currently working on fragile X. This latter group is always
interesting to follow a year after the meeting when one finds many of them are now doing
fragile X research.
David Nelson said that he’d been attending fragile X meetings for over 15 years and
the 2001 Banbury meeting was the best one he'd ever experienced.
C. 2. 3. 2003 Banbury Conference
This meeting, organized by Mark Bear and Michael Tranfaglia, focused on synaptic
function in fragile X syndrome. Emerging data suggest that aspects of Fragile X
syndrome reflect synaptic dysfunction, specifically, weaker excitatory synapses,
increased numbers of long thin dendritic spines, increased susceptibility to network
seizure activity. One suspected function of the fragile X mental retardation protein
(FMRP) is regulation of protein synthesis at synapses, and one synaptic trigger for
protein synthesis is activation of group 1 metabotropic glutamate receptors (mGluRs).
Some documented consequences of mGluR-dependent protein synthesis are long-term'
synaptic depression (LTD), elongated dendritic spines, and epileptiform activity. This
meeting explored the possibility that aspects of Fragile X syndrome could reflect
exaggerated mGluR function occurring in the absence of FMRP as well as broader issues
surrounding dendritic protein synthesis and structural plasticity in Fragile X.
In the introductory session, Katherine Clapp and Michael Tranfaglia provided an
overview of the Fragile X mutation, the consequences in humans, and treatment strategies
tried to date. Mark Bear gave an overview of synaptic transmission and plasticity, and the
mGluR hypothesis of Fragile X.
The first session focused on dendritic RNA, activity-dependent protein synthesis, and
structural plasticity. Jerry Yin discussed how active synapses are "tagged" by activity to
generate synapse-specific changes in protein synthesis and how this mechanism may go
awry in fragile X; prevailing theories of synaptic tagging correlate closely with recent
findings of mechanisms of normal function of dFXRP in drosophila. Laura Antar
described how mRNA is transported from nucleus to synapse, and how this trafficking is
regulated in conjunction with FMRP in response to synaptic activity. Oswald Steward
described the targeting of mRNA to synaptic sites on dendrites and mechanisms of LTD.
Justin Fallon discussed translational regulation of the Fragile X message, and how FMRP
may be involved in regulation of translation of a discrete subset of dendritic mRNAs.
Ivan Jeanne Weiler presented data on activity-dependent regulation of synaptic mRNA
translation. Peter Vanderklish described relationships between dendritic translation and
synaptic structure, focusing on IRES dependent translation and LTP; he has demonstrated
that agonism of group I mGluR’s can induce the long, thin dendritic spines which are
characteristic of fragile X and other developmental disorders. Bill Greenough outlined
structural consequences of the absence of FMRP, including apparent abnormalities in
dendritic pruning seen in examination of whisker barrels. Ben Oostra presented new data
on findings of cerebellar ataxia and inclusion bodies, both in premutation males and in
premutation male knockout mice. Finally, Edouard Khandjian presented data on a
xenopus model for fragile X, and the insights which this may offer in understanding the
transport functions of FMRP.
The next session focused on the metabotropic glutamate receptors (mGluRs). Ever since
the original discovery by Weiler and Greenough that mGluR activation triggers local
synthesis of FMRP, there has been an intriguing thread linking the biology of mGluRs,
synaptic protein synthesis regulation, and Fragile X. Robert Wong presented one recently
characterized consequence of mGluR-dependent protein synthesis, the induction and
maintenance of epileptiform activity in the hippocampus. He showed data indicating
abnormal epileptiform activity in FMR1 knockout mice as compared to wildtype,
correlating with the human phenotype of seizure disorder. Robert Bauchwitz then
discussed one of the more robust phenotypes in the Fmrl knockout mouse, audiogenic
seizure, and presented data showing that administration of the mGluR5 antagonists
MPEP rescues this phenotype. Finally, an extensive discussion of mGluR behavioral
pharmacology was presented by Will Spooren, with particular emphasis on the anxiolytic
effects of mGluR5 antagonists; a discussion of medicinal chemistry and
pharmacokinetics of mGlur5 antagonists was presented by Fabrizio Gasparini, one of the
pioneers in this field.
Next followed a session on AMPA receptor regulation at the synapse. Synaptic
expression of AMPARs is regulated by activity and is believed to be the basis for
synaptic plasticity that is important for refining cortical circuits during development and
storing information in adults. Robert Malinow began by discussing the molecular basis of
AMPA receptor regulation during bidirectional synaptic plasticity. Kim Huber discussed
the effect of the loss of synaptic AMPARs that accompanies induction of two forms of
LTD in the hippocampus, one that is mGluR and protein-synthesis dependent, and the
other that is not. David Linden described the role of mGluRs (primarily mGluR1) and
protein synthesis in regulation of AMPARs that accompanies LTD in the cerebellum.
Peter Carlen presented biochemical data suggesting reduced AMPAR expression in the
cortex of the FMRl knockout mouse. John Larson described a role for AMPAR
regulation in olfactory learning, and his development of an olfactory descrimination assay
of cognitive function in the fmr1 knockout mouse. Finally, Elizabeth Berry-Kravis
discussed her ongoing phase II clinical trial of a drug, Ampakine CX516, that enhances
AMPAR function for the treatment of Fragile X syndrome.
The final session brought together new information about the biology of FMRP,
beginning with a talk by Steve Warren on microarray studies of gene expression in fragile
X, highlighting potentially significant targets of FMRP which could affect CNS function,
and on new findings on the fragile X permutation syndrome. Jennifer Darnell presented
evidence of recognition sequences which determine FMRP binding and, presumably,
regulation. Andrea Beckel-Mitchener discussed specific target mRNAs identified by her
group, and their potential function, with particular focus on decreases in levels of the
glucocorticoid receptor found in the KO mouse. Daniella Zarnescu presented new data on
modifiers of drosophila and their potential significance for our understanding of the
human disease; she focused on one modifier, Penelope, which is a known cancer
suppressor gene. Amy Caudy described new findings of RNA interference and its
possible role in the pathophysiology of fragile X. Finally, David Nelson summarized a
broad range of studies in his research group which focus on identifying the basic
functions of FMRP and describing a behavioral phenotype of the knockout mouse.
Convergent technologies in molecular, cell, and neurobiology have resulted in significant
new insights into fragile X. This meeting concluded with a discussion of the prospects for
developing new and effective treatments for the disorder and specific steps which might
now be taken to facilitate the process.
C. 2.4. 2004 Banbury Conference
In April 2004, the 5th annual Fragile X research meeting was held at the Banbury Center.
This year’s focus was pharmacological treatments for Fragile X: which existing drugs
and experimental new compounds might be further evaluated for treating Fragile X
syndrome. The participants were roughly equally drawn from the pharmaceutical
industry, representing seven different companies including Novartis, Addex, Lilly,
Merck, and Hoffman LaRoche, and the university-based basic research community. The
previous meetings in this series have focused on basic research describing the behavioral,
molecular, morphological and pharmacological characteristics of human fragile X
syndrome and its animal models. The 2004 meeting focused upon putting this basic
knowledge to use to address translational questions: What would be necessary to go from
the basic research, including that emanating from study of human clinical syndromes
with similar symptoms, to the development and assessment of pharmacological
treatments for fragile X syndrome? To help in both planning and running the meeting,
we recruited Dr. Will Spooren, a drug development scientist of Hoffman LaRoche
(Project Leader, F. Hoffmann-La Roche, Pharma Research Basel Discovery –
Neuroscience, Psychiatry Disease Area), who served as conference co-chair with PI
William Greenough. Dr. Spooren, in turn, recruited key researchers from the
pharmaceutical industry with specializations in drug development, receptor systems and
psychiatric aspects of a range of drug classes. The meeting had a "cut to the chase"
treatment development orientation and thus focused on drugs and drug classes which
show promise as potential treatments for fragile X.
Much discussion centered on receptor/ transmitter systems that current research indicates
are impaired or otherwise implicated in fragile X syndrome, particularly those involving
glutamate and GABA, and compounds that target those systems. By the end of the
meeting, it had become clear that more work is needed to design good clinical trials to
effectively test drug treatments for fragile X. Several exciting new collaborations
between industry and university scientists were established at the meeting, and several
drug trials in Fragile X patients are now being planned.
In the introductory session, chaired by Dr. Elizabeth Berry-Kravis, a parent perspective
of the fragile X syndrome was provided by Katherine Clapp of FRAXA Research
Foundation, in an effort to familiarize scientists new to the field with the clinical
presentation of fragile X. This was followed by an overview of phenotype and of
patterns of medication use by Don Bailey of the University of North Carolina, Chapel
Hill. His group has conducted the longest longitudinal studies of fragile X children, and
has systematically examined the prevalence of autism and autistic symptoms in fragile X
children.
Dr. Randi Hagerman from the M.I.N.D. Institute at the University of California, Davis
discussed psychopharmacological interventions in fragile X, with an emphasis on types
of behavioral problems encountered in the pediatric clinic. A review of the few clinical
trials done to date in fragile X was included. This was followed by Stephen T. Warren of
Emory University who described molecular mechanisms of the fragile X phenotypes; his
research group is studying several model systems, including mice and drosophila, and
has developed phenotypes which may be useful for testing putative therapeutic agents.
The second session focused on the technology of mouse models, both for fragile X in
particular, and for neuropsychiatric disorders in general. Representing pharmaceutical
industry research, Thomas Steckler of Johnson & Johnson discussed pitfalls of attempting
to model complex human conditions like depression and anxiety in mice, and his
development team’s experience with CRF antagonists. Just as depression and anxiety are
typically stress-related disorders, fragile X has also been reported to involve exaggerated
cortisol responses to stress. This implies that agents like CRF antagonists, which may
modulate the HPA axis, may have applications in the treatment of fragile X. The next
presentation, by Richard E. Paylor of Baylor College of Medicine, dealt with the
behavioral phenotype of fmr1 KO mice and the role of genetic background; a behavioral
phenotype has been surprisingly difficult to obtain in the KO mouse, and many of the
differences found to date have been highly strain-dependent. This has made evaluation of
potential pharmacologic treatments especially difficult.
Ben A. Oostra of Erasmus Universiteit, Rotterdam then presented evidence of enhanced
LTD at enlarged Purkinje cell spines in the cerebellum, which causes motor learning
deficits in fmr1 KO mice. This phenotype manifests as aberrant eye-blink conditioning,
an assay which can also be utilized in human subjects as a potential outcome measure for
clinical trials of investigational agents. Miklos Toth of Cornell University Medical
College then discussed his studies of the hyperactivity of fragile X mice to sensory
stimuli, as evidenced by increased startle response and increased sensitivity to audiogenic
seizures. He is using pharmacologic probes to elucidate causes of network
hyperexcitability and alterations in FMRP-target expression.
The third session focused on functional consequences of the fragile X mutation in the
nervous system. Robert Wong of SUNY Downstate discussed the role of metabotropic
glutamate receptors in epileptogenesis in fragile X syndrome. He has found that neural
circuits in fmr1 KO mouse hippocampal slices show markedly increased epileptogenesis
compared to normal littermates, that this increase is mediated by mGluR5, and that this
aspect of neurophysiology can be normalized by administration of the mGluR5
antagonist MPEP. Addressing a similar question in intact mice, Robert P. Bauchwitz of
Columbia University described enhanced susceptibility to audiogenic seizure in all strains
of KO mice examined, but with significant variability depending of genetic background.
In all strains, audiogenic seizure could be prevented by administration of MPEP, but with
wide variation in ED50.
Peter W. Vanderklish of Scripps Research Institute then presented his findings on
regulatory interactions between synaptic structure and local translation. He has
demonstrated that activation of group I mGluRs can induce long, thin dendritic spines
over short time periods---morphology reminiscent of that seen in fragile X; he has been
investigating the specific events in translational regulation at the dendrite which control
this process, and how they may be altered in fragile X. He was followed by Eric Klann
of Baylor College of Medicine, discussing regulation of translation signaling pathways
during mGlur-LTD in fmr1 knockout mice. He has quantified many elements of the
mGluR signaling pathways and shown how these respond to synaptic activity in normal
and fmr1 KO mice. J. Julius Zhu, from the University of Virginia School of Medicine,
then presented on Ras signaling of excitatory synapses of FMR1 knockout mice; precise
alterations in elements of signaling pathways in fragile X have been described in his
work, offering potential targets for drug discovery. Finally Mark Bear, of MIT, described
his original findings of enhanced mGluR-LTD in the fmr1 KO mouse, then elaborated by
discussing mechanisms in various brain regions which could explain other aspects of the
fragile X phenotype.
Session 4 was chaired by two representatives of the pharmaceutical industry, Will
Spooren of Hoffmann-LaRoche and Graeme Bilbe, Global Head of Neuroscience
Research for Novartis. This session highlighted possible “drugable” targets in fragile X,
and explored paths to development of viable treatment strategies. Michael R. Tranfaglia
MD, Medical Director of FRAXA Research Foundation, outlined the use of available
medications in the treatment of fragile X. Graeme Bilbe then presented an overview of
the drug discovery process, with an emphasis on specific needs of the fragile X field,
followed by presentations addressing specific neurotransmitter systems. Vincent Mutel
of Addex Pharmaceuticals discussed development of positive and negative allosteric
modulators of mGluR’s, and their potential uses in psychiatry. Michael P. Johnson of Eli
Lilly discussed his experiences in development of mGluR2/3 agonists for treatment of
anxiety, and their potential application to treatment of fragile X and related disorders.
Fabrizio Gasparini of Novartis then presented “Allosteric Modulation for the mGlu
Receptors: Antagonists, Positive Modulators, Neutral Ligands” which included a brief
history of the search for specific mGluR5 antagonists.
The series of presentations continued with Will Spooren discussing Neurokinin 3 (NK3)
receptors as a new target for the treatment of psychosis. Gerard R. Dawson of Merck
presented on the role of GABA-A receptor subtypes in anxiety and cognition. Bernhard
Bettler of Universitat Basel discussed GABA-B receptors as therapeutic targets for CNS
disorders, and Carl Dobkin, New York State Institute for Basic Research, presented his
findings of seizure susceptibility of the fragile X mouse and alterations of the
GABAergic system. To conclude the day, a lengthy discussion involving the fragile X
experts and the pharmaceutical researchers highlighted potentially fruitful areas for future
exploration, and spawned several collaborations to investigate specific neurotransmitter
systems in fragile X.
Session 5 had a distinct clinical/therapeutic focus. Thomas Jongens of U. Penn presented
evidence of a robust cognitive and behavioral phenotype in dFXR null mutant
drosophila; he further described rescue of the phenotype using the mGluR5 antagonist
MPEP and lithium, which inhibits several elements of the mGluR signaling pathways.
Peng Jin of Emory also noted a distinct phenotype in drosophila, but one of reduced
survival of dFXR null mutants on a common kind of laboratory fly food. Even though
this phenotype is not obviously neural, it is nevertheless rescued by administration of
MPEP, and may serve as the basis for a higher throughput drug screen. Herman
Westenberg of Utrecht University then presented findings from studies of the role of
dopamine in anxiety disorders, especially OCD and social phobia. These disorders are
highly relevant to the clinical presentation of fragile X, and may warrant investigation of
dopaminergic systems. Elizabeth Berry-Kravis concluded the formal presentations with a
description of her ongoing trial of CX516, a compound that facilitates AMPA receptor
transmission.
The final session was an open discussion moderated by Michael Tranfaglia of FRAXA
and Will Spooren, with the express purpose of defining strategies to develop treatments
for fragile X. The expertise of pharmaceutical industry scientists combined with the
experience of fragile X researchers to generate a list of important interim goals, such as
devising improved outcome measures to assess efficacy in fragile X clinical trials.
Summary: Convergent technologies in molecular, cell, and neurobiology have resulted in
significant new insights into fragile X. At least some existing drugs may be ready for
preliminary evaluation in fragile X syndrome and animal models of it. This meeting
concluded with a discussion of the prospects for developing new and effective treatments
for the disorder and specific steps which might now be taken to facilitate the process. One
point of agreement was the importance of clear, ideally quantifiable descriptions of the
phenotype of the syndrome for use in evaluating the efficacy of drug treatments. It was
decided that this would be one of the topics of the next meeting.
C. PRELIMINARY STUDIES/PROGRESS REPORT
C.1. How the conference has evolved across the years
As can be seen, the conferences have become increasingly sophisticated over the years,
reflecting the rapidly growing knowledge of the genetic, molecular, cellular,
pharmacological and behavioral aspects of FXS and its animal models. Many findings
were first communicated at this conference before they were published, including the
drosophila dFMR brain phenotype, the existence and characteristics of mRNA binding
partners for FMRP, and the apparently restorative effects on some aspects of phenotype
associated with the administration of the mGluR5 antagonist MPEP. The scope has also
broadened, with the inclusion of pharmacologists involved in the development or testing
of potential therapeutic agents. The increased focus on ways in which the basic research
knowledge being presented at the meeting can aid in the development of therapeutic
interventions parallels the call for more translationally-oriented research in the NIH
funding portfolio. The most recent meeting illustrates this trend in a particularly dramatic
manner, with basic researchers presenting details of molecular, structural and behavioral
aspects of the FXS and mouse model phenotype that could be valuable in the assessment
of the efficacy of potential therapeutic drugs.
C.2. New scientists attracted to FXS meeting attendance (all, but Katie probably has
the most complete records [and memories])
The Banbury meeting has been very successful in attracting newcomers to the field of
fragile X research, as well as in focusing research in the FXS area on brain
mechanisms involved in the syndrome, which seems the best possibility for
developing treatments for the most salient symptoms of the disorder. Among
scientists who became (at least part-time) fragile X researchers subsequent to
attending a Banbury Conference are Mark Bear, Os Steward,
C.3. Involvement of young, minority and female scientists (Katie again has the best
records)
Minority scientists who have participated in the meeting include Jennifer Hill Karrer,
University of Kansas, and Joanella Morales, Baylor College of Medicine. Young female
scientists who have participated in the meeting include: Esther Nimchinsky Cold Spring
Harbor Laboratory; Laura Antar, Albert Einstein College of Medicine; Kim Huber, UT
Southwestern Medical Center, Dallas; Stephanie Ceman, now at University of Illinois;
Jennifer Darnell, Rockefeller University; Claudia Bagni, University of Rome; Flora
Tassone, UC Davis; Karen Zito, CHSL; Daniela Zarnescu, Emory University; Julie
Lauterborn, Univ. Calif. Irvine; Michelle Adams, Brown University; Yue Feng, Emory
University; Peng Jin, Emory University; Andrea Beckel-Mitchener, now at NIMH; Sally
Till, Columbia University; and Mikiko Siomi, University of Tokushima; Isabel Boutet,
University of Toronto. Young male scientists include Julius Zhu, University of Virginia
School of Medicine; Kenneth J. Mack, University of Wisconsin (Mayo Clinic); John
Larson, Univ. of Illinois, Chicago; Gary Bassell, Albert Einstein College of Medicine;
Robert P. Bauchwitz, Columbia University; Sean McBride and Jason Dictenberg, both
at Albert Einstein College of Medicine, and Francois Bolduc, CSHL. On average well
over 15 % of attendees at the meetings fall into one or more of these classes (young,
female or minority, as can be seen in the appended lists of participants for each meeting),
and a number of those listed above have attended more than a single meeting. More
senior females are also very well represented. The meeting has provided an excellent
informal environment for younger, female and minority scientists to get to know both
their peers in the fragile X area as well as the leaders in the field. Many of them were in
their earliest years as faculty or at earlier career stages at the time that they first attended
the meeting and have now matured into tenured faculty or for those who attended as
postdocs or graduate students, into junior faculty or comparable positions.
Collaborations
C.4. Comments from past attendees including Collaborations resulting from
interactions at the 2005 meeting
C.5.
D. CONFERENCE PLANS AND METHODS
D.1. Proposed Conference Series
We propose to continue the Banbury meetings, one meeting per year for the next
5 years, beginning in 2006. Meetings will usually be held in March or April, although
accommodations may sometimes need to be made to the Banbury Center schedule. The
Banbury site is ideal for a meeting of this sort, although the meeting room could be a
small bit larger. The problem of the 41st chair – or whom we cannot invite for reasons of
space -- sometimes looms large, but the intimate nature of the meeting, in which virtually
everyone present has the chance to participate in informal discussion with everyone else,
would erode if the meeting were substantially larger.
D.2. Plans for the 2006 Meeting
OK—what do we want to do here? Do you think we have a mandate?
D.3. Guidelines for Future Meetings
A valuable aspect of the Banbury meetings, judging by both comments at our
concluding discussions and the comments of participants (Section C), is that, while the
meeting has featured certain themes, such as “delineating the phenotype” in the 2005
meeting (Sec. B.2.5), it remains very general in approach, with behavioral, clinical,
pharmacological, cell biological and molecular approaches, and others, all wellrepresented. It is our proposal to maintain this property of the meeting, largely by
including past attendees of the meeting in the planning process, both during and at the
end of the meeting and in the course of organizing the meeting during the year prior to its
occurrence.
A suggestion of a number of participants over the years has been that we include a
session or at least a few presentations on methods that are in use in other fields that could
benefit the FXS research area. Dr. Porges attended the last meeting at the suggestion of
Bill Greenough and Elizabeth Berry-Kravis in response to suggestions that prior meetings
were relatively lacking in individuals specialized in measurement of aspects of
physiological phenotype in humans. For the next meeting Eric Klann has suggested that
we have a presentation on proteomic methods that could help in delineating the
expression profile of FXS and its models at the protein level.
D.3.1. Selection of annual topics
The evolution of the meeting, which has been very much participant-driven, was
described in Sec. C.1. With verbal input at the meeting from members of the group and
from comments received both from individuals who have attended the previous meetings
and from those who have not (not all of it positive—see comments by Ben A. Oostra in
Sec. C.4.), the general development of the theme laid out in the discussion at the prior
meeting is generated and then individuals with interests most closely related to the topic
are contacted, asked if they would be willing to participate in the upcoming meeting, and
asked what they would talk about with respect to the developing theme. They are also
queried as to individuals that would contribute appropriately to the theme. Thus the
construction of the meeting is a iterative process which depends heavily upon the
directions suggested at previous meetings.
D.3.2. Administrative organization
While the PI’s have played a central role in the organization of meetings to date,
the administrative input has been largely in the nature of a “committee of the whole,”
with perhaps 10-15 relatively regular participants serving as a functional “board. As
noted above, at the end of each meeting, a final discussion covers future directions and
topics for the next meeting. This has largely served as the basis upon which the next
meeting is formulated. Typically the PI’s on the grant have twisted the arms of one or
more of the people closest to the suggested topic to join them in organizing the next
year’s meeting. These individuals then formulate an outline of the meeting with
relatively frequent telephone and email contact with both past attendees and individuals
who have been mentioned as appropriate to a planned topic. This has allowed us to both
draw upon the expertise of individuals who have not attended previous meetings and to
include in our meetings individuals who might be persuaded by the meeting’s content to
devote some future effort to FXS research.
For example, with regard to proteomics methodologies, mentioned above, Donald
Hunt, Chemistry, University of Virginia has been suggested as a good consultant and
possible meeting participant. Hunt has pioneered Mass spectrometric analysis of the
contents of mixtures of relatively large numbers of proteins, a technique that, across its
variants, might be very valuable in determining differences in expression levels of both
the genes whose mRNAs bind to FMRP and of other proteins whose expression levels are
secondarily affected by these primary expression level differences.
D.3.3. Program advisory committee
We actually specified a committee of this sort in the last proposal.
Functionally we may have had this but we really didn’t use it in any very specific
way. Question—do we want to propose having one, and, if so, who should be on it?
D.3.4. What else?
E. HUMAN SUBJECTS RESEARCH
N/A
F. VERTEBRATE ANIMALS
N/A
G. LITERATURE CITED
H. CONSORTIUM/CONTRACTUAL ARRANGEMENTS
N/A
I. RESOURCE SHARING
N/A
J. CONSULTANTS
N/A