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Toward fulfilling the promise of molecular medicine in fragile x syndrome





Toward Fulfilling the Promise of Molecular Medicine in Fragile X Syndrome Dilja D. Krueger and Mark F. Bear The Picower Institute for Learning and Memory, Howard Hughes Medical Institute,Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology,Cambridge, Massachusetts 02139; email: mbear@mit.edu Annu. Rev. Med. 2011. 62:411–29 First published online as a Review in Advance on FMRP, metabotropic glutamate receptor, autism, mental retardation, November 19, 2010 protein synthesis, long-term depression The Annual Review of Medicine is online atmed.annualreviews.org This article's doi: Fragile X syndrome (FXS) is the most common inherited form of men- tal retardation and a leading known cause of autism. It is caused by  2011 by Annual Reviews.
loss of expression of the fragile X mental retardation protein (FMRP), All rights reserved an RNA-binding protein that negatively regulates protein synthesis.
In neurons, multiple lines of evidence suggest that protein synthesis by Copenhagen University on 06/03/11. For personal use only.
at synapses is triggered by activation of group 1 metabotropic gluta-mate receptors (Gp1 mGluRs) and that many functional consequencesof activating these receptors are altered in the absence of FMRP. These Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org observations have led to the theory that exaggerated protein synthesisdownstream of Gp1 mGluRs is a core pathogenic mechanism in FXS.
This excess can be corrected by reducing signaling by Gp1 mGluRs, andnumerous studies have shown that inhibition of mGluR5, in particular,can ameliorate multiple mutant phenotypes in animal models of FXS.
Clinical trials based on this therapeutic strategy are currently underway. FXS is therefore poised to be the first neurobehavioral disorder inwhich corrective treatments have been developed from the bottom up:from gene identification to pathophysiology in animals to novel ther-apeutics in humans. The insights gained from FXS and other autism-related single-gene disorders may also assist in identifying molecularmechanisms and potential treatment approaches for idiopathic autism.
has been slow and difficult owing to the daunt- Combining insights obtained from basic bio- ing biological complexity of the brain and thecomplex genetics that underlie most of these ASD: autism
logical research with genetic and clinical find- spectrum disorder ings can paint a detailed picture of the molecular diseases (1, 2). However, in recent years therehave been significant advances in understand- FXS: fragile X
events that result in disease, and suggest ways these mechanisms can be targeted with correc- ing the pathophysiology of several single-gene tive interventions. Not surprisingly, application disorders of brain development associated with of such "molecular medicine" approaches to intellectual impairment and autism (see side- psychiatric and neurodevelopmental disorders bar "The Promise of Molecular Medicine in
Brain Disorders" and Figure 1). The encour-
aging prospects for developing new corrective
treatments based on these insights have gen-
THE PROMISE OF MOLECULAR MEDICINE
erated considerable excitement, particularly in IN BRAIN DISORDERS
light of reports that the prevalence of autismspectrum disorders (ASDs) is rising sharply (3).
Understanding the genetic basis of psychiatric and neurological In this review, we describe recent progress in disorders eventually will lead to insights into how the brain func- the study of one of these disorders, fragile X tions differently in these diseases. Understanding their patho- syndrome (FXS), and discuss the implications physiology, in turn, will suggest molecular targets for therapeutic of these data for autism and other related neu- interventions. This process begins with careful phenotypic strat- ification of patients followed by gene-variant discovery efforts.
Psychiatry, in particular, faces major obstacles in applying thisstrategy because (a) many behavioral disorders fall on a spectrum, MIXING THE STREAMS
making diagnosis and stratification difficult; (b) there is often a OF GENETICS AND
large contribution of environmental influences to disease pro- gression and outcome; and (c) most disorders do not have a single FRAGILE X SYNDROME
major genetic cause but are the result of a plethora of individ-ual mutations and gene copy number variations (2). Single-gene Fragile X Syndrome
disorders such as FXS are therefore particularly valuable as mod- FXS, originally known as Martin-Bell syn- els for more genetically complex disorders such as autism. Once drome (4), was first described in 1943 as an a disease-associated gene has been identified, animal models of X-linked form of inherited mental retardation the disorder can be generated through genetic manipulation to (Figure 2) (5). The first evidence regarding the
reproduce the underlying genetic deficits. These animal models by Copenhagen University on 06/03/11. For personal use only.
molecular origin of FXS was obtained in 1969, enable further study of the cellular, physiological, and behav- when an unusual constriction, or fragile site, ioral consequences of aberrant gene expression. It is here that was observed at the end of the X chromosome Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org basic neurobiology research becomes an essential partner in the in several affected individuals (6, 7). It was not drug discovery process, allowing the observations made in animal until 1991, however, that the fragile site was models to be interpreted in the context of a vast background of mapped to a specific location in the genome knowledge on brain structure and function. Based on the con- (8). This fragile site coincided with an ex- vergence of information from these sources, hypotheses can be panded CGG trinucleotide repeat in the 5 un- formulated and tested to generate a plausible model of disease translated region of a novel gene, subsequently pathophysiology. This model, in turn, can be used to identify po- named fragile X mental retardation 1 (FMR1).
tential drug targets, which then provide the basis for the develop- Trinucleotide repeat expansions consisting of ment of novel therapeutic strategies that can finally be validated >200 CGG repeats were found to cause hy- in clinical trials. Although the causes vary, many autism spec- permethylation of the surrounding sequence, trum disorders may share pathophysiological mechanisms with resulting in transcriptional silencing of FMR1 FXS and therefore respond to the same treatments.
(for an extensive review of this subject, seeReference 5). In 1993, the protein encoded Krueger · Bear Target ID
The promise of molecular medicine in psychiatric and neurodevelopmental disorders (see sidebar "ThePromise of Molecular Medicine in Brain Disorders" for explanation).
by FMR1, fragile X mental retardation protein conclusions regarding the precise nature of this (FMRP), was characterized and found to be an role (see 14 for a review of the early literature); RNA binding protein that is expressed predom- however, there is now broad support for the hy- inantly in the brain and testes (9). Shortly there- pothesis that FMRP functions as a translational after, an Fmr1 knockout mouse model (Fmr1 repressor of target mRNAs (15–17), as we dis- KO) was generated, in which part of the Fmr1 cuss further below. In addition to its role in reg- gene was deleted to mimic the loss of FMRP ulating protein synthesis, FMRP has also been expression seen in FXS (10). Since then, these implicated in the transport and localization of by Copenhagen University on 06/03/11. For personal use only.
Fmr1 KO mice and other animal models have mRNAs to dendrites and synapses. The mech- been extensively characterized, and in conjunc- anisms by which FMRP may regulate protein Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org tion with further in vitro studies of FMRP func- synthesis and transport remain under active in- tion, they have been instrumental in providing vestigation, and a detailed discussion of the cur- insights into potential roles for FMRP in the rent state of this field can be found elsewhere brain as well as the pathophysiology of FXS.
The observation that FMRP may be linked to the protein synthesis machinery, together FMRP Function and Loss
with the fact that both FMRP and mRNA are of Function in FXS
expressed in the dendrites and dendritic spines One important clue regarding the function of of neurons, suggested that it may play an impor- FMRP came from the observation that it is as- tant role in local protein synthesis at synapses FMRP: fragile X
sociated with polyribosomes, implying a role (18, 19). These findings were of particular in- mental retardation in the regulation of protein synthesis (11–13).
terest in light of the increasing evidence linking Early biochemical studies yielded contradictory local protein synthesis to synapse maturation www.annualreviews.org • Molecular Medicine in Fragile X Genetics and molecular biology
Basic neurobiology and pharmacology
Martin-Bell syndrome Watson & Crick publish structure of DNA Description of ocular dominance plasticity in visual cortex (36) Constriction on X chromosome identified in FXS (6) Conditions identified to express "marker X" fragile site (7) BCM theory of cortical plasticity proposes homosynaptic LTD (37) "Metabotropic" action of glutamate described (31) mGluRs discovered (32), hypothesizedto trigger LTD (38) Group 1 mGluR sequenced (33) Homosynaptic LTD discovered (39) FMR1 gene identified (8) mGluR stimulation induces FMRP synthesis FMRP protein described (9) Fmr1 KO mouse model generated (10) mGluR-dependent protein synthesis prolongs epileptiform bursts (49) mGluR5 inhibitor MPEP described (67) Protein synthesis-dependent mGluR-LTDdescribed (44) 2002 mGluR-LTD exaggerated in Fmr1 KO mice (17),
mGluR theory of fragile X proposed (52) 2005 MPEP ameliorates fragile X phenotypes in
animal models (68, 72) 2007 Genetic reduction of mGluR5 corrects
multiple Fmr1 KO phenotypes in mice (48) 2009 Clinical trials initiated using mGluR5
inhibitors to treat FXS (83) by Copenhagen University on 06/03/11. For personal use only.
Some milestones in defining the pathophysiology of fragile X syndrome (FXS). The current therapeutic efforts in FXS originate fromthe mixing of two independent lines of research: genetic research on FXS (left timeline, yellow) and basic neurobiology research onmGluR-dependent synaptic plasticity (right timeline, blue). The discovery that mGluR-LTD is exaggerated in Fmr1 knockout (KO) Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org mice (17) led to the mGluR theory of FXS pathophysiology, culminating in the initiation of clinical trials to test the efficacy ofmGluR5 antagonists in the treatment of FXS. Numbers in parentheses are reference citations. Fmr1, fragile X mental retardation 1;FMRP, fragile X mental retardation protein; mGluR, metabotropic glutamate receptor; LTD, long-term depression; MPEP, 2-methyl-6-(phenylethynyl)-pyridine.
and synaptic plasticity (20), and it was hypoth- spines (21–23). Cultured neurons from Fmr1 esized that loss of FMRP expression might KO mice mimic this phenotype, displaying an result in disruptions of synaptic structure and increased number of structural synapses (24).
function. Consistent with this notion, one of In addition, loss of FMRP in animal models has the most prominent morphological phenotypes also been shown to affect synaptic plasticity: observed in both FXS patients and Fmr1 KO Fmr1 KO mice show exaggerated forms of mice is an increase in dendritic spine density and long-term depression (LTD) in hippocampus the presence of abnormally long and tortuous (17) and cerebellum (25), discussed in further Krueger · Bear detail below. Moreover, several groups have visual cortex. Temporarily degrading image reported alterations in long-term potentiation formation in one eye sets in motion synaptic (LTP) in the cortex and hippocampus of Fmr1 changes in the visual cortex that render neu- KO mice (26–30). Together, these findings rons unresponsive to the deprived eye (36).
suggested that the absence of FMRP may Various theories of synaptic modification were glutamate receptor alter synaptic plasticity throughout the brain, developed to account for these and related which may be important in the pathogenesis of modifications. The influential BCM theory (37) posited that the loss of strength of deprived-eyesynapses was not caused by the loss of activityfrom the deprived retina but rather by the pres- Metabotropic Glutamate Receptors
ence of stochastic afferent activity (registered in the cortex as glutamate release) that no longer It was shortly after the discovery that FMRP correlates with strong postsynaptic responses.
plays a role in protein synthesis that the FXS On the basis of this idea and the observation field crossed paths with emerging lines of re- that glutamate-stimulated PI turnover was search on metabotropic glutamate receptors exaggerated in visual cortex at the age of max- and activity-dependent synaptic plasticity, lead- imal plasticity, the hypothesis was put forth ing to the discoveries that gave rise to current that Gp1 mGluRs might serve as a trigger for therapeutic efforts in FXS (Figure 2).
synaptic weakening (38). Homosynaptic LTD, triggered by weak activation of glutamate (mGluRs) are G protein–coupled receptors receptors, was subsequently discovered in the that link to intracellular signaling pathways, CA1 region of hippocampus (39), and one type including the Gq/PLC pathway [group 1 of homosynaptic LTD was later shown to be (Gp1) mGluRs] and Gi/Go pathways (groups 2 triggered by activation of mGluR5 (40–42) and and 3 mGluRs). Their existence was predicted to require synaptic protein synthesis (43, 44).
in 1986, when it was observed that agonists For a recent detailed review of the molecular of glutamate receptors, thought to be ion mechanisms underlying mGluR-LTD, see channels exclusively at the time, could also Reference 45.
stimulate phosphatidylinositide (PI) turnover(31). In 1988, the first mGluR mRNAs wereisolated (32), and the first corresponding The mGluR Theory
gene was cloned in 1991 (33). These findings of Fragile X Syndrome
by Copenhagen University on 06/03/11. For personal use only.
caused a major shift in the way people thought In 1997, while conducting a screen for synaptic about glutamate as a neurotransmitter (34), mRNAs that are translated in response to Gp1 suggesting that it may act as a neuromodulator mGluR activation, Weiler et al. demonstrated Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org in addition to its role in fast excitatory neuro- that FMRP is synthesized following applica- transmission. In 1993, Weiler & Greenough tion of the receptor agonist DHPG (dihydrox- presented the first evidence that one conse- yphenylglycine) in synaptoneurosomes (46). As quence of activating Gp1 mGluRs, comprising described above, another line of investigation mGluR1 and mGluR5, is increased protein showed that DHPG, acting through mGluR5, synthesis at synapses (35).
can trigger LTD that requires translation of The explosion of knowledge about glu- preexisting mRNA (40, 44). Thus, FMRP was tamate receptors in the 1980s also made at the top of the list of candidate proteins that possible the formulation of detailed hypotheses are synthesized in response to mGluR5 activa- about how excitatory synapses in the brain tion to stabilize LTD. The simple hypothesis are bidirectionally modified by experience that FMRP is required for mGluR-LTD was to store information. One powerful in vivo tested using the Fmr1 KO mouse (17). The re- model of experience-dependent plasticity is the sults showed, however, that instead of impaired www.annualreviews.org • Molecular Medicine in Fragile X LTD, there was exaggerated LTD in the Fmr1 mutant phenotypes in FXS. This latter predic- tion generated considerable excitement in the These findings did not fit the prevailing FXS field because it hinted at the possibility of model of the time, in which synaptic protein a targeted treatment strategy that did not rely synthesis was impeded by the loss of FMRP in on replacement of the FMRP molecule itself.
FXS (46), nor the hypothesis that FMRP sta-bilizes LTD. Rather, the data were consistentwith the notion that FMRP, by binding directly Testing the mGluR Theory:
to synaptic mRNAs, functions as a repressor Evidence for Altered Consequences
of synaptic protein synthesis (15, 16). Thus, of mGluR Activation
Huber et al. (17) suggested a model in which Following the original discovery of exagger- FMRP normally serves to limit expression of ated mGluR-LTD, a wave of studies focused LTD by inhibiting mGluR-dependent transla- on further investigating this and other mGluR- tion of other synaptic mRNAs encoding the hy- related phenomena in Fmr1 KO mice. In agree- pothetical "LTD protein(s)." According to this ment with the assumptions of the theory, idea, FMRP synthesis in response to mGluR5 mGluR1-dependent cerebellar LTD was ob- activation normally serves as an important served to be exaggerated in the KO (25), as was brake on synthesis of other proteins—in the mGluR1/5-dependent prolongation of epilep- absence of FMRP, there is runaway or poorly tiform bursts in area CA3 of hippocampus (53).
regulated synaptic protein synthesis. The hy- One of the mechanisms underlying mGluR- pothesis that cerebral protein synthesis is ele- LTD is the protein synthesis–dependent loss vated in FXS was later confirmed by metabolic of surface AMPA (alpha-amino-3-hydroxy-5- experiments in the Fmr1 KO mouse, both in methyl-4-isoxazole-propionate) receptors (43), vivo and in vitro (47, 48).
and it was hypothesized that the excess LTD in Gp1 mGluRs participate in many brain cir- the Fmr1 KO mice might be due to an excess cuits and serve diverse functions in addition to internalization of AMPA receptors (52). This LTD. By the end of 2002, there were indi- hypothesis was confirmed using knockdown of cations in the literature that some other last- FMRP levels by short interfering RNAs in hip- ing consequences of activating mGluR1 and pocampal cultures (54).
mGluR5 require mRNA translation (49–51), A particularly striking discovery was that consistent with the early biochemical finding hippocampal mGluR-LTD in the Fmr1 KO that mGluR activation stimulates protein syn- mice no longer requires acute stimulation of by Copenhagen University on 06/03/11. For personal use only.
thesis (35). The realization that many of the protein synthesis at the time of induction, symptoms of FXS might plausibly be explained consistent with the notion that "LTD proteins" by excessive protein synthesis downstream of are constitutively overexpressed and no longer Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org mGluR1/5 led to a formal proposal entitled rate-limiting for LTD in the KO (55, 56).
"the mGluR theory of fragile X," which was These findings inspired examination of another first publicly presented in 2002 and published mGluR- and protein synthesis–dependent phe- in 2004 (52).
nomenon that occurs at the same population The mGluR theory made two important of hippocampal synapses, called LTP priming predictions that could and would be extensively (30). Priming is an enhancement of LTP that tested in future experiments. First, it implied occurs when Gp1 mGluRs are briefly and that other consequences of mGluR activation weakly stimulated prior to delivery of LTP- should be altered in the absence of FMRP.
inducing tetanic stimulation (51). Like mGluR- Second, and more significantly, it suggested LTD, LTP priming normally requires mGluR- that a reduction of mGluR activity might re- dependent translation of mRNA in wild-type store normal synaptic protein synthesis in the mice but not in the Fmr1 KO (30). Because it absence of FMRP and therefore reverse some is not known if "LTP priming proteins" and Krueger · Bear "LTD proteins" are the same or different, it in Fmr1 KO mice. A number of interesting is probably better to conceptualize these prod- candidates have been proposed to date, includ- ucts of mGluR-stimulated mRNA translation ing MAP1B, eEF1A, Arc, CaMKIIα, PSD-95, as "plasticity gating proteins." These gating SAPAP3, and APP (for recent detailed reviews, proteins are apparently overexpressed in the see 18, 63). The roles of these candidate pro- Fmr1 KO, leading to diverse consequences.
teins in mGluR-related phenotypes in Fmr1 A number of studies have also investigated KO mice are currently under investigation.
the role of signaling pathways downstream ofGp1 mGluRs in protein synthesis and LTDphenotypes in Fmr1 KO mice. mGluR5 recep- Reversal of FXS-Related Phenotypes
tors were shown to be less tightly coupled with by Reduction or Antagonism
synapses and Homer scaffolding proteins in of Gp1 mGluRs
Fmr1 KO mice (57), and the Homer-dependent Experiments investigating the effects of mGluR activation of mTOR (mammalian target of ra- inhibition on phenotypes induced by the loss of pamycin) signaling in response to mGluR5 FMRP have two essential benefits: They test stimulation was absent altogether (58). Other the validity of the biological principles under- groups have reported a slight basal increase in lying the mGluR theory of fragile X, and they ERK (extracellular signal-regulated kinase) ac- provide information on the potential for target- tivity (56), an aberrant mGluR-induced inac- ing mGluRs as a therapeutic strategy in FXS.
tivation of ERK (59), a basal increase in the Accordingly, a significant body of literature has Akt/mTOR pathway (60) and an excess of PI3K emerged on this topic in the past five years.
(phosphoinositide 3-kinase) activity (61). How- In an extensive proof-of-principle study, ever, many of these signaling alterations ap- Dolen et al. used a genetic strategy to inves- pear to be highly dependent on the experimen- tigate the effect of reducing mGluR5 levels tal preparation and are not observed under all on FXS-related phenotypes in Fmr1 KO conditions. For example, neither Akt/mTOR mice (48). Mice heterozygous for the Grm5 nor ERK signaling pathways were altered in gene (which encodes mGluR5) were crossed hippocampal slices confirmed to have increased with Fmr1 KO mice, and the resulting 50% basal protein synthesis in the Fmr1 KO (62).
reduction in mGluR5 protein levels led to These data suggest that altered Akt/mTOR and the correction (or prevention) of 7 out of ERK signaling may not be a cause (but may the 8 FXS-related phenotypes assessed. A be a consequence) of aberrant protein synthe- significant decrease was observed in the induc- by Copenhagen University on 06/03/11. For personal use only.
sis in Fmr1 KO mice. We favor the hypothesis tion of audiogenic seizures, a model for the that the protein synthesis phenotype is due to epilepsy frequently observed in FXS patients.
hypersensitivity of the mRNA translation ma- In addition, the increase in dendritic spines Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org chinery to normal levels of mGluR signaling, seen in visual cortex of Fmr1 KO mice, the rather than hyperactivity of the mGluR signal- increase in protein synthesis in hippocampus, ing pathways themselves (62). It is likely, how- and the increase in total body weight observed ever, that the aberrant signaling pathways ob- in young Fmr1 KO mice were all reversed.
served in some preparations do contribute to Moreover, the genetic reduction of mGluR5 Fmr1 KO phenotypes, regardless of whether corrected the excessive hippocampal LTD this is proximal or distal to excess global protein and abnormal experience-dependent plasticity of visual cortex. Finally, the cross rescued Finally, an important line of research has fo- an enhanced extinction phenotype in an cused on identifying target proteins whose syn- inhibitory avoidance paradigm, a mouse model thesis is regulated by both mGluR5 and FMRP for learning and memory. The only phenotype and that are differentially expressed basally not rescued in this study was macroorchidism, and/or in response to mGluR stimulation suggesting that other pathways (possibly www.annualreviews.org • Molecular Medicine in Fragile X mGluR1) are involved in the testicular phenotypes in dFmr1 mutant flies, including phenotype. Similarly, a genetic approach was abnormalities in behavioral and structural used in a Drosophila model of FXS, in which the measures of courtship-related learning and Drosophila homolog of the Fmr1 gene, known as memory (72), deficits in olfactory memory dFmr1 or dfxr1, was disrupted. Using a double (73), and increased embryonic lethality due to KO of dFmr1 and the only Drosophila mGluR, excitotoxicity (74). Finally, one group gener- known as dmGluRA, it was shown that dFmr1 ated Fmr1 knockdown zebrafish embryos using and dmGluRA pathways converge to regulate the morpholino antisense oligonucleotide multiple phenotypes including glutamate technology. By developing these embryos in receptor trafficking, synaptic plasticity, presy- medium containing MPEP, the authors were naptic ultrastructure, and coordinated motor able to reverse disruptions in neurite morphol- behavior (64–66). Based on these observations, ogy in the hindbrain and spinal cord of the the authors concluded that loss of the receptor embryos, as well as in craniofacial development at least partially corrects defects caused by im- (75). Together, these data provide compelling paired translational regulation and vice versa.
evidence that manipulating mGluR5 signaling The genetic rescue experiments provide can reverse fragile X–related phenotypes across essential validation, not only for the theory species, indicating that mGluR5 may indeed but also for the concept that drugs targeting provide a viable target for the treatment of FXS.
mGluR5 could treat multiple aspects of the It is worth emphasizing that the interaction disease. The therapeutic potential of mGluR5 between FMRP and mGluRs seems to be highly inhibitors has also been investigated on vari- conserved in evolution, appearing across the ous phenotypes in FXS animal models. Most of phylogenetic tree from invertebrates to mam- these studies have relied on MPEP [2-methyl- mals. This interplay between a repressor and 6-(phenylethynyl)-pyridine], a potent negative an activator of protein synthesis may thus rep- allosteric modulator of mGluR5 that crosses the resent an essential core mechanism by which blood-brain barrier. MPEP has proven to be synaptic plasticity is regulated at glutamater- a critical tool for studying mGluR5 function gic synapses, which in turn may explain how (67). In the first study to investigate the effects its disruption can cause such widespread and of pharmacological antagonism, acute adminis- severe pathological alterations in FXS. This tration of MPEP in Fmr1 KO mice was found striking degree of evolutionary conservation to reduce the abnormal response of these mice also boosts confidence that pharmacological ap- in an open field test, an experimental measure proaches that have been successful in animals by Copenhagen University on 06/03/11. For personal use only.
commonly used to assess anxiety-like pheno- have great potential to succeed in humans.
types in mouse models (68). In addition, likethe genetic rescue, MPEP significantly reduced Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org the probability of audiogenic seizures in these Limitations of the mGluR Theory
Although a large number of studies have Subsequently, other groups have demon- provided evidence in support of the mGluR strated that MPEP reverses a large number of theory, not all findings are consistent with phenotypes in Fmr1 KO mice, including pro- the simple notion that excessive mGluR- longed epileptiform discharges in hippocampal dependent protein synthesis and synaptic slices (53), deficits in prepulse inhibition of star- plasticity in the absence of FMRP accounts for tle (69), decreased mRNA granule expression mutant phenotypes in the Fmr1 KO mouse.
(70), excess protein synthesis in hippocampal For example, some of the proposed FMRP slices (62), increased density of dendritic target proteins do not show the expected basal filopodia in hippocampal cultures (69), and hy- upregulation in KO mice. The synaptic scaffold peractivity of glycogen synthase kinase-3 (71).
protein PSD-95, in particular, was reported Similarly, MPEP was able to reverse several to be downregulated in hippocampus owing to Krueger · Bear an alteration in mRNA stability, with mGluR alternative is the "genetic rescue" approach, stimulation resulting in stabilization of PSD-95 implemented by crossing Grm5 heterozygotes mRNA in wild-type but not in KO mice (76).
with Fmr1 KO mice (48). This approach can Additionally, in cortex (28) and amygdala (77), overcome pharmacokinetic limitations, but forms of LTP that depend on mGluR5 activa- only a single "dose" can be tested, namely a tion in wild-type mice were found to be absent, 50% reduction in mGluR5 protein. Novel rather than exaggerated, in the KO. Further- pharmacological agents with long-lasting more, it was observed that MPEP injected once pharmacokinetic properties will be neces- daily for several days during early postnatal de- sary to fully address these issues, and their velopment actually accentuated the immature development is currently under way.
appearance of dendritic spines imaged in vivo Although the mGluR theory may with- in somatosensory cortex of KO mice (78).
stand the challenges presented by the afore- In interpreting these studies, it is important mentioned studies, it nevertheless seems very to distinguish manifestations of an ongoing ex- likely that FMRP has functions that are unre- cess of mGluR signaling from the manifesta- lated to mGluRs. Accordingly, it will be just tions of synaptic development that has been al- as informative to identify phenotypes that are tered because of elevated mGluR signaling. For not corrected by mGluR1/5 antagonism as to example, the absence of mGluR5-dependent investigate those that are, since this distinction LTP in the cortex could reflect the fact that this may lead to important mechanistic insights that LTP mechanism has already been saturated in will further direct the development of success- vivo as a consequence of exaggerated mGluR5 ful therapeutic strategies.
function during development. In the case of theamygdala, a substantial deficit in basal transmis-sion was also reported at the same synapses that Testing the mGluR Theory
showed impaired LTP. Reduced synaptic con- in Humans
nectivity might have caused the defective LTP Encouraged by the exciting findings arising and might have arisen as a consequence of in- from preclinical research, a number of clini- creased mGluR5-dependent protein synthesis cal trials have been initiated to test the efficacy during the development of amygdala circuitry.
of compounds directly or indirectly related to These alternative interpretations will need to mGluR signaling in treating FXS (80, 81). To be explored before rejecting the relevance of date, none of these compounds are specifically the mGluR theory to the fragile X LTP phe- approved for the treatment of FXS, but promis- by Copenhagen University on 06/03/11. For personal use only.
notypes in cortex and amygdala.
ing preliminary results have been obtained.
It is also important to understand the lim- The first Gp1 mGluR inhibitor to be tested itations of the tools that are available to inhibit in clinical trials was fenobam, a compound that Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org mGluR5 signaling. Most pharmacological was originally developed as an anxiolytic with studies published to date have relied on the an unknown molecular target and was subse- mGluR5 negative allosteric modulator MPEP.
quently demonstrated to be a selective mGluR5 However, MPEP is an extremely short-acting antagonist (82). An open-label phase II clini- antagonist in vivo, with maximal receptor cal trial was recently completed, in which 12 occupancy lasting only ∼15 min in mouse adult patients with FXS received a single dose brain (79). It is therefore virtually impossible to of fenobam to assess drug safety, pharmacoki- study the drug's effect on phenotypes that may netics, and a small number of cognitive and be- require chronic mGluR5 antagonism during havioral effects. In this trial, fenobam was re- brain development. Single MPEP injections ported to reduce anxiety and hyperarousal and will not produce chronic inhibition and will to improve prepulse inhibition of startle and likely cause transient rebound increases in accuracy on a continuous performance task (a mGluR5 function as the drug wears off. An measure of sustained attention and impulsivity) www.annualreviews.org • Molecular Medicine in Fragile X in a subset of patients (80, 83). Although these tic release of glutamate and hence the activation results are encouraging, it is important to note of postsynaptic mGluR5. It has been previously that the study was not performed blind and was shown that GABA-B receptor agonists such as not placebo controlled. The study also revealed baclofen inhibit glutamate release, and baclofen highly variable plasma levels of fenobam after has been shown to reduce audiogenic seizures oral dosing, making this compound problem- in Fmr1 KO mice (86). Based on this informa- atic as a potential therapeutic and an inadequate tion, a placebo-controlled double-blind phase agent to test the mGluR theory in humans.
II study was conducted using arbaclofen (also In another small open-label trial, three known as STX209), the R-isomer of baclofen.
young adult patients with FXS were treated This study has been completed and it is antici- with acamprosate, a drug with mGluR antag- pated that the results will soon be available.
onist properties that is approved for mainte- Finally, clinical trials are also under way for nance of abstinence from alcohol (84). In all minocycline, a tetracycline analog that (among three patients, acamprosate was associated with other things) inhibits matrix metalloproteinase- improved linguistic communication and global 9 (MMP-9). It was previously shown that lev- clinical benefit as assessed by the CGI-I (clinical els of MMP-9 are elevated in Fmr1 KO mice, global impression-improvement) scale.
possibly as a consequence of excess mGluR5 New, highly potent and selective mGluR5 signaling, and that minocycline reverses several negative allosteric modulators have shown phenotypes in these mice (87).
promising results in preclinical studies and
are currently in clinical trials in FXS (see
http://www.clinicaltrials.gov). These include
Window of Opportunity for
STX107 (Seaside Therapeutics, phase I trial initiated in the United States), AFQ056 An important issue for the design of successful (Novartis, phase II trial recently completed treatment strategies concerns the developmen- in France, Italy, and Switzerland), and tal time window available for intervention.
RO4917523 (Hoffman-LaRoche, phase II trial Mutations such as those underlying FXS initiated in the United States). The results of presumably alter the trajectory of normal brain these studies are anxiously awaited, as they development by disrupting the mechanisms clearly represent the best tests to date of the of activity-dependent circuit formation and applicability of the mGluR theory to humans.
plasticity (Figure 3a). The key question then
In addition to targeting mGluRs di- is whether these alterations are reversible, by Copenhagen University on 06/03/11. For personal use only.
rectly, another approach to reducing excessive or whether the aberrant connectivity, once mGluR-mediated plasticity is to target the sig- established, results in a permanent dysfunction naling pathways downstream or upstream of that cannot be influenced by subsequent phar- Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org mGluRs. This approach is exemplified by the macological interventions. Given that most recent interest in lithium, which is already clin- neurodevelopmental disorders are currently ically approved for the treatment of mood dis- not diagnosed until well after the onset of orders. Lithium targets multiple intracellular symptoms, this issue will play an essential role signaling pathways, including phospholipase C in determining treatment outcomes and the and glycogen synthase kinase-3, which have need for newborn screening to facilitate early been linked to Gp1 mGluR signaling and FXS, respectively (71, 85). In a pilot trial on 15 We can consider several potential scenarios patients with FXS, lithium treatment for two for the efficacy of drugs after symptom onset months was found to have positive effects on be- (Figure 3b). The most optimistic possibility is
havioral adaptive skills and one cognitive mea- that intervention after symptom onset results sure (85). A second approach that is currently in near-complete reversal of associated phe- in clinical trials aims at reducing the presynap- notypes. This scenario is conceivable, e.g., if Krueger · Bear the molecular mechanisms affected are required express the same combination of symptoms. In only for acute synapse function or for the re- order to optimize therapeutic strategies, it will versible strengthening or weakening of previ- therefore be necessary to characterize each of ously existing connections. The pessimistic sce- these phenotypes individually and identify the nario is that, following an initial window of underlying pathogenic mechanisms and devel- opportunity, pharmacological approaches can opmental time course. Extensive research in an- no longer alter the course or progression of imal models will be crucial in addressing these the disorder. This may occur either because important questions and should help guide of large-scale irreversible structural alterations, human clinical trial design.
such as aberrant axonal pathfinding or dendriticbranching, or because the pathogenic molecule no longer plays a role in the later stages of de- Normal development velopment or in adulthood. An intermediate,but still hopeful, scenario is that intervention Accumulated deficit can slow or prevent progression of symptoms, although it may not fully correct previously es- Fragile X syndrome tablished impairments. At the molecular level,this scenario would be likely if structural ab- normalities occurred locally (e.g., the develop- ment of aberrant dendritic spines) and could becompensated for by local adjustments followingdrug administration.
It should also be considered that most neu- Optimistic scenario: rodevelopmental disorders manifest as a com- reversal of deficit plex combination of symptoms, each of whichmay involve different molecular mechanismsand developmental trajectories. Moreover, notall individuals with a given disorder will always Pessimistic scenario: Prospects for the treatment of a developmental missed critical period brain disorder. (a) Divergence of brain maturation by Copenhagen University on 06/03/11. For personal use only.
in normal development versus development infragile X syndrome (FXS). This continuous Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org divergence results in an accumulated deficit inindividuals with FXS that increases with age.
(b) Prospects for treatment of this accumulateddeficit in FXS. Three scenarios are conceivable with respect to interventions that occur followingsymptom onset: (i ) The optimistic view assumes Hopeful scenario: that pharmacological intervention after symptom no further insult onset results in near-complete reversal of associatedphenotypes. (ii ) The pessimistic view is that after aninitial window of opportunity, pharmacologicalapproaches can no longer alter the course of thedisorder. (iii ) The intermediate (but still hopeful)view suggests that pharmacological intervention canslow or prevent progression of symptoms, althoughit may not fully correct previously establishedimpairments.
www.annualreviews.org • Molecular Medicine in Fragile X autism with extreme macrocephaly. A mouse APPROACHES IN OTHER
model in which PTEN was deleted in a subset of neurons in cortex and hippocampus displays TSC: tuberous
sclerosis complex
neuron hypertrophy resulting in disruptionsof hippocampal structure, as well as increased Although mGluR-based therapeutics for FXS are among the first targeted treatments to reach susceptibility to seizures and increased anxiety clinical trials for any ASD-related disorder, im- (91). Chronic (four- to six-week) administra- portant progress has recently also been made tion of rapamycin was able to reverse all of in other single-gene neurodevelopmental dis- these phenotypes in the mice, again suggesting orders associated with autism (88).
that mTOR might be a relevant drug targetfor certain forms of autism (91). Since PTENmutations are also associated with a greatly in- Tuberous Sclerosis Complex
creased susceptibility to certain cancers, clinicaltrials are currently under way to examine the Tuberous sclerosis complex (TSC) is a mul- effects of rapamycin on tumor growth in cancer tisystem disorder characterized by tumorous patients with PTEN mutations (http://www.
growths, or hamartomas, in organs including clinicaltrials.gov). However, studies to exam-
the kidneys, lung, heart, brain, and liver (89).
ine the effects of rapamycin on the cognitive Clinical manifestations related to the brain and behavioral deficits associated with PTEN can include mental retardation, autism, and mutations have yet to be initiated.
epilepsy. TSC is caused by heterozygous muta-tions in either the TSC1 or TSC2 gene, both ofwhich encode proteins that are negative regula- Neurofibromatosis Type 1
tors of the mTOR intracellular signaling path- Neurofibromatosis type 1 (NF1) is a neurode- way. The mTOR pathway has been linked to velopmental disorder associated with cognitive the control of protein synthesis, and disruption impairments, including difficulties with visu- of this control is thought to underlie the neu- ospatial skills and executive function, as well ropsychiatric phenotypes observed in TSC.
as an increased incidence of autism (88, 92).
Mice with a heterozygous mutation in the NF1 is caused by mutations in the gene encod- Tsc2 gene mimic several of the cognitive deficits ing neurofibromin, which inhibits p21Ras func- observed in human TSC patients. Using this tion. Mice with a heterozygous deletion of the mouse model, it was shown that brief treatment Nf1 gene show an increase in the phosphoryla- with rapamycin, an inhibitor of the mTOR by Copenhagen University on 06/03/11. For personal use only.
tion of ERK1/2, one of the downstream targets complex, rescues deficits in context discrimi- of p21Ras signaling, as well as deficits in vi- nation and spatial learning (90). Clinical tri- suospatial attention, spatial learning, prepulse Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org als of rapamycin, also known as sirolimus, are inhibition, and hippocampal LTP (92). Brief under way in children with TSC, although to administration of the farnesyl transferase in- our knowledge there are no trials specifically hibitor lovastatin, which (among other things) investigating the effects of rapamycin on the decreases p21Ras activity by blocking its far- cognitive and behavioral phenotypes in TSC nesylation and membrane association, was able to reverse all of these phenotypes, indicat-ing that lovastatin may be a viable treatmentfor NF1. Clinical trials to investigate the ef- fects of lovastatin on visual spatial learning and Mutations in the phosphatase and tensin memory in children with NF1 have been initi- homolog (PTEN) gene, another negative reg- ulator of the Akt/mTOR signaling pathway, it should be noted that simvastatin, a related have also been linked to a number of cases of farnesyl transferase inhibitor, was previously Krueger · Bear found to have no effect on cognitive functioning disorders for which few mechanistic details are in children with NF1 (93).
currently available. Idiopathic autism has beennotoriously difficult to study owing to the lack RTT: Rett syndrome
of suitable animal models. By comparing the molecular mechanisms underlying different Rett syndrome (RTT) is a severe ASD that single-gene disorders, it may be possible to also includes intellectual disabilities as well as discover commonalities and general principles motor symptoms such as ataxia, dystonia, and that might hold true even for those cases respiratory dysfunction (88, 94). RTT is caused in which no specific genetic cause has been by mutations of the X-chromosomal gene en- identified. One such key principle arose from coding the transcriptional regulator MeCP2, the observation that many of the single-gene and deletion of this gene in mice causes a severe disorders appear to affect key regulators of neurological phenotype related to the symp- protein synthesis, suggesting that it may be the toms observed in RTT patients, including gait dysregulation of protein synthesis itself that abnormalities, respiratory dysfunction, hind represents one final common pathogenic mech- limb clasping, and decreased survival. Two anism (96). According to this theory, aberrant recent studies have sought to reverse these phe- synaptic protein synthesis caused by mutations notypes using a genetic and a pharmacological or copy-number variations in regulatory signal- strategy, respectively. In the former, MeCP2 ing pathways may lead to changes in synaptic function was restored during or following onset connectivity and function, which subsequently of symptoms using a tamoxifen-inducible Cre- result in the cognitive and behavioral deficits LoxP strategy, resulting in the robust reversal that are observed in ASDs. This notion has re- of several associated phenotypes (94). This cently been supported by clinical studies linking is an extremely important proof of principle autism to mutations in the gene encoding the that the course of this disease can be arrested translation initiation factor eIF4E (97) and to and even reversed with manipulations begun copy-number variations in GTPase/Ras signal- in late adolescence (the optimistic scenario in ing pathways (98) that may be involved in the Figure 3). In the second study, mice were
regulation of synaptic protein synthesis. Based treated with insulin-like growth factor 1 on these findings, treatments that successfully (IGF-1), based on previous observations that target protein synthesis pathways in the single- another growth factor, brain-derived neu- gene disorders mentioned above, including rotrophic factor (BDNF), was a key target of mGluR5 modulators, may very well have by Copenhagen University on 06/03/11. For personal use only.
MeCP2 regulation (95). IGF-1 administration broader therapeutic applications in idiopathic improved several phenotypes in the MeCP2 KO mice, including locomotor activity, res- However, it is also clear that altered synap- Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org piratory function, dendritic spine density, and tic protein synthesis is not the only mechanism survival. Neither of these rescue strategies has by which autism-related mutations can cause resulted in the initiation of clinical trials to abnormalities in synaptic function. For exam- date, but they suggest that the development of ple, a key role is also emerging for mutations in targeted treatments for RTT may be a feasible structural proteins that are involved in synap- goal in the future.
tic development or function, including thesynaptic cell adhesion molecules neuroligin-3,neuroligin-4 and neurexin-1, and the synaptic scaffolding protein SHANK3 (for recent de- In addition to the providing the basis for the tailed reviews, see 99–101). While structural development of targeted treatments for specific proteins themselves are unlikely to represent developmental disorders, the above studies useful targets for traditional small-molecule are also yielding key insights into related drug therapies, it may ultimately be possible to www.annualreviews.org • Molecular Medicine in Fragile X target common downstream consequences of It is already clear that diverse molecular these mutations, such as a shift in the balance mechanisms can contribute to the synaptic ab- of excitatory and inhibitory synaptic transmis- normalities that underlie ASDs. In order to de- sion. Indeed, the first studies investigating such sign appropriate therapeutic strategies for id- strategies in mouse models are beginning to iopathic autism, it will be critical to identify emerge (102), although significant further pre- biomarkers that report the pathophysiological clinical research is likely to be necessary before processes at work in the brains of the affected clinical trials can be considered.
1. FXS, the most common inherited form of mental retardation and a leading known cause of autism, is caused by loss of expression of the RNA-binding protein FMRP.
2. The mGluR theory of fragile X states that mGluRs and FMRP act in functional oppo- sition to regulate protein synthesis at the synapse, with mGluRs stimulating and FMRPrepressing this protein synthesis.
3. Inhibition or reduction of mGluR5 in animal models of FXS has been shown to reverse a wide range of phenotypes, suggesting that antagonism of mGluRs may provide aneffective strategy in the treatment of FXS.
4. Clinical trials have been initiated to test the efficacy of mGluR5 antagonists and other pharmacological interventions related to mGluR function in individuals with FXS.
5. Significant progress has also been made in understanding the molecular mechanisms un- derlying other single-gene disorders, including TSC, NF1, and RTT, and in developingtherapeutic approaches that are being tested in clinical trials.
6. All of these disorders involve mutations on pathways related to the regulation of protein synthesis, suggesting that this may be a common pathogenic mechanism that may alsobe relevant to idiopathic autism.
by Copenhagen University on 06/03/11. For personal use only.
1. Is it possible to identify additional novel therapeutic targets for FXS based on insights into the molecular mechanisms of FMRP function at the synapse? Can these targets be Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org validated in animal models of FXS and subsequently in clinical trials? 2. What is the developmental window of opportunity for the pharmacological treatment of FXS? Can existing symptoms be corrected or improved, or is it necessary to initiatetreatment early in pre- or postnatal development prior to phenotype onset? Is newbornscreening for early intervention warranted? 3. Can we gain new insights into idiopathic autism from comparing and contrasting the molecular mechanisms underlying different autism-associated single-gene disorders?Can this knowledge be used to develop novel treatment strategies for at least a sub-set of individuals with ASDs? 4. What peripheral biomarkers will best reflect brain pathophysiology and inform clinical trials and treatments for autism of unknown etiology? Krueger · Bear Mark Bear has a financial interest in Seaside Therapeutics, Inc.
The authors thank Emily Osterweil for valuable discussions and the FRAXA Research Foundation,the National Institute of Mental Health, and the National Institute of Child Health and HumanDevelopment for funding support.
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Role of Postmarketing Surveillance in Contemporary Medicine Janet Woodcock, Rachel E. Behrman, and Gerald J. Dal Pan p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
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State of the Art in Therapeutic Hypothermia Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org Therapeutic Potential of Lung Epithelial Progenitor Cells Derived from Embryonic and Induced Pluripotent Stem Cells Therapeutics Development for Cystic Fibrosis: A Successful Model for a Multisystem Genetic Disease Early Events in Sexual Transmission of HIV and SIV and Opportunities for Interventions HIV Infection, Inflammation, Immunosenescence, and Aging The Increasing Burden of HIV-Associated Malignancies in Resource-Limited Regions Biliary Atresia: Will Blocking Inflammation Tame the Disease? Advances in Palliative Medicine and End-of-Life Care Clostridium difficile and Methicillin-Resistant Staphylococcus aureus: Emerging Concepts in Vaccine Development Antiestrogens and Their Therapeutic Applications in Breast Cancer and Other Diseases Mechanisms of Endocrine Resistance in Breast Cancer Jacob Laubach, Paul Richardson, and Kenneth Anderson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 249
Muscle Wasting in Cancer Cachexia: Clinical Implications, Diagnosis, and Emerging Treatment Strategies Shontelle Dodson, Vickie E. Baracos, Aminah Jatoi, William J. Evans, David Cella, James T. Dalton, and Mitchell S. Steiner p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 265
Pharmacogenetics of Endocrine Therapy for Breast Cancer Therapeutic Approaches for Women Predisposed to Breast Cancer by Copenhagen University on 06/03/11. For personal use only.
New Approaches to the Treatment of Osteoporosis Annu. Rev. Med. 2011.62:411-429. Downloaded from www.annualreviews.org Regulation of Bone Mass by Serotonin: Molecular Biology and Therapeutic Implications Alpha-1-Antitrypsin Deficiency: Importance of Proteasomal and Autophagic Degradative Pathways in Disposal of LiverDisease–Associated Protein Aggregates Hepcidin and Disorders of Iron Metabolism Interactions Between Gut Microbiota and Host Metabolism Predisposing to Obesity and Diabetes Giovanni Musso, Roberto Gambino, and Maurizio Cassader p p p p p p p p p p p p p p p p p p p p p p p p p p p p 361
The Brain-Gut Axis in Abdominal Pain Syndromes Cognitive Therapy: Current Status and Future Directions Toward Fulfilling the Promise of Molecular Medicine in Fragile X Syndrome Stress- and Allostasis-Induced Brain Plasticity Update on Sleep and Its Disorders A Brain-Based Endophenotype for Major Depressive Disorder Cumulative Index of Contributing Authors, Volumes 58–62 p p p p p p p p p p p p p p p p p p p p p p p p p p p 475
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  • Role of Postmarketing Surveillance in Contemporary Medicine
  • Genome-Wide Association Studies: Results from the First Few Years and Potential Implications for Clinical Medicine
  • Imaging of Atherosclerosis
  • Novel Oral Factor Xa and Thrombin Inhibitors in the Managementof Thromboembolism
  • The Fabry Cardiomyopathy: Models for the Cardiologist
  • Kawasaki Disease: Novel Insights into Etiology and Genetic Susceptibility
  • State of the Art in Therapeutic Hypothermia
  • Therapeutic Potential of Lung Epithelial Progenitor Cells Derivedfrom Embryonic and Induced Pluripotent Stem Cells
  • Therapeutics Development for Cystic Fibrosis: A Successful Model for a Multisystem Genetic Disease
  • Early Events in Sexual Transmission of HIV and SIV and Opportunities for Interventions
  • HIV Infection, Inflammation, Immunosenescence, and Aging
  • The Increasing Burden of HIV-Associated Malignancies in Resource-Limited Regions
  • Biliary Atresia: Will Blocking Inflammation Tame the Disease?
  • Advances in Palliative Medicine and End-of-Life Care
  • Clostridium difficile and Methicillin-Resistant Staphylococcus aureus: Emerging Concepts in Vaccine Development
  • Antiestrogens and Their Therapeutic Applications in Breast Cancer and Other Diseases
  • Mechanisms of Endocrine Resistance in Breast Cancer
  • Muscle Wasting in Cancer Cachexia: Clinical Implications, Diagnosis, and Emerging Treatment Strategies
  • Pharmacogenetics of Endocrine Therapy for Breast Cancer
  • Therapeutic Approaches for Women Predisposed to Breast Cancer
  • New Approaches to the Treatment of Osteoporosis
  • Regulation of Bone Mass by Serotonin: Molecular Biology and Therapeutic Implications
  • Alpha-1-Antitrypsin Deficiency: Importance of Proteasomal and Autophagic Degradative Pathways in Disposal of Liver Disease–Associated Protein Aggregates
  • Hepcidin and Disorders of Iron Metabolism
  • Interactions Between Gut Microbiota and Host Metabolism Predisposing to Obesity and Diabetes
  • The Brain-Gut Axis in Abdominal Pain Syndromes
  • Cognitive Therapy: Current Status and Future Directions
  • Toward Fulfilling the Promise of Molecular Medicine in Fragile X Syndrome
  • Stress- and Allostasis-Induced Brain Plasticity
  • Update on Sleep and Its Disorders
  • A Brain-Based Endophenotype for Major Depressive Disorder
  • Source: http://www.medico-innovation.dk/wp-content/uploads/2011/07/Fulfilling-the-promise-of-molecular-medicine.pdf

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