Sensitive time-windows for susceptibility in neurodevelopmental disorders

Opinion

Sensitive time-windows for susceptibility in neurodevelopmental disorders Rhiannon M. Meredith, Julia Dawitz and Ioannis Kramvis Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research (CNCR), VU University Amsterdam, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

Many neurodevelopmental disorders (NDDs) are characterized by age-dependent symptom onset and regression, particularly during early postnatal periods of life. The neurobiological mechanisms preceding and underlying these developmental cognitive and behavioral impairments are, however, not clearly understood. Recent evidence using animal models for monogenic NDDs demonstrates the existence of time-regulated windows of neuronal and synaptic impairments. We propose that these developmentally-dependent impairments can be unified into a key concept: namely, time-restricted windows for impaired synaptic phenotypes exist in NDDs, akin to critical periods during normal sensory development in the brain. Existence of sensitive time-windows has significant implications for our understanding of early brain development underlying NDDs and may indicate vulnerable periods when the brain is more susceptible to current therapeutic treatments. Synaptic pathology and neurodevelopmental disorders Development of cognitive functions requires the formation and refinement of synaptic networks of neurons in the brain. In the mammalian cortex, late embryonic and early postnatal stages of development are the periods of greatest synaptic density with significant structural and functional synaptic plasticity [1,2]. Commonly observed in both patients and animal models for many NDDs with early childhood onset are increased filopodia-to-spine ratios and abnormal protrusion densities, interpreted as a more immature synaptic network [3–5]. Interestingly, in many such disorders the underlying signaling pathways interact with the Rho family of GTPase proteins [3,6] (Figure 1). Of these, Ras homolog gene family member-A (RhoA), Rasrelated C3 botulinum toxin substrate-1 (Rac1), and cell division control protein-42 homolog (Cdc42) take part in regulating spine morphology and distribution along the dendrite, through interactions with F-actin at the cytoskeleton [7,8]. Mutations of proteins within Rho signaling pathways including fragile-X mental retardation protein (FMRP) (Glossary), tuberous sclerosis complex (TSC) and oligophrenin1 (OPHN1) are linked to different NDDs with prominent intellectual disability [3]. Corresponding author: Meredith, R.M. ([email protected]). Keywords: synapse; neurodevelopmental disorder; autism; intellectual disability; critical period; sensitive time-window.

Abnormalities in synaptic phenotypes in NDDs are found throughout different brain regions and are already present during embryonic and early postnatal stages [9]. These observations support the hypothesis that abnormal patterns of spine maturation and distribution are not necessarily a consequence of impaired cognitive and sensory processing. Rather, they indicate initial aberrations in mechanisms regulating synaptic circuitry at presymptomatic stages, before significant cognitive and behavioral dysfunction. In this article we present the idea that there are sensitive time-windows for impaired synaptic phenotypes in NDDs. We evaluate data across different developmental periods from animal models for these disorders and propose underlying causal mechanisms for their time-regulated phenotypes. The existence of transient phenotypes has implications both for our understanding of key regulators at specific developmental stages and for the potential of timely therapeutic interventions in NDDs with pronounced childhood onset. Transient age-dependent phenotypes of neuronal disorders Clinically, the onset and progression of NDDs with intellectual disabilities and autism phenotypes can be striking. In their emergence during early childhood, NDDs are often characterized by a series of missed developmental milestones, regression of speech and motor function, and impaired social interactions [10,11]. Using genetic mouse and Glossary Amblyopia (‘lazy eye’): loss in one eye of the ability to see details. This is the most common cause of visual problems in children. Treatment must occur at a young age, within the critical period of the visual system, to successfully correct the impairment. Critical period: a regulated time-window during which sensory experience and intrinsic neuronal activity provide information that is essential for normal development and refinement of neural circuits. FMRP (fragile X mental retardation protein): silenced or downregulated in fragile X syndrome (FXS). MeCP2 (methyl CpG binding protein 2): shows altered expression in Rett Syndrome. Sensitive time-window: a restricted period, caused by a gene misregulated in a NDD, during which the synaptic phenotype is vulnerable to impairment. Synaptic phenotype: a measurement of synaptic morphology or functional response at identified synaptic connections in neural circuits. TSC1/2 (tuberous sclerosis complex proteins 1 and 2): proteins mutated in the tuberous sclerosis complex (TSC) disorder.

0166-2236/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.tins.2012.03.005 Trends in Neurosciences, June 2012, Vol. 35, No. 6

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Glutamatergic axonal projection

Glial cell projection

Gabaergic axonal projection

Dendrite, mature spine

TSC1

mTOR

RHEB

TSC2

RAC1 OPHN1 4EBP

CDC42 RHOA

CYFIP

eIF4E

FMRP

PAK FMRP Granule

mRNA Translation

LIMK1

F-Actin Key:

GABAARs

mGluRs

GABA

NMDARs

AMPARs

Glutamate transporters

Glutamate

TRENDS in Neurosciences

Figure 1. Misregulated mechanisms underlying spine morphology in NDDs. Several proteins implicated in monogenic NDDs (highlighted in red) are linked to the regulation of the synaptic cytoskeleton via F-actin through different Rho-mediated signaling pathways (highlighted in green). Mutations in OPHN1, TSC1/2, FMRP, p21-activated kinase (PAK) are directly linked to human NDDs of intellectual disability. For instance, point mutations in OPHN1 and a PAK isoform are linked to non-syndromic mental retardation [3], whereas mutations or altered expression of TSC1/2 and FMRP are linked to TSC and FXS, respectively [13,100]. Cytoplasmic interacting protein (CYFIP) and LIM-domain kinase 1 (LIMK1) are known to interact with FMRP and PAK, respectively [105]. LIMK1 is one of many dysregulated proteins contributing to the NDD Williams syndrome [6]. Mouse models are available for all highlighted (red) proteins and reveal specific synaptic and behavioral deficits [3,12,13,100]. Local protein synthesis in synapses, dendrites and glia is also regulated by proteins such as TSC1/2 and the FMRP/CYFIP complex [38,40,105,106]. Abbreviations: 4EBP, 4E binding protein; eIF4E, eukaryotic translation initiation factor 4E.

fly models for specific NDDs, many cellular and synaptic alterations in the mature brain have been uncovered that address mechanisms underlying symptomatic stages for specific disorders. However, it is not clear which changes 336

occur presymptomatically or what triggers are responsible for the onset of behavioral and cognitive impairments. Here, we use recent data predominantly from the fragile-X mental retardation gene 1 (Fmr1) knockout (KO)

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Refs Layer 4

[22]

Layer 4

Layer 2/3 pyramidals

[23]

Layer 4

Fast-spiking neurons

[73]

Thalamus

Δω

Layer 2/3 pyramidals

[20]

Layer 5 pyramidals

[18]

Layer 5 pyramidals

[19]

Layer 4 synaptoneurosomes

[22]

P7

P14

P21

P25-28

P75

Postnatal age (days) Key:

Synaptic connectivity/function

Δω

Synaptic plasticity

GluN1 NMDARs Spine morphology

Significant change from WT Same as WT TRENDS in Neurosciences

Figure 2. Developmentally-regulated time-windows in the Fmr1-KO mouse model. Several morphological and functional impairments in synapse properties are observed in the somatosensory cortex [19] and, specifically, the barrel region of the Fmr1-KO model for FXS [18,20,22,23,73]. Many of these functional alterations are transient, occurring only during restricted periods of early postnatal development. Another developmentally regulated phenotype is spine morphology in layer 5 pyramidal neurons, where the impairment disappears at P28 but reappears in later adulthood around P75 [18,19].

mouse models for fragile-X syndrome (FXS), a monogenic intellectual disability with partial concomitance for autism disorders [12,13]. We highlight and discuss data where many altered synaptic phenotypes observed in monogenic NDDs are regulated by developmental age and some occur only transiently in specific neural circuits. Synapse pathology is a prevalent phenotype in NDDs in both juveniles and adults [6] and is well-characterized in FXS patients and in the Fmr1-KO mouse [14,15]. FMRP regulates local mRNA translation in response to synaptic metabotropic glutamate receptor 5 (mGluR5) stimulation [16]. Immature spine morphologies and higher protrusion densities observed in FXS patients and mice led to the ‘pruning’ hypothesis that FMRP refines synapses during normal maturation [17]. In Fmr1-KO barrel cortex, spine protrusions are longer in layer 5 pyramidal neurons during postnatal day (P)7 and P14 [18] (Figure 2). Intriguingly, at P28 this phenotype disappears, only to return by P73–76 [19]. Furthermore, spine stabilization is delayed in Fmr1-KO mouse layer 2–3 pyramidal neurons,

although abnormally high spine-turnover is persistent from 2 wks onwards [20,21]. Alterations in synaptic function and plasticity are also observed in the Fmr1-KO barrel cortex. During the first postnatal week, thalamic inputs to layer-4 barrel cortex show enhanced NMDA/AMPA ratios and altered synaptic plasticity, indicating developmental delays in functional maturation [22]. However, these changes in synaptic strength are transient, being no different from wild-type (WT) littermates by P14. At this time-point, connectivity between layers 4 and 2/3 in barrel cortex is significantly decreased and layer 4 axonal branching is more diffuse in the Fmr1-KO mouse [23]. Similarly to thalamocortical synapses, these phenotypic alterations are transient and appear normal 1 wk later. The effects of FMRP absence on the bidirectional relationship between spine morphology and functional plasticity are difficult to separate causally ([14] for detailed discussion). Whether immature morphology prevents subsequent plasticity or whether a change in plasticity disrupts corresponding shape changes in 337

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Table 1. Evidence for transient and developmentally-regulated phenotypic impairments in mouse and fly models for FXSa Neural circuit Mouse Somatosensory cortex

Molecular/functional changes

Developmental time

Refs.

Spine turnover downregulation

Rapid decrease seen in WT from around P7 to P10–12 is delayed Maturation shifted by 3 days to P7 More prevalent at P7 Peak plasticity shifted by 3–4 days to around P7 Defective around P14, normalized by P21 Abnormal around P7, normalizes around P28 Abnormal at P73–76

[20]

Glutamatergic signaling – NMDA/AMPA ratio Silent synapses Developmental window for synaptic plasticity Projections from layer 4 to 3 Spine morphology Hippocampal formation

Frontal brain regions

Auditory startle circuitry b Anterior piriform cortex Spinal motor neurons Drosophila sLNvs Mushroom body NMJ

Spine length

Abnormal increase at P10, normalized by P20, decreased at P30, normalized by P40 Impaired at P12–16, normalized at P40–50

[22]

[23] [18] [19] [107]

NMDA/AMPA ratio NMDA-dependent LTP Connectivity and synaptic dynamics GABAAR a1 subunit expression GABAAR b2 subunit expression GABAAR d1 subunit expression, GABA-T and SSADH Acoustic startle response

Defective around P12–21, normalized by P21–36 Reduced in early postnatal development, normalized in adult Normal at P5, reduced from P12 through adulthood Normal in early postnatal development and in adulthood, decreased at P12 Normal up to around P28, arrested from P28 onwards

[24] [80]

LTP induced by theta bursts

Normal up to P180, increasingly impaired thereafter

[109]

Dendritic length and arborization

Subtle disturbances varying in a complex transient manner

[110]

Circadian rhythmicity and elaboration of synaptic architecture Axon formation and branching Magnitude and fidelity of calcium signals Synapse elaboration

Consistently disturbed. Window for rescue by re-expression of dFMRP only between pupal days 3 and 4 Normal up to pupal day 4, longer and more branches thereafter Incrementally defective with age progression Disturbed; window for full rescue by transient re-expression of dFMRP during early development

[49]

[48]

[108]

[111] [112] [113]

a

Abbreviations: dFMRP, Drosophila fragile X mental retardation protein; FXS, fragile X syndrome; GABAAR, GABAA receptor; GABA-T, GABA transaminase; LTP, long-term potentiation; NMJ, neuromuscular junction; P, postnatal day; sLNVs, small ventrolateral neurons; SSADH, succinic semialdehyde dehydrogenase; WT, wild-type.

b

Startle reflex mediated via synaptic circuitry between sensory neurons, the cochlea, caudal pons and spinal motor neurons.

developing Fmr1-KO neurons could be revealed by observing newly formed and mature spines before and after synaptic plasticity paradigms. Transiently altered synaptic phenotypes are not limited to barrel cortex (Table 1). In medial prefrontal cortex (mPFC), layer 5 pyramidal neurons are hyperconnected and show slower synapse recovery after stimulation in Fmr1-KO mice at P12–19 but not at P20-P36 [24]. Changes in higher-order cognitive regions including mPFC could be a precursor for later attentional and executive-function deficits in Fmr1-KO mice [25] and FXS patients [26]. The occurrence of time-windows for altered synaptic phenotypes in two different neocortical regions indicate that this phenomenon is applicable to other affected brain regions in FXS and potentially to other NDDs. Supporting this, transient delays in establishing correct NMDA/AMPA ratios are also observed at synapses on layer 2/3 pyramidal mPFC neurons in a valproic acid rat model of autism before P21 but not later [27]. The relevance of these time-windows in NDDs remains unknown. The transient appearance of synaptic phenotypes probably reflects changes during crucial developmental stages of synaptic network formation. These occur when the system is being challenged, as documented in sensory cortex during normal brain maturation [28–30]. 338

Sensitive time-windows for synaptic disruption in sensory cortex Cortical circuitry is fine-tuned by sensory experience in early life, and this refinement is proposed to be governed by synaptic plasticity mechanisms [29,31]. Such circuits display heightened sensitivity and enhanced synaptic modulation to incoming sensory information during restricted periods of early development, a so-called ‘critical period’ [32]. At the synaptic level, plasticity is sensitive to developmental age in rodents: for thalamic inputs to sensory cortex, synapse strength can be modulated during the first postnatal week, after which this critical period for plasticity closes [28,33]. Sensitive periods are also observed for synapse plasticity between cortical layers 4 to 2/3 and within 2/3 [30,34]. In Fmr1-KO barrel cortex, transient impairments at thalamocortical and cortico–cortical synapses that are observed at weeks 1 and 2 respectively (Figure 2), coincide with the critical periods for synaptogenesis. Furthermore, the barrel cortex has restricted time-periods for responseremodeling following sensory deprivation [35,36] and these are also aberrant in the Fmr1-KO mouse. Both spine formation/elimination and synaptic connectivity are less sensitive to modulation in young Fmr1-KO mice following whisker deprivation [21,23] (but see [22] for

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(b)

(c)

Δ synaptic phenotype

(a)

NDD gene and targets expressed outside of critical period Developmental time

Key:

NDD gene and targets expressed around critical period onset Developmental time

WT profile

NDD profile

NDD gene and targets expressed during and/or after critical period Developmental time

Sensitive time window TRENDS in Neurosciences

Figure 3. Sensitive time-windows, synaptic phenotypes and NDD gene targets. Sensitive time-windows exist in neural circuits, during which gene targets implicated in NDDs are normally expressed. Misregulation of these genes can affect multiple synaptic phenotypes during a restricted developmental period. The effect upon synaptic phenotypes is dependent upon the temporal expression of these NDD genes and their targets. (a) Expression outside a critical period of development will have no effect upon synaptic phenotypes. (b,c) A temporal expression pattern that overlaps with the onset (b) or closure (c) of a known critical period can alter the synaptic phenotype during that developmental time-window.

thalamocortical synapses). We propose that sensitive timewindows exist during development in NDDs wherein synaptic phenotypes are more susceptible to alteration, and these can also lead to developmental impairments in critical periods. In the following section we elaborate on potential mechanisms underlying these transient synaptic phenotypes during sensitive time-windows and how they could be misregulated during critical periods in NDD models. Potential neurobiological mechanisms regulating sensitive time-windows in NDDs The transiently disrupted phenotypes in NDD models preceding later behavioral impairments are probably mediated by molecular and cellular mechanisms that underlie sensitive time-windows. These windows may be mediated by neurodevelopmental genes and the pathways they regulate. In addition, considerable evidence points towards an imbalance between inhibitory and excitatory signaling in patients with NDDs [37]. Alterations in glutamatergic and GABAergic signaling, either via direct NDD gene regulation or in a compensatory manner, are known to impair critical periods [31], and may thus influence sensitive timewindows in NDDs. Although the discussions here are limited to neuronal mechanisms, we acknowledge that disruption of non-neuronal cell functions also plays a key role in many NDDs. For example, restricted loss of methyl CpG binding protein 2 (MeCP2), FMRP, or TSC protein-1 (TSC1) in astrocytes leads to abnormal dendritic growth, altered hippocampal circuitry, and seizures, respectively [38–40], and can create sensitive time-windows for synaptic phenotypes. Misregulated gene expression patterns Gene expression is, both temporally and spatially, a highly regulated process. Expression patterns of particular genes at specific developmental stages and within particular brain regions are key for the establishment and maturation of intra- and inter- layer-specific circuits [41]. In FXS, downregulated expression or absence of the Fmr1 mRNA

and its product FMRP may underlie the transient and developmentally regulated phenotypes (Table 1). In mice and humans, FMRP expression occurs in a developmentally-regulated pattern throughout the body with significant levels in cortex and hippocampus [42], as early as 18 wks prenatally in humans [43]. In mouse barrel cortex, peak expression of FMRP occurs between P7 and P14, decreasing again by P21 [22]. This timing coincides with the critical period of enhanced barrel-cortex synapse formation and plasticity during development [30], and also with transient phenotypic deficits in Fmr1-KO mice (Figure 2). In mouse hippocampus, peak FMRP expression occurs at P7 and decreases until P28 [44]. This period overlaps with significantly more filopodia than stubby mature spines in Fmr1-KO hippocampal cultures at days in vitro (DIV) 16 (of neurons prepared from P0 rats) and DIV21 [of neurons prepared from embryonic-day 18 (E18) rats] [45,46]. Also during this time-period, smaller AMPAmediated synaptic currents are observed in Fmr1-KO hippocampal slices compared to at later developmental stages [47,48]. If FMRP acts to stabilize and refine spines during maturation [14,17], its absence during these ‘peak’ expression periods may account for the abundance of immature spines with smaller synaptic currents. Failure to stabilize and enable normal spine maturation during hippocampal and barrel cortex circuit refinement in the Fmr1KO mouse could underlie later circuitry impairments. Correlative expression profiles do not prove a causal link between misregulation of single genes and synaptic impairments. In the genetically more-accessible model, Drosophila melanogaster, restricted reintroduction of the FMRP homolog (dFMRP) only during late brain development (i.e. during the circuit-refinement phase) was observed to rescue the dendritic phenotype in small ventrolateral neurons within circuits important for circadianclock function [49]. Reintroduction during early development (i.e. during the circuit-formation phase) or adulthood did not rescue this phenotype [49]. Such findings establish a causal relationship between dFMRP presence during a restricted developmental time-window and normal circuit 339

Opinion development. Confirmation of the causal relationship between timely expression of mammalian FMRP and correct synaptic phenotypes is imperative. This can be investigated by timely deletion or re-expression of the Fmr1 gene using pharmacologically-inducible promoters to direct Cre recombinase expression in conditional Fmr1-KO mice [12]. A similar approach has recently been used to demonstrate the ongoing role of MeCP2 in maintaining brain function in adulthood, its removal affecting mRNA expression and inducing behavioral deficits [50]. Similarly to FMRP, MeCP2 also exhibits distinct development-dependent expression profiles in different mouse brain regions [51]. MeCP2 expression coincides with excitatory synapse formation in many regions, including cortex, hippocampus, cerebellum and olfactory receptor neurons [51,52]. In mouse cerebellum, onset of synaptogenesis in Purkinje and neighboring granule cells coincides with upregulated MeCP2 expression in a cell type-specific manner at P6 and P21, respectively [51]. Following olfactory bulb and epithelial injury, MeCP2 expression is upregulated in mouse olfactory receptor neurons and precedes subsequent terminal differentiation and synaptogenesis of the newly generated projections [53]. At the synapse, the absence of MeCP2 caused differential alterations in NMDA receptor (NMDAR) expression patterns during peak synaptogenesis periods at P14 but also later at P49 compared to WT [54]. In addition to synaptic receptors, MeCP2 effects upon functional and structural plasticity could be mediated via brain-derived neurotrophic factor (BDNF), whose expression it regulates [52]. Supporting this, BDNF overexpression in MeCP2-deficient mice rescued specific behavioral and synaptic phenotypes [55]. Aberrant dendritic spine morphology is a prominent phenotype of Fmr1-KO mice [14], and FMRP alters expression of cytoskeletal hippocampal proteins in mouse brain at P14 [56]. Of these proteins, FMRP regulates the microtubule-associated protein-1B (MAP1B) [57]. Furthermore, it co-precipitates with MAP1B in messenger ribonucleoprotein complexes [44]. In WT hippocampus, expression levels of MAP1B decrease from P5 onwards, but in Fmr1-KO mice MAP1B expression peaks at P10 and its subsequent downregulation is delayed [44]. Thus, modified synaptic and cytoskeletal protein expression via misregulated NDD genes can result in developmentally delayed impairments in synaptic phenotypes during key periods of synaptogenesis (Figure 3). There are many mRNA targets of FMRP [57]. Likewise, a plethora of genes are regulated by MeCP2, and TSC1/2 acts via the Ras-family small GTPase (Ras homolog enriched in brain) Rheb to control the vital cell-growth regulator mTOR (mammalian target of rapamycin) [58–60]. Their absence causes misregulated expression of downstream targets during maturation, and this can cause the abnormal synaptic phenotypes observed. Thus in NDDs the temporal profiles of gene targets may reveal the dependence of a synaptic network upon a misregulated gene at that time-point. These gene products would act as crucial ‘hubs’ in protein signaling networks that govern development of specified neural circuits during their formation and refinement. Furthermore, misregulated timing of a specific connection, such as for long-range thalamocortical projections, could explain why 340

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specific behavioral impairments relying upon these projections are affected in that syndrome. Aberrant excitatory mechanisms Many misregulated proteins implicated in NDDs, including Rho GTPases, are localized in dendritic spines and can influence glutamatergic synaptic function and plasticity [4,14] (Figure 1). Cortical expression and kinetics of NMDARs are indicative of synapse maturity, and change significantly during the first postnatal weeks in mice [61,62]. The NMDAR subunits GluN1, GluN2A and GluN2B are a key group of synaptic-protein targets of FMRP [51]. GluN2A expression is upregulated from P7 onwards in mouse cortex and hippocampus [63] and is negatively regulated by FMRP through microRNA association [64]. In young Fmr1-KO neurons, increased silent synapses, enhanced NMDA/ AMPA ratios and impaired NMDAR-dependent plasticity are all prevalent [48,65,66] (but see evidence for unaltered NMDA/AMPA ratios in Fmr1-KO mouse hippocampal cultures in [67]). Thus, altered FMRP levels during the first postnatal week could cause a delayed enhancement of NMDA-mediated synaptic currents observed in young Fmr1-KO mice (Figure 2) [22,48]. Emergence of timingdependent plasticity rules in the cortex correlates with altered glutamate receptor expression and synaptogenesis [34,68]. Misregulation of the expression of NMDARs would alter links to intracellular signaling mechanisms and synaptic receptor kinetics, perturbing the timing precision required for plasticity [68]. However, absence of the GluN2A subunit does not alter the closure of the critical period in thalamocortical synapses [69]. Thus, disruption of normal NMDAR expression in Fmr1-KO mice [22] could disrupt excitatory plasticity, but additional mechanisms (such as GABAergic inhibition) may mediate altered critical periods. Altered maturation of GABAergic inhibition Homeostatic scaling mechanisms in response to altered excitation in the brain could promote compensatory inhibitory changes, as seen, for example, in vitro via regulation of the Mecp2 gene [70]. However, direct inhibitory changes are also observed in NDDs. Irregularities at the molecular, cellular, and functional levels of the GABAergic machinery occur in both FXS patients and Fmr1-KO mice [71–75]. An increased incidence of seizures in FXS patients (18% compared with <1% prevalence in the general population [76]), a greater susceptibility to audiogenic seizures in Fmr1-KO mice [77], and epileptiform activity in KO brain slices [78], further support a dysregulation in excitatory/inhibitory balance. Alterations in GABAA and GABAB receptors have been shown to be associated with such seizure activity in Fmr1-KO mice [77,78]. Changes in GABAergic signaling can influence the temporal dynamics of sensory critical periods during early postnatal development [32,79]. We propose that these changes underlie some of the impaired phenotypes observed in NDDs. In the cortex of developing Fmr1-KO mice, a1 GABAA receptor (GABAAR) subunit expression is transiently downregulated at P5 and P12, and GABAAR subunit b2 expression is attenuated from P12 onwards [80]. Attenuated functional inhibition at P14–16 and P25– 31 is also observed [73], and in young adult Fmr1-KO

Opinion cortex reduced expression of GABAAR subunits is reported at P56–84 [71]. The GABAAR a1 subunit is a key subunit of a majority of GABAARs, and a1-containing GABAARs have been shown to be crucial for correct spine maturation and stabilization of the extracellular matrix around parvalbumin interneurons [32,81]. In the visual cortex, sustained reduction in GABAAR-mediated signaling indefinitely postpones the critical-period onset [82], and expression of the GABAAR a1 subunit drives cortical plasticity during this critical period [83]. Thus, cortical reduction of key GABAAR subunits early in development in the FXS brain could bestow similar effects on the timing of critical periods and alter synaptic phenotypes. Indeed, in Fmr1-KO mice, monocular deprivation causes delayed hyperplastic reactions relative to WT, suggestive of an enduring immaturity of the underlying networks [84], although it is unclear if this is due to GABAergic dysfunction. Given high MeCP2 expression during cortical synaptogenesis [51], and recapitulation of Rett-syndrome phenotypes by GABAergic-cell restricted loss of MeCP2 [50,85], we speculate that critical periods for vision and remodeling in the cortex following sensory deprivation will be altered in MeCP2 KO mice. Indeed, from P21 onwards during the critical visual period for experience-dependent synapse refinement, retinogeniculate projections into visual thalamus in MeCP2 KO mice are significantly weaker, receive larger afferent innervation and show abnormal remodeling following light deprivation [86]. In visual cortex, genetically engineered overexpression of BDNF from P2 onwards enhances maturation of GABAergic inhibition, prematurely ending the critical period [87]. Thus, misregulated inhibition in MeCP2 mutant mice could directly alter cortical critical periods via BDNF-mediated mechanisms. Experiments to this end could discern between the role of MeCP2 in either delaying critical-period onset (Figure 3b) or in eliciting closure of critical periods (Figure 3c). Altering the excitatory/inhibitory balance in the mPFC leads to impairments in social behaviors and information processing [88]. The early dysregulation in GABAAR subunit expression [80] could underlie later impairments in synaptic plasticity and mPFC-dependent working memory in Fmr1-KO mice [25,65]. Presymptomatic excitatory/inhibitory changes are also observed in the cortex of MeCP2 KO mice [89]. Lack of MeCP2 specifically in GABAergic neurons restricted to the forebrain recapitulates some of the phenotypes observed with global MeCP2 deletion, including repetitive behaviors and altered social interactions [85]. Imbalanced inhibition in mPFC circuitry early in development could potentially cause subsequent symptomatic cognitive deficits in many NDDs. Implications of time-windows in developmental brain disorders The existence of impaired transient phenotypes in NDDs has implications for our understanding of developing brain circuits. Identification of sensitive time-windows where synaptic phenotypes are dysregulated in monogenic NDDs reveals a key role for specific genes at particular neurocircuit developmental stages. Given the aberrant developmental expression patterns of many genes in neurodevelopmental

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and autism disorders [90], there are likely to be additional defects in neurocircuit formation that have yet to be determined. The regional localization of proteins including FMRP and MeCP2 and their temporal developmental expression patterns may give clues as to why different brain regions are affected more strongly in some disorders than others. Should the misregulated NDD gene be the sole regulator in a synaptic protein network it is likely that brain deficits would be so great as to be embryonically lethal. However, in many disorders, the transient nature of some impairments indicate that building synaptic networks is by no means a rigid process and that compensatory pathways that regulate and rescue synapse maturation are activated, albeit with a delay. This delay may have consequences for critical periods of successively developing connected synaptic circuits. If developmental checkpoints are featured in normal brain development [91], delays in synapse maturation in NDDs may serve as a ‘biomarker’ for the underlying disorder that will ultimately result in aberrations in neural circuits and cognitive dysfunction. Why are many synaptic phenotypes transiently altered in NDDs? Impairments in synaptic phenotypes may only arise when a system is challenged and coincide with peak expression-levels of NDD gene-regulated synaptic proteins. At another developmental stage, when a NDD-regulated mechanism occurs outside the critical period, no misregulation will be seen (Figure 3a). The consequences of sensitive time-windows of synaptic maturation are not clearly understood, but we speculate that they have an effect upon critical periods and can underlie some of the later behavioral deficits observed in NDDs. Given the time-regulated impairments in NDDs, are there optimal time-windows in early development for therapeutic interventions? Recent studies have demonstrated success in rescuing deficits during adulthood in NDD mouse models, including models for Rett syndrome [92], Down syndrome [93], TSC disorder [94], and FXS [46,65,95]. In Fmr1-KO mice, rescue strategies include targeting mGluR5 receptors [46,84] or behavioral enrichment [65,96]. Chronic inhibition of mGluR5 corrects many behavioral, synaptic and protein synthesis phenotypes of Fmr1-KO mice following intervention in young adulthood [95]. In young FXS adults, clinical studies with mGluR5 antagonists have so far shown reduction in stereotypic behaviors and irritability in young FXS adults [97,98]. Improvements in cognitive and learning behaviors occur following treatment in mouse models of Down syndrome and TSC [93,94]. If the synaptic networks are more sensitive to intervention during early development, as we propose here, how is it that the symptoms can be rescued in adult mouse models? First, although many symptoms in the mouse can be reversed, the rescue is often partial and some phenotypes continue to be impaired [84,92,99]. Indeed, corrections of sensory-cortex synapse morphology [84,96], normalized cellular plasticity [92,94], and improved motor skills [98,99] in mouse models do not address whether cognitive impairments prominently associated with NDDs are rescued in every syndrome. Furthermore, partial phenotypic adult rescues in these models may arise from compensatory mechanisms that ease the measurable symptoms but do not completely overcome them. 341

Opinion Partial rescue could also be explained by the dual role of some NDD-associated proteins, such as MeCP2, in both circuit-development and subsequent circuit-maintenance phases [50]. Rescue of MeCP2 activity during maintenance occurs in adult stages whereas the underlying developmental deficits will remain. For NDDs such as FXS and Down syndrome in which multiple downstream targets are dysregulated, a promising but difficult approach is to prevent the trigger or onset phase of the disorder instead of rescuing multiple (and partly undetermined) pathways that are affected later on. By contrast, the downstream target of TSC, mTOR, is intact but overactive [100]. Treatment with rapamycin in adult TSC2-heterozygous mutant mice efficiently dampens mTOR activation and can rescue learning deficits and synaptic plasticity phenotypes [94]. Earlier intervention at P7–9 in TSC1-KO mice is also effective for abnormal behaviors, body weight, neuron size and myelination, but not for the phenotypic traits of neuronal dendrite orientation or spine density in the cortex [99]. However, partial rescue may occur because early developmental hyperactivation of mTOR could leave a long-lasting and irreversible imprint on synaptic networks, especially during critical periods of sensory integration, that are resistant to changes in later life. Current intervention in clinical trials for NDDs such as FXS [97] occurs after the major remodeling and plasticity periods in brain development. In human sensory developmental disorders such as amblyopia, early treatment before adolescence is essential to correct visual processing impairments [101]. Following our proposal that early timewindows exist for synaptic and neuronal deficits in NDDs, we hypothesize more prominent rescue during earlier presymptomatic stages when the neuronal networks are forming and are more sensitive to modulation. Indeed, early pharmacological mGluR5 blockade in Fmr1-KO pups recently showed greater improvement in spine morphology compared to adult mice [102]. However, key to effective therapy are reliable biomarkers for neonatal or early childhood diagnosis. Early genetic testing, brain imaging in young children [103], or brain functional measurements during social tasks [104], may in turn extend the currently known sensitive time-windows in NDDs. Concluding remarks Based on recently published findings, we propose the existence of sensitive windows of susceptibility for synaptic impairments in models of NDDs. Developmentally-regulated alterations in synaptic phenotypes and their consequences have not yet been fully explored (Box 1). However, we think they illustrate the dependence of specific neurocircuits during development and refinement on identified proteins misregulated in NDDs. Unraveling temporal and spatial expression profiles of genes misregulated in NDDs will further our knowledge regarding which neurocircuits and underlying behaviors are more susceptible to impairments in these disorders. To explore the existence of sensitive windows, time-regulated manipulation of gene expression in juvenile and adult animal models for monogenic NDD syndromes is crucial, similar to the approach used to elucidate the ongoing role for MeCP2 in maintaining healthy adult brain function [50]. Misregulated expression in specific cell types in identified neurocircuits, such as 342

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Box 1. Outstanding questions  Can treatment at early presymptomatic stages in animal models for NDDs prevent or ease the later synaptic, neuronal, and behavioral impairments?  Are all sensory critical periods equally misregulated in mouse models for a specific NDD? Are there different susceptibilities for auditory, visual and somatosensory neurocircuits that reflect the degree of impairments observed in patients?  If one critical period is missed or delayed during formation of a layer-specific connection in a network, does the network overcome this misregulated connectivity or plasticity window?  In monogenic NDDs, does the severity of misregulating one particular time-window for synaptic establishment during development correlate with the importance of that gene for that synaptic circuit?  Why do critical periods close in brain development? And what underlies the regression of some altered synaptic phenotypes in Fmr1-KO mice?  Can the concept of susceptible time-windows be applied to other NDDs, including schizophrenia and Tourette’s syndrome?

neurotransmitter-specific long-range projections to frontal cortical regions, would broaden our knowledge on whether attentional and executive-function deficits – common in many NDDs – arise from misregulation of specific neuronal pathways. A direct implication of the existence of early time-windows for phenotypic impairments is that they may provide hot-spots for early periods of therapeutic treatment. By comparing outcomes of treatment regimes during young presymptomatic stages and during adulthood in NDD mouse models, the potential translational benefit from such an approach can be assessed. If genes directly misregulated in NDDs play a key role in neurocircuit formation and synaptogenesis during early postnatal development, these identified circuits are likely to be more susceptible to early pharmaceutical and behavioral therapeutic interventions at this age. Knowledge of these early time-windows in NDDs may therefore act as a guide for future clinical interventions. This is likely to be an especially fruitful avenue for monogenic syndromes, such as FXS and TSC, but is also likely to have important implications for other developmental disorders including ASDs. Acknowledgments The authors are supported by the Nederlandse Organisatie voor Wetenschappelijke Onderzoek (NWO #917.10.372) and by the European Commission Seventh Framework Programme grant agreement FP7People-ITN-2008-238055 (‘BrainTrain’ project). We would like to thank Guus Smit, Mustafa Sahin, Femke DeVrij and Anis Contractor for their constructive comments on earlier versions of the manuscript.

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