Astrocyte-neuron lactate transport is required for long-term memory formation.

We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation.

The human-specific CASP4 gene product contributes to Alzheimer-related synaptic and behavioural deficits.

Recent studies have indicated that innate immune signalling molecules are involved in late-onset Alzheimer's disease (LOAD) risk. Amyloid beta (Aß) accumulates in AD brain, and has been proposed to act as a trigger of innate immune responses. Caspase-4 is an important part of the innate immune response. We recently characterized transgenic mice carrying human CASP4, and observed that the mice manifested profound innate immune responses to lipopolysaccharide (LPS). Since these inflammatory processes are important in the aetiology of AD, we have now analysed the correlation of expression of caspase-4 in human brain with AD risk genes, and studied caspase-4 effects on AD-related phenotypes in APPswe/PS1deltaE9 (APP/PS1) mice. We observed that the expression of caspase-4 was strongly correlated with AD risk genes including TYROBP, TREM2, CR1, PSEN1, MS4A4A and MS4A6A in LOAD brains. Caspase-4 expression was upregulated in CASP4/APP/PS1 mice in a region-specific manner, including hippocampus and prefrontal cortex. In APP/PS1 mice, caspase-4 expression led to impairments in the reversal phase of a Barnes maze task and in hippocampal synaptic plasticity, without affecting soluble or aggregated Aß levels. Caspase-4 was expressed predominantly in microglial cells, and in the presence of CASP4, more microglia were clustered around amyloid plaques. Furthermore, our data indicated that caspase-4 modulates microglial cells in a manner that increases proinflammatory processes. We propose that microglial caspase-4 expression contributes to the cognitive impairments in AD, and that further study of caspase-4 will enhance our understanding of AD pathogenesis and may lead to novel therapeutic targets in AD.

VGF and Its C-Terminal Peptide TLQP-62 Regulate Memory Formation in Hippocampus via a BDNF-TrkB-Dependent Mechanism.

Regulated expression and secretion of BDNF, which activates TrkB receptor signaling, is known to play a critical role in cognition. Identification of additional modulators of cognitive behavior that regulate activity-dependent BDNF secretion and/or potentiate TrkB receptor signaling would therefore be of considerable interest. In this study, we show in the adult mouse hippocampus that expression of the granin family gene Vgf and secretion of its C-terminal VGF-derived peptide TLQP-62 are required for fear memory formation. We found that hippocampal VGF expression and TLQP-62 levels were transiently induced after fear memory training and that sequestering secreted TLQP-62 peptide in the hippocampus immediately after training impaired memory formation. Reduced VGF expression was found to impair learning-evoked Rac1 induction and phosphorylation of the synaptic plasticity markers cofilin and synapsin in the adult mouse hippocampus. Moreover, TLQP-62 induced acute, transient activation of the TrkB receptor and subsequent CREB phosphorylation in hippocampal slice preparations and its administration immediately after training enhanced long-term memory formation. A critical role of BDNF-TrkB signaling as a downstream effector in VGF/TLQP-62-mediated memory consolidation was further revealed by posttraining activation of BDNF-TrkB signaling, which rescued impaired fear memory resulting from hippocampal administration of anti-VGF antibodies or germline VGF ablation in mice. We propose that VGF is a critical component of a positive BDNF-TrkB regulatory loop and, upon its induced expression by memory training, the TLQP-62 peptide rapidly reinforces BDNF-TrkB signaling, regulating hippocampal memory consolidation. SIGNIFICANCE STATEMENT: Identification of the cellular and molecular mechanisms that regulate long-term memory formation and storage may provide alternative treatment modalities for degenerative and neuropsychiatric memory disorders. The neurotrophin BDNF plays a prominent role in cognitive function, and rapidly and robustly induces expression of VGF, a secreted neuronal peptide precursor. VGF knock-out mice have impaired fear and spatial memory. Our study shows that VGF and VGF-derived peptide TLQP-62 are transiently induced after fear memory training, leading to increased BDNF/TrkB signaling, and that sequestration of hippocampal TLQP-62 immediately after training impairs memory formation. We propose that TLQP-62 is a critical component of a positive regulatory loop that is induced by memory training, rapidly reinforces BDNF-TrkB signaling, and is required for hippocampal memory consolidation.

A critical role for human caspase-4 in endotoxin sensitivity.

Response to endotoxins is an important part of the organismal reaction to Gram-negative bacteria and plays a critical role in sepsis and septic shock, as well as other conditions such as metabolic endotoxemia. Humans are generally more sensitive to endotoxins when compared with experimental animals such as mice. Inflammatory caspases mediate endotoxin-induced IL-1ß secretion and lethality in mice, and caspase-4 is an inflammatory caspase that is found in the human, and not mouse, genome. To test whether caspase-4 is involved in endotoxin sensitivity, we developed a transgenic mouse expressing human caspase-4 in its genomic context. Caspase-4 transgenic mice exhibited significantly higher endotoxin sensitivity, as measured by enhanced cytokine secretion and lethality following LPS challenge. Using bone marrow-derived macrophages, we then observed that caspase-4 can support activation of caspase-1 and secretion of IL-1ß and IL-18 in response to priming signals (LPS or Pam3CSK4) alone, without the need for second signals to stimulate the assembly of the inflammasome. These findings indicate that the regulation of caspase-1 activity by human caspase-4 could represent a unique mechanism in humans, as compared with laboratory rodents, and may partially explain the higher sensitivity to endotoxins observed in humans. Regulation of the expression, activation, or activity of caspase-4 therefore represents targets for systemic inflammatory response syndrome, sepsis, septic shock, and related disorders.

CCAAT enhancer binding protein δ plays an essential role in memory consolidation and reconsolidation.

A newly formed memory is temporarily fragile and becomes stable through a process known as consolidation. Stable memories may again become fragile if retrieved or reactivated, and undergo a process of reconsolidation to persist and strengthen. Both consolidation and reconsolidation require an initial phase of transcription and translation that lasts for several hours. The identification of the critical players of this gene expression is key for understanding long-term memory formation and persistence. In rats, the consolidation of inhibitory avoidance (IA) memory requires gene expression in both the hippocampus and amygdala, two brain regions that process contextual/spatial and emotional information, respectively; IA reconsolidation requires de novo gene expression in the amygdala. Here we report that, after IA learning, the levels of the transcription factor CCAAT enhancer binding protein δ (C/EBPδ) are significantly increased in both the hippocampus and amygdala. These increases are essential for long-term memory consolidation, as their blockade via antisense oligodeoxynucleotide-mediated knockdown leads to memory impairment. Furthermore, C/EBPδ is upregulated and required in the amygdala for IA memory reconsolidation. C/EBPδ is found in nuclear, somatic, and dendritic compartments, and a dendritic localization of C/EBPδ mRNA in hippocampal neuronal cultures suggests that this transcription factor may be translated at synapses. Finally, the induction of long-term potentiation at CA3-CA1 synapses by tetanic stimuli in acute slices, a cellular model of long-term memory, leads to an accumulation of C/EBPδ in the nucleus. We conclude that the transcription factor C/EBPδ plays a critical role in memory consolidation and reconsolidation.

Reduced excitatory neurotransmission and mild autism-relevant phenotypes in adolescent Shank3 null mutant mice.

Mutations in the synaptic scaffolding protein gene SHANK3 are strongly implicated in autism and Phelan-McDermid 22q13 deletion syndrome. The precise location of the mutation within the Shank3 gene is key to its phenotypic outcomes. Here, we report the physiological and behavioral consequences of null and heterozygous mutations in the ankyrin repeat domain in Shank3 mice. Both homozygous and heterozygous mice showed reduced glutamatergic transmission and long-term potentiation in the hippocampus with more severe deficits detected in the homozygous mice. Three independent cohorts were evaluated for magnitude and replicability of behavioral endophenotypes relevant to autism and Phelan-McDermid syndrome. Mild social impairments were detected, primarily in juveniles during reciprocal interactions, while all genotypes displayed normal adult sociability on the three-chambered task. Impaired novel object recognition and rotarod performance were consistent across cohorts of null mutants. Repetitive self-grooming, reduced ultrasonic vocalizations, and deficits in reversal of water maze learning were detected only in some cohorts, emphasizing the importance of replication analyses. These results demonstrate the exquisite specificity of deletions in discrete domains within the Shank3 gene in determining severity of symptoms.

Synaptic loss and retention of different classic cadherins with LTP-associated synaptic structural remodeling in vivo.

Cadherins are synaptic cell adhesion molecules that contribute to persistently enhanced synaptic strength characteristic of long-term potentiation (LTP). What is relatively unexplored is how synaptic activity of the kind that induces LTP-associated remodeling of synapse structure affects localization of cadherins, particularly in mature animals in vivo, details which could offer insight into how different cadherins contribute to synaptic plasticity. Here, we use a well-described in vivo LTP induction protocol that produces robust synaptic morphological remodeling in dentate gyrus of adult rats in combination with confocal and immunogold electron microscopy to localize cadherin-8 and N-cadherin at remodeled synapses. We find that the density and size of cadherin-8 puncta are significantly diminished in the potentiated middle molecular layer (MML) while concurrently, N-cadherin remains tightly clustered at remodeled synapses. These changes are specific to the potentiated MML, and occur without any change in density or size of synaptophysin puncta. Thus, the loss of cadherin-8 probably represents selective removal from synapses rather than overall loss of synaptic junctions. Together, these findings suggest that activity-regulated loss and retention of different synaptic cadherins could contribute to dual demands of both flexibility and stability in synapse structure that may be important for synaptic morphological remodeling that accompanies long-lasting plasticity.

Persistence of coordinated long-term potentiation and dendritic spine enlargement at mature hippocampal CA1 synapses requires N-cadherin.

Persistent changes in spine shape are coupled to long-lasting synaptic plasticity in hippocampus. The molecules that coordinate such persistent structural and functional plasticity are unknown. Here, we generated mice in which the cell adhesion molecule N-cadherin was conditionally ablated from postnatal, excitatory synapses in hippocampus. We applied to adult mice of either sex a combination of whole-cell recording, two-photon microscopy, and spine morphometric analysis to show that postnatal ablation of N-cadherin has profound effects on the stability of coordinated spine enlargement and long-term potentiation (LTP) at mature CA1 synapses, with no effects on baseline spine density or morphology, baseline properties of synaptic neurotransmission, or long-term depression. Thus, N-cadherin couples persistent spine structural modifications with long-lasting synaptic functional modifications associated selectively with LTP, revealing unexpectedly distinct roles at mature synapses in comparison with earlier, broader functions in synapse and spine development.

Extracellular proteolysis by matrix metalloproteinase-9 drives dendritic spine enlargement and long-term potentiation coordinately.

Persistent dendritic spine enlargement is associated with stable long-term potentiation (LTP), and the latter is thought to underlie long-lasting memories. Extracellular proteolytic remodeling of the synaptic microenvironment could be important for such plasticity, but whether or how proteolytic remodeling contributes to persistent modifications in synapse structure and function is unknown. Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is activated perisynaptically after LTP induction and required for LTP maintenance. Here, by monitoring spine size and excitatory postsynaptic potentials (EPSPs) simultaneously with combined 2-photon time-lapse imaging and whole-cell recordings from hippocampal neurons, we find that MMP-9 is both necessary and sufficient to drive spine enlargement and synaptic potentiation concomitantly. Both structural and functional MMP-driven forms of plasticity are mediated through beta1-containing integrin receptors, are associated with integrin-dependent cofilin inactivation within spines, and require actin polymerization. In contrast, postsynaptic exocytosis and protein synthesis are both required for MMP-9-induced potentiation, but not for initial MMP-9-induced spine expansion. However, spine expansion becomes unstable when postsynaptic exocytosis or protein synthesis is blocked, indicating that the 2 forms of plasticity are expressed independently but require interactions between them for persistence. When MMP activity is eliminated during theta-stimulation-induced LTP, both spine enlargement and synaptic potentiation are transient. Thus, MMP-mediated extracellular remodeling during LTP has an instructive role in establishing persistent modifications in both synapse structure and function of the kind critical for learning and memory.

Requirement for protein synthesis at developing synapses.

Activity and protein synthesis act cooperatively to generate persistent changes in synaptic responses. This forms the basis for enduring memory in adults. Activity also shapes neural circuits developmentally, but whether protein synthesis plays a congruent function in this process is poorly understood. Here, we show that brief periods of global or local protein synthesis inhibition decrease the synaptic vesicles available for fusion and increase synapse elimination. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a critical target; its levels are controlled by rapid turnover, and blocking its activity or knocking it down recapitulates the effects of protein synthesis inhibition. Mature presynaptic terminals show decreased sensitivity to protein synthesis inhibition, and resistance coincides with a developmental switch in regulation from CaMKII to PKA (protein kinase A). These findings demonstrate a novel mechanism regulating presynaptic activity and synapse elimination during development, and suggest that protein translation acts coordinately with activity to selectively stabilize appropriate synaptic interactions.

The neurotrophin-inducible gene Vgf regulates hippocampal function and behavior through a brain-derived neurotrophic factor-dependent mechanism.

VGF is a neurotrophin-inducible, activity-regulated gene product that is expressed in CNS and PNS neurons, in which it is processed into peptides and secreted. VGF synthesis is stimulated by BDNF, a critical regulator of hippocampal development and function, and two VGF C-terminal peptides increase synaptic activity in cultured hippocampal neurons. To assess VGF function in the hippocampus, we tested heterozygous and homozygous VGF knock-out mice in two different learning tasks, assessed long-term potentiation (LTP) and depression (LTD) in hippocampal slices from VGF mutant mice, and investigated how VGF C-terminal peptides modulate synaptic plasticity. Treatment of rat hippocampal slices with the VGF-derived peptide TLQP62 resulted in transient potentiation through a mechanism that was selectively blocked by the BDNF scavenger TrkB-Fc, the Trk tyrosine kinase inhibitor K252a (100 nm), and tPA STOP, an inhibitor of tissue plasminogen activator (tPA), an enzyme involved in pro-BDNF cleavage to BDNF, but was not blocked by the NMDA receptor antagonist APV, anti-p75(NTR) function-blocking antiserum, or previous tetanic stimulation. Although LTP was normal in slices from VGF knock-out mice, LTD could not be induced, and VGF mutant mice were impaired in hippocampal-dependent spatial learning and contextual fear conditioning tasks. Our studies indicate that the VGF C-terminal peptide TLQP62 modulates hippocampal synaptic transmission through a BDNF-dependent mechanism and that VGF deficiency in mice impacts synaptic plasticity and memory in addition to depressive behavior.

The extracellular protease matrix metalloproteinase-9 is activated by inhibitory avoidance learning and required for long-term memory.

Matrix metalloproteinases (MMPs) are a family of extracellularly acting proteolytic enzymes with well-recognized roles in plasticity and remodeling of synaptic circuits during brain development and following brain injury. However, it is now becoming increasingly apparent that MMPs also function in normal, nonpathological synaptic plasticity of the kind that may underlie learning and memory. Here, we extend this idea by investigating the role and regulation of MMP-9 in an inhibitory avoidance (IA) learning and memory task. We demonstrate that following IA training, protein levels and proteolytic activity of MMP-9 become elevated in hippocampus by 6 h, peak at 12-24 h, then decline to baseline values by approximately 72 h. When MMP function is abrogated by intrahippocampal infusion of a potent gelatinase (MMP-2 and MMP-9) inhibitor 3.5 h following IA training, a time prior to the onset of training-induced elevation in levels, IA memory retention is significantly diminished when tested 1-3 d later. Animals impaired at 3 d exhibit robust IA memory when retrained, suggesting that such impairment is not likely attributed to toxic or other deleterious effects that permanently disrupt hippocampal function. In anesthetized adult rats, the effective distance over which synaptic plasticity is impaired by a single intrahippocampal infusion of the MMP inhibitor of the kind that blocks IA memory is approximately 1200 microm. Taken together, these data suggest that IA training induces a slowly emerging, but subsequently protracted period of MMP-mediated proteolysis critical for enabling long-lasting synaptic modification that underlies long-term memory consolidation.

Matrix metalloproteinase-9 is required for hippocampal late-phase long-term potentiation and memory.

Matrix metalloproteinases (MMPs) are extracellular proteases that have well recognized roles in cell signaling and remodeling in many tissues. In the brain, their activation and function are customarily associated with injury or pathology. Here, we demonstrate a novel role for MMP-9 in hippocampal synaptic physiology, plasticity, and memory. MMP-9 protein levels and proteolytic activity are rapidly increased by stimuli that induce late-phase long-term potentiation (L-LTP) in area CA1. Such regulation requires NMDA receptors and protein synthesis. Blockade of MMP-9 pharmacologically prevents induction of L-LTP selectively; MMP-9 plays no role in, nor is regulated during, other forms of short-term synaptic potentiation or long-lasting synaptic depression. Similarly, in slices from MMP-9 null-mutant mice, hippocampal LTP, but not long-term depression, is impaired in magnitude and duration; adding recombinant active MMP-9 to null-mutant slices restores the magnitude and duration of LTP to wild-type levels. Activated MMP-9 localizes in part to synapses and modulates hippocampal synaptic physiology through integrin receptors, because integrin function-blocking reagents prevent an MMP-9-mediated potentiation of synaptic signal strength. The fundamental importance of MMP-9 function in modulating hippocampal synaptic physiology and plasticity is underscored by behavioral impairments in hippocampal-dependent memory displayed by MMP-9 null-mutant mice. Together, these data reveal new functions for MMPs in synaptic and behavioral plasticity.

N-cadherin regulates ingrowth and laminar targeting of thalamocortical axons.

Thalamocortical axons are precisely targeted to cortical layer IV, but the identity of specific molecules that govern the establishment of laminar specificity in the thalamocortical projection has been elusive. In this study, we test the role of N-cadherin, a homophilic cell adhesion molecule, in laminar targeting of thalamocortical axons using cocultured thalamic and cortical slice explants exposed to N-cadherin function-blocking antibodies or inhibitory peptides. In untreated cocultures, labeled thalamocortical axons normally grow to and stop in layer IV, forming terminal-like arbors. In the N-cadherin-blocked cocultures, thalamic axons reach layer IV by growing through deep layers at the same rate as those in the untreated cocultures, but instead of terminating in layer IV, they continue growing uninterruptedly through layer IV and extend into supragranular layers to reach the outermost cortical edge, where some form terminal-like arbors in this aberrant laminar position. In cocultures in which the cortical slice is taken at an earlier maturational stage, one that corresponds to a time when thalamic axons are normally growing through deep layers before the emergence of layer IV from the cortical plate, thalamic axon ingrowth through deep layers is significantly attenuated by N-cadherin blocking reagents. These data indicate that N-cadherin has multifaceted roles in establishing the thalamocortical projection, governing aspects of both thalamic axon ingrowth and laminar targeting by acting as a layer IV stop signal, which progressively change in parallel with the maturational state of the cortex.

Altered Abeta formation and long-term potentiation in a calsenilin knock-out.

Calsenilin has been identified as a presenilin-binding protein, a transcription factor regulating dynorphin expression, and a beta-subunit of Kv4 channels and could, thus, be a multifunctional protein. To study these functions of calsenilin in vivo and to determine the neuroanatomical expression pattern of calsenilin, we generated mice with a disruption of the calsenilin gene by the targeted insertion of the beta-galactosidase gene. We found that calsenilin expression (as represented by beta-galactosidase activity) is very restricted but overlaps better with that of presenilins and Kv4 channels than with dynorphin, suggesting that calsenilin may regulate presenilin and Kv4 channels in brain. Abeta peptide levels are reduced in calsenilin knock-out mice, demonstrating that calsenilin affects presenilin-dependent gamma-cleavage in vivo. Furthermore, long-term potentiation (LTP) in dentate gyrus of hippocampus, in which calsenilin is strongly and selectively expressed, is enhanced in calsenilin knock-out mice. This enhancement of LTP coincides with a downregulation of the Kv4 channel-dependent A-type current and can be mimicked in wild-type animals by a Kv4 channel blocker. The data presented here show that lack of calsenilin affects both Abeta formation and the A-type current. We suggest that these effects are separate events, caused by a common mechanism possibly involving protein transport.

The cadherin family of cell adhesion molecules: multiple roles in synaptic plasticity.

Cadherins are cell adhesion molecules that are critically important for establishing brain structure and connectivity during early development. They are enriched at synapses and, by virtue of a number of properties including homophilic recognition and molecular diversity, have been implicated in the generation of synaptic specificity. Cadherins also participate in remodeling synaptic architecture and modifying the strength of the synaptic signal, thereby retaining an active role in synaptic structure, function, and plasticity, which extends beyond initial development. Cadherins have been implicated in the induction of long-term potentiation (LTP) of hippocampal synaptic strength, a cellular model for learning and memory. LTP is associated with the synthesis and recruitment of N-cadherin to newly forming synaptic junctions, induces molecular changes to N-cadherin indicative of augmented adhesive force, and can be prevented when cadherin adhesion is blocked. NMDA receptor activation, which is critically required for synaptic plasticity, may provide a signal that regulates the molecular configuration of synaptic N-cadherin, and therefore the strength of adhesion across the synaptic cleft. Additionally, there exists at the synapse a pool of surface cadherins that is untethered to the actin cytoskeleton and capable of a rapid and reversible dispersion along the plasmalemma under conditions of strong activity. These observations suggest that synaptic activity dynamically regulates both the strength and the localization of cadherin-cadherin bonds across the synaptic junctional interface, changes that may be crucial for regulating synaptic plasticity.

Axonal cap-dependent translation regulates presynaptic p35.

Axonal growth cones synthesize proteins during development and in response to injury in adult animals. Proteins locally translated in axons are used to generate appropriate responses to guidance cues, contribute to axon growth, and can serve as retrograde messengers. In addition to growth cones, mRNAs and translational machinery are also found along the lengths of axons where synapses form en passant, but contributions of intra-axonal translation to developing synapses are poorly understood. Here, we engineered a subcellular-targeting translational repressor to inhibit mRNA translation in axons, and we used this strategy to investigate presynaptic contributions of cap-dependent protein translation to developing CNS synapses. Our data show that intra-axonal mRNA translation restrains synaptic vesicle recycling pool size and that one target of this regulation is p35, a Cdk5 activating protein. Cdk5/p35 signaling regulates the size of vesicle recycling pools, p35 levels diminish when cap-dependent translation is repressed, and restoring p35 levels rescues vesicle recycling pools from the effects of spatially targeted translation repression. Together our findings show that intra-axonal synthesis of p35 is required for normal vesicle recycling in developing neurons, and that targeted translational repression provides a novel strategy to investigate extrasomal protein synthesis in neurons.

Insulin-like growth factor-1 rescues synaptic and motor deficits in a mouse model of autism and developmental delay.

BACKGROUND: Haploinsufficiency of SHANK3, due to either hemizygous gene deletion (termed 22q13 deletion syndrome or Phelan-McDermid syndrome) or to gene mutation, accounts for about 0.5% of the cases of autism spectrum disorder (ASD) and/or developmental delay, and there is evidence for a wider role for SHANK3 and glutamate signaling abnormalities in ASD and related conditions. Therapeutic approaches that reverse deficits in SHANK3-haploinsufficiency may therefore be broadly beneficial in ASD and in developmental delay. FINDINGS: We observed that daily intraperitoneal injections of human insulin-like growth factor 1 (IGF-1) over a 2-week period reversed deficits in hippocampal α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) signaling, long-term potentiation (LTP), and motor performance that we had previously reported in Shank3-deficient mice. Positive effects were observed with an IGF-1 peptide derivative as well. CONCLUSIONS: We observed significant beneficial effects of IGF-1 in a mouse model of ASD and of developmental delay. Studies in mouse and human neuronal models of Rett syndrome also show benefits with IGF-1, raising the possibility that this compound may have benefits broadly in ASD and related conditions, even with differing molecular etiology. Given the extensive safety data for IGF-1 in children with short stature due to primary IGF-1 deficiency, IGF-1 is an attractive candidate for controlled clinical trials in SHANK3-deficiency and in ASD.

Haploinsufficiency of Cyfip1 produces fragile X-like phenotypes in mice.

BACKGROUND: Copy number variation (CNV) at the 15q11.2 region, which includes a gene that codes for CYFIP1 (cytoplasmic FMR1 interacting protein 1), has been implicated in autism, intellectual disability and additional neuropsychiatric phenotypes. In the current study we studied the function of Cyfip1 in synaptic physiology and behavior, using mice with a disruption of the Cyfip1 gene. METHODOLOGY/PRINCIPAL FINDINGS: We observed that in Cyfip1 heterozygous mice metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) induced by paired-pulse low frequency stimulation (PP-LFS) was significantly increased in comparison to wildtype mice. In addition, mGluR-LTD was not affected in the presence of protein synthesis inhibitor in the Cyfip1 heterozygous mice, while the same treatment inhibited LTD in wildtype littermate controls. mGluR-agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD was also significantly increased in hippocampal slices from Cyfip1 heterozygous mice and again showed independence from protein synthesis only in the heterozygous animals. Furthermore, we observed that the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin was only effective at reducing mGluR-LTD in wildtype animals. Behaviorally, Cyfip1 heterozygous mice showed enhanced extinction of inhibitory avoidance. Application of both mGluR5 and mGluR1 antagonist to slices from Cyfip1 heterozygous mice reversed the increase in DHPG-induced LTD in these mice. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that haploinsufficiency of Cyfip1 mimics key aspects of the phenotype of Fmr1 knockout mice and are consistent with the hypothesis that these effects are mediated by interaction of Cyfip1 and Fmrp in regulating activity-dependent translation. The data provide support for a model where CYFIP1 haploinsufficiency in patients results in intermediate phenotypes increasing risk for neuropsychiatric disorders.

Optimizing the phenotyping of rodent ASD models: enrichment analysis of mouse and human neurobiological phenotypes associated with high-risk autism genes identifies morphological, electrophysiological, neurological, and behavioral features.

BACKGROUND: There is interest in defining mouse neurobiological phenotypes useful for studying autism spectrum disorders (ASD) in both forward and reverse genetic approaches. A recurrent focus has been on high-order behavioral analyses, including learning and memory paradigms and social paradigms. However, well-studied mouse models, including for example Fmr1 knockout mice, do not show dramatic deficits in such high-order phenotypes, raising a question as to what constitutes useful phenotypes in ASD models. METHODS: To address this, we made use of a list of 112 disease genes etiologically involved in ASD to survey, on a large scale and with unbiased methods as well as expert review, phenotypes associated with a targeted disruption of these genes in mice, using the Mammalian Phenotype Ontology database. In addition, we compared the results with similar analyses for human phenotypes. FINDINGS: We observed four classes of neurobiological phenotypes associated with disruption of a large proportion of ASD genes, including: (1) Changes in brain and neuronal morphology; (2) electrophysiological changes; (3) neurological changes; and (4) higher-order behavioral changes. Alterations in brain and neuronal morphology represent quantitative measures that can be more widely adopted in models of ASD to understand cellular and network changes. Interestingly, the electrophysiological changes differed across different genes, indicating that excitation/inhibition imbalance hypotheses for ASD would either have to be so non-specific as to be not falsifiable, or, if specific, would not be supported by the data. Finally, it was significant that in analyses of both mouse and human databases, many of the behavioral alterations were neurological changes, encompassing sensory alterations, motor abnormalities, and seizures, as opposed to higher-order behavioral changes in learning and memory and social behavior paradigms. CONCLUSIONS: The results indicated that mutations in ASD genes result in defined groups of changes in mouse models and support a broad neurobiological approach to phenotyping rodent models for ASD, with a focus on biochemistry and molecular biology, brain and neuronal morphology, and electrophysiology, as well as both neurological and additional behavioral analyses. Analysis of human phenotypes associated with these genes reinforced these conclusions, supporting face validity for these approaches to phenotyping of ASD models. Such phenotyping is consistent with the successes in Fmr1 knockout mice, in which morphological changes recapitulated human findings and electrophysiological deficits resulted in molecular insights that have since led to clinical trials. We propose both broad domains and, based on expert review of more than 50 publications in each of the four neurobiological domains, specific tests to be applied to rodent models of ASD.