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Intimate partner violence (IPV) increases risk of traumatic brain injury (TBI). Physical assaults increase in frequency and intensity during pregnancy. The consequences of TBI during pregnancy (gravida TBI; gTBI) on offspring development is unknown, for which stress and inflammation during pregnancy worsen fetal developmental outcomes. We hypothesized that gTBI would lead to increased anxiety- and depression-related behavior, altered inflammatory responses and gut pathology, and distorted brain circuitry in mixed-sex offspring compared to mice born to control mothers. Pregnant dams received either diffuse TBI or sham injury (control) 12 days post-coitum. We found that male gTBI offspring were principal drivers of the gTBI effects on health, physiology, and behavior. For example, male, but not female, gTBI offspring weighed significantly less at weaning compared to male control offspring. At post-natal day (PND) 28, gTBI offspring had significantly weaker intralaminar connectivity onto layer 5 pre-frontal pyramidal neurons compared to control offspring. Neurological performance on anxiety-like behaviors was decreased, with only marginal differences in depressive-like behaviors, for gTBI offspring compared to control offspring. At PND42 and PND58, circulating neutrophil and monocyte populations were significantly smaller in gTBI male offspring than control male offspring. In response to a subsequent inflammatory challenge at PND75, gTBI offspring had significantly smaller circulating neutrophil populations than control offspring. Anxiety-like behaviors persisted during the immune challenge in gTBI offspring. However, spleen immune response and gut histology showed no significant differences between groups. The results compel further studies to determine the full extent of gTBI on fetal and maternal outcomes.

Mutations in genes encoding amyloid precursor protein (APP) and presenilins (PSs) cause familial forms of Alzheimer's disease (AD), a neurodegenerative disorder strongly associated with aging. It is currently unknown whether and how AD risks affect early brain development, and to what extent subtle synaptic pathology may occur prior to overt hallmark AD pathology. Transgenic mutant APP/PS1 over-expression mouse lines are key tools for studying the molecular mechanisms of AD pathogenesis. Among these lines, the 5XFAD mice rapidly develop key features of AD pathology and have proven utility in studying amyloid plaque formation and amyloid β (Aβ)-induced neurodegeneration. We reasoned that transgenic mutant APP/PS1 over-expression in 5XFAD mice may lead to neurodevelopmental defects in early cortical neurons, and performed detailed synaptic physiological characterization of layer 5 (L5) neurons from the prefrontal cortex (PFC) of 5XFAD and wild-type littermate controls. L5 PFC neurons from 5XFAD mice show early APP/Aβ immunolabeling. Whole-cell patch-clamp recording at an early post-weaning age (P22-30) revealed functional impairments; although 5XFAD PFC-L5 neurons exhibited similar membrane properties, they were intrinsically less excitable. In addition, these neurons received smaller amplitude and frequency of miniature excitatory synaptic inputs. These functional disturbances were further corroborated by decreased dendritic spine density and spine head volumes that indicated impaired synapse maturation. Slice biotinylation followed by Western blot analysis of PFC-L5 tissue revealed that 5XFAD mice showed reduced synaptic AMPA receptor subunit GluA1 and decreased synaptic NMDA receptor subunit GluN2A. Consistent with this, patch-clamp recording of the evoked L23L5 synaptic responses revealed a reduced AMPA/NMDA receptor current ratio, and an increased level of AMPAR-lacking silent synapses. These results suggest that transgenic mutant forms of APP/PS1 overexpression in 5XFAD mice leads to early developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.

Depression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.

The molecular regulation of the temporal dynamics of circuit maturation is a key contributor to the emergence of normal structure–function relations. Developmental control of cortical MET receptor tyrosine kinase, expressed early postnatally in subpopulations of excitatory neurons, has a pronounced impact on the timing of glutamatergic synapse maturation and critical period plasticity. Here, we show that using a controllable overexpression (cto-Met) transgenic mouse, extending the duration of MET signaling after endogenous Met is switched off leads to altered molecular constitution of synaptic proteins, persistent activation of small GTPases Cdc42 and Rac1, and sustained inhibitory phosphorylation of cofilin. These molecular changes are accompanied by an increase in the density of immature dendritic spines, impaired cortical circuit maturation of prefrontal cortex layer 5 projection neurons, and altered laminar excitatory connectivity. Two photon in vivo imaging of dendritic spines reveals that cto-Met enhances de novo spine formation while inhibiting spine elimination. Extending MET signaling for two weeks in developing cortical circuits leads to pronounced repetitive activity and impaired social interactions in adult mice. Collectively, our data revealed that temporally controlled MET signaling as a critical mechanism for controlling cortical circuit development and emergence of normal behavior.

Central tolerance prevents autoimmunity, but also limits T cell responses to potentially immunodominant tumor epitopes with limited expression in healthy tissues. In peripheral APCs, γ-IFN–inducible lysosomal thiol reductase (GILT) is critical for MHC class II–restricted presentation of disulfide bond–containing proteins, including the self-antigen and melanoma Ag tyrosinase-related protein 1 (TRP1). The role of GILT in thymic Ag processing and generation of central tolerance has not been investigated. We found that GILT enhanced the negative selection of TRP1-specific thymocytes in mice. GILT expression was enriched in thymic APCs capable of mediating deletion, namely medullary thymic epithelial cells (mTECs) and dendritic cells, whereas TRP1 expression was restricted solely to mTECs. GILT facilitated MHC class II–restricted presentation of endogenous TRP1 by pooled thymic APCs. Using bone marrow chimeras, GILT expression in thymic epithelial cells (TECs), but not hematopoietic cells, was sufficient for complete deletion of TRP1-specific thymocytes. An increased frequency of TRP1-specific regulatory T (Treg) cells was present in chimeras with increased deletion of TRP1-specific thymocytes. Only chimeras that lacked GILT in both TECs and hematopoietic cells had a high conventional T/Treg cell ratio and were protected from melanoma challenge. Thus, GILT expression in thymic APCs, and mTECs in particular, preferentially facilitates MHC class II–restricted presentation, negative selection, and increased Treg cells, resulting in a diminished antitumor response to a tissue-restricted, melanoma-associated self-antigen.

Mutations in genes encoding amyloid precursor proteins and presenilins lead to increased β-amyloid (Aβ) production and cause familial Alzheimer’s disease (AD), a neurodegenerative disorder often associates with aging and features synapse loss and impaired synaptic plasticity. Aβ deposition is a pathological hallmark of AD. It is currently unknown whether and how AD risk alleles affects development of brain circuit function, and whether subtle synaptic pathology occurs prior to overt Aβ deposition. Transgenic mutated APP/PS1 over-expression mice lines are key tools to study molecular mechanisms of AD pathogenesis. Among these mice lines, the 5XFAD mice rapidly recapitulate key features of AD pathology, and have proven utility in studying amyloid plaque formation and Aβ-induced neurodegeneration. We reason that transgenic mutant APP/PS1 over-expression may lead to neurodevelopmental defects in early cortical neurons as a result of continuous APP/Aβ expression from early life. We first explored age-dependent plasticity changes in prefrontal cortical circuits in the 5XFAD mice, and found that at an early age (6-8 wks old) that does not produce overt impairment of hippocampus LTP, layer (L) 5 neuron circuit plasticity, measured by long term potentiation (LTP), is impaired. In addition, L5 neurons, which are reportedly vulnerable cortical neuron populations, show reduced mEPSC amplitude and frequency in early postweaning ages, indicating impaired synaptic transmission as a result of transgenic APP/Aβ overloading during early postnatal development. These functional changes were corroborated by the morphological findings of smaller, sparser dendritic spines on L5 pyramidal neurons, indicative of impaired prefrontal circuit function. Lastly, laser scanning photostimulation (LSPS) combined with glutamate uncaging revealed that L2/3 > L5 cortical connectivity was decreased at this early age. Our results suggest 5XFAD transgenic mutant APP/PS1 over expression causes developmental defects of cortical circuits, which could contribute to the age-dependent synaptic pathology and neurodegeneration later in life.

As more genes conferring risks to neurodevelopmental disorders are identified, translating these genetic risk factors into biological mechanisms that impact the trajectory of the developing brain is a critical next step. Here, we report that disrupted signaling mediated MET receptor tyrosine kinase (RTK), an established risk factor for autism spectrum disorders, in the developing hippocampus glutamatergic circuit leads to profound deficits in neural development, synaptic transmission, and plasticity. In cultured hippocampus slices prepared from neonatal mice, pharmacological inhibition of MET kinase activity suppresses dendritic arborization and disrupts normal dendritic spine development. In addition, single-neuron knockdown (RNAi) or overexpression of Met in the developing hippocampal CA1 neurons leads to alterations, opposite in nature, in basal synaptic transmission and long-term plasticity. In forebrain-specific Met conditional knockout mice (Metfx/fx ;emx1cre ), an enhanced long-term potentiation (LTP) and long-term depression (LTD) were observed at early developmental stages (P12-14) at the Schaffer collateral to CA1 synapses compared with wild-type littermates. In contrast, LTP and LTD were markedly reduced at young adult stage (P56-70) during which wild-type mice show robust LTP and LTD. The altered trajectory of synaptic plasticity revealed by this study indicate that temporally regulated MET signaling as an intrinsic, cell autonomous, and pleiotropic mechanism not only critical for neuronal growth and functional maturation, but also for the timing of synaptic plasticity during forebrain glutamatergic circuits development.

The activation of microglia and the various substances they produce have been linked to the pathologic development of Parkinson's disease (PD), but the precise role of microglia in PD remains to be defined. The survival of microglia depends on colony-stimulating factor 1 receptor (CSF1R) signaling, and CSF1R inhibition results in rapid elimination of microglia in the central nervous system. Using a mouse PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment, we showed that the depletion of microglia via the CSF1R inhibitor PLX3397 exacerbated the impairment of locomotor activities and the loss of dopaminergic neurons. Further, depletion of microglia augmented the production of inflammatory mediators and infiltration of leukocytes in the brain after MPTP exposure. Microglia depletion-induced aggravation of MPTP neurotoxicity was also seen in lymphocyte-deficient mice. In addition, the depletion of microglia did not affect the production of brain-derived neurotrophic factor, but it dramatically augmented the production of inflammatory mediators by astrocytes after MPTP treatment. Our findings suggest microglia play a protective role against MPTP-induced neuroinflammation and dopaminergic neurotoxicity.-Yang, X., Ren, H., Wood, K., Li, M., Qiu, S., Shi, F.-D., Ma, C., Liu, Q. Depletion of microglia augments the dopaminergic neurotoxicity of MPTP.

SUMMARY Projection neuron subtype identities in the cerebral cortex are established by expressing pan-cortical and subtype-specific effector genes that execute terminal differentiation programs bestowing neurons with a glutamatergic neuron phenotype and subtype-specific morphology, physiology, and axonal projections. Whether pan-cortical glutamatergic and subtype-specific characteristics are regulated by the same genes or controlled by distinct programs remains largely unknown. Here, we show that FEZF2 functions as a transcriptional repressor, and it regulates subtype-specific identities of both corticothalamic and subcerebral neurons by selectively repressing expression of genes inappropriate for each neuronal subtype. We report that TLE4, specifically expressed in layer 6 corticothalamic neurons, is recruited by FEZF2 to inhibit layer 5 subcerebral neuronal genes. Together with previous studies, our results indicate that a cortical glutamatergic identity is specified by multiple parallel pathways active in progenitor cells, whereas projection neuron subtype-specific identity is achieved through selectively repressing genes associated with alternate identities in differentiating neurons., Graphical Abstract, In brief Layer 6 corticothalamic and layer 5 subcerebral projection neurons connect the cerebral cortex to the thalamus and brainstem and spinal cord, respectively. Tsyporin et al. report that the transcription factor FEZF2 specifies the development of these neuronal identities by repressing expression of genes associated with alternate projection neuron subtypes.

The key developmental milestone events of the human brain, such as neurogenesis, synapse formation, maturation, and plasticity are determined by a myriad of molecular signaling events, including those mediated by a number of receptor tyrosine kinases and their cognate ligands. Aberrant or mistimed brain development and plasticity can lead to maladaptive changes, such as dysregulated synaptic connectivity and breakdown of circuit functions necessary for cognition and adaptive behaviors, which are hypothesized pathophysiologies of many neurodevelopmental and neuropsychiatric disorders. Here we review recent literature that supports autism spectrum disorder (ASD) as a likely result of aberrant synapse development due to mistimed maturation and plasticity. We focus on MET receptor tyrosine kinase, a prominent genetic risk factor for autism, and discuss how a pleiotropic molecular signaling system engaged by MET exemplifies a genetic program that controls cortical circuit development and plasticity by modulating anatomical and functional connectivity of cortical circuits, thus conferring genetic risk for neurodevelopmental disorders.

Normal development of cortical circuits, including experience-dependent cortical maturation and plasticity, requires precise temporal regulation of gene expression and molecular signaling. Such regulation, and the concomitant impact on plasticity and critical periods, is hypothesized to be disrupted in neurodevelopmental disorders. A protein that may serve such a function is the MET receptor tyrosine kinase, which is tightly regulated developmentally in rodents and primates, and exhibits reduced cortical expression in autism spectrum disorder and Rett Syndrome. We found that the peak of MET expression in developing mouse cortex coincides with the heightened period of synaptogenesis, but is precipitously down-regulated prior to extensive synapse pruning and certain peak periods of cortical plasticity. These results reflect a potential on-off regulatory synaptic mechanism for specific glutamatergic cortical circuits in which MET is enriched. In order to address the functional significance of the ‘off’ component of the proposed mechanism, we created a controllable transgenic mouse line that sustains cortical MET signaling. Continued MET expression in cortical excitatory neurons disrupted synaptic protein profiles, altered neuronal morphology, and impaired visual cortex circuit maturation and connectivity. Remarkably, sustained MET signaling eliminates monocular deprivation-induced ocular dominance plasticity during the normal cortical critical period; while ablating MET signaling leads to early closure of critical period plasticity. The results demonstrate a novel mechanism in which temporal regulation of a pleiotropic signaling protein underlies cortical circuit maturation and timing of cortical critical period, features that may be disrupted in neurodevelopmental disorders.

SHANK3 mutations, including de novo deletions, have been associated with autism spectrum disorders (ASD). However, the effects of SHANK3 loss of function on neurodevelopment remain poorly understood. Here we generated human induced pluripotent stem cells (iPSC) in vitro, followed by neuro-differentiation and lentivirus-mediated shRNA expression to evaluate how SHANK3 knockdown affects the in vitro neurodevelopmental process at multiple time points (up to 4 weeks). We found that SHANK3 knockdown impaired both early stage of neuronal development and mature neuronal function, as demonstrated by a reduction in neuronal soma size, growth cone area, neurite length and branch numbers. Notably, electrophysiology analyses showed defects in excitatory and inhibitory synaptic transmission. Furthermore, transcriptome analyses revealed that multiple biological pathways related to neuron projection, motility and regulation of neurogenesis were disrupted in cells with SHANK3 knockdown. In conclusion, utilizing a human iPSC-based neural induction model, this study presented combined morphological, electrophysiological and transcription evidence that support that SHANK3 as an intrinsic, cell autonomous factor that controls cellular function development in human neurons.

Human genetic studies established MET gene as a risk factor for autism spectrum disorders. We have previously shown that signaling mediated by MET receptor tyrosine kinase, expressed in early postnatal developing forebrain circuits, controls glutamatergic neuron morphological development, synapse maturation, and cortical critical period plasticity. Here we investigated how MET signaling affects synaptic plasticity, learning and memory behavior, and whether these effects are age-dependent. We found that in young adult (postnatal 2–3 months) Met conditional knockout (Metfx/fx:emx1cre, cKO) mice, the hippocampus exhibits elevated plasticity, measured by increased magnitude of long-term potentiation (LTP) and depression (LTD) in hippocampal slices. Surprisingly, in older adult cKO mice (10–12 months), LTP and LTD magnitudes were diminished. We further conducted a battery of behavioral tests to assess learning and memory function in cKO mice and littermate controls. Consistent with age-dependent LTP/LTD findings, we observed enhanced spatial memory learning in 2–3 months old young adult mice, assessed by hippocampus-dependent Morris water maze test, but impaired spatial learning in 10–12 months mice. Contextual and cued learning were further assessed using a Pavlovian fear conditioning test, which also revealed enhanced associative fear acquisition and extinction in young adult mice, but impaired fear learning in older adult mice. Lastly, young cKO mice also exhibited enhanced motor learning. Our results suggest that a shift in the window of synaptic plasticity and an age-dependent early cognitive decline may be novel circuit pathophysiology for a well-established autism genetic risk factor.

As more genes conferring risks to neurodevelopmental disorders are identified, translating these genetic risk factors into biological mechanisms that impact the trajectory of the developing brain is a critical next step. Here, we report that disrupted signaling mediated MET receptor tyrosine kinase (RTK), an established risk factor for autism spectrum disorders, in the developing hippocampus glutamatergic circuit leads to profound deficits in neural development, synaptic transmission, and plasticity. In cultured hippocampus slices prepared from neonatal mice, pharmacological inhibition of MET kinase activity suppresses dendritic arborization and disrupts normal dendritic spine development. In addition, single-neuron knockdown (RNAi) or overexpression of Met in the developing hippocampal CA1 neurons leads to alterations, opposite in nature, in basal synaptic transmission and long-term plasticity. In forebrain-specific Met conditional knockout mice (Met(fx/fx);emx1(cre)), an enhanced long-term potentiation (LTP) and long-term depression (LTD) were observed at early developmental stages (P12–14) at the Schaffer collateral to CA1 synapses compared with wild-type littermates. In contrast, LTP and LTD were markedly reduced at young adult stage (P56–70) during which wild-type mice show robust LTP and LTD. The altered trajectory of synaptic plasticity revealed by this study indicate that temporally regulated MET signaling as an intrinsic, cell autonomous, and pleiotropic mechanism not only critical for neuronal growth and functional maturation, but also for the timing of synaptic plasticity during forebrain glutamatergic circuits development.

SHANK3 mutations, including de novo deletions, have been associated with autism spectrum disorders (ASD). However, the effects of SHANK3 loss of function on neurodevelopment remain poorly understood. Here we generated human induced pluripotent stem cells (iPSC) in vitro, followed by neuro-differentiation and lentivirus-mediated shRNA expression to evaluate how SHANK3 knockdown affects the in vitro neurodevelopmental process at multiple time points (up to 4 weeks). We found that SHANK3 knockdown impaired both early stage of neuronal development and mature neuronal function, as demonstrated by a reduction in neuronal soma size, growth cone area, neurite length and branch numbers. Notably, electrophysiology analyses showed defects in excitatory and inhibitory synaptic transmission. Furthermore, transcriptome analyses revealed that multiple biological pathways related to neuron projection, motility and regulation of neurogenesis were disrupted in cells with SHANK3 knockdown. In conclusion, utilizing a human iPSC-based neural induction model, this study presented combined morphological, electrophysiological and transcription evidence that support that SHANK3 as an intrinsic, cell autonomous factor that controls cellular function development in human neurons.

Degrading vision by one eye during a developmental critical period yields enduring deficits in both eye dominance and visual acuity. A predominant model is that ‘reactivating’ ocular dominance (OD) plasticity after the critical period is required to improve acuity in amblyopic adults. However, here we demonstrate that plasticity of eye dominance and acuity are independent, and restricted by the nogo-66 receptor (ngr1) in distinct neuronal populations. Ngr1 mutant mice display greater excitatory synaptic input onto both inhibitory and excitatory neurons with restoration of normal vision. Deleting ngr1 in excitatory cortical neurons permits recovery of eye dominance but not acuity. Reciprocally, deleting ngr1 in thalamus is insufficient to rectify eye dominance but yields improvement of acuity to normal. Abolishing ngr1 expression in adult mice also promotes recovery of acuity. Together, these findings challenge the notion that mechanisms for OD plasticity contribute to the alterations in circuitry that restore acuity in amblyopia.

The MET receptor tyrosine kinase (RTK), implicated in risk for autism spectrum disorder (ASD) and in functional and structural circuit integrity in humans, is a temporally and spatially regulated receptor enriched in dorsal pallial-derived structures during mouse forebrain development. Here we report that loss or gain of function of METin vitroorin vivoleads to changes, opposite in nature, in dendritic complexity, spine morphogenesis, and the timing of glutamatergic synapse maturation onto hippocampus CA1 neurons. Consistent with the morphological and biochemical changes, deletion ofMetin mutant mice results in precocious maturation of excitatory synapse, as indicated by a reduction of the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of AMPA receptors at synaptic sites. Thus, MET-mediated signaling appears to serve as a mechanism for controlling the timing of neuronal growth and functional maturation. These studies suggest that mistimed maturation of glutamatergic synapses leads to the aberrant neural circuits that may be associated with ASD risk.

Human hypothalamic hamartoma (HH) is a rare subcortical lesion associated with treatment-resistant epilepsy. Cellular mechanisms responsible for epileptogenesis are unknown. We hypothesized that neuronal gap junctions contribute to epileptogenesis through synchronous activity within the neuron networks in HH tissue. We studied surgically resected HH tissue with Western-blot analysis, immunohistochemistry, electron microscopy, biocytin microinjection of recorded HH neurons, and microelectrode patch clamp recordings with and without pharmacological blockade of gap junctions. Normal human hypothalamus tissue was used as a control. Western blots showed increased expression of both connexin-36 (Cx36) and connexin-43 (Cx43) in HH tissue compared with normal human mammillary body tissue. Immunohistochemistry demonstrated that Cx36 and Cx43 are expressed in HH tissue, but Cx36 was mainly expressed within neuron clusters while Cx43 was mainly expressed outside of neuron clusters. Gap-junction profiles were observed between small HH neurons with electron microscopy. Biocytin injection into single recorded small HH neurons showed labeling of adjacent neurons, which was not observed in the presence of a neuronal gap-junction blocker, mefloquine. Microelectrode field recordings from freshly resected HH slices demonstrated spontaneous ictal/interictal-like discharges in most slices. Bath-application of gap-junction blockers significantly reduced ictal/interictal-like discharges in a concentration-dependent manner, while not affecting the action-potential firing of small gamma-aminobutyric acid (GABA) neurons observed with whole-cell patch-clamp recordings from the same patient's HH tissue. These results suggest that neuronal gap junctions between small GABAergic HH neurons participate in the genesis of epileptic-like discharges. Blockade of gap junctions may be a new therapeutic strategy for controlling seizure activity in HH patients., Highlights • Neuronal-type functional gap junctions are present in HH tissue. • Expression of neuronal-type gap junctions is more abundant in HH tissue relative to normal hypothalamus (mammillary body). • In HH slices, pharmacological block of neuronal-type gap junctions significantly reduces seizure-like discharges. This paper evaluates the role that gap junctions contribute to seizure activity utilized human hypothalamic hamartoma (HH) tissue after surgical resection. We find that 1) gap junctions are present in HH tissue, 2) gap junctions are more abundant in HH tissue relative to normal hypothalamus (mammillary body), and 3) pharmacological blockade of gap junctions in freshly-resected HH tissue slices can decrease the seizure-like discharges. These results provide evidence that gap junctions participate in the generation of seizures from HH tissue and suggest further research into the possibility that gap junction blocking medications may improve seizures in patients with HH.

The closure of developmental critical periods consolidates neural circuitry but also limits recovery from early abnormal sensory experience. Degrading vision by one eye throughout a critical period both perturbs ocular dominance (OD) in primary visual cortex and impairs visual acuity permanently. Yet understanding how binocularity and visual acuity interrelate has proven elusive. Here we demonstrate the plasticity of binocularity and acuity are separable and differentially regulated by the neuronal nogo receptor 1 (NgR1). Mice lacking NgR1 display developmental OD plasticity as adults and their visual acuity spontaneously improves after prolonged monocular deprivation. Restricting deletion of NgR1 to either cortical interneurons or a subclass of parvalbumin (PV)-positive interneurons alters intralaminar synaptic connectivity in visual cortex and prevents closure of the critical period for OD plasticity. However, loss of NgR1 in PV neurons does not rescue deficits in acuity induced by chronic visual deprivation. Thus, NgR1 functions with PV interneurons to limit plasticity of binocularity, but its expression is required more extensively within brain circuitry to limit improvement of visual acuity following chronic deprivation.

The generation of induced tissue-specific stem cells has been hampered by the lack of well-established methods for the maintenance of pure tissue-specific stem cells like the ones we have for embryonic stem (ES) cell cultures. Using a cocktail of cytokines and small molecules, we demonstrate that primitive neural stem (NS) cells derived from mouse ES cells and rat embryos can be maintained. Furthermore, using the same set of cytokines and small molecules, we show that induced NS (iNS) cells can be generated from rat fibroblasts by forced expression of the transcriptional factors Oct4, Sox2 and c-Myc. The generation and long-term maintenance of iNS cells could have wide and momentous implications.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (>3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for >3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Domoic acid (DOM) is known to cause hippocampal neuronal damage and produces amnesic effects. We examined synaptic plasticity changes induced by DOM exposure in rat hippocampal CA1 region. Brief bath application of DOM to hippocampal slices produces a chemical form of long-term potentiation (LTP) of CA1 field synaptic potentials. The potentiation cannot be blocked by NMDA receptor antagonist MK-801 but can be blocked by the calcium-calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-62 or cAMP-dependent protein kinase (PKA) inhibitor H-89. DOM-potentiated slices show decreased autophosphorylated CaMKII (p-Thr286), an effect that is also dependent on the activity of CaMKII and PKA. Increased phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit GluR1 (p-Ser831) was seen in DOM-potentiated slices. Therefore, aberrant regulation of CaMKII and GluR1 phosphorylation occurs after DOM application. In addition, tetanus-induced LTP as well as the increase of phosphorylation of CaMKII (p-Thr286) were reduced in DOM-potentiated slices. Compared with brief exposure, slices recovering from prolonged exposure did not show potentiation or altered levels of CaMKII (p-Thr286) or GluR (p-Ser831). However, decreased phosphorylation of GluR1 at Ser845 was seen. These results describe a new chemical form of LTP and uncover novel molecular changes induced by DOM. The observed impairment of tetanus LTP and misregulation of CaMKII and GluR1 phosphorylation may partially account for DOM neurotoxicity and underlie the molecular basis for DOM-induced memory deficit.

HuD is a neuronal RNA-binding protein associated with the stabilization of mRNAs for GAP-43 and other neuronal proteins that are important for nervous system development and learning and memory mechanisms. To better understand the function of this protein, we gener- ated transgenic mice expressing human HuD (HuD-Tg) in adult forebrain neurons. We have previously shown that expression of HuD in adult den- tate granule cells results in an abnormal accumulation of GAP-43 mRNA via posttranscriptional mechanisms. Here we show that this mRNA accu- mulation leads to the ectopic expression of GAP-43 protein in mossy fibers. Electrophysiological analyses of the mossy fiber to CA3 synapse of HuD-Tg mice revealed increases in paired-pulse facilitation (PPF) at short interpulse intervals and no change in long-term potentiation (LTP). Pre- synaptic calcium transients at the same synapses exhibited faster time constants of decay, suggesting a decrease in the endogenous Ca 21 buffer capacity of mossy fiber terminals of HuD-Tg mice. Under resting condi- tions, GAP-43 binds very tightly to calmodulin sequestering it and then releasing it upon PKC-dependent phosphorylation. Therefore, subsequent studies examined the extent of GAP-43 phosphorylation and its associa- tion to calmodulin. We found that despite the increased GAP-43 expres- sion in HuD-Tg mice, the levels of PKC-phosphorylated GAP-43 were decreased in these animals. Furthermore, in agreement with the increased proportion of nonphosphorylated GAP-43, HuD-Tg mice showed increased binding of calmodulin to this protein. These results suggest that a significant amount of calmodulin may be trapped in an inactive state, unable to bind free calcium, and activate downstream signaling path- ways. In conclusion, we propose that an unregulated expression of HuD disrupts mossy fiber physiology in adult mice in part by altering the expression and phosphorylation of GAP-43 and the amount of free cal- modulin available at the synaptic terminal. V C 2008 Wiley-Liss, Inc.

Reelin signaling through the low-density lipoprotein receptor family members, apoliproprotein E receptor 2 (apoER2) and very-low-density lipoprotein receptor (VLDLR), plays a pivotal role in dictating neuronal lamination during embryonic brain development. Recent evidence suggests that this signaling system also plays a role in the postnatal brain to modulate synaptic transmission, plasticity, and cognitive behavior, mostly likely due to a functional coupling with N-methyl-d-aspartate (NMDA) receptors. In this study, we investigated the effects of reelin on the maturation of CA1 glutamatergic function using electrophysiological and biochemical approaches. In cultured hippocampal slices, reelin treatment increased the amplitude of AMPAR-mediated miniature excitatory postsynaptic currents and the evoked AMPA/NMDA receptor current ratios. In addition, reelin treatment also reduced the number of silent synapses, facilitated a developmental switch from NR2B to NR2A of NMDARs, and increased surface expression of AMPARs in CA1 tissue. In cultured hippocampal neurons from reeler embryos, reduced numbers of AMPAR subunit GluR1 and NMDAR subunit NR1 clustering were observed compared with those obtained from wild-type embryos. Supplementing reelin in the reeler culture obliterated these genotypic differences. These results demonstrate that reelin- and lipoprotein receptor-mediated signaling may operate during developmental maturation of hippocampal glutamatergic function and thus represent a potential important mechanism for controlling synaptic strength and plasticity in the postnatal hippocampus.

This review briefly discusses key recent research literature on Angelman Syndrome (AS), a rare genetic disorder of neurodevelopmental origin. Dysfunction/inactivation of the maternal UBE3A gene and its surrounding chromosome regions has been identified as the causative factor for AS. The human UBE3A gene is located within human chromosome 15q11-13 and encodes an E3 ubiquitin-protein ligase (UBE3A, also called E6 associated protein, E6-AP). Due to genetic imprinting of the paternal copy of UBE3A gene in many brain regions, loss of function of a single maternal copy of UBE3A is highly penetrant and pathogenic. Most of the deficits seen in AS patients have been reproduced in Ube3a gene maternal deficiency mice (‘AS mice’, Ube3am−/p+), thus enabling mechanistic interrogations of AS pathogenesis and therapeutic explorations using mice models. Here we briefly discuss recent advances on AS etiology and identify some challenges in translating mechanistic insights into potential therapeutic interventions. Experimental evidence collected so far indicate impaired maternal UBE3A in neurons may contribute to AS deficiency by influencing multifaceted neural developmental processes including cell survival, synaptic transmission, signal transduction, gene expressions.

The developmental lamination of the hippocampus and other cortical structures requires a signaling cascade initiated by reelin and its receptors, apoER2 (apolipoprotein E receptor 2) and VLDLR (very-low-density lipoprotein receptor). However, the functional significance of continued reelin expression in the postnatal brain remains poorly understood. Here, we show that reelin application to adult mice hippocampal slices leads to enhanced glutamatergic transmission mediated by NMDA receptors (NMDARs) and AMPA receptors (AMPARs) through distinct mechanisms. Application of recombinant reelin enhanced NMDAR-mediated currents through postsynaptic mechanisms, as revealed by the variance-mean analysis of synaptic NMDAR currents, assessment of spontaneous miniature events, and the levels of NMDAR subunits at synaptic surface. In comparison, nonstationary fluctuation analysis of miniature AMPAR currents and quantification of synaptic surface proteins revealed that reelin-induced enhancement of AMPAR responses was mediated by increased AMPAR numbers. Reelin enhancement of synaptic NMDAR currents was abolished when receptor-associated protein (RAP) or the Src inhibitor 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1) was bath applied and was abrogated by including PP1 in the recording electrodes. In comparison, including RAP or an inactive PP1 analog PP3 in the recording electrode was without effect. Interestingly, the increased AMPAR response after reelin application was not blocked by PP1 but was blocked by the phosphoinositide-3′ kinase (PI3K) inhibitors wortmannin and LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride]. Furthermore, reelin-induced, PI3K-dependent AMPAR surface insertion was also observed in cultured hippocampal neurons. Together, these results reveal a differential functional coupling of reelin signaling with NMDAR and AMPAR function and define a novel mechanism for controlling synaptic strength and plasticity in the adult hippocampus.

Human hypothalamic hamartoma (HH) is a rare developmental malformation often characterized by gelastic seizures, which are refractory to medical therapy. Ictal EEG recordings from the HH have demonstrated that the epileptic source of gelastic seizures lies within the HH lesion itself. Recent advances in surgical techniques targeting HH have led to dramatic improvements in seizure control, which further supports the hypothesis that gelastic seizures originate within the HH. However, the basic cellular and molecular mechanisms of epileptogenesis in this subcortical lesion are poorly understood. Since 2003, Barrow Neurological Institute has maintained a multidisciplinary clinical program to evaluate and treat patients with HH. This program has provided a unique opportunity to investigate the basic mechanisms of epileptogenesis using surgically resected HH tissue. The first report on the electrophysiological properties of HH neurons was published in 2005. Since then, ongoing research has provided additional insights into the mechanisms by which HH generate seizure activity. In this review, we summarize this progress and propose a cellular model that suggests that GABA-mediated excitation contributes to epileptogenesis in HH lesions.

There has been a surge of diagnosis of autism spectrum disorders (ASD) over the past decade. While large, high powered genome screening studies of children with ASD have identified numerous genetic risk factors, research efforts to understanding how each of these risk factors contributes to the development autism has met with limited success. Revealing the mechanisms by which these genetic risk factors affect brain development and predispose a child to autism requires mechanistic understanding of the neurobiological changes underlying this devastating group of developmental disorders at multifaceted molecular, cellular and system levels. It has been increasingly clear that the normal trajectory of neurodevelopment is compromised in autism, in multiple domains as much as aberrant neuronal production, growth, functional maturation, patterned connectivity, and balanced excitation and inhibition of brain networks. Many autism risk factors identified in humans have been now reconstituted in experimental mouse models to allow mechanistic interrogation of the biological role of the risk gene. Studies utilizing these mouse models have revealed that underlying the enormous heterogeneity of perturbed cellular events, mechanisms directing synaptic and circuit assembly may provide a unifying explanation for the pathophysiological changes and behavioral endophenotypes seen in autism, although synaptic perturbations are far from being the only alterations relevant for ASD. In this review, we discuss synaptic and circuit abnormalities obtained from several prevalent mouse models, particularly those reflecting syndromic forms of ASD that are caused by single gene perturbations. These compiled results reveal that ASD risk genes contribute to proper signaling of the developing gene networks that maintain synaptic and circuit homeostasis, which is fundamental to normal brain development.

To the Editor: In our manuscript entitled “Single-neuron RNA-Seq: technical feasibility and reproducibility,” published online on July 6th, 2012, we have made a few serious mistakes which need to be corrected: We referenced several points and quoted statements from a recent study by Okaty et al., but we neither cited the paper nor placed quotation marks on the statements. The full citation should be “Okaty, B. W., Sugino, K., and Nelson, S. B. (2011). Cell type-specific transcriptomics in the brain. J. Neurosci. 31, 6939–6943.” We request to add a citation after “The analysis of each single-cell transcriptome consists of several independent steps” in the first paragraph of Results, as “The analysis of each single-cell transcriptome consists of several independent steps (Okray et al., 2011),” given that these points were previously raised by Okaty et al. We request to add a citation to the first paragraph of Introduction, as “studies of gene expression and function in the brain were restricted to a relatively small number of genes (Luo and Geschwind, 2001; Zhang et al., 2002; Okaty et al., 2011),” given that this points was recently reviewed in the Okaty et al., paper. More importantly, we have made a mistake in quoting sentence from Okray et al., without quotation or citation. We wish to change the following sentence: Moreover, gene expression may be regulated in opposing directions in different cell types, thereby appearing static in composite data. To Moreover, as previously discussed by Okaty et al., “gene expression may be regulated in opposing directions in different cell types, thereby appearing static in composite data (Okaty et al., 2011).” We apologize to the readers of Frontiers in Genetics and to the authors of the Okaty et al., manuscript, for our failure to correct these mistakes during the review process of our manuscript.

In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions.

Understanding brain function involves improved knowledge about how the genome specifies such a large diversity of neuronal types. Transcriptome analysis of single neurons has been previously described using gene expression microarrays. Using high-throughput transcriptome sequencing (RNA-Seq), we have developed a method to perform single-neuron RNA-Seq. Following electrophysiology recording from an individual neuron, total RNA was extracted by aspirating the cellular contents into a fine glass electrode tip. The mRNAs were reverse transcribed and amplified to construct a single neuron cDNA library, and subsequently subjected to high-throughput sequencing. This approach was applicable to both individual neurons cultured from embryonic mouse hippocampus, as well as neocortical neurons from live brain slices. We found that the average pairwise Spearman’s rank correlation coefficient of gene expression level expressed as RPKM (reads per kilobase of transcript per million mapped reads) was 0.51 between five cultured neuronal cells, whereas the same measure between three cortical layer V neurons in situ was 0.25. The data suggest that there may be greater heterogeneity of the cortical neurons, as compared to neurons in vitro. The results demonstrate the technical feasibility and reproducibility of RNA-Seq in capturing a part of the transcriptome landscape of single neurons, and confirmed that morphologically identical neurons, even from the same region, have distinct gene expression patterns.

Local hyperconnectivity in the neocortex is a hypothesized pathophysiological state in autism spectrum disorder (ASD).MET, a receptor tyrosine kinase that regulates dendrite and spine morphogenesis, has been established as a risk gene for ASD. Here, we analyzed the synaptic circuit organization of identified pyramidal neurons in the anterior frontal cortex of mice with a dorsal pallium-derived, conditional knock-out (cKO) ofMet. Synaptic mapping by glutamate uncaging identified layer 2/3 as the main source of local excitatory input to layer 5 projection neurons in controls. In both cKO and heterozygotes, this pathway was stronger by a factor of ∼2. This increase was both sublayer and projection-class specific, restricted to corticostriatal neurons in upper layer 5B and not neighboring corticopontine neurons. Paired recordings in cKO slices demonstrated increased unitary connectivity. We propose that excitatory hyperconnectivity in specific neocortical microcircuits constitutes a physiological basis for Met-mediated ASD risk.

Background The limbic system-associated membrane protein (LAMP) promotes development of neurons of limbic origin. We have previously shown that genetic deletion of LAMP results in heightened reactivity to novelty and reduced anxiety-like behaviors in mice. Here, we demonstrate a critical role of LAMP in hippocampal-dependent synaptic physiology and behavior. Methods We tested spatial memory performance, hippocampal synaptic plasticity, and stress-related modalities in Lsamp −/− mice and their littermate control mice. Results Lsamp −/− mice exhibit a pronounced deficit in spatial memory acquisition and poorly sustained CA1 long-term potentiation. We found reduced expression of mineralocorticoid receptor (MR) transcripts in the hippocampus and reduction in the corticosterone-induced, MR-mediated nongenomic modulatory effects on CA1 synaptic transmission. Importantly, the impaired long-term potentiation in Lsamp −/− mice can be rescued by stress-like levels of corticosterone in a MR-dependent manner. Conclusions Our study reveals a novel functional relationship between a cell adhesion molecule enriched in developing limbic circuits, glucocorticoid receptors, and cognitive functioning.

Genetic studies have demonstrated an important role for proprotein convertase subtilisin/kexin type 9 (PCSK9) as a determinant of plasma cholesterol levels. However, the underlying molecular mechanism is not completely understood. To this end, we have generated a mammalian cell expression system for human PCSK9 and its mutants and produced transgenic mice expressing human PCSK9. HEK293T cells transfected with the human PCSK9 DNA construct expressed and secreted PCSK9 and displayed decreased LDLR levels; functional PCSK9 protein was purified from the conditioned medium. In vitro studies showed that PCSK9 self-associated in a concentration-, temperature-, and pH-dependent manner. A mixture of PCSK9 monomers, dimers, and trimers displayed an enhanced LDLR degrading activity compared to monomeric PCSK9. A gain-of-function mutant, D374Y, displayed greatly increased self-association compared to wild-type PCSK9. Moreover, we demonstrated that the catalytic domain of PCSK9 is responsible for the self-association. Self-association of PCSK9 was enhanced by incubation with mouse apoE-/- VLDL and inhibited by incubation with both human and mouse HDL. When PCSK9 protein was incubated with total serum, it partially associated with LDL and HDL but not with VLDL. In transgenic mice, PCSK9 also associated with LDL and HDL but not with VLDL. We conclude that self-association is an intrinsic property of PCSK9, correlated to its LDLR-degrading activity and affected by plasma lipoproteins. These results provide a basis for developing strategies to manipulate PCSK9 activity in the circulation for the treatment of hypercholesterolemia.

Human apoE is a multifunctional and polymorphic protein synthesized and secreted by liver, brain, and tissue macrophages. Here we show that apoE isoforms and mutants expressed through lentiviral transduction display cell-specific differences in secretion efficiency. Whereas apoE3, apoE4, and a natural mutant of apoE4 (apoE-Cys(142)) were efficiently secreted from macrophages, apoE2 and a non-natural apoE mutant (apoE-Cys(112)/Cys(142)) were retained in the perinuclear region and only minimally secreted. The secretory block for apoE2 in macrophages was not affected by the ablation of LDLR (low density lipoprotein receptor), ABCA-1, or SR-BI (scavenger receptor class B type I) but was released in the absence of low density lipoprotein receptor related protein (LRP). In co-immunoprecipitation experiments, an anti-apoE antibody pulled down two times more LRP in apoE2-transduced macrophages than in apoE3-expressing macrophages. Non-reducing SDS-PAGE/Western blot analyses showed that macrophage apoE2 is mostly dimeric and multimeric, whereas apoE3 is predominantly monomeric. ApoE2 retention and multimer formation also occurred in human macrophages derived from the monocyte cell line THP-1. These results were specific for macrophages, as in transduced mouse primary hepatocytes: 1) ApoE2 was secreted as efficiently as apoE3 and apoE4; 2) all isoforms were exclusively in monomeric form; 3) there was no co-immunoprecipitation of apoE and LRP. A microsomal triglyceride transfer protein (MTP) inhibitor nearly deleted apoB100 secretion from hepatocytes without affecting apoE secretion. These data show that macrophages retain apoE2, a highly expressed protein carried by about 8% of the human population. Given the role of locally produced apoE in regulating cholesterol efflux, modulating inflammation, and controlling oxidative stress, this unique property of apoE2 may have important impacts on atherogenesis.

SummaryApolipoprotein E receptor 2 (Apoer2), a member of the LDL receptor gene family, and its ligand Reelin control neuronal migration during brain development. Apoer2 is also essential for induction of long-term potentiation (LTP) in the adult brain. Here we show that Apoer2 is present in the postsynaptic densities of excitatory synapses where it forms a functional complex with NMDA receptors. Reelin signaling through Apoer2 markedly enhances LTP through a mechanism that requires the presence of amino acids encoded by an exon in the intracellular domain of Apoer2. This exon is alternatively spliced in an activity-dependent manner and is required for Reelin-induced tyrosine phosphorylation of NMDA receptor subunits. Mice constitutively lacking the exon perform poorly in learning and memory tasks. Thus, alternative splicing of Apoer2, a novel component of the NMDA receptor complex, controls the modulation of NMDA receptor activity, synaptic neurotransmission, and memory by Reelin.

6q23.3 (AHI1*) Jouberin; interacts with b-catenin in cilia Joubert syndrome. (Reduced brain and body size. Cerebellar, retinal, and kidney defects. Most die by P10. Neuron-specific loss leads to depressive-like phenotypes.)7q35-q36.1 (CNTNAP2*)Caspr2, a neurexin family member; clusters voltage-gated K+ channels Recessive EPI syndrome, ASD, ADHD, TS, OCD. (Neuronal migration defects. Reduced GABAergic neurons and decreased cortical synchrony. Seizures. Deficits in social, repetitive behaviors, and USV.) 9q34.13 (TSC1) Hamartin, a growth inhibitory protein that negatively regulates the mTOR pathwayTuberous Sclerosis type I. (Liver and neural tube defects. Die by E12. Abnormal kidney and liver growth in heterozygotes. Variable brain structure, function and behavior abnormalities in conditional mutants. Die at various postnatal ages. Neuron-specific loss causes abnormal spine morphology and cortical excitability. Loss of LTD.)10q23.31 (PTEN) Protein tyrosine phosphatase; negatively regulates the mTOR pathwayCowden disease. (Placenta and germ cell defects. Die by E9.5. Neuron-specific loss alters synaptic physiology. Heterozygotes have prostate, skin and colon defects, and spontaneous tumors. Macrocephaly, neuronal hypertrophy, abnormal social interaction, and increased survival in conditional mutants.)11q13.4 (DHCR7) Final enzyme in cholesterol biosynthetic pathwaySmith-Lemli-Opitz syndrome. (Craniofacial and lung abnormalities. Die by P1. Abnormal cholesterol regulation and enlarged bladders. Hypomorphic mutants are viable and fertile. Compound mutants have fused toes, enlarged ventricles, and 25% embryonic lethality.) 12p13.33 (CACNA1C)a-1 subunit of a voltage-dependent Ca2+ channelTimothy syndrome. (Die embryonically. Impaired pancreatic function. Motor defects and antidepressant-like behavior in heterozygotes; anxiety-like deficits in females. Neuron-specific loss, impaired cognition and LTP.) 15q11.2 (UBE3A) Ubiquitination ligase; targets protein degradation systemAngelman syndrome. (Small brain, seizure susceptibility, motor and learning deficits. Reduced spine density and impaired LTP. Impaired synapse maturation and plasticity.) 16p13.3 (TSC2) Tuberin; which negatively regulates the mTOR pathwayTuberous Sclerosis type II. (Heart, neural tube, and motor defects. Purkinje cell death. Die by E12. Various tumors and axon guidance defects in heterozygotes. Dominant-negative mutant has enhanced anxiety-like behaviors; motor, learning, social behavior deficits.) 17q11.2 (NF1) Neurofibromin; a GTPase activator and negative regulator of RAS signaling Neurofibromatosis. (Macrocephaly, small eyes, and heart defects. Delayed organ development. Embryonic lethal. Increased astrocytes and tumor susceptibility. LTP and learning and memory deficits in heterozygotes.) Xp21.2 (DMD) Dystrophin; cytoskeletal protein bridging ECMDuchenne muscular dystrophy. (Muscle and heart defects in hemizygous males and homozygous females. Reduced fertility. Abnormal retinal electrophysiology and synapse organization, density, and maturation.)Xp21.3 (ARX) Aristaless-related homeobox protein TFLIS, XLID, EPI, ASD. (Hemizygous males die perinatally. Decreased inhibitory synaptic transmission. Males hemizygous for point mutations or triple repeat expansions have seizures. Defects in behavior and GABAergic neuron generation and migration.)Xq27.3 (FMR1) Fragile X mental retardation protein; an RNA-binding protein that traffics mRNA Fragile X syndrome. (Seizures. Enlarged testes in males. Learning and social behavior defects. Dendritic spine abnormalities. Enhanced LTD and impaired LTP. Altered cortical drive and E/I neuronal cortical networks.) Xq28 (MECP2) MeCP2; involved in transcriptional regulation and chromatin organizationRett syndrome. (Brain, breathing, and motor defects in hemizygous males. Mild cognitive and anxiety-like phenotypes in heterozygous females. Various conditional loss and postnatal reduction mimic null phenotypes in adult hemizygous males. Impaired excitatory synapses and spine morphology. Increased neuronal connectivity.)