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Dietary restriction (DR) and rapamycin extend healthspan and life span across multiple species. We have recently shown that DR in progeroid DNA repair-deficient mice dramatically extended healthspan and trippled life span. Here, we show that rapamycin, while significantly lowering mTOR signaling, failed to improve life span nor healthspan of DNA repair-deficient Ercc1∆/− mice, contrary to DR tested in parallel. Rapamycin interventions focusing on dosage, gender, and timing all were unable to alter life span. Even genetically modifying mTOR signaling failed to increase life span of DNA repair-deficient mice. The absence of effects by rapamycin on P53 in brain and transcription stress in liver is in sharp contrast with results obtained by DR, and appoints reducing DNA damage and transcription stress as an important mode of action of DR, lacking by rapamycin. Together, this indicates that mTOR inhibition does not mediate the beneficial effects of DR in progeroid mice, revealing that DR and rapamycin strongly differ in their modes of action.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function.

Half a century ago, Harry Angelman reported three patients with overlapping clinical features, now well known as Angelman syndrome. Angelman syndrome is caused by mutations affecting the maternally inherited UBE3A gene, which encodes an E3-ubiquitin ligase that is critical for typical postnatal brain development. Emerging evidence indicates that UBE3A plays a particularly important role in the nucleus. However, the critical substrates that are controlled by UBE3A remain elusive, which hinders the search for effective treatments. Moreover, given the multitude of signalling mechanisms that are derailed, it is unlikely that targeting a single pathway is going to be very effective. Therefore, expectations are very high for approaches that aim to restore UBE3A protein levels. A particular promising strategy is an antisense oligonucleotide approach, which activates the silenced paternal UBE3A gene. When successful, such treatments potentially offer a disease-modifying therapy for Angelman syndrome and several other neurodevelopmental disorders.

Descriptions of the clinical features of Angelman syndrome (AS) have mainly been focused on children. Here, we describe t

The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This singe amino-acid deletion is shared with apes and Old World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This hUBE3A-Iso2 isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a si

Objective: Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder that is associated with cognitive disabilities. Based on studies involving animals, the hypothesized cause of these disabilities results from increased activity of inhibitory interneurons that decreases synaptic plasticity. We obtained transcranial magnetic stimulation (TMS)-based measures of cortical

Background: Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of functional ubiquitin protein ligase E3A (UBE3A). In neurons, UBE3A expression is tightly regulated by a mechanism of imprinting which suppresses the expression of the paternal UBE3A allele. Promising treatment strategies for AS are directed at activating paternal UBE3A gene expression. However, for such strategies to be successful, it is important to know when such a treatment should start, and how much UBE3A expression is needed for normal embryonic brain development. Methods: Using a conditional mouse model of AS, we further delineated the critical period for UBE3A expression during early brain development. Ube3a gene expression was induced around the second week of gestation and mouse phenotypes were assessed using a behavioral test battery. To investigate the requirements of embryonic UBE3A expression, we made use of mice in which the paternal Ube3a allele was deleted. Results: We observed a full behavioral rescue of the AS mouse model phenotypes when Ube3a gene reactivation was induced around the start of the last week of mouse embryonic development. We found that full silencing of the paternal Ube3a allele was not completed till the first week after birth but that deletion of the paternal Ube3a allele had no significant effect on the assessed phenotypes. Limitations: Direct translation to human is limited, as we do not precisely know how human and mouse brain development aligns over gestational time. Moreover, many of the assessed phenotypes have limited translational value, as the underlying brain regions involved in these tasks are largely unknown. Conclusions: Our findings provide further important insights in the requirement of UBE3A expression during brain development. We found that loss of

Background: Loss of functional UBE3A, an E3 protein ubiquitin ligase, causes Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, speech impairment, epilepsy, movement or balance disorder, and a characteristic behavioral pattern. We identified a novel UBE3A sequence variant in a large family with eight affected individuals, who did not meet the clinical AS criteria. Methods: Detailed clinical examination and genetic analysis was performed to establish the phenotypic diversity and the genetic cause. The function of the mutant UBE3A protein was assessed with respect to its subcellular localization, stability, and E3 ubiquitin ligase activity. Results: All eight affected individuals showed the presence of a novel maternally inherited UBE3A sequence variant (NM_130838.4(UBE3A):c.1018-1020del, p.(Asn340del), which is in line with a genetic AS diagnosis. Although they presented with moderate to severe intellectual disability, the phenotype did not match the clinical criteria for AS. In line with this, functional analysis of the UBE3A p.Asn340del mutant protein revealed no major deficits in UBE3A protein localization, stability, or E3 ubiquitin ligase activity. Conclusion: The p.(Asn340del) mutant protein behaves distinctly different from previously described AS-linked missense mutations in UBE3A, and causes a phenotype that is markedly different from AS. This study further extends the range of phenotypes that are associated with UBE3A loss, duplication, or mutation.

The UBE3A gene is part of the chromosome 15q11-q13 region that is frequently deleted or duplicated, leading to several neurodevelopmental disorders (NDD). Angelman syndrome (AS) is caused by the absence of functional maternally derived UBE3A protein, while the paternal UBE3A gene is present but silenced specifically in neurons. Patients with AS present with severe neurodevelopmental delay, with pronounced motor deficits, absence of speech, intellectual disability, epilepsy, and sleep problems. The pathophysiology of AS is still unclear and a treatment is lacking. Animal models of AS recapitulate the genotypic and phenotypic features observed in AS patients, and have been invaluable for understanding the disease process as well as identifying apropriate drug targets. Using these AS mouse models we have learned that loss of UBE3A probably affects many areas of the brain, leading to increased neuronal excitability and a loss of synaptic spines, along with changes in a number of distinct behaviours. Inducible AS mouse models have helped to identify the critical treatment windows for the behavioral and physiological phenotypes. Additionally, AS mouse models indicate an important role for the predominantly nuclear UBE3A isoform in generating the characteristic AS pathology. Last, but not least, the AS mice have been crucial in guiding Ube3a gene reactivation treatments, which present a very promising therapy to treat AS.

Purpose: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder associated with cognitive deficits. The NF1 cognitive phenotype is generally considered to be highly variable, possibly due to the observed T2-weighted hyperintensities, loss of heterozygosity, NF1-specific genetic modifiers, or allelic imbalance. Methods: We investigated cognitive variability and assessed the contribution of genetic factors by performing a retrospective cohort study and a monozygotic twin case series. We included data of 497 children with genetically confirmed NF1 and an IQ assessment, including 12 monozygotic twin and 17 sibling sets. Results: Individuals carrying an NF1 chromosomal microdeletion showed significant lower full-scale IQ (FSIQ) scores than individuals carrying intragenic pathogenic NF1 variants. For the intragenic subgroup, the variability in cognitive ability and the correlation of IQ between monozygotic NF1 twin pairs or between NF1 siblings is similar to the general population. Conclusions: The variance and heritability of IQ in individuals with NF1 are similar to that of the general population, and hence mostly driven by genetic background differences. The only factor that significantly attenuates IQ in NF1 individuals is the NF1 chromosomal microdeletion genotype. Implications for clinical management are that individuals with intragenic NF1 variants that score <1.5–2 SD below the mean of the NF1 population should be screened for additional causes of cognitive disability.

Ca2+/calmodulin-dependent protein kinase II (CAMK2) is a key player in synaptic plasticity and memory formation. Mutations in Camk2a or Camk2b cause intellectual disability in humans, and severe plasticity and learning deficits in mice, indicating unique functions for each isoform. However, considering the high homology between CAMK2A and CAMK2B, it is conceivable that for critical functions, one isoform compensates for the absence of the other, and that the full functional spectrum of neuronal CAMK2 remains to be revealed.Here we show that germline as well as adult deletion of both CAMK2 isoforms in male or female mice is lethal. Moreover, Ca2+-dependent activity as well as autonomous activity of CAMK2 is essential for survival. Loss of both CAMK2 isoforms abolished LTP, whereas synaptic transmission remained intact. The double-mutants showed no gross morphological changes of the brain, and in contrast to the long-considered role for CAMK2 in the structural organization of the postsynaptic density (PSD), deletion of both CAMK2 isoforms did not affect the biochemical composition of the PSD. Together, these results reveal an essential role for CAMK2 signaling in early postnatal development as well as the mature brain, and indicate that the full spectrum of CAMK2 requirements cannot be revealed in the single mutants because of partial overlapping

Ca2+/calmodulin-dependent protein kinase II (CAMK2) is a key player in synaptic plasticity and memory formation. Mutations in Camk2a or Camk2b cause intellectual disability in humans, and severe plasticity and learning deficits in mice, indicating unique functions for each isoform. However, considering the high homology between CAMK2A and CAMK2B, it is conceivable that for critical functions, one isoform compensates for the absence of the other, and that the full functional spectrum of neuronal CAMK2 remains to be revealed. Here we show that germline as well as adult deletion of both CAMK2 isoforms in male or female mice is lethal. Moreover, Ca2+-dependent activity as well as autonomous activity of CAMK2 is essential for survival. Loss of both CAMK2 isoforms abolished LTP, whereas synaptic transmission remained intact. The double-mutants showed no gross morphological changes of the brain, and in contrast to the long-considered role for CAMK2 in the structural organization of the postsynaptic density (PSD), deletion of both CAMK2 isoforms did not affect the biochemical composition of the PSD. Together, these results reveal an essential role for CAMK2 signaling in early postnatal development as well as the mature brain, and indicate that the full spectrum of CAMK2 requirements cannot be revealed in the single mutants because of partial overlapping functions of CAMK2A and CAMK2B. SIGNIFICANCE STATEMENT CAMK2A and CAMK2B have been studied for over 30 years for their role in neuronal functioning. However, most studies were performed using single knock-out mice. Because the two isoforms show high homology with respect to structure and function, it is likely that some redundancy exists between the two isoforms, meaning that for critical functions CAMK2B compensates for the absence of CAMK2A and vice versa, leaving these functions to uncover. In this study, we generated Camk2a/Camk2b double-mutant mice, and observed that loss of CAMK2, as well as the loss of Ca2+-dependent

This study presents a broad overview of health issues and psychomotor development of 100 children with Angelman syndrome (AS), seen at the ENCORE Expertise Center for AS in Rotterdam, the Netherlands. We aimed to further delineate the phenotype of AS, to evaluate the association of the phenotype with genotype and other determinants such as epilepsy and to get insight in possible targets for intervention. We confirmed the presence of a more severe phenotype in the 15q11.2-q13 deletion subtype. Novel findings were an association of (early onset of) epilepsy with a negative effect on development, a high occurrence of nonconvulsive status epilepticus, a high rate of crouch gait in the older children with risk of deterioration of mobility, a relatively low occurrence of microcephaly, a higher mean weight for height in all genetic subtypes with a significant higher mean in the nondeletion children, and a high occurrence of hyperphagia across all genetic subtypes. Natural history data are needed to design future trials. With this large clinical cohort with structured prospective and multidisciplinary follow-up, we provide unbiased data on AS to support further intervention studies to optimize outcome and quality of life of children with AS and their family.

Background: Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutations affecting UBE3A gene expression. Previous studies in mice revealed distinct critical periods during neurodevelopment in which reactivation of Ube3a gene expression can prevent the onset of behavioral deficits. Whether UBE3A is required for brain function throughout life is unknown. Here, we address the importance of maintaining UBE3A expression after normal brain development. Findings: Using a conditional mouse, we deleted the Ube3a gene at three ages spanning brain maturation. We assessed the consequences of Ube3a gene deletion by testing the mice in behavioral tasks previously shown to produce robust phenotypes in AS model mice. Early embryonic deletion of Ube3a recapitulated all behavioral deficits of AS mice. In contrast, Ube3a gene deletion at 3 or 12 weeks of age did not have a significant effect on most behavioral tasks and did not increase seizure sensitivity. Conclusions: Taken together, these results emphasize that UBE3A critically impacts early brain development, but plays a more limited role in adulthood. Our findings provide important considerations for upcoming clinical trials in which UBE3A gene expression is reactivated and suggest that even transient UBE3A reinstatement during a critical window of early development is likely to prevent most adverse Angelman syndrome phenotypes. However, sustained UBE3A expression into adulthood is probably needed for optimal clinical benefit.

Objective: An epilepsy mouse model for Tuberous Sclerosis Complex (TSC) was developed and validated to investigate the mechanisms underlying epileptogenesis. Furthermore, the possible antiepileptogenic properties of commonly used antiepileptic drugs (AEDs) and new compounds were assessed. Methods: Tsc1 deletion was induced in CAMK2A-expressing neurons of adult mice. The antiepileptogenic properties of commonly used AEDs and inhibitors of the mTOR pathways were assessed by EEG recordings and by molecular read outs. Results: Mice developed epilepsy in a narrow ti

The abundantly expressed calcium/calmodulin-dependent protein kinase II (CAMK2), alpha (CAMK2A), and beta (CAMK2B) isoforms are essential for learning and memory formation. Recently, a de novo candidate mutation (p.Arg292Pro) in the gamma isoform of CAMK2 (CAMK2G) was identified in a patient with severe intellectual disability (ID), but the mechanism(s) by which this mutation causes ID is unknown. Here, we identified a second, unrelated individual, with a de novo CAMK2G p.Arg292Pro mutation, and used in vivo and in vitro assays to assess the impact of this mutation on CAMK2G and neuronal function. We found that knockdown of CAMK2G results in inappropriate precocious neuronal maturation. We further found that the CAMK2G p.Arg292Pro mutation acts as a highly pathogenic gain-of-function mutation, leading to increased phosphotransferase activity and impaired neuronal maturation as well as impaired targeting of the nuclear CAMK2G isoform. Silencing the catalytic site of the CAMK2G p.Arg292Pro protein reversed the pathogenic effect of the p.Arg292Pro mutation on neuronal maturation, without rescuing its nuclear targeting. Taken together, our results reveal an indispensable function of CAMK2G in neurodevelopment and indicate that the CAMK2G p.Arg292Pro protein acts as a pathogenic gain-of-function mutation, through constitutive activity toward cytosolic targets, rather than impaired targeting to the nucleus.

Background: Angelman syndrome (AS) is a neurodevelopmental disorder caused by mutations affecting UBE3A function. AS is characterized by intellectual disability, impaired motor coordination, epilepsy, and behavioral abnormalities including autism spectrum disorder features. The development of treatments for AS heavily relies on the ability to test the efficacy of drugs in mouse models that show reliable, and preferably clinically relevant, phenotypes. We previously described a number of behavioral paradigms that assess phenotypes in the domains of motor performance, repetitive behavior, anxiety, and seizure susceptibility. Here, we set out to evaluate the robustness of these phenotypes when tested in a standardized test battery. We then used this behavioral test battery to assess the efficacy of minocycline and levodopa, which were recently tested in clinical trials of AS. Methods: We combined data of eight independent experiments involving 111 Ube3a mice and 120 wild-type littermate control mice. Using a meta-analysis, we determined the statistical power of the subtests and the effect of putative confounding factors, such as the effect of sex and of animal weight on rotarod performance. We further assessed the robustness of these phenotypes by comparing Ube3a mutants in different genetic backgrounds and by comparing the behavioral phenotypes of independently derived Ube3a-mutant lines. In addition, we investigated if the test battery allowed re-testing the same animals, which would allow a within-subject testing design. Results: We find that the test battery is robust across different Ube3a-mutant lines, but confirm and extend earlier studies that several phenotypes are very sensitive to genetic background. We further found that the audiogenic seizure susceptibility phenotype is fully reversible upon pharmacological treatment and highly suitable for dose-finding studies. In agreement with the clinical trial results, we found that minocycline and levodopa treatment

The neurodevelopmental disorder Angelman syndrome (AS) is characterized by intellectual disability, motor dysfunction, distinct behavioral aspects, and epilepsy. AS is caused by a loss of the maternally expressed UBE3A gene, and many of the symptoms are recapitulated in a Ube3a mouse model of this syndrome. At the cellular level, changes in the axon initial segment (AIS) have been reported, and changes in vesicle cycling have indicated the presence of presynaptic deficits. Here we studied the role of UBE3A in the auditory system by recording synaptic transmission at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) through in vivo whole cell and juxtacellular recordings. We show that MNTB principal neurons in Ube3a mice exhibit a hyperpolarized resting membrane potential, an increased action potential (AP) amplitude and a decreased AP half width. Moreover, both the pre- and postsynaptic AP in the calyx of Held synapse of Ube3a mice showed significantly faster recovery from spike depression. An increase in AIS length was observed in the principal MNTB neurons of Ube3a mice, providing a possible substrate for these gain-of-function changes. Apart from the effect on APs, we also observed that EPSPs showed decreased short-term synaptic depression (STD) during long sound stimulations in AS mice, and faster recovery from STD following these tones, which is suggestive of a presynaptic gain-of-function. Our findings thus provide in vivo evidence that UBE3A plays a critical role in controlling synaptic transmission and excitability at excitatory synapses.

In a non-selected sample of children with Neurofibromatosis type 1 (NF1) the prevalence rate of autism spectrum disorder (ASD) and predictive value of an observational (ADOS)—and questionnaire-based screening instrument were assessed. Complete data was available for 128 children. The prevalence rate for clinical ASD was 10.9%, which is clearly higher than in the general population. This prevalence rate is presumably more accurate than in previous studies that examined children with NF1 with an ASD presumption or solely based on screening instruments. The combined observational- and screening based classifications demonstrated the highest positive predictive value for DSM-IV diagnosis, highlighting the importance of using both instruments in children with NF1.

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10−5 and c.2702T > G [p.V901G], MAF 2.51 × 10−3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05–13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10−8), viability (P = 8.9 × 10−7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10−4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10−5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

RASopathies, characterized by germline mutations in genes encoding proteins of the RAS-ERK signaling pathway, show overlapping phenotypes, which manifest themselves with a varying severity of intellectual disability. However, it is unclear to what extent they share the same downstream pathophysiology that underlies the cognitive deficits. Costello syndrome (CS) is a rare RASopathy caused by activating mutations in the HRAS gene. Here we investigated the mechanisms underlying the cognitive deficits of HRas G12V/G12V mice. HRas G12V/G12V mice showed robust upregulation of ERK signaling, neuronal hypertrophy, increased brain volume, spatial learning deficits, and impaired mGluR-dependent long-term depression (LTD). In contrast, long-term potentiation (LTP), which is affected in other RASopathy mouse models was unaffected. Treatment with lovastatin, a HMG-CoA-Reductase inhibitor which has been shown to rescue the behavioral phenotypes of mouse models of NF1 and Noonan syndrome, was unable to restore ERK signaling and the cognitive deficits of HRas G12V/G12V mice. Administration of a potent mitogen-activated protein kinase (MEK) inhibitor rescued the ERK upregulation and the mGluR-LTD deficit of HRas G12V/G12V mice, but failed to rescue the cognitive deficits. Taken together, this study indicates that the fundamental molecular and cellular mechanisms underlying the cognitive aspects of different RASopathies are remarkably distinct, and may require disease specific treatments.

De novo mutations in specific mTOR pathway genes cause brain overgrowth in the context of intellectual disability (ID). By analyzing 101 mMTOR-related genes in a large ID patient cohort and two independent population cohorts, we show that these genes modulate brain growth in health and disease. We report the mTOR activator gene RHEB as an ID gene that is associated with megalencephaly when mutated. Functional testing of mutant RHEB in vertebrate animal models indicates pathway hyperactivation with a concomitant increase in cell and head size, aberrant neuronal migration, and induction of seizures, concordant with the human phenotype. This study reveals that tight control of brain volume is exerted through a large community of mTOR-related genes. Human brain volume can be altered, by either rare disruptive events causing hyperactivation of the pathway, or through the collective effects of common alleles.

__Background:__ Tuberous sclerosis complex (TSC) is a rare monogenic disorder characterized by benign tumors in multiple organs as well as a high prevalence of epilepsy, intellectual disability and autism. TSC is caused by inactivating mutations in the TSC1 or TSC2 genes. Heterozygocity induces hyperactivation of mTOR which can be inhibited by mTOR inhibitors, such as rapamycin, which have proven efficacy in the treatment of TSC-associated symptoms. The aim of the present study was (1) to identify molecular changes associated with social and cognitive deficits in the brain tissue of Tsc1 +/- mice and (2) to investigate the molecular effects of rapamycin treatment, which has been shown to ameliorate genotype-related behavioural deficits. __Methods:__ Molecular alterations in the frontal cortex and hippocampus of Tsc1 +/- and control mice, with or without rapamycin treatment, were investigated. A quantitative mass spectrometry-based shotgun proteomic approach (LC-MSE) was employed as an unbiased method to detect changes in protein levels. Changes identified in the initial profiling stage were validated using selected reaction monitoring (SRM). Protein Set Enrichment Analysis was employed to identify dysregulated pathways. __Results:__ LC-MSE analysis of Tsc1 +/- mice and controls (n = 30) identified 51 proteins changed in frontal cortex and 108 in the hippocampus. Bioinformatic analysis combined with targeted proteomic validation revealed several dysregulated molecular pathways. Using targeted assays, proteomic alterations in the hippocampus validated the pathways "myelination", "dendrite," and "oxidative stress", an upregulation of ribosomal proteins and the mTOR kinase. LC-MSE analysis was also employed on Tsc1 +/- and wildtype mice (n = 34) treated with rapamycin or vehicle. Rapamycin treatment exerted a stronger proteomic effect in Tsc1 +/- mice with significant changes (mainly decreased expression) in 231 and 106 proteins, respectively. The cellular pathways "oxi

Memories are encoded within sparsely distributed neuronal ensembles. However, the defining cellular properties of neurons within a memory trace remain incompletely understood. Using a fluorescence-based Arc reporter, we were able to visually identify the distinct subset of lateral amygdala (LA) neurons activated during auditory fear conditioning. We found that Arc-expressing neurons have enhanced intrinsic excitability and are preferentially recruited into newly encoded memory traces. Furthermore, synaptic potentiation of thalamic inputs to the LA during fear conditioning is learning-specific, postsynaptically mediated and highly localized to Arc-expressing neurons. Taken together, our findings validate the immediate-early gene Arc as a molecular marker for the LA neuronal ensemble recruited during fear learning. Moreover, these results establish a model of fear memory formation in which intrinsic excitability determines neuronal selection, whereas learning-related encoding is governed by synaptic plasticity.

Objective: To evaluate the appropriateness of cognitive and behavioral outcome measures in clinical trials in neurofibromatosis type 1 (NF1) by analyzing the degree of deficits compared to reference groups, test-retest reliability, and how scores correlate between outcome measures. Methods: Data were analyzed from the Simvastatin for cognitive deficits and behavioral problems in patients with neurofibromatosis type 1 (NF1-SIMCODA) trial, a randomized placebo-controlled trial of simvastatin for cognitive deficits and behavioral problems in children with NF1. Outcome measures were compared with age-specific reference groups to identify domains of dysfunction. Pearson r was computed for before and after measurements within the placebo group to assess test-retest reliability. Principal component analysis was used to identify the internal structure in the outcome data. Results: Strongest mean score deviations from the reference groups were observed for full-scale intelligence (-1.1 SD), Rey Complex Figure Test delayed recall (-2.0 SD), attention problems (-1.2 SD), and social problems (-1.1 SD). Long-term test-retest reliability were excellent for Wechsler scales (r > 0.88), but poor to moderate for other neuropsychological tests (r range 0.52-0.81) and Child Behavioral Checklist subscales (r range 0.40-0.79). The correlation structure revealed 2 strong components in the outcome measures behavior and cognition, with no correlation between these components. Scores on psychosocial quality of life correlate strongly with behavioral problems and less with cognitive deficits. Conclusions: Children with NF1 show distinct deficits in multiple domains. Many outcome measures showed weak test-retest correlations over the 1-year trial period. Cognitive and behavioral outcomes are complementary. This analysis demonstrates the need to include reliable outcome measures on a variety of cognitive and behavioral domains in clinical trials for NF1.

Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2-/- mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2-/- MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC.

Defects in the rat sarcoma viral oncogene homolog (Ras)/extracellular-signal-regulated kinase and the phosphatidylinositol 3-kinase-mammalian target of rapamycin (mTOR) signaling pathways are responsible for several neurodevelopmental disorders. These disorders are an important cause for intellectual disability; additional manifestations include autism spectrum disorder, seizures, and brain malformations. Changes in synaptic function are thought to underlie the neurological conditions associated with these syndromes. We therefore studied morphology and in vivo synaptic transmission of the calyx of Held synapse, a relay synapse in the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem, in mouse models of tuberous sclerosis complex (TSC), Fragile X syndrome (FXS), Neurofibromatosis type 1 (NF1), and Costello syndrome. Calyces from both Tsc1+1- and from Fmr1 knock-out (KO) mice showed increased volume and surface area compared to wild-type (WT) controls. In addition, in Fmr1 KO animals a larger fraction of calyces showed complex morphology. In MNTB principal neurons of Nf1+/- mice the average delay between EPSPs and APs was slightly smaller compared to WT controls, which could indicate an increased excitability. Otherwise, no obvious changes in synaptic transmission, or short-term plasticity were observed during juxtacellular recordings in any of the four lines. Our results in these four mutants thus indicate that abnormalities of mTOR or Ras signaling do not necessarily result in changes in in vivo synaptic transmission.

In addition to its well established role in motor coordination, the cerebellum has been hypothesized to be involved in the control of cognitive and emotional functions. Although a cerebellar contribution to nonmotor functions has been supported by recent studies in human and monkey, it remains to be clarified with an in-depth, systematic approach in mouse mutants. Here we subjected four different cerebellar cell-specific mouse lines whereby the excitatory or inhibitory input to Purkinje cells (PCs) and/or PC postsynaptic plasticity were compromised, to a wide battery of standard cognitive and emotional tests. The four lines, which have all been shown to suffer from impaired motor learning without being ataxic, were tested for social behavior using a sociability task, for spatial navigation using the Morris watermaze, for fear responses using contextual and cued conditioning, and general anxiety using the open-field task. None of the four cerebellum-specific genetic perturbations showed significantly impaired cognitive or emotional behavior. In fact, even without correction for multiple comparisons, only 5 of 154 statistical comparisons showed a marginally significant deficit. Therefore, our data indicate that none of the perturbations of cerebellar functioning studied here affected the cognitive or emotional tests we used. This suggests that there may be a differentia

The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.

Brain development in neurodevelopmental disorders has been considered to comprise a sequence of critical periods, and abnormalities occurring during early development have been considered irreversible in adulthood. However, findings in mouse models of neurodevelopmental disorders, including fragile X, Rett syndrome, Down syndrome, and neurofibromatosis type I suggest that it is possible to reverse certain molecular, electrophysiological, and behavioral deficits associated with these disorders in adults by genetic or pharmacological manipulations. Furthermore, recent studies have suggested that critical period-like plasticity can be reactivated in the adult brain by environmental manipulations or by pharmacotherapy. These studies open up a tantalizing possibility that targeted pharmacological treatments in combination with regimes of training or rehabilitation might alleviate or reverse the symptoms of neurodevelopmental disorders even after the end of critical developmental periods. Even though translation from animal experimentation to clinical practice is challenging, these results suggest a rational basis for treatment of neurodevelopmental disorders in adulthood.

Background: Recent studies have suggested that deregulated AKT1 signaling is associated with schizophrenia. We hypothesized that if this is indeed the case, we should observe both decreased AKT1 expression as well as deregulation of AKT1 regulated pathways in Peripheral Blood M

22q11 Deletion Syndrome (22q11DS) is associated with dysmorphology and a high prevalence of schizophrenia-like symptoms. Several genes located on chromosome 22q11 have been linked to schizophrenia. The deletion is thought to disrupt the expression of multiple genes involved in maturation and development of neurons and neuronal circuits, and neurotransmission. We investigated whole-genome gene expression of Peripheral Blood Mononuclear Cells (PBMC's) of 8 22q11DS patients and 8 age- and gender-matched controls, to (1) investigate the expression levels of 22q11 genes and (2) to investigate whether 22q11 genes participate in functional genetic networks relevant to schizophrenia. Functional relationships between genes differentially expressed in patients (as identified by Locally Adaptive Statistical procedure (LAP) or satisfying p<0.05 and fold-change >1.5) were investigated with the Ingenuity Pathways Analysis (IPA). 14 samples (7 patients, 7 controls) passed quality controls. LAP identified 29 deregulated genes. Pathway analysis showed 262 transcripts differentially expressed between patients and controls. Functional pathways most disturbed were cell death, cell morphology, cellular assembly and organization, and cell-to-cell signaling. In addition, 10 canonical pathways were identified, among which the signal pathways for Natural Killer-cells, neurotrophin/Trk, neuregulin, axonal guidance, and Huntington's disease. Our findings support the use of 22q11DS as a research model for schizophrenia. We identified decreased expression of several genes (among which COMT, Ufd1L, PCQAP, and GNB1L) previously linked to schizophrenia as well as involvement of signaling pathways relevant to schizophrenia, of which Neurotrophin/Trk and neuregulin signaling seems to be especially notable.

The aim of this study was to quantify the frequently observed problems in motor control in Neurofibromatosis type 1 (NF1) using three tasks on motor performance and motor learning. A group of 70 children with NF1 was compared to age-matched controls. As expected, NF1 children showed substantial problems in visuo-motor integration (Beery VMI). Prism-induced hand movement adaptation seemed to be mildly affected. However, no significant impairments in the accuracy of simple eye or hand movements were observed. Also, saccadic eye movement adaptation, a cerebellum dependent task, appeared normal. These results suggest that the motor problems of children with NF1 in daily life are unlikely to originate solely from impairments in motor learning. Our findings, therefore, do not support a general dysfunction of the cerebellum in children with NF1.

Ras homolog enriched in brain (Rheb) couples growth factor signaling to activation of the target of rapamycin complex 1 (TORC1). To study its role in mammals, we generated a Rheb knockout mouse. In contrast to mTOR or regulatory-associated protein of mTOR (Raptor) mutants, the inner cell mass of Rheb-/-embryos differentiated normally. Nevertheless, Rheb-/-embryos died around midgestation, most likely due to impaired development of the cardiovascular system. Rheb-/-embryonic fibroblasts showed decreased TORC1 activity, were smaller, and showed impaired proliferation. Rheb heterozygosity extended the life span of tuberous sclerosis complex 1-deficient (Tsc1-/-) embryos, indicating that there is a genetic interaction between the Tsc1 and Rheb genes in mouse.

Cerebellar motor learning is required to obtain procedural skills. Studies have provided supportive evidence for a potential role of kinase-mediated long-term depression (LTD) at the parallel fiber to Purkinje cell synapse in cerebellar learning. Recently, phosphatases have been implicated in the induction of potentiation of Purkinje cell activities in vitro, but it remains to be shown whether and how phosphatase-mediated potentiation contributes to motor learning. Here, we investigated its possible role by creating and testing a Purkinje cell-specific knockout of calcium/calmodulin-activated protein-phosphatase-2B (L7-PP2B). The selective deletion of PP2B indeed abolished postsynaptic long-term potentiation in Purkinje cells and their ability to increase their excitability, whereas LTD was unaffected. The mutants showed impaired "gain-decrease" and "gain-increase" adaptation of their vestibulo-ocular reflex (VOR) as well as impaired acquisition of classical delay conditioning of their eyeblink response. Thus, our data indicate that PP2B may indeed mediate potentiation in Purkinje cells and contribute prominently to cerebellar motor learning.

Synaptic gain control and information storage in neural networks are mediated by alterations in synaptic transmission, such as in long-term potentiation (LTP). Here,weshowusingboth in vitroandin vivo recordingsfromthe rat cerebellum that tetanization protocols for the induction of LTP at parallel fiber (PF)-to-Purkinje cell synapsescanalsoevokeincreases in intrinsic excitability. Thisformof intrinsic plasticity shares with LTP a requirement for the activation of protein phosphatases 1, 2A, and 2B for induction. Purkinje cell intrinsic plasticity resembles CA1 hippocampal pyramidal cell intrinsic plasticity in that it requires activity of protein kinase A(PKA) and casein kinase 2 (CK2) and is mediated by a downregulation of SK-type calcium-sensitive K conductances. In addition, Purkinje cell intrinsic plasticity similarly results in enhanced spine calcium signaling. However, there are fundamental differences: first, while in the hippocampus increases in excitability result in a higher probability for LTP induction, intrinsic plasticity in Purkinje

We uncovered a role for ERK signaling in GABA release, long-term potentiation (LTP), and learning, and show that disruption of this mechanism accounts for the learning deficits in a mouse model for learning disabilities in neurofibromatosis type I (NF1). Our results demonstrate that neurofibromin modulates ERK/synapsin I-dependent GABA release, which in turn modulates hippocampal LTP and learning. An Nf1 heterozygous null mutation, which results in enhanced ERK and synapsin I phosphorylation, increased GABA release in the hippocampus, and this was reversed by pharmacological downregulation of ERK signaling. Importantly, the learning deficits associated with the Nf1 mutation were rescued by a subthreshold dose of a GABAAantagonist. Accordingly, Cre deletions of Nf1 showed that only those deletions involving inhibitory neurons caused hippocampal inhibition, LTP, and learning abnormalities. Importantly, our results also revealed lasting increases in GABA release triggered by learning, indicating that the mechanisms uncovered here are of general importance for learning.

Germline mutations in SPRED1, a negative regulator of Ras, have been described in a neurofibromatosis type 1 (NF1)-like syndrome (NFLS) that included learning difficulties in some affected individuals. NFLS belongs to the group of phenotypically overlapping neurocardio-facial-cutaneous syndromes that are all caused by germ line mutations in genes of the Ras/mitogen-activated protein kinase extracellular signal-regulated kinase (ERK) pathway and that present with some degree of learning difficulties or mental retardation. We investigated hippocampus-dependent learning and memory as well as synaptic plasticity in Spred1-/-mice, an animal model of this newly discovered human syndrome. Spred1-/-mice show decreased learning and memory performance in the Morris water maze and visual-discrimination T-maze, but normal basic neuromotor and sensory abilities. Electrophysiological recordings on brain slices from these animals identified defects in short- and long-term synaptic hippocampal plasticity, including a disequilibrium between long-term potentiation (LTP) and long-term depression in CA1 region. Biochemical analysis, 4 h after LTP induction, demonstrated increased ERK-phosphorylation in Spred1-/-slices compared with those of wild-type littermates. This indicates that deficits in hippocampusdependent learning and synaptic plasticity induced by SPRED1 deficiency are related to hyperactivation of the Ras/ERK pathway. Copyright

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.