text:
Research ArticleAutism
Increased Gene Dosage of Ube3a Results in Autism Traits and Decreased Glutamate Synaptic Transmission in Mice
Stephen E. P. Smith1,
Yu-Dong Zhou1,
Guangping Zhang1,
Zhe Jin1,
David C. Stoppel1,2 and
Matthew P. Anderson1,2,*
1Beth Israel Deaconess Medical Center, Departments of Pathology and Neurology, Harvard Medical School, Boston, MA 02215, USA.
2Program in Neuroscience, Harvard Medical School, Boston, MA 02215, USA.
↵*To whom correspondence should be addressed. E-mail: Matthew_Anderson{at}bidmc.harvard.edu
Stephen E. P. Smith
Yu-Dong Zhou
Guangping Zhang
Zhe Jin
David C. Stoppel
Matthew P. Anderson
For correspondence: Matthew_Anderson@bidmc.harvard.edu
People with autism spectrum disorder are characterized by impaired social interaction, reduced communication, and increased repetitive behaviors. The disorder has a substantial genetic component, and recent studies have revealed frequent genome copy number variations (CNVs) in some individuals. A common CNV that occurs in 1 to 3% of those with autism—maternal 15q11-13 duplication (dup15) and triplication (isodicentric extranumerary chromosome, idic15)—affects several genes that have been suggested to underlie autism behavioral traits. To test this, we tripled the dosage of one of these genes, the ubiquitin protein ligase Ube3a, which is expressed solely from the maternal allele in mature neurons, and reconstituted the three core autism traits in mice: defective social interaction, impaired communication, and increased repetitive stereotypic behavior. The penetrance of these autism traits depended on Ube3a gene copy number. In animals with increased Ube3a gene dosage, glutamatergic, but not GABAergic, synaptic transmission was suppressed as a result of reduced presynaptic release probability, synaptic glutamate concentration, and postsynaptic action potential coupling. These results suggest that Ube3a gene dosage may contribute to the autism traits of individuals with maternal 15q11-13 duplication and support the idea that increased E3A ubiquitin ligase gene dosage results in reduced excitatory synaptic transmission.
Autism spectrum disorders (ASDs) are estimated to affect 1 in 110 individuals and are clinically defined by three core traits: impaired social interaction, reduced communication, and increased repetitive, stereotyped behaviors (1). Despite high heritability revealed by sibling, twin, and family studies (2), the diagnosis is based solely on behavioral criteria. The phenotypic heterogeneity and frequent medical comorbidities that characterize the disorder also present significant challenges for animal modeling and translational research. Existing mouse models of syndromic neurodevelopmental disorders such as Rett syndrome, fragile X, and tuberous sclerosis have proven invaluable for investigations of these specific conditions, but these conditions display incomplete autism penetrance and include other neurologic and pathologic comorbidities.
Recent studies have established the presence of a high rate of small genomic DNA copy number variations (CNVs) in ASD, present in 10 to 20% of cases (3–7). The altered gene dosages resulting from these CNVs may explain a significant proportion of autism cases. Maternally inherited 15q11-13 duplications and triplications are among the most common genomic CNVs identified in patients with autism (1 to 3%) (3, 8). Individuals with one extra maternal 15q11-13 copy, inverted duplication (dup15), display partial autism penetrance, whereas individuals with two extra copies resulting from an isodicentric extranumerary chromosome (idic15) display near-complete autism penetrance (8). Paternally inherited duplications (with rare exceptions) are not associated with autism (8). These observations suggested to us that the dosage of an imprinted gene or genes within the duplicated region underlies the autism risk in these patients.
E3 ubiquitin-protein ligase, Ube3a (also known as E6-AP), is the only gene within the 15q11-13 duplicated segment consistently expressed solely from the maternal allele in mature neurons (9), making it a likely candidate to mediate the autism phenotype. CNVs of other E3 ligase genes have recently been identified in ASD (3). Moreover, inactivating mutations or deletions of Ube3a cause Angelman syndrome, a neurological disorder characterized by intellectual disability, hypotonia, and seizures (10, 11).
The Ube3a imprinting seen in humans is preserved in mice, and inheritance of a maternal allele deletion is sufficient to reconstitute many of the features of Angelman syndrome including seizures, defective motor performance, impaired contextual fear learning, defective synaptic long-term potentiation, and reduced dendritic spine density (10, 12, 13). The molecular mechanisms by which Ube3a deficiency in Angelman syndrome causes cognitive impairments are not fully understood. Nevertheless, the Angelman mouse model displays a significant increase in the phosphorylation of hippocampal α-calcium/calmodulin-dependent protein kinase II (αCaMKII), specifically at the constitutively active site Thr286 and the autoinhibitory site Thr305 (14, 15). Furthermore, by crossing the Angelman mouse model to mice with an αCaMKII-T305V/T306A mutant knock-in that prevents inhibitory autophosphorylation of αCaMKII, the incidence of seizures was decreased, and the deficits in motor function, learning, and synaptic plasticity were partially reversed (15). More recently, the Angelman mouse model was shown to display impaired experience-dependent maturation of visual cortex, and defects in synaptic plasticity were rescued by dark rearing (16, 17). However, the proteins ubiquitinated by Ube3a that might mediate these behavioral and synaptic defects have not been identified. Ube3a (E6-AP) was originally discovered to ubiquitinate and promote degradation of p53, through which it plays a pathogenic role in human papillomavirus–induced cervical epithelium neoplasia (18). More recently, Ube3a was shown to ubiquitinate and promote degradation of two important neuronal proteins: Arc and Ephexin5 (19, 20).
On the basis of these effects of maternal Ube3a deficiency on behavior and neuronal function, we hypothesized that increased Ube3a dosage in individuals with 15q11-13 duplication might underlie autism behavioral traits. Thus, we predicted that increased Ube3a gene dosage might produce autism-related behavioral traits in mice. Because autism behavioral traits are weakly penetrant in individuals with dup15, but highly penetrant in idic15 (8), we compared these traits in mice with a doubling or tripling of the normal Ube3a gene dosage expressed in mature neurons, aimed at modeling dup15 and idic15, respectively.
Genetically increasing Ube3a gene copy number increases protein levels
We hypothesized that Ube3a mediates the autism-related behavioral phenotypes associated with dup15 and idic15 because of its known roles in neurologic function and because Ube3a is the only gene in the region known to be expressed exclusively from the maternal chromosome (Fig. 1A). Using bacterial artificial chromosome (BAC) recombineering techniques (21), we inserted a 162-kb segment of mouse chromosome 7 (syntenic to human chromosome 15), containing the entire 78-kb exon-intron coding sequence of Ube3a as well as its 63-kb 5′ and 21-kb 3′ extragenic sequences, into FVB embryos to generate transgenic mice, which were subsequently bred to produce single (1×Tg) and double (2×Tg) copy transgenic animals. A 3×FLAG tag followed by two stop codons was inserted in-frame in exon 12 [isoforms 2 and 3, long form (L)] or in exon 8 [isoform 1, short form (S)] to produce the full-length FLAG-tagged transgenic protein (figs. S1 and S2A). Transgenic mice carrying the exon 12 FLAG insert (isoforms 2 and 3, L) are used throughout the paper, unless otherwise noted. Two independent transgenic founder lines of the exon 12 insert (L) with independent integration sites were compared to control for any potential insertion-site effects (Ube3a, founder lines 1 and 2); line 1 is used throughout, except where otherwise noted.
Expression of functional, full-length Ube3a gene BAC transgene Ube3a protein in the native expression pattern. (A) Schematic representation of the human genes located between breakpoint (BP) 1 and BP3 in the 15q11-13 region. Paternally expressed genes are blue, maternally expressed genes are red, and the location of the genomic DNA contained in the BAC is green. (B) Quantification of Ube3a protein in maternal Ube3a knockout (KO), wild-type (WT), 1×Tg, and 2×Tg Ube3a transgenic mice (total brain protein, Ube3a antibody) (ANOVA: F(3,24) = 26.95; *P < 0.05; **P < 0.001 by Dunnett's post hoc, n = 4 to 11). (C) Western blot of Arc assessing degradation in vitro by transgenic Ube3a isoforms 2 and 3 (Ube3a-L) and isoform 1 (Ube3a-S). Ube3a was immunoprecipitated with FLAG antibody from total brain (ANOVA: F(2,6) = 9.93; *P < 0.05 by Dunnett's post hoc, n = 3 independent in vitro assays). (D) Western blot of Arc in the barrel cortex in WT and 2×Tg mice. n = 10 to 12. *P = 0.03, two-tailed, unpaired t test. (E to G) Double immunofluorescence staining for total Ube3a (red) and Ube3a-FLAG transgene (green) (colocalization is shown in yellow) in (E) hippocampus and cortex, (F) superficial layers of the barrel cortex, and (G) CA3 of the hippocampus. Scale bars, 500 μm (E) and 100 μm [(F) and (G)].
Ube3a 1× and 2× transgenic mice were identified by semiquantitative polymerase chain reaction (PCR) (fig. S2B) and confirmed by Western blot to express two and three times more Ube3a protein in whole-brain lysates than wild-type mice, respectively (Fig. 1B). The endogenous Ube3a gene is expressed only from the maternal chromosome in neurons, explaining this dose-dependent effect of adding extra Ube3a copies. But the Ube3a transgene expresses independent of parent of origin as a result of the lack of the antisense transcript initiation site underlying imprinting (22) (fig. S2C). Transgene expression is also independent of sex (fig. S2D). We concluded that the Ube3a transgene generated functional protein because FLAG antibody immunoprecipitated long (L), but not short (S), FLAG-tagged Ube3a isoform degraded exogenous recombinant Arc, a known Ube3a target (19) in vitro (Fig. 1C). Further, Ube3a (2×) transgene decreased endogenous Arc in the barrel cortex (Fig. 1D). Immunofluorescence staining for FLAG in the Ube3a-FLAG transgenic animals recapitulated the native Ube3a staining pattern seen in wild-type animals and matched previously reported Ube3a expression patterns (17, 23) (fig. S3). Further, dual immunofluorescence staining to FLAG and Ube3a showed complete overlap in cortex, hippocampus, and thalamus, indicating that the transgenic protein expresses in all cells expressing native Ube3a (Fig. 1, E to G).
Increasing Ube3a gene dosage impairs social behavior
Impaired social interaction is a hallmark of autism. We assessed a measure of this trait in juvenile transgenic mice, using a three-chamber social interaction test (24) in which mice are first acclimated to the arena and then choose between a social chamber containing a caged and sex- and age-matched wild-type stranger mouse and a chamber with an empty cage (Fig. 2A). Wild-type mice displayed normal social preference, spending more time on the side of the apparatus containing the probe mouse (Fig. 2B) and more time interacting with the probe mouse (Fig. 2C). Ube3a transgenic mice with three times the normal amounts of Ube3a protein (2×Tg) failed to show a social preference in either measure, investigating the empty and mouse-occupied cages equally. Ube3a transgenic mice with twice the normal amount of Ube3a (1×Tg) showed an intermediate phenotype, failing to show a preference for the social zone but showing a significant preference to interact with the cage occupied by a mouse (Fig. 2, B and C). Comparisons across genotypes are not standard (24) but resulted in the same conclusions (table S1). Plotting the middle zone time as a function of genotype shows the preference for the outer two zones containing the novel objects (cages) in all strains (Fig. 2B). We then performed a different version of the three-chambered social interaction test on a new cohort of adult mice with acclimation of the mice to both the arena and the cages (Fig. 2D) (25). In this paradigm, wild-type and Ube3a (1×) transgenic mice displayed a more robust preference for the side containing the novel mouse (Fig. 2E). Ube3a (2×Tg) transgenic mice again showed no preference for the side containing the mouse, whether assessed as time in the social zone (Fig. 2E) or in close proximity to the probe mouse (Fig. 2F). Ube3a transgenic mice carrying two copies of the inactive short form (2×S) also displayed a normal social preference (Fig. 2, E and F, and table S1). This second social behavior experimental design permitted us to add a novel object rather than the probe mouse to the cage, revealing that Ube3a (2×Tg) transgenic mice, like controls, displayed a normal preference for this immobile novel object (Fig. 2G). Two independent Ube3a (2×Tg) transgenic founders (lines 1 and 2) displayed the same lack of preference for interaction with the novel mouse, eliminating transgene insertion effects (Fig. 2H). The Ube3a (2×Tg) transgene also abolished the social preference equally in males and females (fig. S4A).
Effects of increased Ube3a gene dosage on mouse social behavior. (A) Diagram of three-chamber social interaction test with acclimation to arena only and choice between a novel container containing a novel mouse and a novel empty container. (B) Time in social, middle, or opposite zone in juvenile WT, 1×Tg, and 2×Tg mice (*P = 0.0162 comparing within-genotype "Social" and "Opposite" by t test). (C) Time spent interacting with either the caged mouse (social) or the empty cage (opposite). (*P = 0.0157 and 0.0186, respectively, t test). n(WT, 1×, 2×) = 11, 15, and 12. (D) Diagram of a modified three-chambered social interaction test with acclimation to arena and cages (left) and a test session (right) in which the mouse chooses between exploring a familiar empty cage or a cage containing a novel mouse. (E) Time in social, middle, or opposite zone in adult WT, 1×Tg-L, 2×Tg-L, and 2×Tg-S mice. *P < 0.002, t test. (F) Time in area proximal to the enclosures (dark circles, "Close" zone). *P < 0.005, t test. n(WT, 1×, 2×) = 17, 10, and 15. (G) Time spent in proximity to a caged object after acclimation to chamber and cages, which tests for novel object exploration. *P < 0.03, t test, comparing within-genotype "Object" and "Opposite"; n = 11 to 13. (H) Time spent in social and opposite zone for independent Ube3a transgenic founder lines 1 (Fd1) and 2 (Fd2) (n = 10 and 5) and WT littermates (n = 7 and 4). **P < 0.01; ****P < 0.001, t test. Color code (used throughout): WT (black), single-transgenic long-form Ube3a (1×, blue), double-transgenic long-form Ube3a (2×, red), and double-transgenic short-form "inactive" Ube3a (2×S, green).
One potential explanation for the lack of social preference could be motor deficits or increased generalized anxiety. However, in the open field test, Ube3a (2×) transgenic mice displayed mobility that was similar to that of controls (Fig. 3A). The number of entries into the center and time spent in the center of the field also did not differ from that of controls (Fig. 3, B and C); anxious mice tend to avoid the center space in an open field. In the elevated plus maze, Ube3a (2×) transgenic mice explored the open arms similar to controls (Fig. 3, D to F), again providing no evidence of an anxiety-like phenotype. Motor performance on the accelerating rotorod in adulthood was also similar across genotypes (Fig. 3G). Orienting motor responses during postnatal development were also unaffected (fig. S5). Finally, novel object exploration (Fig. 3, H and I) and short-term object memory (Fig. 3, H and J) were similar across genotypes. The results indicate that a tripling of Ube3a gene dosage impairs social interaction, a potential correlate of the social behavior deficits found in human autism, whereas a doubling of Ube3a gene dosage causes a more limited social behavior deficit only during competition between a mouse and a novel object.
Effects of increased Ube3a gene dosage on anxiety-like and exploratory behavior, rotorod motor performance, or short-term object memory. (A to C) The open field tests exploratory behavior, comparing (A) distance traveled, (B) numbers of entries into the center, and (C) time spent in the center of an open field in double-transgenic long-form Ube3a (2×, red) and WT littermates. (D to F) The elevated plus maze tests anxiety-like behavior, assessing (D) number of entries into the open arms, (E) percentage of entries into the open arms, and (F) percent time spent in the open arms (NS, not significant). (G) The accelerating rotorod assesses motor performance in mice, comparing time to fall during three sequential days. (H to J) Short-term memory test in mice is diagrammed (H) and examines (I) number of object exploratory sniffs with first exposure to two novel objects during acclimation phase of memory test (left) and (J) number of object exploratory sniffs after one object is replaced by a new novel object (right), the memory phase of the test. (*P < 0.05, within genotype, t test). For complete statistics, see table S1.
Increasing Ube3a gene dosage impairs communication and increases repetitive behavior
Defective communication is the second diagnostic criteria of autism. Thus, we assessed whether increased Ube3a gene dosage impairs communicative ultrasonic vocalizations in mice (Fig. 4, A to D). We measured two types of social behavior–relevant vocalizations in adult rodents: vocalizations generated by same-sex pairs encountering each other for the first time and vocalizations generated by sexually experienced males exposed to female urine.
Effect of increased Ube3a gene dosage on ultrasonic vocalizations and repetitive self-grooming. (A) Number of social ultrasonic vocalizations made by pairs of genotype- and sex-matched mice (Kruskal-Wallis test: H = 7.76, df = 2, P = 0.021, *P < 0.05 by Dunn's multiple comparison post hoc, n = 8 to 14 pairs). (B) Ultrasonic vocalization responses of male mice to female urine as scored by number (ANOVA: F(2,22) = 4.52, P = 0.023, *P < 0.05 by Dunnett's post hoc) and time spent vocalizing (ANOVA: F(2,22) = 5.31, P = 0.013, *P < 0.05 by Dunnett's post hoc). n = 7 to 11. (C and D) Vocalization types, representative examples, and distribution in urine-exposed males, distinguished by shape and harmonics. n.s., not significant by ANOVA. (E) Time spent sniffing in the olfactory habituation/dishabituation test (genotype: two-way ANOVA: F(1,132) = 2.723, P = 0.12, n = 7). (F) Ultrasonic vocalizations of pups during acute maternal separation (ANOVA: F(2,262) = 0.87, P = 0.42, n = 10 to 14). (G) Repetitive self-grooming (ANOVA: F(2,34) = 5.41, P = 0.0095, **P < 0.01 by Dunnett's post hoc, n = 11 to 12). (H) Repetitive self-grooming in independent Ube3a transgenic founder lines Fd1 (nWT, 2× = 10 and 14) and Fd2 (nWT, 2× = 3,5). *P = 0.01; **P = 0.004, t test.
Social stimulus–induced vocalizations were assessed when two age-, sex-, and genotype-matched, nonlittermate stranger mice were paired. Wild-type mice emitted vocalizations that were markedly reduced by the 2×, but not the 1×, Ube3a transgene dosage (Fig. 4A). Ube3a 2× transgene effects were most pronounced in females, because female pairs vocalized with a much greater frequency than males (fig. S4). As previously reported (26), female urine elicited a diverse repertoire of vocalizations in sexually experienced, wild-type males (Fig. 4, B to D). In this test, the number of vocalizations and the time spent vocalizing were strongly reduced in both 1× and 2× Ube3a transgenic mice (Fig. 4B). Despite the large decrease of vocalization quantity, other vocalization features were unaffected: average peak frequency (67 ± 1, 67 ± 2, and 69 ± 2 kHz, respectively) and call type distribution (Fig. 3D). To control for a potential confounding deficit in olfaction, we tested mice in an olfactory habituation/dishabituation test, which confirmed the ability of transgenic animals to respond normally to novel scents presented on a cotton swab (Fig. 4E). There could also be a general deficit in the ability to vocalize. However, arguing against this possibility is the observation that pup vocalizations during a 5-min separation from their mother, an aversive stimulus (27), were unaffected by the increased Ube3a gene dosage (Fig. 4F). The results indicate that ultrasonic vocalizations generated in a social context are impaired by increases of Ube3a gene dosage.
The third hallmark autism trait is repetitive, stereotyped behaviors such as body rocking, hand flapping, or self-injurious behavior. These behaviors have been assumed to be equivalent to repetitive self-grooming in mice (24, 28, 29). Self-grooming was increased by three times in Ube3a (2×Tg) transgenic mice, but was unaffected in Ube3a (1×Tg) compared to wild types (Fig. 4G). We ruled out the possibility that these effects were caused by disruptions from the transgene insertion by showing that two independent Ube3a transgene founder lines 1 and 2 produced the same increase of self-grooming relative to that of control littermates (Fig. 4H). Increased self-grooming was also observed when male and female Ube3a (2×Tg) transgenic mice were separately examined (fig. S4). Transgenic mice carrying a 2× gene dosage of the enzymatically inactive Ube3a [the short (S) form] (2×S) exhibited normal grooming (Fig. 4G). In summary, tripling Ube3a gene dosage expressed in neurons caused impairment of all three measures of autistic-like behavior—decreased social interaction and vocalization and excessive repetitive grooming behavior. Doubling the Ube3a gene dosage caused a more limited phenotype in mice, selectively impairing social interactions during novel object competition (not prehabituated to the cages) and male vocalizations to a female urine stimulus.
Increased Ube3a gene dosage impairs glutamatergic synaptic transmission
Ube3a is present in the cytoplasm, the nucleus, and at distinct dendritic puncta colocalized with the synapse marker postsynaptic density protein 95 (PSD-95) in mouse cortical neuron primary cultures (fig. S3, I to Q). We hypothesized that increasing Ube3a gene dosage might alter evoked excitatory and/or inhibitory postsynaptic currents (EPSCs and IPSCs) in cortical pyramidal neurons. We measured somatosensory whisker barrel cortex layer 2/3 pyramidal neurons, because the neurons are highly active [Ube3a is an activity-induced gene (19)], strongly express Ube3a (Fig. 1F and fig. S3, A and B) (19, 23), and have been previously studied in other mouse models of ASD. These neurons reportedly displayed increased GABAergic transmission in a mouse carrying the autism-associated neuroligin 3 (NLGN3) point mutation (30) and decreased GABAergic transmission because of GABAergic neuron deletion in the MeCP2 mouse model of Rett syndrome (31) without affecting glutamatergic synaptic transmission.
Synaptic currents were measured by whole-cell, voltage-clamp recordings in acute brain slices. Slices from Ube3a (2×Tg) transgenic mice displayed strong suppression of EPSC amplitude (Fig. 5A). By contrast, IPSC amplitude was not significantly reduced (Fig. 5B). We measured spontaneous IPSCs and EPSCs and found reduced spontaneous EPSC amplitude and frequency (fig. S6A). Spontaneous IPSCs had reduced amplitudes but unaltered frequencies (fig. S6B). Thus, increasing Ube3a gene dosage markedly reduced glutamatergic synaptic transmission but had more limited effects on GABAergic transmission.
Effect of increased (2×Tg) Ube3a gene dosage on excitatory and inhibitory synaptic transmission in slices from somatosensory whisker barrel cortex layer 2/3 pyramidal neurons. Black, WT; red, Ube3a 2×Tg. (A) Evoked EPSC traces and graph (ANOVA: F(1,97) = 41.45, **P < 0.001, n = 6 to 8). (B) Evoked IPSC traces and graph (ANOVA: F(1,83) = 0.03, P = 0.8551, n = 6). (C) mEPSC traces (top) and cumulative frequency plots of amplitude (bottom, left) and interevent interval (bottom, right) (Kolmogorov-Smirnov test: **P < 0.01, ***P < 0.001; n = 9 to 11). (Insets) Median mEPSC amplitude (left; P < 0.01, t test) and frequency (right; P < 0.01, t test; n = 9 to 11). (D) mIPSC traces and cumulative amplitude (left) and frequency (right) plots (P > 0.05, Kolmogorov-Smirnov test; n = 9 to 11). (Insets) Median mIPSC amplitude (left) and frequency (right) compared across genotypes (P > 0.05, t test).
To address the mechanism of this effect, we recorded miniature EPSCs (mEPSCs) and miniature IPSCs (mIPSCs), with action potentials inhibited. Ube3a (2×) strongly suppressed mEPSC amplitude and frequency (Fig. 5C). By contrast, and unlike the Nlgn3 and Mecp2 mutant mice (30, 31), excess Ube3a failed to significantly alter mIPSC frequency or amplitude (Fig. 5D). To be sure that the reduced mEPSC frequency was not a result of reduced mEPSC amplitude (falling below detection threshold), we clamped the cells at −80 mV to increase amplitude. Again, we found reduced mEPSC amplitude and frequency (fig. S6C). These decreases in mEPSC amplitude, typically due to reduced postsynaptic AMPA receptors, and the decreased mEPSC frequency, typically due to reduced synapse number or presynaptic release probability, suggested that increased Ube3a gene dosage may regulate glutamate synaptic transmission at both pre- and postsynaptic sites.
Increasing Ube3a gene dosage reduces presynaptic glutamate release via two mechanisms
Synaptic glutamate release (R) is governed by the equation R = Npq, where N is the number of release sites, p is the probability of release, and q is the quantal size. We suspected that a change in synapse number might contribute to the decreased glutamate release, a mechanism analogous to the maternal Ube3a knockout mice that displayed fewer dendritic spines (12, 17). Therefore, we looked for changes in glutamate synapse number in layer 2/3 barrel cortex using three independent measures: counting asymmetric synapses using electron microscopy (Fig. 6A); counting the number of puncta with colocalized pre- and postsynaptic markers, VGlut1 and PSD-95, in thin (5 μm) sections by dual immunofluorescence staining (Fig. 6B); and counting dendritic spines in Golgi-stained sections (Fig. 6C). By all measures, synapse number was unaltered by the increased Ube3a (2×) gene dosage. Furthermore, the number of release sites, assessed electrophysiologically (see below), was unaltered (fig. S7). Thus, changes in N failed to explain the reduced mEPSC frequency.
Effect of increased (2×Tg) Ube3a gene dosage on glutamate synapse number and postsynaptic glutamate receptor currents. Black, WT; red, Ube3a 2×Tg. (A) Synapse number, as measured by electron microscopy (P = 0.67, t test; n = 3 animals per group; 28 to 32 micrographs per animal). (B) Colocalized immunostaining of presynaptic (VGlut1) and postsynaptic (PSD-95) synaptic markers (scale bar, 1 μm; P = 0.8706, t test; n = 4 animals per group; eight or more micrographs per animal). (C) Spine number per dendrite length, as shown by Golgi staining (apical: P = 0.64; basal: P = 0.77, t test; n = 4 animals per group; ≥10 dendrites per animal). (D) Glutamate ionophoresis–induced AMPA and NMDA currents (AMPA: P = 0.46, t test; n = 5 to 6; NMDA: P = 0.97, t test; n = 3 to 6). (E) Fiber stimulation–evoked AMPA/NMDA traces (above, unscaled; below, scaled to WT) and ratio (P = 0.54, t test; n = 6 to 8).
Presynaptic release probability (p), another potential cause of decreased mEPSC frequency, was assessed with a repeated minimum stimulation protocol (fig. S7, A to D). Release probability was significantly reduced in Ube3a (2×) transgenic mice (fig. S7D). Paired-pulse ratio, increased at low-release probability synapses, was also increased in Ube3a (2×) transgenic mice (Fig. 7A). Both measures suggested that increasing Ube3a gene dosage decreases the release probability (p) of cortical glutamate synapses and so reduced mEPSC frequency, contributing to the lowered efficacy of glutamatergic synaptic transmission.
Effect of increased (2×Tg) Ube3a gene dosage on glutamate synapse release probability, synaptic glutamate concentration, and ES coupling. Black, WT; red, Ube3a 2×Tg. (A) Representative unscaled paired-pulse traces (right), traces scaled to match first pulse to WT (left), and bar graph (P = 0.028, t test; n = 7 to 10). (B) Representative traces of unscaled (above) and scaled-to-WT (below) evoked EPSCs with (dotted lines) or without (solid lines) γ-DGG and graph (right) (*P = 0.0127, t test; n = 7 to 8). (C) Representative voltage tracings (left) and graph (right) assessing action potential firing to a 5-ms EPSC-like somatic current (0 to 640 pA, 40-pA steps). **P = 0.003, χ2 test (n = 14 to 17).
Quantal size (q) can be altered by pre- or postsynaptic changes, and reduced mEPSC amplitude often results from decreased postsynaptic AMPA receptor density. To directly measure the postsynaptic glutamate receptor responses, we applied small puffs of glutamate to the neuron and recorded postsynaptic AMPA and N-methyl-D-aspartate (NMDA) currents. Glutamate iontophoresis induced AMPA and NMDA receptor currents that were similar in amplitude across genotypes (Fig. 6D). In separate experiments, fiber stimulation–evoked AMPA and NMDA currents displayed a similar NMDA/AMPA ratio (Fig. 6E). Using antagonists to isolate mEPSC AMPA or NMDA currents, we showed that the decay time kinetics of these currents did not differ among genotypes (fig. S7, E and F), revealing no evidence of altered receptor subunit composition. Additionally, the quantity of glutamate receptor subunits and other synaptic proteins, when measured by Western blot of microdissected barrel cortex, were unaltered (fig. S8). The results suggested that Ube3a does not reduce glutamatergic synaptic transmission and quantal size (q) by decreasing the number of postsynaptic AMPA or NMDA glutamate receptors.
The reduced mEPSC amplitude with preserved postsynaptic glutamate receptor function suggested a possible decrease in synaptic glutamate concentration. To test this hypothesis, we applied a weak glutamate receptor antagonist, γ-DGG (γ-d-glutamylglycine), to assess for a glutamate concentration–dependent decrease of synaptic transmission. A greater percent inhibition by γ-DGG indicates a lower synaptic glutamate concentration (32). γ-DGG did indeed reduce EPSC amplitude to a greater extent in Ube3a (2×) transgenic than in wild-type neurons, indicating that increased Ube3a gene dosage decreased synaptic glutamate concentration (Fig. 7B). The decreased synaptic glutamate could not be explained by reduced synaptic vesicle diameter [measured by electron microscopy: wild type, 37.40 ± 0.22 nm; Ube3a (2×), 37.37 ± 0.19 nm] or reduced glutamate transporter protein (VGlut1 or EAAT1-3) (fig. S9).
Ube3a reduces postsynaptic excitability to phasic excitatory synapse-like stimuli
Effective glutamatergic synaptic transmission also depends on the efficacy with which EPSCs couple to the firing of postsynaptic action potentials. This EPSC-spike coupling was assessed with short (5 ms) EPSC-like current injections directly into the patch-clamped neuron, bypassing the defects already shown to be present at the synaptic inputs. This measure assesses the intrinsic excitability of a neuron, compared to the EPSC measures, which assess synaptic inputs from surrounding neurons. Ube3a (2×) transgenic mice displayed impaired ES coupling (Fig. 7C). By contrast, action potential threshold, capacitance, and resting membrane potential were unaltered, whereas peak firing rate showed some evidence of a decrease (fig. S10). The results indicate that in addition to the strong impairment of glutamatergic synaptic transmission, the ability of these synaptic currents to couple to the firing of a postsynaptic action potential is concurrently suppressed by increased Ube3a gene dosage.
In summary, excess Ube3a acts at multiple, distinct sites within the pre- and postsynaptic compartments of the neuronal circuitry to impair excitatory synaptic transmission, causing decreased presynaptic vesicle release probability, decreased synaptic glutamate concentration, and decreased postsynaptic EPSC-spike coupling. By contrast, glutamate synapse densities and postsynaptic glutamate receptors were unaltered and GABAergic synaptic transmission showed only minor changes.
To date, several ASD mouse models—based on rare single gene point mutations [for example, NLGN3 (30, 33) and Shank3 (34–37)], syndromic disorders with partial autism penetrance [for example, tuberous sclerosis, fragile X, and Rett syndrome; see (38, 39)], mouse social behavior screens [for example, BTBR mouse (40) and MALTT mouse (41)], or immune models (25, 42)—have phenocopied a subset of the behavioral components of ASD. Our mouse model of ASD with extra copies of the Ube3a gene, which was designed to reproduce in part a common known risk factor for ASD (15q11-13 duplication present in 1 to 3% of ASD cases), displayed strong penetrance of three core autism-related behavior traits and to date has not displayed other major confounding traits or comorbidities [no evidence of the following: anxiety (elevated plus maze, open field test, social avoidance); motor behavior deficits (rotorod performance, motor milestones); memory deficits (object memory); or sensory behavior deficits (olfactory response, novel object recognition)].
Individuals with idic15 show typical autism traits such as reduced speech, reduced socialization including reciprocity and eye contact, and repetitive stereotyped behaviors. There is also some evidence for regression in idic15 with better eye contact and vocalizations in infants and toddlers than in older children (8). There are also reports of delayed echolalia, pronoun reversal, and stereotyped utterances. A recently recognized feature of individuals with idic15 is comorbid epilepsy and sudden unexpected death in epilepsy (SUDEP) (8). Individuals with idic15 also display moderate to severe learning deficits that correlated in one study to the severity of the comorbid epilepsy (43). Our mice, which carry the idic15-equivalent increase in Ube3a gene dosage, showed behavioral correlates of the typical autism traits, but epilepsy and detailed learning and memory testing have not yet been performed.
We directly compared mice with Ube3a gene duplication and triplication and found gene-dosage effects on autism-related trait penetrance as observed in humans (8). Although gene dosage does not necessarily predict mRNA and protein dosage, studies in transformed idic15 lymphocytes and two idic15 postmortem brain samples indicate that the increase of Ube3a mRNA and protein is generally proportional to the increase of chromosomal gene dosage (44, 45). In our mice, a threefold increase of brain Ube3a protein, as would be predicted for patients with idic15, caused strong autism trait penetrance and reconstituted surrogates of all three core autism-related behavioral traits. By contrast, a twofold increase of brain Ube3a protein, as would be predicted for patients with dup15, associated with weaker autism penetrance and generated only a subset of the behavioral defects (most strongly reduced male vocalizations to female urine). These dose-dependent effects of increased Ube3a gene copies may explain the difficulty in finding autism traits in a recently developed mouse model aimed at reconstituting the maternally inherited autism disorder associated with dup15 (46). In this report, the authors used an elegant chromosome-engineering technique to replicate dup15 in mice (spanning the full set of genes within the syntenic region of chromosome 7) and demonstrated the expected maternal- and paternal-specific gene expression patterns of the imprinted chromosomal region. Yet, despite successful reconstitution of the typical expression pattern of Ube3a in brain tissues and the expected doubling of brain Ube3a mRNA when inheriting this duplication from the mother, autism-related behavioral traits were not observed with maternally inherited 15q11-13 duplications. Instead, they reported some behavioral deficits with paternally inherited 15q11-13 duplication. Our finding that a triplication of Ube3a gene dosage is necessary to reconstitute the full set of autism-like traits in mice may explain the lack of phenotype in this mouse model. Triplication of the maternally inherited Ube3a gene cannot be generated with this chromosome engineering technique, a limitation overcome by our BAC transgenic approach.
The 15q11-13 duplicated region contains at least 30 characterized genes, several previously proposed to underlie the autism phenotype. ATP10A is of interest because early studies suggested that, like Ube3a, it is expressed exclusively from the maternal chromosome (47, 48), although this has since been refuted (46, 49). Other genes within the duplicated genomic region, such as GABAA (γ-aminobutyric acid type A) receptor subunits β3, α5, and γ3 and cytoplasmic FMRP-interacting protein 1 (CYFIP1), have also been proposed to mediate the autism risk (8, 50). None are imprinted in a way that readily explains the selective association of autism with maternally inherited duplications, although their expression may be regulated in more complex, as yet undefined, ways (51). Although we cannot rule out a contribution from these other genes, our results indicate that increased dosage of the Ube3a gene alone is sufficient to replicate the three core autism-related traits in mice. Because our study did not systematically examine all possible genes within the 15q11-13 duplicated region, we cannot eliminate the possibility that increases or even decreases of expression of other genes could contribute.
The glutamatergic synaptic defects we report in these mice with increased Ube3a gene dosage are not those predicted from simply inverting the effects previously observed in the Angelman syndrome mouse model with a heterozygous, maternally inherited Ube3a knockout. For example, Yashiro et al. (17) reported that a loss of maternal Ube3a reduces mEPSC frequency in visual cortex layer 2/3 pyramidal neurons, an effect we also report with increased Ube3a gene dosage in comparable neurons of the somatosensory cortex. Similarly, Greer et al. (19) reported that a loss of maternal Ube3a reduces glutamatergic synaptic transmission, evoked AMPA/NMDA current ratio, and mEPSC amplitude in hippocampal CA1 pyramidal neurons. They attributed this effect to increases of the Ube3a target Arc that is known to regulate AMPA receptor trafficking and would deplete synaptic AMPA receptors when increased (52). Although we confirmed that increasing Ube3a gene dosage partially reduces Arc, the predicted increase in AMPA currents was not observed. Loss of maternal Ube3a was also reported to decrease dendritic spine density, potentially explaining the reduced mEPSC frequency reported in visual cortex (12, 17). Yet, increasing Ube3a gene dosage reduced mEPSC frequency, but did not increase or decrease dendritic spine density or glutamate synapse density. Although both high and low levels of Ube3a associate with human neurologic diseases, our findings suggest that the molecular and circuit mechanisms leading to Angelman syndrome and idic15 autism may be quite different.
Comparison of the circuit and behavioral defects in this Ube3a (2×) transgenic mouse model with those of other models of ASD may yield insights into shared pathophysiological mechanisms. A recently published report of a Shank3-deficient mouse model of ASD (36) indicated that the animals display increased anxiety (elevated plus maze), potentially explaining defective social interaction. In the three-chambered social interaction test, these mice avoided the chamber containing the probe mouse, consistent with reduced social interaction due to increased fear/anxiety. In contrast, our ASD mice with increased Ube3a gene dosage showed no evidence of anxiety behavior and did not avoid the social chamber side. Similarly, other mouse models including the Shank3 (36) and Angelman syndrome mice (10, 53) display impaired motor function on the accelerating rotorod, another potential behavioral confound. Ube3a (2×) transgenic mice displayed no defects in accelerating rotorod performance or other motor milestones. These differences further support the recognized heterogeneity of ASD.
Differences are also emerging in the types of circuit defects found in ASD mouse models. For example, mice with knock-in of an ASD-associated neuroligin 3 point mutation show increased GABAergic synaptic transmission, predicting decreased cortical circuit excitability (29, 30). By contrast, increasing Ube3a gene dosage reduced circuit excitability by reducing glutamatergic synaptic transmission. These distinct circuit defects are consistent with the notion that different therapeutic targets may be necessary to treat subtypes of ASD.
Ube3a is an E3 ubiquitin ligase, a class of protein that provides substrate specificity to the ubiquitin protein degradation system. Many tens of targets of Ube3a have been identified in cell culture systems (54, 55), in Drosophila (56, 57), and recently in mouse brain (19, 20). Our initial screen of potential Ube3a targets only revealed changes in Arc. The pre- and postsynaptic functional defects in glutamate synapses produced by excess Ube3a suggest that several distinct unidentified ubiquitination targets could be regulating excitatory synapse function. Recent studies also indicate that ubiquitination, rather than promoting proteasome degradation, can instead sometimes increase protein stability or alter protein trafficking (58, 59). Alternatively, ubiquitin ligase activity may not be critical to the effects of excess Ube3a on behavior and neuronal circuit function because the protein can also act as steroid hormone transcriptional coactivator independent of its E3 ligase activity (60, 61). The potential important effects of Ube3a on gene expression are supported by its strong nuclear localization.
The translational value of this new mouse model of ASD lies in its construct and face validity, because it adds excess copies of one gene found within a common CNV region found in nonsyndromic ASD and displays correlates of three core ASD behavioral traits. The predictive validity of this model has not yet been tested due to the lack of drug treatments for the human condition. Current therapies for ASD largely target the comorbid neurologic (epilepsy) and neuropsychiatric (anxiety) conditions. The quantitative measures of social behavior and communication disrupted in this murine model of ASD can serve as a valuable system for preclinical testing of candidate drugs. Further, the detailed mechanistic analysis of neuronal circuit defects made possible by such mouse models of ASD will provide a strong foundation for the rational design of therapeutics aimed at bolstering specific defects in circuit function.
Generation of mice
Using BAC recombineering techniques, we inserted a 162-kb segment of mouse chromosome 7, containing the entire 78-kb exon-intron coding sequence of Ube3a as well as its 63-kb 5′ and 21-kb 3′ sequences, into FVB embryos to generate transgenic mice (fig. S1). A 3×FLAG tag followed by two stop codons was inserted in-frame after exon 12 to generate two independent founder mice. Transgenic founder offspring were then crossed to produce Ube3a 1× or 2× transgenic mice. Genotyping was accomplished by semiquantitative PCR of sequences flanking the FLAG-tagged Ube3a gene with the native gene as an internal control. Primers and detailed protocols are available in the Supplementary Materials and Methods.
Ube3a protein overexpression and function
Ube3a expression was confirmed by Western blot of cortical lysates with both anti-FLAG M2 antibody (Sigma) and anti-Ube3a (BD Biosciences). The ubiquitin ligase activity of Ube3a was assayed by an in vitro target protein degradation assay. Ube3a was immunoprecipitated with anti-FLAG M2 antibodies and protein G magnetic beads (NEB) and eluted in nondenaturing conditions with a 3× FLAG peptide (Sigma). Recombinant Arc (1 μg) (BD Biosciences) was added to 10 μl of immunoprecipitated Ube3a in the presence of 50 ng of E1 and 100 ng of UbcH7 enzymes and 4 μg of hemagglutinin-ubiquitin (Boston BioProducts) (19). Western blots were probed with anti-Arc (Santa Cruz) and quantified with ImageJ (National Institutes of Health). Detailed protocols are available in the Supplementary Materials and Methods.
Three-chamber social interaction testing
Separate cohorts of mice were tested in the three-chamber social test as either juveniles (3 to 4 weeks) or adults (8 to 12 weeks) following previously published protocols (24, 25). For the juvenile test, after a 10-min acclimation period in an empty chamber, a stranger wild-type mouse was placed in a small enclosure in one of the outer chambers, and an empty enclosure was placed in the opposite side. The round wire enclosure (a pencil holder, Office Depot) allowed visual, olfactory, and tactile interaction. The test session lasted 10 min. For the adult test, the enclosures were present during the acclimation, and sessions lasted 5 min. Therefore, in the juvenile test, the comparison was between a novel mouse and a novel object (the enclosure), whereas the adult test compared a novel mouse with a familiar container. To control for novel object preference in the adult, we repeated the test but placed a novel object (a striped plastic cup) into one of the two enclosures a week after the initial test.
Mice were placed in a clean cage in a fume hood in their home room and were allowed to acclimate for 10 min. Mice were then video-recorded for 10 min, and the time spent grooming was measured by an experienced observer (29).
For urine-induced vocalizations, male mice were single-housed for several days and then exposed to brief (5 min) social interactions with both male and female mice for 4 days before the test. On the fifth day, mice were placed in a small plastic box inside a larger soundproof container. A cotton swab dipped in freshly collected urine pooled from at least 10 females from at least five different cages was suspended from the top of the smaller box, so that the tip was about 5 cm above the floor. An ultrasonic microphone recorded vocalizations and fed data into a computer running Avisoft-Recorder (Avisoft Bioacoustics), which automatically counted the vocalizations over the 5-min test period. For social vocalizations, sex-, age-, and genotype-matched, nonlittermate mice that had never encountered each other before were placed in a small plastic enclosure simultaneously (to avoid resident-intruder aggression), and the number of vocalizations and time spent vocalizing were recorded automatically (UltraVox, Noldus) for 5 min.
EPSCs were recorded in voltage-clamp mode with cesium-based artificial intracellular fluid and regular artificial cerebrospinal fluid (ACSF). A bipolar platinum/iridium-stimulating electrode (CE2C55, FHC Inc.) was placed at layer 2/3 of the barrel cortex 200 μm away from the recording site. A glass pipette filled with 0.5 mM bicuculline methiodide in ACSF that locally inhibited GABAergic transmission was placed above the soma of the cell being recorded. IPSCs were recorded at a holding potential of +10 mV in the presence of bath 10 μM DNQX (6,7-dinitroquinoxaline-2,3-dione) and 50 μM APV [(2R)-amino-5-phosphonopentanoate]. mEPSCs and mIPSCs were recorded at −60 or −80 mV and +10 mV, respectively. Detailed protocols are available in the Supplementary Materials and Methods.
Glutamate iontophoresis. Pyramidal neurons were voltage-clamped at −70 or +40 mV in the presence of 1 μM tetrodotoxin and 100 μM picrotoxin. Iontophoretically applied glutamate (10 mM sodium glutamate in 10 mM Hepes, pH 7.4) was delivered through glass pipettes (4 to 6 megohms when filled with normal internal solution) placed 1 to 2 μm away from the main apical shaft (~15 to 20 μm from cell body).
Minimal stimulation and estimation of vesicle glutamate content. The vesicle glutamate content was estimated by the relative inhibition of mean single fiber EPSC amplitude by the fast off-rate, non-NMDA receptor blocker γ-DGG (300 μM). The higher the percentage inhibition by γ-DGG, the lower the concentration of synaptic glutamate. Detailed protocols are available in the Supplementary Materials and Methods.
For behavior analysis, comparisons between two groups used two-tailed unpaired Student's t test. Comparisons among multiple groups used one-way analysis of variance (ANOVA) with Dunnett's post hoc test comparing each genotype to wild type; nonsignificant comparisons are not stated in the manuscript. Comparisons involving multiple independent variables used two-way ANOVAs. Nonnormal data (social vocalizations) were tested with the Kruskal-Wallis test followed by Dunn's multiple comparison post hoc test comparing each genotype to wild type. For electrophysiological data, two-tailed unpaired Student's t test was used to compare group means. Kolmogorov-Smirnov test was used to compare cumulative distributions. Mantel-Haenszel χ2 test was used to compare the ES coupling data. Unbalanced two-way ANOVA was used to compare group variance. Tukey's honestly significant difference test was used to perform post-ANOVA pairwise comparisons. n is the number of mice analyzed (behavior and synapse counting), number of pairs of mice analyzed (social vocalizations), or number of cells analyzed (electrophysiology). All data are presented as means ± SEM. P < 0.05 was considered statistically significant.
Fig. S1. Recombineering a C-terminal FLAG tag into the three isoforms of the wild-type Ube3a gene (162 kb, BAC vector).
Fig. S2. Design and expression of Ube3a BAC transgene.
Fig. S3. Distribution of transgenic and native Ube3a protein expression in the brain.
Fig. S4. Gender-specific effects of increased Ube3a gene dosage on social, repetitive, and communication behaviors.
Fig. S5. Effects of increased Ube3a gene dosage on developmental milestones.
Fig. S6. Effects of increased Ube3a gene dosage on spontaneous EPSCs, spontaneous IPSCs, and mEPSCs at −80 mV in layer 2/3 pyramidal neurons from the barrel cortex.
Fig. S7. Effects of increased Ube3a gene dosage on release probability, readily releasable pool size, and AMPA and NMDA kinetics.
Fig. S8. Effects of increased Ube3a gene dosage on synaptic and other neuronal proteins.
Fig. S9. Effects of increased Ube3a gene dosage on quantity of proteins regulating synaptic glutamate concentration.
Fig. S10. Effects of increased Ube3a gene dosage on other biophysical properties of layer 2/3 barrel cortex pyramidal neurons.
Table S1. Effects of increased Ube3a gene dosage on behavior: statistical analyses.
S. E. Levy,
D. S. Mandell,
R. T. Schultz
, Autism. Lancet 374, 1627–1638 (2009).
B. S. Abrahams,
D. H. Geschwind
, Advances in autism genetics: On the threshold of a new neurobiology. Nat. Rev. Genet. 9, 341–355 (2008).
J. T. Glessner,
K. Wang,
G. Cai,
O. Korvatska,
C. E. Kim,
S. Wood,
H. Zhang,
A. Estes,
C. W. Brune,
J. P. Bradfield,
M. Imielinski,
E. C. Frackelton,
J. Reichert,
E. L. Crawford,
J. Munson,
P. M. Sleiman,
R. Chiavacci,
K. Annaiah,
K. Thomas,
C. Hou,
W. Glaberson,
J. Flory,
F. Otieno,
M. Garris,
L. Soorya,
L. Klei,
J. Piven,
K. J. Meyer,
E. Anagnostou,
T. Sakurai,
R. M. Game,
D. S. Rudd,
D. Zurawiecki,
C. J. McDougle,
L. K. Davis,
J. Miller,
D. J. Posey,
S. Michaels,
A. Kolevzon,
J. M. Silverman,
R. Bernier,
R. T. Schultz,
G. Dawson,
T. Owley,
W. M. McMahon,
T. H. Wassink,
J. A. Sweeney,
J. I. Nurnberger,
H. Coon,
J. S. Sutcliffe,
N. J. Minshew,
S. F. Grant,
M. Bucan,
E. H. Cook,
J. D. Buxbaum,
B. Devlin,
G. D. Schellenberg,
H. Hakonarson
, Autism genome-wide copy number variation reveals ubiquitin and neuronal genes. Nature 459, 569–573 (2009).
E. M. Morrow,
S. Y. Yoo,
S. W. Flavell,
T. K. Kim,
Y. Lin,
R. S. Hill,
N. M. Mukaddes,
S. Balkhy,
G. Gascon,
A. Hashmi,
S. Al-Saad,
J. Ware,
R. M. Joseph,
R. Greenblatt,
D. Gleason,
J. A. Ertelt,
K. A. Apse,
A. Bodell,
J. N. Partlow,
B. Barry,
H. Yao,
K. Markianos,
R. J. Ferland,
M. E. Greenberg,
C. A. Walsh
, Identifying autism loci and genes by tracing recent shared ancestry. Science 321, 218–223 (2008).
D. Pinto,
A. T. Pagnamenta,
R. Anney,
D. Merico,
R. Regan,
J. Conroy,
T. R. Magalhaes,
C. Correia,
J. Almeida,
E. Bacchelli,
G. D. Bader,
A. J. Bailey,
G. Baird,
A. Battaglia,
T. Berney,
N. Bolshakova,
S. Bölte,
P. F. Bolton,
T. Bourgeron,
S. Brennan,
J. Brian,
S. E. Bryson,
A. R. Carson,
G. Casallo,
J. Casey,
B. H. Chung,
L. Cochrane,
C. Corsello,
A. Crossett,
C. Cytrynbaum,
M. de Jonge,
R. Delorme,
I. Drmic,
E. Duketis,
F. Duque,
P. Farrar,
B. A. Fernandez,
S. E. Folstein,
E. Fombonne,
C. M. Freitag,
J. Gilbert,
C. Gillberg,
J. Goldberg,
A. Green,
J. Green,
S. J. Guter,
H. Hakonarson,
E. A. Heron,
M. Hill,
R. Holt,
J. L. Howe,
G. Hughes,
V. Hus,
R. Igliozzi,
C. Kim,
S. M. Klauck,
V. Kustanovich,
C. M. Lajonchere,
J. A. Lamb,
M. Laskawiec,
M. Leboyer,
A. Le Couteur,
B. L. Leventhal,
A. C. Lionel,
X. Q. Liu,
C. Lord,
L. Lotspeich,
S. C. Lund,
E. Maestrini,
W. Mahoney,
C. Mantoulan,
C. R. Marshall,
H. McConachie,
J. McGrath,
A. Merikangas,
O. Migita,
G. K. Mirza,
S. F. Nelson,
C. Noakes,
A. Noor,
G. Nygren,
G. Oliveira,
K. Papanikolaou,
J. R. Parr,
B. Parrini,
T. Paton,
A. Pickles,
M. Pilorge,
C. P. Ponting,
A. Poustka,
F. Poustka,
A. Prasad,
J. Ragoussis,
K. Renshaw,
J. Rickaby,
W. Roberts,
K. Roeder,
B. Roge,
M. L. Rutter,
L. J. Bierut,
J. P. Rice,
J. Salt,
K. Sansom,
D. Sato,
R. Segurado,
A. F. Sequeira,
L. Senman,
N. Shah,
V. C. Sheffield,
I. Sousa,
O. Stein,
N. Sykes,
V. Stoppioni,
C. Strawbridge,
R. Tancredi,
K. Tansey,
B. Thiruvahindrapduram,
A. P. Thompson,
S. Thomson,
A. Tryfon,
J. Tsiantis,
H. Van Engeland,
J. B. Vincent,
F. Volkmar,
S. Wallace,
Z. Wang,
C. Webber,
R. Weksberg,
K. Wing,
K. Wittemeyer,
B. L. Yaspan,
L. Zwaigenbaum,
R. M. Cantor,
M. L. Cuccaro,
S. Ennis,
L. Gallagher,
D. H. Geschwind,
M. Gill,
J. L. Haines,
J. Hallmayer,
A. P. Monaco,
J. I. Nurnberger Jr..,
A. D. Paterson,
M. A. Pericak-Vance,
P. Szatmari,
A. M. Vicente,
V. J. Vieland,
E. M. Wijsman,
S. W. Scherer,
C. Betancur
, Functional impact of global rare copy number variation in autism spectrum disorders. Nature 466, 368–372 (2010).
J. Sebat,
B. Lakshmi,
D. Malhotra,
J. Troge,
C. Lese-Martin,
T. Walsh,
B. Yamrom,
S. Yoon,
A. Krasnitz,
J. Kendall,
A. Leotta,
D. Pai,
R. Zhang,
Y. H. Lee,
J. Hicks,
S. J. Spence,
A. T. Lee,
K. Puura,
T. Lehtimäki,
D. Ledbetter,
P. K. Gregersen,
J. Bregman,
V. Jobanputra,
W. Chung,
D. Warburton,
M. C. King,
D. Skuse,
T. C. Gilliam,
K. Ye,
M. Wigler
, Strong association of de novo copy number mutations with autism. Science 316, 445–449 (2007).
L. A. Weiss,
Y. Shen,
J. M. Korn,
D. E. Arking,
D. T. Miller,
R. Fossdal,
E. Saemundsen,
H. Stefansson,
M. A. Ferreira,
T. Green,
O. S. Platt,
D. M. Ruderfer,
C. A. Walsh,
D. Altshuler,
A. Chakravarti,
R. E. Tanzi,
K. Stefansson,
S. L. Santangelo,
J. F. Gusella,
P. Sklar,
B. L. Wu,
M. J. Daly
Autism Consortium, Association between microdeletion and microduplication at 16p11.2 and autism. N. Engl. J. Med. 358, 667–675 (2008).
A. Hogart,
D. Wu,
J. M. LaSalle,
N. C. Schanen
, The comorbidity of autism with the genomic disorders of chromosome 15q11.2-q13. Neurobiol. Dis. 38, 181–191 (2010).
U. Albrecht,
B. M. Cattanach,
C. V. Beechey,
D. Armstrong,
G. Eichele,
A. L. Beaudet
, Imprinted expression of the murine Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nat. Genet. 17, 75–78 (1997).
Y. H. Jiang,
C. M. Atkins,
J. L. Noebels,
J. D. Sweatt,
, Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation. Neuron 21, 799–811 (1998).
T. Kishino,
M. Lalande,
J. Wagstaff
, UBE3A/E6-AP mutations cause Angelman syndrome. Nat. Genet. 15, 70–73 (1997).
S. V. Dindot,
B. A. Antalffy,
M. B. Bhattacharjee,
, The Angelman syndrome ubiquitin ligase localizes to the synapse and nucleus, and maternal deficiency results in abnormal dendritic spine morphology. Hum. Mol. Genet. 17, 111–118 (2008).
Y. Pan,
L. Zhu,
L. Landa,
J. Yoo,
C. Spencer,
I. Lorenzo,
M. Brilliant,
J. Noebels,
, Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3. PLoS One 5, e12278 (2010).
E. J. Weeber,
Y. Elgersma,
A. W. Varga,
Y. Carrasquillo,
S. E. Brown,
J. M. Christian,
B. Mirnikjoo,
A. Silva,
A. L. Beaudet,
J. D. Sweatt
, Derangements of hippocampal calcium/calmodulin-dependent protein kinase II in a mouse model for Angelman mental retardation syndrome. J. Neurosci. 23, 2634–2644 (2003).
G. M. van Woerden,
K. D. Harris,
M. R. Hojjati,
R. M. Gustin,
S. Qiu,
R. de Avila Freire,
E. J. Weeber
, Rescue of neurological deficits in a mouse model for Angelman syndrome by reduction of αCaMKII inhibitory phosphorylation. Nat. Neurosci. 10, 280–282 (2007).
M. Sato,
M. P. Stryker
, Genomic imprinting of experience-dependent cortical plasticity by the ubiquitin ligase gene Ube3a. Proc. Natl. Acad. Sci. U.S.A. 107, 5611–5616 (2010).
K. Yashiro,
T. T. Riday,
K. H. Condon,
A. C. Roberts,
D. R. Bernardo,
R. Prakash,
R. J. Weinberg,
M. D. Ehlers,
B. D. Philpot
, Ube3a is required for experience-dependent maturation of the neocortex. Nat. Neurosci. 12, 777–783 (2009).
M. Scheffner,
J. M. Huibregtse,
R. D. Vierstra,
P. M. Howley
, The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75, 495–505 (1993).
P. L. Greer,
R. Hanayama,
B. L. Bloodgood,
A. R. Mardinly,
D. M. Lipton,
E. C. Griffith,
Z. Waldon,
R. Maehr,
H. L. Ploegh,
S. Chowdhury,
P. F. Worley,
J. Steen,
M. E. Greenberg
, The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating Arc. Cell 140, 704–716 (2010).
S. S. Margolis,
J. Salogiannis,
C. Mandel-Brehm,
Z. P. Wills,
L. Hu,
J. B. Bikoff,
H. Y. Ho,
M. J. Soskis,
M. Sahin,
, EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation. Cell 143, 442–455 (2010).
Y. D. Zhou,
S. Lee,
Z. Jin,
M. Wright,
S. E. Smith,
M. P. Anderson
, Arrested maturation of excitatory synapses in autosomal dominant lateral temporal lobe epilepsy. Nat. Med. 15, 1208–1214 (2009).
M. Landers,
D. L. Bancescu,
E. Le Meur,
C. Rougeulle,
H. Glatt-Deeley,
C. Brannan,
F. Muscatelli,
M. Lalande
, Regulation of the large (~1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn. Nucleic Acids Res. 32, 3480–3492 (2004).
T. J. Bichell,
M. Bubser,
J. Daily,
I. Filonova,
D. Mrelashvili,
A. Y. Deutch,
R. J. Colbran,
K. F. Haas
, Tissue-specific variation of Ube3a protein expression in rodents and in a mouse model of Angelman syndrome. Neurobiol. Dis. 39, 283–291 (2010).
J. N. Crawley
, Mouse behavioral assays relevant to the symptoms of autism. Brain Pathol. 17, 448–459 (2007).
J. Li,
K. Garbett,
K. Mirnics,
P. H. Patterson
, Maternal immune activation alters fetal brain development through interleukin-6. J. Neurosci. 27, 10695–10702 (2007).
T. E. Holy,
Z. Guo
, Ultrasonic songs of male mice. PLoS Biol. 3, e386 (2005).
G. Ehret
, Infant rodent ultrasounds—A gate to the understanding of sound communication. Behav. Genet. 35, 19–29 (2005).
J. Blundell,
C. A. Blaiss,
M. R. Etherton,
F. Espinosa,
K. Tabuchi,
C. Walz,
M. F. Bolliger,
T. C. Südhof,
C. M. Powell
, Neuroligin-1 deletion results in impaired spatial memory and increased repetitive behavior. J. Neurosci. 30, 2115–2129 (2010).
C. M. Powell,
T. C. Sudhof
, Mouse neurexin-1α deletion causes correlated electrophysiological and behavioral changes consistent with cognitive impairments. Proc. Natl. Acad. Sci. U.S.A. 106, 17998–18003 (2009).
R. E. Hammer,
X. Liu,
, A neuroligin-3 mutation implicated in autism increases inhibitory synaptic transmission in mice. Science 318, 71–76 (2007).
H. T. Chao,
H. Chen,
R. C. Samaco,
M. Xue,
M. Chahrour,
J. L. Neul,
S. Gong,
H. C. Lu,
N. Heintz,
M. Ekker,
J. L. Rubenstein,
C. Rosenmund,
H. Y. Zoghbi
, Dysfunction in GABA signalling mediates autism-like stereotypies and Rett syndrome phenotypes. Nature 468, 263–269 (2010).
G. Liu,
S. Choi,
R. W. Tsien
, Variability of neurotransmitter concentration and nonsaturation of postsynaptic AMPA receptors at synapses in hippocampal cultures and slices. Neuron 22, 395–409 (1999).
K. K. Chadman,
M. L. Scattoni,
S. E. Boltuck,
S. U. Gandhy,
, Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice. Autism Res. 1, 147–158 (2008).
M. A. Bangash,
J. M. Park,
T. Melnikova,
D. Wang,
S. K. Jeon,
D. Lee,
S. Syeda,
J. Kim,
M. Kouser,
J. Schwartz,
Y. Cui,
X. Zhao,
H. E. Speed,
S. E. Kee,
J. C. Tu,
J. H. Hu,
R. S. Petralia,
D. J. Linden,
A. Savonenko,
B. Xiao,
P. F. Worley
, Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism. Cell 145, 758–772 (2011).
X. Wang,
P. A. McCoy,
R. M. Rodriguiz,
H. S. Je,
C. J. Kim,
J. Berrios,
J. S. Colvin,
D. Bousquet-Moore,
G. Wu,
B. D. Philpot,
W. C. Wetsel,
Y. H. Jiang
, Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3. Hum. Mol. Genet. 20, 3093–3108 (2011).
J. Peça,
C. Feliciano,
J. T. Ting,
W. Wang,
M. F. Wells,
T. N. Venkatraman,
C. D. Lascola,
Z. Fu,
G. Feng
, Shank3 mutant mice display autistic-like behaviours and striatal dysfunction. Nature 472, 437–442 (2011).
O. Bozdagi,
D. Papapetrou,
D. L. Dickstein,
N. Takahashi,
Y. Kajiwara,
M. Yang,
A. M. Katz,
M. J. Harris,
R. Saxena,
J. L. Silverman,
J. N. Crawley,
Q. Zhou,
P. R. Hof,
J. D. Buxbaum
, Haploinsufficiency of the autism-associated Shank3 gene leads to deficits in synaptic function, social interaction, and social communication. Mol. Autism 1, 15 (2010).
C. E. Bakker,
C. Verheij,
R. Willemsen,
R. van der Helm,
F. Oerlemans,
M. Vermey,
A. Bygrave,
A. T. Hoogeveen,
B. A. Oostra,
E. Reyniers,
K. De Boule,
R. D'Hooge,
P. Cras,
D. van Velzen,
G. Nagels,
J.-J. Martin,
P. P. De Deyn,
J. K. Darby,
P. J. Willems
The Dutch-Belgian Fragile X Consorthium
C. E. Bakker, C. Verheij, R. Willemsen, R. van der Helm, F. Oerlemans, M. Vermey, A. Bygrave, A. T. Hoogeveen, B. A. Oostra, E. Reyniers, K. De Boule, R. D'Hooge, P. Cras, D. van Velzen, G. Nagels, J.-J. Martin, P. P. De Deyn, J. K. Darby, P. J. Willems, Fmr1 knockout mice: A model to study fragile X mental retardation. Cell 78, 23–33 (1994). Medline
M. Shahbazian,
J. Young,
L. Yuva-Paylor,
B. Antalffy,
R. Paylor,
H. Zoghbi
, Mice with truncated MeCP2 recapitulate many Rett syndrome features and display hyperacetylation of histone H3. Neuron 35, 243–254 (2002).
H. G. McFarlane,
G. K. Kusek,
J. L. Phoenix,
V. J. Bolivar,
, Autism-like behavioral phenotypes in BTBR T+tf/J mice. Genes Brain Behav. 7, 152–163 (2008).
S. M. Hamilton,
C. M. Spencer,
W. R. Harrison,
L. A. Yuva-Paylor,
D. F. Graham,
R. A. Daza,
R. F. Hevner,
P. A. Overbeek,
R. Paylor
, Multiple autism-like behaviors in a novel transgenic mouse model. Behav. Brain Res. 218, 29–41 (2011).
L. Shi,
S. H. Fatemi,
R. W. Sidwell,
, Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring. J. Neurosci. 23, 297–302 (2003).
N. R. Dennis,
M. W. Veltman,
R. Thompson,
E. Craig,
N. S. Thomas
, Clinical findings in 33 subjects with large supernumerary marker(15) chromosomes and 3 subjects with triplication of 15q11-q13. Am. J. Med. Genet. A 140, 434–441 (2006).
C. A. Baron,
C. G. Tepper,
S. Y. Liu,
R. R. Davis,
N. J. Wang,
N. C. Schanen,
J. P. Gregg
, Genomic and functional profiling of duplicated chromosome 15 cell lines reveal regulatory alterations in UBE3A-associated ubiquitin–proteasome pathway processes. Hum. Mol. Genet. 15, 853–869 (2006).
K. N. Leung,
D. J. Wu,
J. Driscoll,
R. O. Vallero,
J. M. LaSalle
, Chromosome 15q11–13 duplication syndrome brain reveals epigenetic alterations in gene expression not predicted from copy number. J. Med. Genet. 46, 86–93 (2009).
J. Nakatani,
K. Tamada,
F. Hatanaka,
S. Ise,
H. Ohta,
K. Inoue,
S. Tomonaga,
Y. Watanabe,
Y. J. Chung,
R. Banerjee,
K. Iwamoto,
T. Kato,
M. Okazawa,
K. Yamauchi,
K. Tanda,
K. Takao,
T. Miyakawa,
A. Bradley,
T. Takumi
, Abnormal behavior in a chromosome-engineered mouse model for human 15q11-13 duplication seen in autism. Cell 137, 1235–1246 (2009).
A. Kashiwagi,
M. Meguro,
H. Hoshiya,
M. Haruta,
F. Ishino,
T. Shibahara,
M. Oshimura
, Predominant maternal expression of the mouse Atp10c in hippocampus and olfactory bulb. J. Hum. Genet. 48, 194–198 (2003).
K. Mitsuya,
M. Nakao,
I. Kondo,
S. Saitoh,
, A novel maternally expressed gene, ATP10C, encodes a putative aminophospholipid translocase associated with Angelman syndrome. Nat. Genet. 28, 19–20 (2001).
T. Kayashima,
K. Yamasaki,
K. Joh,
T. Yamada,
T. Ohta,
K. Yoshiura,
N. Matsumoto,
Y. Nakane,
T. Mukai,
N. Niikawa,
T. Kishino
, Atp10a, the mouse ortholog of the human imprinted ATP10A gene, escapes genomic imprinting. Genomics 81, 644–647 (2003).
Y. Nishimura,
C. L. Martin,
A. Vazquez-Lopez,
A. I. Alvarez-Retuerto,
M. Sigman,
C. Steindler,
S. Pellegrini,
S. T. Warren,
, Genome-wide expression profiling of lymphoblastoid cell lines distinguishes different forms of autism and reveals shared pathways. Hum. Mol. Genet. 16, 1682–1698 (2007).
R. P. Nagarajan,
K. A. Patzel,
D. H. Yasui,
, 15q11-13 GABAA receptor genes are normally biallelically expressed in brain yet are subject to epigenetic dysregulation in autism-spectrum disorders. Hum. Mol. Genet. 16, 691–703 (2007).
J. D. Shepherd,
G. Rumbaugh,
N. Plath,
D. Kuhl,
R. L. Huganir,
, Arc/Arg3.1 mediates homeostatic synaptic scaling of AMPA receptors. Neuron 52, 475–484 (2006).
K. Miura,
E. Li,
H. Webber,
P. Dikkes,
G. L. Holmes,
, Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice. Neurobiol. Dis. 9, 149–159 (2002).
A. Mishra,
N. R. Jana
, Regulation of turnover of tumor suppressor p53 and cell growth by E6-AP, a ubiquitin protein ligase mutated in Angelman mental retardation syndrome. Cell. Mol. Life Sci. 65, 656–666 (2008).
S. A. Mulherkar,
J. Sharma,
, The ubiquitin ligase E6-AP promotes degradation of α-synuclein. J. Neurochem. 110, 1955–1964 (2009).
F. Ferdousy,
W. Bodeen,
K. Summers,
O. Doherty,
O. Wright,
N. Elsisi,
G. Hilliard,
J. M. O'Donnell,
L. T. Reiter
, Drosophila Ube3a regulates monoamine synthesis by increasing GTP cyclohydrolase I activity via a non-ubiquitin ligase mechanism. Neurobiol. Dis. 41, 669–677 (2011).
L. T. Reiter,
T. N. Seagroves,
M. Bowers,
E. Bier
, Expression of the Rho-GEF Pbl/ECT2 is regulated by the UBE3A E3 ubiquitin ligase. Hum. Mol. Genet. 15, 2825–2835 (2006).
L. Hicke,
R. Dunn
, Regulation of membrane protein transport by ubiquitin and ubiquitin-binding proteins. Annu. Rev. Cell Dev. Biol. 19, 141–172 (2003).
D. Mukhopadhyay,
H. Riezman
, Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315, 201–205 (2007).
Z. Nawaz,
D. M. Lonard,
C. L. Smith,
E. Lev-Lehman,
S. Y. Tsai,
M. J. Tsai,
B. W. O'Malley
, The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol. Cell. Biol. 19, 1182–1189 (1999).
S. Ramamoorthy,
Z. Nawaz
, E6-associated protein (E6-AP) is a dual function coactivator of steroid hormone receptors. Nucl. Recept. Signal. 6, e006 (2008).
Acknowledgments: We thank X. Wang and O. DiStefano for technical assistance, D. Brown for electron microscopy technical assistance, J. N. Crawley for information on FVB mouse vocalizations, M. W. Anderson for comments on the manuscript, and E. Maratos-Flier for providing behavioral equipment and advice. Funding: This work was supported in part by the U.S. National Institute of Neurological Disorders and Stroke grants R21NS070295, R01 NS057444, and K02 NS054674-03 (M.P.A.); Nancy Lurie Marks Family Foundation (M.P.A.); Autism Speaks/U.S. National Alliance for Autism Research (M.P.A.); U.S. National Institute of Mental Health National Research Service Award Fellowship F32 MH087085 (S.E.P.S.); and Beth Israel Deaconess Medical Center. Author contributions: M.P.A., S.E.P.S., and Y.-D.Z. designed the study. M.P.A. and S.E.P.S. wrote the paper. S.E.P.S., Y.-D.Z., G.Z., and M.P.A. analyzed the data. S.E.P.S. did behavioral and protein studies. Y.-D.Z. performed all electrophysiology experiments except miniature postsynaptic currents and action potential coupling experiments done by G.Z. Z.J. engineered the BAC constructs. D.C.S. did the synaptic density measures. Competing interests: The authors declare that they have no competing interests. A patent has been filed by Beth Israel Deaconess Medical Center on the animal model of autism reported in this paper with M.P.A. as inventor.
You are going to email the following Increased Gene Dosage of Ube3a Results in Autism Traits and Decreased Glutamate Synaptic Transmission in Mice
By Stephen E. P. Smith, Yu-Dong Zhou, Guangping Zhang, Zhe Jin, David C. Stoppel, Matthew P. Anderson
Science Translational Medicine 05 Oct 2011 : 103ra97
Tripling the dosage of an autism- and Angelman syndrome–related gene in mice results in reduced glutamatergic synaptic transmission and autism behavioral traits.

redpajama_set_name: RedPajamaCommonCrawl


text:
From 1G to 5G: How innovations in cellular have shaped our lives
Article by: Gigamon CTO Shehzad Merchant.
Each generation of cellular networks has fundamentally changed our world. 1G brought us the brick phone utilising analogue technology in the '80s. For the first time, phones became truly mobile, albeit being bulky and expensive, and limited to voice features.
In the '90s, 2G brought us digital handsets with SMS and text messaging, which made communication on-the-go possible. Phone sizes and prices shrank, and adoption grew, although mostly for business and professional use.
In the early 2000s, 3G brought us online with mobile broadband integrating voice, video and data. 3G made it popular for not just adults but also teens, to use cell phones, driving mass adoption.
The decade from 2010 to 2020 has been characterised by the use of 4G, which significantly increased the speed and capacity of cellular networks. It gave rise to an entirely new generation of applications, which fundamentally changed how we do business and communicate.
The rise of ride-sharing services, live-streaming video, and a host of other applications were all possible due to 4G. Entire industries such as the gig economy were created as a result of 4G. During the pandemic this year, 4G has made remote learning and telemedicine a reality.
As we look to the present decade from 2020 onwards, 5G will be at the forefront. The race for 5G is not about merely deploying new infrastructure, but getting the first-mover advantage in who can build and take the leadership role in the host of new applications and services that 5G will enable.
It is this race that has captured the minds of all – from nation-states to entrepreneurs.
5G brings with it significant changes. It provides improvements in many ways – up to 10x faster than 4G, up to 10 times lower latency than 4G, up to 100 times more devices than 4G, and up to 90% lower energy consumption compared to 4G.
Here are the new technologies that will be required to work in tandem with 5G for rollout to be successful:
Millimetre-wave
This is an entirely new band of spectrum being opened up for 5G – from 6GHz all the way up to 300GHz. It is a higher frequency spectrum that can drive higher capacity as well as higher bandwidth.
Initially, only specific bands within this spectrum will be utilised, in conjunction with lower and mid-frequency bands from 600MHz to 3GHz.
Massive MIMO
MIMO stands for multiple input, multiple output and refers to the antenna technology that is utilised to drive better coverage.
The higher frequency bands provide an advantage in that they require smaller antennas. This makes it feasible to pack more antennas on a base station, which in turn drives much higher capacity. 5G will be able to pack around 256 antennas per base station, significantly higher than the dozen or so with 4G.
Small cells and beamforming
Higher frequencies lead to higher propagation loss. With the higher density of MIMO antennas, there can also be increased signal interference. And so the goal in 5G is to develop many smaller cells that would then be served by the high-density MIMO antennas.
The smaller cell size addresses the issues around propagation loss. At the same time, beamforming technology allows the 5G signals to minimise interference by selectively targetting devices.
Network slicing
5G will support network slicing, which is the ability to carve off portions of the 5G network for specific use cases. For example, carving off a slice for first responders, or carving off slices for ultra-low latency applications such as autonomous driving.
Mobile edge computing
This is the key to reducing end-to-end latency for applications. With mobile edge computing, applications can run in data centres closer to the 5G network, thereby significantly reducing the end-to-end latency of applications.
Control and user plane separation (CUPS)
By separating the user data from the control plane processing, user plane traffic can be switched locally without having to backhaul traffic, thereby lowering latency and increasing throughput.
The journey to 5G will require careful planning both on the network operator side and the companies leveraging and building applications over a 5G infrastructure.
Ensuring visibility into the applications and the underlying network traffic will be vital in determining whether SLAs are being met for critical application needs that depend on aspects such as guaranteed latency, among others. Planning for this upfront will help speed up the journey and reduce delays in deploying both the infrastructure and the applications.
As with each generation of cellular technology, 5G deployment and adoption will occur over an extended period, perhaps several years. But with it will come an entirely new breed of applications and services that can take advantage of the lower latency, higher speeds and additional 5G capabilities.
Yes, we will be able to download movies much faster - it may take only 5-10 seconds for a full HD movie download. But that is a very limiting way to think about 5G. New applications, like fully autonomous driving, drone-based emergency response, customised multi-dimensional content-rich news delivery, and many more advancements, become possible with 5G.
As these new applications start taking root, many of the traditional paradigms and businesses may begin seeing disruption. Organisations should look to the future in terms of disruption rather than incrementalism when thinking of how to take advantage of these aspects of 5G.
Frost & Sullivan: "Massive growth" ahead for 5G enterprise market
F5 to pay US$500m for edge app platform vendor
Vertiv anticipates data centre 'fourth utility' criticality in 2021
5G flaws allow criminals to steal data, cut access to the web
5G 5G Networks 4G Edge computing 5G infrastructure
ICT trends: What to expect in 2021
It's no secret that the large-scale transition to remote working has sent ripples through every area of ICT. The reality is, this abrupt move has impacted every business, person and household.More
Advancing the emerging technologies of robotic process automation (RPA), advanced AI and more hinge on achieving these competencies, Gartner says.More
ThoughtWorks announces $720 million in investment
The global software consultancy received funds from GIC, Siemens AG, Fidelity Management, Research LLC and Mubadala Investment Company.More


The Dashboard is main area for players as it showcases most of their need to know information.
As the Dashboard is key to squadSet, it will be continually improved to streamline management and your personalize tastes.
The Dashboard is unique to every Player.
A centralized place to view and manage your personal information and get updates.
In addition to the six main area, the Dashboard also offers a snapshot of other areas such as recruitment, contacts, and more.
The Dashboard offers a centralized area not only to view and interact with information but also a way to navigate to more information.


Portrait of Odit Washington. As a young man he was given to James Imbrie, and he died on the Yakima Indian Reservation.
[Front] [stamped] T. J. Ticknet's Co., Yakima, Washington. [Back] [ink] Washington, Odit. With Col. Cornelius in Yakima War, 1856. [pen] This picture was taken about 1900. This Indian boy was given to James Imbrie by the Indians in the Sacramento Valley when he left California to come to Oregon. He lived with the Imbrie family in his young manhood. Died in Yak'?m [sic] on the Indian Reservation.


Memorial in memory of 14 young men from Hvalba and Sandvík, who lost their lives when two boats capsized during a whal hunt in Sandvík in 1915.
Boats are driving a pod of pilot whales into the bay of Vágur in August 2012.
This page was last edited on 23 November 2014, at 04:51.


Thank you for visiting Roter's World.
If you are interested in purchasing any of the pictures featured on this site, please feel free to drop us a line. If you would just like to say "Hi" or make a general comment on Roter's World, we welcome you to do so.


You can customize the bike type, component selection, colors, graphics and geometry of your CIRCA.
The process for getting your own custom CIRCA is centered around personal attention. From start to finish, you'll work directly with our team to develop your bike.
The first step is to find out what type of bike you'd like to build.
Proper fit enhances comfort and performance, leading to a superior cycling experience. Although most people fit well on our stock sizes, we also offer custom geometry when required.
This custom geometry, custom graphics road build was optimized for a petite rider that required an unusually low standover height.
Color is a great way to give your bike personality. In a CIRCA design session, you can pick from a fun palette of durable anodized finishes and get creative with how colors are applied to different parts of the frame to create a bike that's distinctly yours.
Our laser graphics packages range from simple minimalist designs to full custom storytelling.
CIRCA's versatile dropout can handle a variety of drivetrain options, including the Gates Carbon Drive belt system. We'll work with your functional needs and budget to get you going just right.
This space gray road bike with red accents features laser graphics and locally built Chris King hubs.
And finally, there are all of the little things, just how you like 'em. From saddle selection to rain protection and lighting we'll make sure your CIRCA is customized just for you.
Design Brief and Estimate. After your design session, we'll create a design brief that outlines pricing and all of the details of your new bike, including a sketch that ensures that everyone's on the same page.
If you're in Portland, once construction is complete we'll schedule a final fit and delivery session in our studio, where we'll unveil your new bike, receive final payment and check and adjust the fit. You'll take a test spin and we'll review any other important details. And then you're off! If you can't make it to Portland, we have a range of solutions for shipping and final fit.
At CIRCA, we work with you personally to develop a custom bike solution that fits perfectly, looks incredible and delivers the performance you need. CIRCA isn't just a bike shop. We're a unique manufacturer with a core centered around our proprietary, hyper-localized frame building system. From saddle to drivetrain, you're in control. During your private design session in our modern studio, we'll fine tune everything about your bike. We can also source a wide range of component options, from the big guys like Shimano and Campagnolo to the local heroes like Chris King, Paul's and White Industries.
During your bike design sessions, you'll work directly with CIRCA Founder + Designer Rich Fox. Rich will guide you through the design process and help you create your dream custom bike. We begin by discussing the basics like riding style, budget and any other priorities you might have. From there, we move into the details of function, fit and aesthetics, including taking detailed measurements to ensure that your final fit is perfect. The most efficient way to do this is in person at our studio in Northwest Portland, but we can also work remotely, via email or phone.
Laser Graphics: Priced by design. Please inquire for details.


Insurance may vary by location. Please select your state of practice to get started.
Insurance to help you work with confidence.
Insurance to help you live with certainty.
Protection for what's truly important.
You have successfully subscribe to our monthly posts.
As a lawyer, you're fully committed to your clients. You aim to represent them well and fight for their future. That's a huge responsibility. And chances are, you don't take it lightly.
We don't take our responsibilities lightly either.
With customized products ranging from professional liability insurance to cyber coverage, our commitment is to you. Explore our product offerings below and get a free, no-obligation quote today.
Protect your career against claims of negligence and more.
Fill out the form below to talk with a Pearl Insurance representative.


text:
Q: Wix: Can I impose a condition on a RemoveFolderEx element to only run when the program is being uninstalled? I am trying to delete a folder hierarchy using the RemoveFolderEx but only when I'm uninstalling the software.  Currently when I do a reinstall it deletes all the folders as well which deletes all the program created content which is undesirable. This is probably because I've set the software to be uninstalled before being reinstalled
<InstallExecuteSequence>
    <RemoveExistingProducts Before="InstallInitialize" />
</InstallExecuteSequence>

Is it possible to impose a condition on the following element?  Should I wrap it in it's own component then add a condition inside?  I've not been able to get it to work thus far.
<util:RemoveFolderEx On="uninstall" Property="MAINDIR" ></util:RemoveFolderEx>

From another question I know the condition has to be
((NOT UPGRADINGPRODUCTCODE) AND (REMOVE="ALL"))

but i'm not quite 100% on how to apply this to the removefolder element if it's even possible.
I guess I could set the path of the folder to empty on a custom action which was subject to the condition but this just seems hacky.
Cheers.
Neil

EDIT: I've almost got this working by putting the removefolderex into its own component.  Without the condition it works as expected and removes the data directory on uninstall and a reinstall.  When I add the condition in it doesn't run but I'm hoping that this is down to the condition being wrong.
<DirectoryRef Id="DATADIR">
    <Component Id="C.RemoveDataFolder" Guid="myguid" KeyPath="yes">
        <util:RemoveFolderEx On="uninstall" Property="DATADIR" ></util:RemoveFolderEx>
        <Condition>(NOT UPGRADINGPRODUCTCODE) AND (REMOVE="ALL")</Condition>
    </Component>
</DirectoryRef>

Does anyone know if the above declaration ok or will this never work?  Can I apply this kind of condition to a component?

A: RemoveFolderEx is tied to a component, so if it's being removed and RemoveFolderEx/@On="uninstall" then RemoveFolderEx will do its thing. There's no support for adding another condition, but that seems reasonable; please file a feature request so it's on the todo list.

A: The component condition controls whether a component is installed. This condition is false on install thus the component is not installed. Naturally on uninstall it doesn't get removed, thus the remove operation doesn't work. I would go with a custom action that sets the target property conditionally.

redpajama_set_name: RedPajamaStackExchange


Thanks heaps for this post!!!!
Just bought one and was annoyed to find 2 drives. Solved my problem without having to return.
That's awesome i can't thank you enough…. i have used every program i could find out about, trying to fix this issue, everything from partition magic, to acronis true image 2013 to gparted… Thanks again!
Greetings from South Africa, this was also what i was looking for so big thanks for sharing the solution. I am sharing this with others so they know aswell with a link back here ;-).
Hi there, I didn't have any problem with the hard drive but yesterday when I had used the hard drive it was working and all of a sudden it just went off, I checked the plug points and even unpluging the cord from the device and plugging it back on but still it just refuses to come on. I have alot of movies stored on it and some of it which I haven't saved as yet so I am really worried now about all the data i have on it and it just refuses to come on, did anyone encounter this problem and how can I fix it?
Thanks Markus, I have tried that so I guess the hdd's power is shot ;-(!!!!
Hi, there! Greetings from Portugal!
Thanks for the tip, Markus, I will try it.
at 26ºC room temperature, both "HD Tune" and "Hard Disk Sentinel" are reporting 48ºC.
Yesterday, without even using it for reading/writing, temperatures got to 52ºC.
well i live in Europe and the temperature here is cold so i don't have that heat problem. maybe you can try buying small fan to keep the harddisk cool?
Thanks a lot, it's been hours that I'm looking for a soluce, and here you are ! Really, thank you !
Thanks a lot guys. Drove me crazy. Also works without any Problems with the 4 TB Version!
Thanks a lot, your solution works!!!!
Whenever I try to start the Verbatim 3TB Mode Changer program I'm greeted with a window proclaiming i need to update Adobe Flash Player for Internet Explorer.
Do I need to have Internet @£$#€ Explorer installer for this to work?
Yes – thanks for this post – I've had my drive sitting around for a couple of years now as I couldn't get it to work. Now I can use it as the mass backup drive I bought it for.
The only solution that works!
The software must have been removed from the Aussie site now.
Can someone share it from a cloud share or private message, please ?


text:
New York State Retirement Fund – invested in Wynn Resorts – sues Wynn Board of Directors for ignoring CEO Steve Wynn's alleged sexual abuses
The New York State Common Retirement Fund filed a lawsuit against the Board of Directors of Wynn Resorts Ltd.
The complaint, filed by New York State Comptroller Thomas P. DiNapoli, as Trustee of the Retirement Fund, alleges that officers and directors of Wynn Resorts were aware that former CEO Stephen Wynn, 76, made unwanted sexual advances and pressured employees to perform sex acts yet failed to hold him accountable.
The lawsuit was filed in Nevada's Eighth Judicial District's Business Court.
"Wynn Resorts' Board members were complicit in Steve Wynn's pervasive pattern of sexual abuse and harassment, setting a tone at the top that permitted Steve Wynn to wield his power unchecked," the complaint alleges.
DiNapoli said "[The] board was loyal to Mr. Wynn at the expense of the safety of the company's employees and the company's shareholders."
Founded in 2000, Wynn Resorts is a developer, owner, and operator of casino resorts that integrate hotel accommodations and dining, retail, entertainment theaters, and meeting complexes. The company owns and operates Wynn Las Vegas and Encore in Las Vegas, Nevada, and Wynn Macau and the Wynn Palace in China.
A luxury retail Strip-front expansion, Wynn Plaza, is under construction in Las Vegas and is scheduled to debut the third quarter of 2018. Wynn Resorts is currently constructing Wynn Boston Harbor located in Everett, Massachusetts.
According to the lawsuit, the board's failure to act was a breach of fiduciary duties to the company and seeks to have Wynn Resorts' take "necessary actions to … protect Wynn Resorts, its employees, and its stockholders from repeating the harms described" in the complaint.
The NY's Retirement Fund holds $30 million in Wynn Resorts stock [183,800 shares] which is 1.5/10th of one percent of Wynn stock [0.15%]
Wynn stock holdings comprises about 1.5/one hundredth [0.015%] of the Retirement Fund's $200 billion in assets.
The lawsuit alleges the board knew about Wynn's behavior since at least 2016, when allegations surfaced in litigation involving the company and Wynn's ex-wife Elaine Wynn.
Wynn resigned in February in response to a Wall Street Journal article published on January 26, 2018. The article detailed Wynn's alleged history of sexual predation at Wynn Resorts, including an alleged sexual assault of a Wynn Resorts employee on company property in 2005. Later reports revealed Wynn created a clandestine limited liability company, Entity Y, to conceal a $7.5 million settlement paid to the alleged assault victim in 2005.
The Wall Street Journal article reported that Wynn is known for a decades-long pattern of sexual abuse and harassment. Former Wynn Resorts employees described an atmosphere of terror at the company, and several claimed their repeated efforts to notify senior management fell on deaf ears.
The text of the complaint, filed in District Court in Clark Co., Nevada, can be read here http://osc.state.ny.us/press/docs/wynn-complaint.pdf
The action the state is pursuing is termed a "derivative lawsuit" which allows shareholders to take legal action on behalf of a corporation when a company's officers or directors have not met their obligations as fiduciaries of the company.
After experiencing a steady increase in the price of Wynn stock in the year leading up to the public exposure of Wynn, going from under $100 per share to over $200 per share, the stock plunged to about $160 after the public scandal was reported in late January and has been holding steady – trading at $163.20 per share as of the time of publication.
The current Board members are named defendants as well as Wynn and Kimmarie Sinatra, the company's legal counsel and senior vice president.
The board consists of John J. Hagenbuch, Ray R. Irani, Jay L. Johnson, Robert J. Miller, Patricia Mulroy, Clark T. Randt, Jr., Alvin V. Shoemaker, J. Edward Virtue, and D. Boone Wayson.
Wynn currently owns 11.8% of the stock of the company. As of January 25, 2018, that stock had a market value of approximately $2.4 billion.
Two outside law firms – one in New York: Cohen Milstein Sellers & Toll PLLC and one in Nevada: Wolf, Rifkin, Shapiro, Schulman & Rabkin, LLP will represent New York State.
It is unknown how much the lawsuit will cost New York State taxpayers in legal fees.
Lawyers for the two firms are: Nevada: Don Springmeyer, Bradley S. Schrager.
New York: Julie Goldsmith Reiser, Kalpana Kotagal, Elizabeth A. Aniskevich, Eric S. Berelovich.
New York State lawsuit against Wynn Resorts paints gruesome picture of Wynn as predator/opportunist and board as well-rewarded & aware
1/3 of New York State University Foundations operating without oversight, says Comptroller


I'm not much of an Instagramer lately… Maybe it's the way time seems to be flying by or maybe I've not been "into it" lately. I don't know. I didn't even bother putting these pups together.
I have six tabs open on my Safari browser right now and I have so much to do but instead I'm watching JASMINE STAR talk on the Creative Live. I capitalized her name because she's just extraordinarily fabulous! And six tabs doesn't mean that all of them are supposed to be open. I'm just procrastinating and I hate it. I have emails, paperwork, and errands to run and I'm stuck without a car. I don't know about you but when I have a To-do list I always try to do the things that sound more fun to me first and then focus on the other things. So in this case running errands sounds waaaay more fun but since I have no car at the moment, I'm slacking off on doing the rest of my list until I do the fun thing. That's terrible I know.
Remember the 500 words a day? Well I haven't been doing it on the blog but I've been doing it on a word document app that I snagged off of the Apple Market Place and it's working really well. I started to time myself and write for 10 minutes straight, no stopping and it has been an extraordinary experience. I've learned things about myself that I've never knew to be there. For example : In the mornings I type two words slower and have more spelling errors per minute than at any other time of the day. Pretty interesting stuff. And I've learned how my brain jumps from subject to subject within seconds. While I'm finishing one sentence I'm already thinking what my next sentence's subject will be. So weird. But it has been quite a successful way of learning who I am as a writer, photographer, and person. You should try.


text:
Fernando Saraiva Reis (São Paulo, 10 de março de 1990) é um halterofilista do Brasil. Ele ficou em 5° lugar nos Jogos Olímpicos da Rio-2016, ganhou uma medalha de bronze no Campeonato Mundial de 2018 e é tricampeão dos Jogos Pan-Americanos, além de recordista das Américas na categoria acima de 105 kg e, após a reestruturação das categorias de peso em 2018, na categoria acima de 109 kg.

Carreira
Fernando participou dos Jogos Pan-Americanos de 2007 e ficou em décimo lugar (último), na categoria até 94 kg, com 304 kg no total combinado (140 no arranque e 164 no arremesso).

Nos Jogos Sul-Americanos de 2010, Fernando ficou com a medalha de prata, na categoria acima de 105 kg.

No campeonato mundial para juniores (até 20 anos) de 2010, em Sofia, Bulgária, Fernando competiu na categoria acima de 105 kg e conquistou a primeira medalha do Brasil nessa competição — o bronze no arranque, com 171 kg. Ele terminou em quarto no total combinado (372 kg).

Participou do Campeonato Mundial de 2010, aberto, sem limitação de idade, e ficou em 26º lugar.

2011
Em 10 de abril de 2011, Fernando venceu o National Collegiate Championships, em Shreveport, na categoria acima de 105 kg, com 385 kg no total combinado (175 no arranque e 210 no arremesso), todas essas marcas que foram novos recordes brasileiros, mas foi pego no doping, ele testou positivo para a substância metilhexanamina.

Mas ele pode competir nos Jogos Pan-Americanos de Guadalajara, em que conquistou a medalha de ouro (primeira do país na modalidade) ao levantar 410 kg no total (185 kg no arranque e 225 no arremesso), quebrando os recordes dos Jogos Pan-Americanos.

2012
Em 2012, participou dos Jogos Olímpicos de Londres, na categoria acima de 105 kg. Na prova do arranque, fez 178 kg na primeira tentativa, e 180 na segunda. Na terceira, tentou bater seu recorde de 185 kg, mas falhou na tentativa de 186 kg. Já na prova do arremesso, começou bem, fazendo 220 kg na primeira tentativa. Quando tentou os 225 kg, as faixas que protegiam a musculatura de seus joelhos se soltaram. Fernando sofreu uma lesão, não conseguiu levantar os 225 kg e desistiu da última tentativa. Com isso, teve o resultado total de 400 kg. Terminou em quarto no grupo B, e em 12º lugar geral inicialmente, mas foi realocado para 11º após a desclassificação do bielorusso Yauheni Zharnasek por doping em 2016.

2015
Em 2015, Fernando Reis se tornou bicampeão dos Jogos Pan Americanos no levantamento de peso, categoria mais de 105 kg masculino com 427 kg (192+235 kg), recorde do Pan e recorde brasileiro. No Campeonato Mundial de 2015, levantou 425 kg (195 kg + 230 kg) e ficou com a 10ª colocação.

2016
Nos Jogos Olímpicos de Verão de 2016 no Rio de Janeiro, melhorou seu desempenho, terminando em 5º lugar batendo o recorde das 3 Américas (195 kg no arranque e 240 kg no arremesso, total de 435 kg).

2017
No Mundial de 2017, Reis levantou 200 kg na competição de arranque, ficando em 4º lugar nesta prova, e batendo o recorde da América. No arremesso, levantou 240 kg, terminando em sexto lugar. Na competição do total geral, a mais importante, terminou em sexto lugar, com 440 kg, recorde da América e melhor resultado da história do Brasil na competição.

2018
No Pan-Americano de Levantamento de Peso de 2018, ele novamente quebrou seu recorde da América do arranque, levantando 201 kg. No arremesso fez 235 kg, e no total geral, 436 kg, ganhando ouro nas três provas.

No Campeonato Mundial de 2018, realizado em novembro, Reis obteve um histórico 4º lugar para o Brasil no total geral, a melhor posição do país de todos os tempos, com a marca de 436 kg (201 kg no arranco e 235 kg no arremesso). Ele ficou muito perto de obter a medalha de bronze, quando chegou a levantar o peso a 245 kg no arremesso, porém os juízes não consideraram o levantamento válido por causa da posição dos braços. Em 20 de março de 2021, a Federação Internacional de Halterofilismo anunciou que os resultados do uzbeque Rustam Djangabaev no Campeonato Mundial de 2018 foram anulados. Reis foi então promovido à medalha de bronze no total, e em quarto lugar tanto no arranque quanto no arremesso. Essa foi a primeira medalha do Brasil na história do Campeonato Mundial de Halterofilismo.

2019
No Pan de Lima 2019, apesar de ainda estar se recuperando de uma cirurgia no joelho, realizada no final de 2018, ele obteve facilmente o tricampeonato, erguendo 420 kg no total, contra 399 do atleta que obteve a prata.

2021
Fernando ganhou o quarto título no Campeonato Pan-Americano de Santo Domingo em abril e estava classificado para os Jogos Olímpicos de Tóquio. Ele era um dos favoritos de medalha na categoria acima de 109 kg, principalmente pela ausência de atletas de países do Leste Europeu e da ex-União Soviética, que tiveram muitos casos de doping, e como punições, tiveram reduções de vagas, mas Fernando perdeu a vaga após testar positivo para hormônio do crescimento, em exame realizado no dia 11 de junho e foi suspenso oficialmente das Olímpiadas. Ele negou que tenha tomado essas substâncias proibidas e afirmou que um nódulo no cérebro estaria causando a liberação de níveis elevados de hormônio, depois de ter feito exames.
Ele desistiu de se defender da acusação de doping e anunciou sua aposentadoria do levantamento de peso ainda em 2021.

Quadro de resultados

NM = Sem marca (No mark)
DNS = Não largou (Did not start)
NA = Não disponível (No available)
DSQ = Desclassificado (Disqualified)
 *Edição de 2020 realizada em 2021.
Notas
Resultado não oficial
Recorde pan-americano não oficial

Ligações externas
freis.net.br
Site pessoal

Halterofilistas de São Paulo
Halterofilistas nos Jogos Olímpicos de Verão de 2012
Naturais da cidade de São Paulo
Halterofilistas do Brasil nos Jogos Pan-Americanos de 2015
Desportistas do Brasil nos Jogos Pan-Americanos de 2011
Halterofilistas olímpicos do Brasil
Atletas de força de São Paulo

redpajama_set_name: RedPajamaWikipedia


The two curves are clearly quite different and, if the results are to be fitted to the HETP equation, only the data obtained using the exit velocity will give meaningful values for the exclusive dispersion processes. This problem is further emphasized in the graphs shown in figure 10. In figure 10, the individual contributions from the different dispersion processes are obtained by deconvoluting the HETP curve obtained using the average velocity data.