UBE3A encodes ubiquitin protein ligase E3A, and in neurons its expression from the paternal allele is repressed by the UBE3A antisense transcript (UBE3A-ATS). This leaves neurons susceptible to loss-of-function of maternal UBE3A. Indeed, Angelman syndrome, a severe neurodevelopmental disorder, is caused by maternal UBE3A deficiency. A promising therapeutic approach to treating Angelman syndrome is to reactivate the intact paternal UBE3A by suppressing UBE3A-ATS. Prior studies show that many neurological phenotypes of maternal Ube3a knockout mice can only be rescued by reinstating Ube3a expression in early development, indicating a restricted therapeutic window for Angelman syndrome. Here, we report that reducing Ube3a-ATS by antisense oligonucleotides in juvenile or adult maternal Ube3a knockout mice rescues the abnormal electroencephalogram (EEG) rhythms and sleep disturbance, two prominent clinical features of Angelman syndrome. Importantly, the degree of phenotypic improvement correlates with the increase of Ube3a protein levels. These results indicate that the therapeutic window of genetic therapies for Angelman syndrome is broader than previously thought, and EEG power spectrum and sleep architecture should be used to evaluate the clinical efficacy of therapies. UBE3A gene encoding ubiquitin protein ligase E3A . UBE3A-ATS is expressed from the paternally inherited chromosome and localized in the nucleus to repress UBE3A in cis through a transcriptional collision mechanism  . It is cgase E3A . UBE3A i neurons . Imprintechanism . Thus, lg defect .Ube3a loss-of-function mutations have provided insights into Angelman syndrome mechanisms and identified therapeutic strategies. Many disease relevant phenotypes were reported in these Angelman syndrome models  expressing CRISPR/Cas9 to newborn, but not older, maternal Ube3a knockout mice rescues a subset of phenotypes , altered EEG power spectrum, and sleep disturbance. Alterations in EEG power spectrum, particularly an increase in the power of low frequency oscillations , are well documented in both Angelman syndrome patients and rodent models . This brUbe3a-ATS-targeted ASOs in a mouse model of Angelman syndrome. The widely used mouse model is a Ube3a knockout allele  that deletes exon 5 (previously named as exon 2), resulting in a premature stop codon in exon 6  and homozygous (mΔe5/pΔe5Ube3a) mutant mice at a level comparable to wild-type (WT) mice  carries a premature stop codon in exon 7 (mΔe6/p+Ube3a) showed that Ube3a mRNA was diminished in the brain and reduced in the liver as compared to WT mice (mΔe6/p+Ube3a mice were 2–17% of those in WT mice when they were at 6 weeks of age or older (see below). Thus, both Ube3a mRNA and proteins are diminished in the mΔe6/p+Ube3a mouse brains. Furthermore, mΔe6/p+Ube3a mice showed similar rotarod and marble burying phenotypes to previously reported deficits in mΔe5/p+Ube3a mice  mice , possiblΔe5 mice . AlthougΔe5 mice , we sougn exon 7 . RT-ddPC WT mice . Western/p+ mice .Ube3a in mice, we used two mouse-specific ASOs to downregulate the Ube3a-ATS levels. The first one (Ube3a-as) is complementary to a region downstream of the Snord115 small nuclear RNA cluster, and this sequence was also targeted by the ‘ASO B’ used in a previous study (Snord115 RNAs. We first tested these two ASOs in 8-week-old WT mice by administering them via intracerebroventricular (ICV) injection. Both Ube3a-as ASO and Snord115 ASO decreased Ube3a-ATS transcripts and increased Ube3a mRNA levels  carrying a yellow fluorescent protein (YFP)-tagged Ube3a  exhibited strong Ube3a-YFP in the brain, whereas m+/pYFPUbe3a mice receiving the control ASO showed little expression . Brain tissues were harvested from eight different regions of both hemispheres at 3, 6, and 10 weeks post ASO injection . At 3 weeks post ASO injection, Ube3a-as ASO and Snord115 ASO also significantly reduced Ube3a-ATS levels and increased Ube3a mRNA and Ube3a protein levels in mΔe6/p+Ube3a mice , all of which can be examined by chronic video-EEG and electromyogram (EMG) recordings. Thus, to determine if upregulation of paternal Ube3a expression can reverse these phenotypes in mΔe6/p+Ube3a mice, we injected male and female mΔe6/p+Ube3a mice with control, Ube3a-as, or Snord115 ASO and their sex- and age-matched WT littermates with control ASO in parallel at the juvenile  or adult  age. Intracranial EEG from the frontal, somatosensory, and visual cortices and EMG from the neck muscles of each mouse were recorded at 3, 6, and 10 weeks post ASO injection /γ2 can be a robust measure of the effects of Ube3a-as and Snord115 ASOs.We first removed artifacts and then computed the absolute power spectral densities (PSDs) of EEG signals including all brain states . To cont WT mice . Thus, wmepoints . Similarmepoints , but the/p+ mice . These rmΔe6/p+Ube3a mice with Ube3a-as ASO at the juvenile age caused a decrease of the power in the alpha, low beta, and high beta bands and an increase of the power in the high gamma band, particularly at 3 and 6 weeks post ASO injection /γ2, or time in REM sleep. We first averaged the Ube3a protein levels across different brain regions to estimate the overall Ube3a protein levels in each mouse from the Western blot experiments /γ2, and time in REM sleep, we also normalized the data by the means of those in the corresponding control ASO-treated WT mice. For both ASO injection into juvenile and adult mice, the relative power in the theta , alpha , low beta , and high beta  bands negatively correlated with the Ube3a protein levels, whereas the relative power in the low gamma  and high gamma  bands positively correlated with the Ube3a protein levels. Therefore, the power ratio (α + β1 + β2)/γ2 also negatively correlated with the Ube3a protein levels  was observed in both strains  and relatively lower in the gamma bands, although the results were not always statistically significant  and two sgRNAs (10 ng/µl each) targeting the genomic sequences of Ube3a intron 5 (5′-TTACATACCAGTACATGTCTTGG-3′) and intron 6 (5′-TGCTTTCTACCAACTGAGACAGG-3′) were microinjected into WT C57BL/6J (JAX # 000664) zygotes. Founder mice carrying the exon 6 deletion (Δe6Ube3a) were identified by PCR using a pair of primers (5′-TTGAGAACAATGCAAAGGAAAATGA-3′ and 5′-GAGCAAACTGCTGTAGACCC-3′) for the WT allele (747 bp) and a pair of primers (5′-TTGAGAACAATGCAAAGGAAAATGA-3′ and 5′-TGAGGCTGGCTTTCAAGATTCA-3′) for the Δe6 (314 bp) allele. Founder mice were then backcrossed to WT C57BL/6J mice to generate N1 mice. N1 mice carrying the Δe6 allele were identified using the same PCR above. Sequencing of the identified N1 mice confirmed that the sequence chr7:59,275,513–59,277,423 were deleted. Δe6Ube3a mice were backcrossed to WT C57BL/6J mice for at least five generations prior to experiments. Heterozygous female mice carrying the mutation on their paternal chromosome (m+/pΔe6Ube3a) were crossed with WT C57BL/6J mice to generate WT and maternal knockout of Ube3a mice (mΔe6/p+Ube3a).The new Δe5Ube3a mice  were described previously  were described previously  fused to the C terminus of Ube3a. Heterozygous male or female m+/pYFPUbe3a mice were crossed with WT C57BL/6J mice to obtain heterozygous paternal or maternal YFPUbe3a mice (m+/pYFPUbe3a or mYFP/p+Ube3a), respectively. Δe5Ube3a and YFPUbe3a mice were also maintained on the C57BL/6J background.eviously , in whiceviously  and carrMice were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-certified animal facility on a 14/10 hr light/dark cycle. All procedures to maintain and use mice were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine (protocol AN-6544).2O (8.2 µl). The droplets for ddPCR were generated by a Biorad Automated Droplet Generator , followed by PCR reaction using a Thermal Cycler C1000 (Bio-Rad). The plate containing the droplets was read by a QX200 Droplet reader . The primers for detecting different fragments of the Ube3a transcripts are provided in Ube3a transcripts were normalized by the Gapdh levels.Mice were anesthetized and decapitated. Brain and liver tissues were extracted and homogenized with Trizol , followed by RNase-free DNase treatment . RNAs were purified with RNeasy Plus Mini Kit  and reverse transcribed to cDNA by High-Capacity cDNA Reverse Transcription Kit . The cDNA concentration was determined by a Nanodrop (Thermo Fisher). Droplet digital PCR (ddPCR) was prepared by mixing the following reagents in a total volume of 20 µl: 2× QX200 ddPCR EvaGreen supermix , forward and reverse primers , cDNA template , and nuclease-free HO-methoxyethylribose (MOE) gapmer ASOs are 20 nucleotides in length, wherein the central gap segment comprising ten 2′-deoxynucleotides is flanked on the 5′ and 3′ wings by five 2′-MOE modified nucleotides. All internucleoside linkages are phosphorothioate linkages, except the ones shown as ‘o’ in the sequences which are phosphodiester. The sequences of the ASOs are as follows: control ASO, 5′-CCToAoToAoGGACTATCCAoGoGAA-3′; Ube3a-as ASO, 5′-CCoAoGoCoCTTGTTGGATAoToCAT-3′; and Snord115 ASO, 5′-TToGoToAoAGCATCAAAGToAoTGA-3′. Lyophilized ASOs were formulated in phosphate-buffered saline (PBS) without Ca2+ and Mg2+ . ASOs were dissolved in PBS to obtain 50 mg/ml concentrations.Synthesis and purification of all chemically modified oligonucleotides were performed as previously described . The 2′-Mice were anesthetized with isoflurane (1.5–2.5%) in oxygen (1 l/min). The body temperature was monitored and maintained at 37°C using a temperature controller . An incision was made along the midline to expose the skull after the head was fixed in a stereotaxic apparatus. Approximately 0.25-mm-diameter craniotomies were performed with a round bur (0.25-mm-diameter) and a high-speed rotary micromotor  at the injection site (see below). A bevelled 50-µm-diameter glass pipette was used to inject ASOs into the right lateral ventricle according to one of the following sets of coordinates that were normalized by the distance between Bregma and Lambda (DBL). (1) Anterior/posterior (AP): 0.055 of DBL, medial/lateral (ML): 0.238 of DBL, dorsal/ventral (DV): −0.499 of DBL; (2) AP: 0.055 of DBL, ML: 0.238 of DBL, DV: −0.594 of DBL; (3) AP: 0.071 of DBL, ML: 0.238 of DBL, DV, −0.713 of DBL. The results were similar among these three sets of coordinates and were grouped together. ASO solution was injected at a rate of 407 nl/s using an UltraMicroPump III and a Micro4 controller . A total of 10 µl ASO solution (50 mg/ml) was administered for a total dosage of 500 µg/mouse except six mice that were injected with 5 µl ASO solution for a total dosage of 250 µg/mouse at the age of 3 weeks and used in the Western blot experiments. The results from these six mice were similar to other mice  and groumYFP/p+Ube3a mice.Mice were anesthetized and transcardially perfused with PBS (pH 7.4) followed by 4% paraformaldehyde in PBS (pH 7.4). Brains were then post-fixed for overnight in 4% paraformaldehyde at 4°C and sectioned into 40-µm sagittal slices using a vibratome . Brain sections were incubated in blocking solution  for 1 hr at 4°C and then with a primary rabbit monoclonal anti-GFP antibody  that recognizes YFP for overnight at 4°C. Sections were washed with 0.2% Triton X-100 in PBS and then incubated with a goat polyclonal anti-rabbit secondary antibody conjugated with Alexa Flour 647  in blocking solution for 3 hr at room temperature. After antibody staining, sections were incubated with NeuroTrace 435/455 blue fluorescent Nissl stain  in 0.2% Triton X-100 in PBS at room temperature for 1 hr to label neurons. Sections were washed with 0.2% Triton X-100 in PBS and mounted in ProLong Diamond Antifade Mountant . High-resolution (1024 × 1024) single-plane images of the brain sections were acquired on a TCS SP8X Confocal Microscope (Leica) using a ×20 oil objective . Mosaic images were stitched together using LAS X software v3.3.0.16799 (Leica) and visualized and exported by ImageJ 1.53c (NIH). To quantify Ube3a-YFP levels, cell somas were identified and segmented based on fluorescent Nissl stain using an ImageJ plugin, LABKIT . For eacMice were anesthetized and decapitated. The brains were extracted, and different regions were dissected from both hemispheres. The brain tissues from the right hemisphere were used for Western blots and the left hemisphere for RT-qPCR. Tissues were frozen at −80°C until analysis.For Western blots, the brain tissues were homogenized in RIPA buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol, and 1 cOmplete Protease Inhibitor tablet . After homogenization, tissue debris was removed by centrifugation and protein concentrations were determined by Pierce BCA Protein Assay Kit . To measure total Ube3a, 10 μg of proteins per sample were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) with 10% Bis-Tris gels  and transferred onto nitrocellulose membranes. To separate the two Ube3a isoforms, 3 μg of proteins per sample were resolved by SDS–PAGE with 4–12% Tris-Glycine gradient gels  at 200 volts for 90 min. Ube3a was detected by a mouse monoclonal anti-E6AP antibody  that recognizes both Ube3a isoforms 2 and 3. Gapdh was detected by a mouse monoclonal anti-Gapdh antibody . β3 tubulin was detected by a mouse monoclonal anti-β3 tubulin antibody . Primary antibodies were detected by a goat polyclonal anti-mouse antibody conjugated with IRDye 800CW . Proteins were visualized and quantified using an Odyssey CLx Imager and Image Studio Lite version 5.2 (LI-COR Biosciences). To quantify the total Ube3a levels, both Ube3a isoforms 2 and 3 were included. To quantify the levels of each isoform, total Ube3a and isoform 3 were quantified, and the isoform 2 levels were measured by subtracting the isoform 3 levels from the total Ube3a levels. Total Ube3a, Ube3a isoform 2, or Ube3a isoform 3 levels were first normalized by the Gapdh or β3 tubulin levels and then by the average total Ube3a, Ube3a isoform 2, or Ube3a isoform 3 of all WT mice from the same blot, respectively.Ube3a or Ube3a-ATS were normalized by the Gapdh levels and then by the average Ube3a or Ube3a-ATS levels of all WT mice from the same experiment, respectively.For RT-qPCR, the brain tissues were homogenized in RLT buffer  containing 1% (vol/vol) β-mercaptoethanol. Homogenization was performed for 20 s at 6000 rpm using a FastPrep Automated Homogenizer . Total RNA was then purified using the RNeasy 96 Kit  that included an in-column DNA digestion with 50 U of DNase I . RT-qPCR was performed in triplicate with the EXPRESSS One-Step SuperScript qRT-PCR kit . Gene-specific primers and probes are provided in Video-EEG/EMG recordings were performed as previously described . BrieflyPoly-spikes and PSD were analyzed from the same 6 hr of each recording . EEG/EMG traces were visualized in Sirenia Seizure 1.8.2 software (Pinnacle Technology) to identify episodes of poly-spikes and artifacts. An episode of poly-spikes is defined as a cluster of three or more spikes on any of the EEG channels. PSD analyses of EEG data were performed using custom scripts in Python. Prior to PSD calculation, data were detrended by subtracting the mean of the data. The data segments containing artifacts on any of the EEG channels were first excluded, and then an eighth-order Butterworth filter was applied to each channel to bidirectionally notch filter around 60 Hz (±2 Hz bandwidth) to remove power-line noise. The PSDs were then estimated for each channel using a Welch’s periodogram  with a 2https://sleeplearning.ethz.ch) was used for automated sleep scoring  represent independent biological replicates that are the numbers of tested mice. Statistical analyses were performed with Prism 9 (GraphPad Software). Student’s t-test or analysis of variance (ANOVA) with multiple comparison test for all pairs of groups was used to determine if there is a statistically significant difference between two groups or among three or more groups, respectively. One- or two-way ANOVA was applied for one or two independent variables, respectively. Anderson–Darling test, D’Agostino–Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov tests were used to determine if data were normally distributed. Non-parametric Kruskal–Wallis one-way ANOVA with Dunn’s multiple comparison test was used for low- to high-frequency band ratio and poly-spike data. The details of all statistical tests, numbers of replicates, and p values are reported in All reported sample numbers (Δe6Ube3a mice will be available upon request or from the Jackson Laboratory (JAX). All data generated or analyzed during this study are included in the manuscript. Computer codes for EEG power spectrum analyses and sleep scoring are available at GitHub  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Larry Reiter, UT Health Science Center (Reviewer #1). The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Thank you for submitting your article "Antisense oligonucleotide therapy rescues disturbed brain rhythms and sleep in juvenile and adult mouse models of Angelman syndrome" for consideration by Essential revisions:Please address these issues fully in your revised submission:1. Analysis of the particular isoforms of Ube3a deficient mice that are rescued by the ASO treatment is an important component of the analysis and will allow a better understanding of the effects of the ASO treatment in the context of previous studies with developmental rescue. Previous studies have demonstrated the importance of particular isoforms of Ube3a in function. The authors should evaluate the success of the ASO in rescuing expression of these isoforms and discuss these results in the context of the literature.2. We think you must carefully address the concerns about western blots and potential for duplication of lanes/bands in at least one of the blots. It appears that lanes may have been duplicated and reproduced at different exposures.3. It is important to address the possibility that this model with ASO rescue may allow analysis of cognitive deficits and their rescue in the mice. This additional information would broaden the significance of the study because it would either indicate that cognitive function may be improved by ASO treatment at postnatal stages, or not. An example would be examining visuospatial behaviors requiring intact hippocampal plasticity and determining if these are rescued.Reviewer #1 (Recommendations for the authors):The figures, in general, are too crowded and difficult to read. The data in these figures is acceptable, but the format is too busy to actually digest the important points. I suggest they find better ways to show changes in EEG spectra than bar graphs (see below). That said, the findings appear to suggest that at least EEG power and sleep rhythms can be rescued at the juvenile and, to a lesser extent, adult stages. Previous studies have shown that through genetic manipulation, Ube3a can be turned on developmentally in a Ube3a deficient background at embryonic stages with complete rescue of "critical" phenotypes . However, these studies did not examine EEG or sleep disturbance in any detail, and yet claimed that rescue of Ube3a expression after embryonic stages may not have any clinically relevant effects.I think the authors make the case that at least some clinically relevant phenotypes can be rescued using ASO approaches postnatally in Ube3a deficient mice. Given the potential impact of this manuscript and the novel claim that is somewhat against current dogma in the field, i.e. that rescue after the embryonic stage will have some effect, I would suggest the following experiments:1. It is critical to know which isoforms are rescued by the ASO treatments in the new Exon6 deletion mouse model. There has been significant emphasis of isoform III in the literature and the nuclear localization of this isoform as being critical to the rescue of predominant phenotypes  Nature Neuro 22, pages 1235-1247). The authors need to show rescue of this critical isoform postnatally and/or demonstrate that one of the isoforms is rescued to near normal levels by western blot using tissue from various regions of the brain. Figure 3 – supplemental suggests that some isoforms may be specific to the cerebellum, thalamus, and olfactory bulb and that in the thalamus, at least, there is better rescue of the lower molecular weight protein than the larger isoform. Can you explain this? A better investigation of the particular isoforms being rescued is warranted here.2. There is a missed opportunity here to test cognition. Did the investigators test these mice for rescue of cognitive phenotypes? If not, why? Hippocampal plasticity is essential for cognition and can be rescued at any age in Ube3a deficient animals  J Clin Invest. 125: 2069-76 and others – see Elgersma and Sonzogni (2021) Dev Med and Child Neuro 63: 802-807).3. Although additional experiments on the impact of other genes in the 15q11.2 critical region  is beyond the scope of the current study, the authors must discuss individual genes, GABA receptors and HERC2, and their impact or involvement in EEG phenotypes in AS. For example, Frolich et al. found that deletion class individuals differ significantly from UBE3A mutation or imprinting center mutation class by spectral power  Biol Psych 85(9): 752-759).Reviewer #2 (Recommendations for the authors):No effort was made to look at the designed ASO in the original ube3a mouse model. The ability to show good penetration beyond several weeks is an advance in the field and should be built on. However, it is unclear how the newly designed ASO will work in wild type brain or non-neuronal cells. One would expect no change would occur in non-neuronal cells. Many of the rescue experiments appear to be modest but significant differences. The protein rescue appears strong. How about substrates? Is the Ube3a protein functional? Is protein degradation restored? The rescue of the abnormal EEG seems slight and is not different between the control ASO and the ube3a-ASO. Sleep pattern differences look promising and could be expanded upon. The authors report on the finding that poly-spikes are not rescued. It is unclear at this time why this is. It seems that this should be followed up with significant experiments. One possibility is that there is not enough protein being made from the paternal ube3a using the current strategy. The authors could consider repeating their experiments in the original ube3a mouse model to see if similar results are observed. It appears from this study that a best mouse model for AS may not exist. With that in mind, it may be highly beneficial for this group to test several of their ideas on a molecular level and see if in iPSCs derived neurons from AS patients they can observe rescue of cellular and physiological changes.Reviewer #3 (Recommendations for the authors):This is a well-designed and executed study. The team first developed a new mouse model of Angelman syndrome and then leveraged the model to validate the ASO oligonucleotides therapeutic approach to knock down ATS expression and increase UBE3A expression from the paternal locus. They performed patient-relevant functional assays including EEG rhythms, sleep architecture, and epileptiform activity. The longitudinal design is another major strength of the study, in which both juvenile and adult age groups were included and functional tests were performed at 3-, 6- and 10-weeks post ASO injection. The authors also did a thorough analysis of UBE3A expression levels in different brain regions under different conditions. Overall, the study demonstrated a clear correlation between UBE3A expression level and EEG power ratio (a+b1+b2)/g2 and REM sleep. Improved UBE3A expression was able to restore (to certain degrees) EEG spectra and sleep functions. These results support a cause-and-effect relationship. Interestingly, improved UBE3A expression did not significantly reduce poly-spikes. In addition, the manuscript is well written with a good discussion of the findings in the context of the literature and future perspectives.I have several concerns.One concern relates to the Western blot data in Figure 3: there appears to be data duplication and the same blot with different exposures was used for different brain regions. This reviewer believes that it is an honest mistake, so please check each and every blot to ensure the accuracy and fidelity of all data.Regarding single unilateral ICV injection, there must be variations among animals and it would help if the authors could provide additional data to support the effectiveness and efficiency of the procedure.Furthermore, the study focused on EEG activity and sleep, and rightly so. If ASO therapy can reduce sleep disturbance, it would likely improve some aspects of cognitive functions. The cognitive improvement would be an important addition to the present work. Essential revisions:Please address these issues fully in your revised submission:1. Analysis of the particular isoforms of Ube3a deficient mice that are rescued by the ASO treatment is an important component of the analysis and will allow a better understanding of the effects of the ASO treatment in the context of previous studies with developmental rescue. Previous studies have demonstrated the importance of particular isoforms of Ube3a in function. The authors should evaluate the success of the ASO in rescuing expression of these isoforms and discuss these results in the context of the literature.Ube3a knockout mice.We thank the reviewer for this important suggestion, as previous studies showed that the short Ube3a isoform 3 is critical for the pathogenesis of Angelman syndrome. We performed new Western blot experiments to separate the two Ube3a isoforms 2 and 3 that differ by 21 amino acids and about 2-3 kDa. We found that both long and short isoforms were similarly up-regulated by Ube3a-as ASO or Snord115 ASO in different brain regions at 3 weeks post ASO injection into juvenile mice . We also examined two brain regions from 3 weeks post ASO injection into adult mice and found the same result . Therefore, these results are consistent with the rescue of EEG power spectrum and sleep phenotypes in maternal 2. We think you must carefully address the concerns about western blots and potential for duplication of lanes/bands in at least one of the blots. It appears that lanes may have been duplicated and reproduced at different exposures.We thank the reviewer for pointing out this critical issue and have inspected all raw data and figures. We realized that in the previous Figure 3-supplement 1A, both images of the “Olfactory bulb” and “Midbrain + Hindbrain” anti-Ube3a blots from “3 weeks post ASO injection to juvenile mice” came from the olfactory bulb blot. We have identified the process that led to this error.The two original image files assigned with the Image ID #179 and #182 were generated by LI-COR Image Studio software on August 14, 2019. Multiple images  were then exported from the original image files with different exposure settings that were adjusted for individual blots see . Images 3. It is important to address the possibility that this model with ASO rescue may allow analysis of cognitive deficits and their rescue in the mice. This additional information would broaden the significance of the study because it would either indicate that cognitive function may be improved by ASO treatment at postnatal stages, or not. An example would be examining visuospatial behaviors requiring intact hippocampal plasticity and determining if these are rescued.We agree with the reviewers that it would have been informative to test the effect of ASO treatment on cognitive function, as it is severely affected in Angelman patients. However, we chose not to test cognitive function in the mouse models of Angelman syndrome for two reasons.Ube3a knockout mice (m∆e5/p+Ube3a) were tested in Morris water maze and fear conditioning, but the deficits are mild and highly variable across labs and strains . This issue has been discussed and summarized by Sonzogni et al., 2018 and Rotaru, et al., 2020.The first reason is that the cognitive deficit is unfortunately a key phenotype that was not well recapitulated in the mouse models, which does not allow a robust test of the ASO therapy. The original maternal For Morris water maze, Sonzogni et al., 2018 discussed that “we found that a large number of mice are needed to detect significant differences and results varied strongly among experimenters.” For fear conditioning, Sonzogni et al., 2018 discussed that “we have not been able to get consistent results across experiments and experimenters” and “…Collectively, these studies indicate that this phenotype is rather weak, and hence results, obtained with these tests should be interpreted with care.” The Figure 2 of Rotaru, et al., 2020 summarizes the cognitive phenotypes of Angelman mouse models being “not present/mild”. We believe that most likely for this reason, most of the recent studies testing various genetic therapies in Angelman mouse models did not test cognitive function . Judson et al., 2021 did test fear conditioning in the AAV gene replacement experiments, but also stated that “Common learning paradigms in mice – such as Morris water maze and fear conditioning – expose rather mild and inconsistent deficits in the AS model that demand intensive statistical sampling (43). Accordingly, we obtained inconclusive results when fear conditioning adult WT and AS mice treated with PHP.B/hUBE3Aopt or vehicle as neonates . Although mice of each group clearly demonstrated associative learning of the unconditioned foot shock stimulus and conditioned auditory cue , we were underpowered to detect robust deficits in either contextual or cued fear memory in the AS + vehicle group – or rescue thereof .” Note that this experiment used 28, 21, 26, and 26 mice for the 4 groups of mice, yet was still not able to detect the phenotype. It is currently unknown why the cognitive phenotype is mild in the mouse models despite their hippocampal plasticity deficits, but perhaps is due to a compensatory mechanism.m∆e5/p+Ube3a) and our new model (m∆e6/p+Ube3a) in different neurobehaviors including cognitive function tests and found that the two models were very similar. Hence, at the outset of our project, we did not plan to test cognitive function or any other neurobehaviors to avoid overlaps with his work. Our goal was to test the EEG power spectrum and sleep phenotypes, which are clinically highly relevant but not examined in any previous genetic therapy experiments.The second reason is that our colleague Dr. Rodney Samaco in our institute has systematically compared the original model :The figures, in general, are too crowded and difficult to read. The data in these figures is acceptable, but the format is too busy to actually digest the important points. I suggest they find better ways to show changes in EEG spectra than bar graphs (see below). That said, the findings appear to suggest that at least EEG power and sleep rhythms can be rescued at the juvenile and, to a lesser extent, adult stages. Previous studies have shown that through genetic manipulation, Ube3a can be turned on developmentally in a Ube3a deficient background at embryonic stages with complete rescue of "critical" phenotypes . However, these studies did not examine EEG or sleep disturbance in any detail, and yet claimed that rescue of Ube3a expression after embryonic stages may not have any clinically relevant effects.I think the authors make the case that at least some clinically relevant phenotypes can be rescued using ASO approaches postnatally in Ube3a deficient mice. Given the potential impact of this manuscript and the novel claim that is somewhat against current dogma in the field, i.e. that rescue after the embryonic stage will have some effect, I would suggest the following experiments:1. It is critical to know which isoforms are rescued by the ASO treatments in the new Exon6 deletion mouse model. There has been significant emphasis of isoform III in the literature and the nuclear localization of this isoform as being critical to the rescue of predominant phenotypes  Nature Neuro 22, pages 1235-1247). The authors need to show rescue of this critical isoform postnatally and/or demonstrate that one of the isoforms is rescued to near normal levels by western blot using tissue from various regions of the brain. Figure 3 – supplemental suggests that some isoforms may be specific to the cerebellum, thalamus, and olfactory bulb and that in the thalamus, at least, there is better rescue of the lower molecular weight protein than the larger isoform. Can you explain this? A better investigation of the particular isoforms being rescued is warranted here.Ube3a knockout mice.We thank the reviewer for this important suggestion, as previous studies showed that the short Ube3a isoform 3 is critical for the pathogenesis of Angelman syndrome. We performed new Western blot experiments to separate the two Ube3a isoforms 2 and 3 that differ by 21 amino acids and about 2-3 kDa. We found that both long and short isoforms were similarly up-regulated by Ube3a-as ASO or Snord115 ASO in different brain regions at 3 weeks post ASO injection into juvenile mice . We also examined two brain regions from 3 weeks post ASO injection into adult mice and found the same result . Therefore, these results are consistent with the rescue of EEG power spectrum and sleep phenotypes in maternal Ube3a knockout mice and (2) the long and short Ube3a isoforms differ only by 2-3 kDa and were not separated in these blots.In the original and new Figure 3-supplement 1, the additional bands above the Ube3a bands are nonspecific band because (1) their levels do not differ between WT and maternal 2. There is a missed opportunity here to test cognition. Did the investigators test these mice for rescue of cognitive phenotypes? If not, why? Hippocampal plasticity is essential for cognition and can be rescued at any age in Ube3a deficient animals  J Clin Invest. 125: 2069-76 and others – see Elgersma and Sonzogni (2021) Dev Med and Child Neuro 63: 802-807).Ube3a knockout mice. However, we did not test our mice for rescue of cognitive phenotypes for two reasons.The reviewer is correct that hippocampal plasticity can be rescued in adult maternal Ube3a knockout mice (m∆e5/p+Ube3a) were tested in Morris water maze and fear conditioning, but the deficits are mild and highly variable across labs and strains . This issue has been discussed and summarized by Sonzogni et al., 2018 and Rotaru, et al., 2020.The first reason is that the cognitive deficit is unfortunately a key phenotype that was not well recapitulated in the mouse models, which does not allow a robust test of the ASO therapy. The original maternal For Morris water maze, Sonzogni et al., 2018 discussed that “we found that a large number of mice are needed to detect significant differences and results varied strongly among experimenters.” For fear conditioning, Sonzogni et al., 2018 discussed that “we have not been able to get consistent results across experiments and experimenters” and “…Collectively, these studies indicate that this phenotype is rather weak, and hence results, obtained with these tests should be interpreted with care.” The Figure 2 of Rotaru, et al., 2020 summarizes the cognitive phenotypes of Angelman mouse models being “not present/mild”. We believe that most likely for this reason, most of the recent studies testing various genetic therapies in Angelman mouse models did not test cognitive function . Judson et al., 2021 did test fear conditioning in the AAV gene replacement experiments, but also stated that “Common learning paradigms in mice – such as Morris water maze and fear conditioning – expose rather mild and inconsistent deficits in the AS model that demand intensive statistical sampling (43). Accordingly, we obtained inconclusive results when fear conditioning adult WT and AS mice treated with PHP.B/hUBE3Aopt or vehicle as neonates . Although mice of each group clearly demonstrated associative learning of the unconditioned foot shock stimulus and conditioned auditory cue , we were underpowered to detect robust deficits in either contextual or cued fear memory in the AS + vehicle group – or rescue thereof .” Note that this experiment used 28, 21, 26, and 26 mice for the 4 groups of mice, yet was still not able to detect the phenotype. It is currently unknown why the cognitive phenotype is mild in the mouse models despite their hippocampal plasticity deficits, but perhaps is due to a compensatory mechanism.m∆e5/p+Ube3a) and our new model (m∆e6/p+Ube3a) in different neurobehaviors including cognitive function tests and found that the two models were very similar. Hence, at the outset of our project, we did not plan to test cognitive function or any other neurobehaviors to avoid overlaps with his work. Our goal was to test the EEG power spectrum and sleep phenotypes, which are clinically highly relevant but not examined in any previous genetic therapy experiments.The second reason is that our colleague Dr. Rodney Samaco in our institute has systematically compared the original model  is beyond the scope of the current study, the authors must discuss individual genes, GABA receptors and HERC2, and their impact or involvement in EEG phenotypes in AS. For example, Frolich et al. found that deletion class individuals differ significantly from UBE3A mutation or imprinting center mutation class by spectral power  Biol Psych 85(9): 752-759).We agree with the reviewer that this is an important issue and have now expanded the discussion on ASO therapy in deletion Angelman syndrome.Reviewer #2 (Recommendations for the authors):No effort was made to look at the designed ASO in the original ube3a mouse model. The ability to show good penetration beyond several weeks is an advance in the field and should be built on. However, it is unclear how the newly designed ASO will work in wild type brain or non-neuronal cells. One would expect no change would occur in non-neuronal cells.m∆e5/p+Ube3a mice . The upregulation of paternal Ube3a was similar to what we observed in the new m∆e6/p+Ube3a mice. Therefore, we did not test the ASOs in the original m∆e5/p+Ube3a mice again.We previously tested Ube3a-as ASO, which is the better one of the two ASOs used in the current study, in the original Ube3a mRNA levels. We have now included the results in new Figure 2-supplement 1.We tested both Ube3a-as ASO and Snord115 ASO in WT mice prior to the rescue experiments and found that they increased The protein rescue appears strong. How about substrates? Is the Ube3a protein functional? Is protein degradation restored?Ube3a allele produces both Ube3a isoforms with correct sizes  and rescues neurological phenotypes. Hence, we believe that the Ube3a protein is functional. We also think that the dysregulated substrates and protein degradation in m∆e6/p+Ube3a mice should be at least partially restored by Ube3a-as ASO and Snord115 ASO. Although experimentally studying these questions is outside of the scope of our current study, we agree with the reviewer that it is important to test these predictions in the future.Reactivation of paternal The rescue of the abnormal EEG seems slight and is not different between the control ASO and the ube3a-ASO. Sleep pattern differences look promising and could be expanded upon.We agree with the reviewer’s assessment and will plan new projects to further study the sleep phenotypes in the future.The authors report on the finding that poly-spikes are not rescued. It is unclear at this time why this is. It seems that this should be followed up with significant experiments. One possibility is that there is not enough protein being made from the paternal ube3a using the current strategy. The authors could consider repeating their experiments in the original ube3a mouse model to see if similar results are observed. It appears from this study that a best mouse model for AS may not exist. With that in mind, it may be highly beneficial for this group to test several of their ideas on a molecular level and see if in iPSCs derived neurons from AS patients they can observe rescue of cellular and physiological changes.m∆e5/p+Ube3a mice and iPSC derived neurons, all of which will be helpful for our future studies.We agree that it is unclear why the poly-spikes are not rescued by the ASOs. We discussed the two possibilities that suppression of poly-spikes requires up-regulation of Ube3a (1) starting at a younger age and (2) reaching to a higher level than what we have achieved, both of which should be tested in future studies. We also thank the reviewer for other suggestions including testing the original Reviewer #3 (Recommendations for the authors):This is a well-designed and executed study. The team first developed a new mouse model of Angelman syndrome and then leveraged the model to validate the ASO oligonucleotides therapeutic approach to knock down ATS expression and increase UBE3A expression from the paternal locus. They performed patient-relevant functional assays including EEG rhythms, sleep architecture, and epileptiform activity. The longitudinal design is another major strength of the study, in which both juvenile and adult age groups were included and functional tests were performed at 3-, 6- and 10-weeks post ASO injection. The authors also did a thorough analysis of UBE3A expression levels in different brain regions under different conditions. Overall, the study demonstrated a clear correlation between UBE3A expression level and EEG power ratio (a+b1+b2)/g2 and REM sleep. Improved UBE3A expression was able to restore (to certain degrees) EEG spectra and sleep functions. These results support a cause-and-effect relationship. Interestingly, improved UBE3A expression did not significantly reduce poly-spikes. In addition, the manuscript is well written with a good discussion of the findings in the context of the literature and future perspectives.I have several concerns.One concern relates to the Western blot data in Figure 3: there appears to be data duplication and the same blot with different exposures was used for different brain regions. This reviewer believes that it is an honest mistake, so please check each and every blot to ensure the accuracy and fidelity of all data.We thank the reviewer for pointing out this critical issue and have inspected all raw data and figures. We realized that in Figure 3-supplement 1A, both images of the “Olfactory bulb and “Midbrain + Hindbrain” anti-Ube3a blots from “3 weeks post ASO injection to juvenile mice” came from the olfactory bulb blot. We have identified the process that led to this error.The two original image files assigned with the Image ID #179 and #182 were generated by LI-COR Image Studio software on August 14, 2019. Multiple images  were then exported from the original image files with different exposure settings adjusted for individual blots see . Images Regarding single unilateral ICV injection, there must be variations among animals and it would help if the authors could provide additional data to support the effectiveness and efficiency of the procedure.The reviewer is correct that for the ICV injection, there are variations among mice, which is probably best reflected by the Western blot results that the Ube3a levels do vary among mice. However, the experimental variation was minimized by a robust injection protocol. We described the method in detail. We also showed that 250 µg ASO/mouse had similar effects as 500 µg ASO/mouse , suggesting that we have likely reached the maximal effect.Furthermore, the study focused on EEG activity and sleep, and rightly so. If ASO therapy can reduce sleep disturbance, it would likely improve some aspects of cognitive functions. The cognitive improvement would be an important addition to the present work.We agree with the reviewer that it is of high interest to test the effect of ASO treatment on cognitive function. However, we chose not to test cognitive function in the mouse models of Angelman syndrome for two reasons.Ube3a knockout mice (m∆e5/p+Ube3a) were tested in Morris water maze and fear conditioning, but the deficits are mild and highly variable across labs and strains . This issue has been discussed and summarized by Sonzogni et al., 2018 and Rotaru, et al., 2020.The first reason is that the cognitive deficit is unfortunately a key phenotype that was not well recapitulated in the mouse models, which does not allow a robust test of the ASO therapy. The original maternal For Morris water maze, Sonzogni et al., 2018 discussed that “we found that a large number of mice are needed to detect significant differences and results varied strongly among experimenters.” For fear conditioning, Sonzogni et al., 2018 discussed that “we have not been able to get consistent results across experiments and experimenters” and “…Collectively, these studies indicate that this phenotype is rather weak, and hence results, obtained with these tests should be interpreted with care.” The Figure 2 of Rotaru, et al., 2020 summarizes the cognitive phenotypes of Angelman mouse models being “not present/mild”. We believe that most likely for this reason, most of the recent studies testing various genetic therapies in Angelman mouse models did not test cognitive function . Judson et al., 2021 did test fear conditioning in the AAV gene replacement experiments, but also stated that “Common learning paradigms in mice – such as Morris water maze and fear conditioning – expose rather mild and inconsistent deficits in the AS model that demand intensive statistical sampling (43). Accordingly, we obtained inconclusive results when fear conditioning adult WT and AS mice treated with PHP.B/hUBE3Aopt or vehicle as neonates . Although mice of each group clearly demonstrated associative learning of the unconditioned foot shock stimulus and conditioned auditory cue , we were underpowered to detect robust deficits in either contextual or cued fear memory in the AS + vehicle group – or rescue thereof .” Note that this experiment used 28, 21, 26, and 26 mice for the 4 groups of mice, yet was still not able to detect the phenotype. It is currently unknown why the cognitive phenotype is mild in the mouse models despite their hippocampal plasticity deficits, but perhaps is due to a compensatory mechanism.m∆e5/p+Ube3a) and our new model (m∆e6/p+Ube3a) in different neurobehaviors including cognitive function tests and found that the two models were very similar. Hence, at the outset of our project, we did not plan to test cognitive function or any other neurobehaviors to avoid overlaps with his work. Our goal was to test the EEG power spectrum and sleep phenotypes, which are clinically highly relevant but not examined in any previous genetic therapy experiments.The second reason is that our colleague Dr. Rodney Samaco in our institute has systematically compared the original model (Therefore, we hope that the reviewer will agree that testing the effect of ASO treatment on the cognitive function of Angelman mouse models is unlikely to be informative. This would be best tested in a different model that recapitulates the patient phenotype better than the mouse models.


Molecular dynamics simulation analysis revealed more conformational changes in c-Srcclose upon binding to SHP-1, as compared to its active/open conformation that is stabilized by the cooperative binding of the C-SH2 domain and C-terminal tail of SHP-1 to c-Src SH2 and KD. In contrast, c-Srcclose and SHP-1 interaction is mediated by PTP domain-specific WPD-loop (WPDXGXP) and Q-loop (QTXXQYXF) binding to c-Srcclose C-terminal tail residues. The dynamic correlation analysis demonstrated a positive correlation for SHP-1 PTP with KD, SH3, and the C-terminal tail of c-Srcclose. In the case of the c-Srcopen-SHP-1 complex, SH3 and SH2 domains of c-Srcopen were correlated to C-SH2 and the C-terminal tail of SHP-1. Our findings reveal that SHP1-dependent c-Src activation through dephosphorylation relies on the conformational shift in the inhibitory C-terminal tail that may ease the recruitment of the N-SH2 domain to phosphotyrosine residue, resulting in the relieving of the PTP domain. Collectively, this study delineates the intermolecular interaction paradigm and underlying conformational readjustments in SHP-1 due to binding with the c-Src active and inactive state. This study will largely help in devising novel therapeutic strategies for targeting cancer development.SHP-1 is a cytosolic tyrosine phosphatase that is primarily expressed in hematopoietic cells. It acts as a negative regulator of numerous signaling pathways and controls multiple cellular functions involved in cancer pathogenesis. This study describes the binding preferences of SHP-1 (pY536) to c-Src Protein tyrosine phosphorylation is a post-translational modification that plays an essential role in cell growth, proliferation, and differentiation , 2. Tyroin vitro studies, it has been reported that Src phosphorylates SHP-1 (Y536 and Y564 residues) by binding at the NH2-terminal of the SH2 domain in the lymphocytes and platelets cells . , 9. 7, 9ectively .open (SHP-1 activation by phosphorylation at Y536) and c-Srcclose (Y527 dephosphorylation of c-Srcclose). In the c-Srcopen-SHP-1 complex, binding of both SH3 and SH2 domains of c-Src was observed with two tandems (SH2) of SHP-1  c-Srcopen and c-Srcclose  and G151-S248 (SH3 domain), ranging from 0.7–0.75 nm, respectively , S57-D59 (0.38–0.53 nm), G66-E67 (0.31–0.40 nm), and Q81-D90 (0.24–0.40nm) regions, while the c-Srcopen-SHP-1 complex was relatively stable throughout simulation run CX5R(S/T), WPD loop motif (WPDXGXP), and Q loop motif (QTXXQYXF) exhibited more transitions (close-SHP-1 complex. D543-K546 (0.35–1.91 nm) and S556-V577 (0.39–1.64) exhibited major fluctuations in the C-terminal tail  and c-Srcclose (PTR527) with SHP-1 (PTR536) through in silico approaches. Convincingly, in case of c-Srcopen, MD simulation analysis revealed the participation of both SH2 and SH3 domains in binding to the SHP-1 N-SH2 domain. In contrast, an active role of c-Srcclose C-terminal tail was observed in the interaction with SHP-1 PTP domain. Intriguingly, more conformational readjustments were witnessed in c-Srcopen upon binding to SHP-1 (33-RKNQG-37 motif (N-SH2) association with SH3 (D117) and SH2  domains of c-Srcopen induces the recognition-induced conformational changes in the SH2 domain for binding to SHP-1 C-terminal tail  and E517  indicates its potential role in stabilizing c-Srcopen conformation. K298 residue has been reported to regulate c-Src global kinase activity, while K298E mutation results in c-Src closed conformation [Current study characterizes the binding patterns of c-Srcto SHP-1 . SHP-1-snal tail . Consequnal tail . These feraction . As compcessible . Upon suose PTR52 with SHPose PTR52 with SHPectively . Interesormation .SHP1-dependent c-Src activation through pY527 dephosphorylation  relies oclose-SHP-1 complex, significant contributions of WPD-loop (WPDXGXP) and Q-loop (QTXXQYXF) of the SHP-1 PTP domain were observed in the transition of close-to-open conformation. The binding preferences for c-Srcclose C-terminal tail residues  to WPD-loop D419 and SHP-1 PTP domain  play a critical role in promoting the dephosphorylation activity and activation of c-Src  is positively correlated with SHP-1 PTP domain and KD, and movement of SH2-SH3 region is highly correlated to that of SHP-1 C-SH2 domain, suggesting their cooperative role in promoting the c-Src closed conformation  and Q-loop (QTXXQYXF) of the SHP-1 PTP domain were observed in the transition of close-to-open conformation. Conversely, in case of c-Srcopen, both SH2 and SH3 domains are responsible in binding to the SHP-1 N-SH2 domain.Collectively, our findings delineate the comparative analysis of SHP-1 binding to both active and inactive forms of c-Src to uncover the underlying global conformational switches that govern the state transition basis of Src due to individual movements of regulatory domains. Our study will expand the previous knowledge of SHP-1-dependent tyrosine dephosphorylation and add significant information regarding the kinase activation to curb cancer development. In the case of the c-SrcS1 File(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Figopen, (B) c-Srcclose and (C) SHP-1.The Ramachandran plot displays the phi-psi torsion angles for (A) c-Src(TIF)Click here for additional data file.S3 Figopen, (B) c-Srcclose, and (C) SHP-1. Y-axis demonstrates the Verify3D score for each residue, whereas the X-axis represents the residue number.Verify3D plots for (A) c-Src(TIF)Click here for additional data file.S4 Figopen-SHP-1. (C and D) c-Srcclose-SHP-1. SHP-1: N-SH2 in deep green, C-SH2 in deep blue, PTP in pink and C-terminal tail in plum. Open and close conformation of c-Src. Color code: SH3, SH2 domain, and linker region are displayed in chartreuse, orange, and dark magenta colors. Kinase domain, N-lobe, C-lobe, and A-loop are shown in light blue, white, and lime green colors. Gly-loop and C-terminal tail are shown in orange-red and cyan colors, while phosphorylated residues (pY416 and pY527) are shown in the deep pink sphere, respectively.(A and B) c-Src(TIF)Click here for additional data file.S5 Figopen-SHP-1. (C and D) c-Srcclose-SHP-1. SHP-1: N-SH2 in deep green, C-SH2 in deep blue, PTP in pink and C-terminal tail in plum. Open and close conformation of c-Src. Color code: SH3, SH2 domain, and linker region are displayed in chartreuse, orange, and dark magenta colors. Kinase domain, N-lobe, C-lobe, and A-loop are shown in light blue, white, and lime green colors. Gly-loop and C-terminal tail are shown in orange-red and cyan colors, while phosphorylated residues (pY416 and pY527) are shown in the stick, respectively.(A and B) c-Src(TIF)Click here for additional data file.S6 Figopen-SHP-1 (red), (C) c-Srcclose-SHP-1 (blue), (D) c-Srcopen (purple), and (E) c-Srcclose (sky blue), and their replicas are represented in deep pink and orange colors, respectively.Comparative RMSD for (A) apo-SHP-1 (green), (B) c-Src(TIF)Click here for additional data file.S7 Figopen, c-Srcclose, and SHP-1 (100ns) are green, while 300 ns are orange. (A) c-Srcopen (B) c-Srcclose (C) SHP1. RMSD values are labeled in angstrom.The 90 ns for c-Src(TIF)Click here for additional data file.S8 Figopen, (C) c-Srcclose, (D) c-Srcopen- SHP-1, and (E) c-Srcclose-SHP-1 plots.(A) SHP-1, (B) c-Src(TIF)Click here for additional data file.


High temperature compression was conducted at 723 K, the strain rate 0.05/s and 0.005/s and the true strain up to −1.0. Dynamic recrystallization was observed in each specimen and the Ca-free alloys showed dislocation glide at high strain rates and solute drag at low strain rates. When the dislocation glide dominated, basal texture was strengthened. In contrast, solute drag caused non-basal texture development. Precipitation hardening caused AZ61 to have higher flow stress than those of the Ca-free alloys by the PSN effect; its texture was observed separately because the PSN grain growth around the precipitation and orientation was specific, similar to the one developed at the solute atom drag.To enhance the formability of magnesium alloys, inhibition of basal texture development by the particle-stimulated nucleation (PSN) effect has attracted significant interest. However, its contribution to texture development is not easily observed due to the separation of texture from the conventional deformation behavior. This study aims to separate the Ca texture from the deformation behavior of AZX611 alloy and quantify it using scanning electron microscopy with electron backscatter diffraction (SEM-EBSD). Since Ca in the AZ61 magnesium alloy precipitated as Al Although magnesium alloys have a high specific strength and low density, an insufficient slip system of hexagonal close packed (HCP) structure has limited their application as a structural material ,2,3. Morn) became almost 5 or more and the dislocation glide became the major mechanism. In this case, the basal texture was set as a stable orientation and strengthened during the high temperature deformation. In contrast, when the n value neared 3, solute atom drag became the major mechanism and different texture component was developed.It was found that factors such as shearing stress differences, crystal orientation, activated slip systems, and stacking fault energies can affect the stable orientation which can be the major texture component after deformation. It was suggested that there must be a relationship between the strain rate and the mechanism of dislocation movement during the creep mechanism ,12. Whenn value, the application of these conditions is difficult owing to the low deformation rate productivity.This seemed to have been governed by deformation rather than recrystallization. Although the ideal non-basal texture can be developed by changing the On the other hand, many studies have been conducted to change the basal texture during deformation. Among them, element alloying was of high interest because of process convenience and ability to assign various properties. Especially, rare-earth (RE) elements have been reported as a modifier of the basal texture to RE-texture ,14. SimiIn our previous study, through the high temperature annealing in the 773 K, the Ca-based precipitate was spheroidized and distributed to the overall matrix with lowered size. Moreover, magnesium matrix had a higher grain size than the conventional annealing condition ,18. TherThus in this study, using field-emission scanning electron microscope (FE-SEM) and electron back scattered diffraction (EBSD), PSN texture was separated and quantified from the deformation behavior of AZX611 during high temperature deformation.The commercial cast AZ31 (Mg-3.0 wt.% Al-1.0 wt.% Zn), AZ61 (Mg-6.0 wt.% Al-1.0 wt.% Zn), and AZX611 (Mg-6.0 wt.% Al-1.0 wt.% Zn-1.5 wt.% Ca) were used as the starting material. The investigation about the effect of Ca element on phase composition was conducted prior to plane strain compression. A Mg-Ca phase diagram for a state with approximately 6 wt.% Al was obtained using FactSage , a phase diagram analysis software.All alloys were hot rolled at 673 K and almost 30% rolling reduction. After rolling, the alloys were machined to rectangular shape withe 6.7 × 10 × 10 mm. The specimens were sealed in a quartz tube with Ar gas and annealed at 773 K-4 H for stress relaxation and second-phase distribution. After then, they were quenched in water. Most grains of the alloys were fully annealed and equiaxed. To investigate the Ca distribution in the deformation specimen, As-received specimen of AZX611 was observed using field-emission electron probe micro analyzer .−2 s−1 and 5 × 10−3 s−1. The stress was applied to the specimen until the true strain became −0.4, −0.7 and −1.0 each. The restricted plane (the transverse direction (TD) plane of the rolled plate) of each deformed specimens was cut to observe the microstructure, and the compression plane (ND plane of rolled plate) was cut for texture observation.High-temperature plane strain compression was conducted with the universal testing machine and channel die. The compression direction of specimen was along the normal direction of the rolling plate and the elongation of transverse direction was restricted using the channel die. The compression was conducted at 723 K and strain rates of 5 × 102 by controlling the voltage at 243 K. The microstructure and texture were observed using FE-SEM  and EBSD , respectively. The texture analysis using EBSD was conducted under the 8 μm step size to the square area with sides of length 1600 μm of ND plane. About 40,000 points were investigated in each image and only datasets over 90% initial indexing rate were used. In addition, any filter or cleaning was not adopted.They were mechanically grinded using conventional method and electrochemically polished in perchloric acid solution. For electropolishing, a solution of 2~4% concentration of perchloric acid with four-nine purity ethanol was used, and the current density was set to 0.18–0.22 A/cm17Al12 and C15 (Al2Ca) could be formed.17Al12 and Al2Ca forms or decomposes around 543 K and 783 K, respectively. Therefore, AZX611 is composed of two different phases, Mg matrix and Al2Ca precipitates, at 723 K where the compression was conducted. In addition, because there is no solid solution zone of Ca in the matrix, the Al content was recalculated for each phase. As a result, approximately 4 wt.% Al was in the matrix while 2 wt.% Al was in the precipitates.According to the phase diagram, Mg2Ca contributed to precipitation hardening.At the lower strain rate, AZ61 had stress values lower than 20 MPa. Moreover, AZX611 had higher stress values than those of the Ca-free alloys AZ31 and AZ61, even though it has the second highest Al concentration of the three, this mean that AlIn As assumed in Dislocation and twinning operate commonly as mechanism which accepts the deformation in Mg alloy. Especially, twinning allows for further basal slip or recrystallization. However, at the temperature over 708 K, CRSS for non-basal slip becomes lower than twinning and it becomes hard to work . In thisIn the true stress-strain curves of AZ61 compressed with different strain rates, their stress was different along with the strain rate. Different texture component was developed after the true strain up to −1.0. This means that strain rate can affect the mechanism of dislocation movement. It has been reported and discussed that the creep mechanism has high relationship with this phenomenon in AZ80 .When the dislocation glide dominates the deformation, dislocation is able to be inhibited by the grain boundaries and piles up around them. They lead to misorientation accumulation and induce subgrain rotation and grain boundary migration. Eventually, HAGBs will be developed from LAGBs and continuous DRX will occur. Similarly, in On the contrary, peak stress decreased under 20 MPa and the non-basal texture component was developed when the strain rate decreased. This phenomenon was considered a conversion of dislocation mechanism to solute drag as the strain rate decreased. When the solute atom drag dominates the deformation behavior as the strain rate decreases, solute atoms react with the dislocation by relaxing the stress field. This eliminates the driving force, enabling the formation of new grain boundaries from conventional ones. This implies that the non-basal texture might be developed by the deformation rather than recrystallization. During the microstructure change by DRX under the dislocation glide mechanism, the basal textures maintained their components and strengthened during the deformation process. It was implied that in this condition, grains with the [0001] orientation were set as the stable orientation and grain growth was directed toward this orientation.To be set as the stable orientation, two independent conditions must be fulfilled: (1) arrangement of the grains to have a low Taylor factor which do not change during the deformation, and (2) every slip system activated in the same family during deformation should equilibrate under stress to prevent crystal rotation.Although CRSS values become almost similar at 723 K, the Schmidt factor and its stress are applied highly on <c + a> slip systems because of the geometrical structure of HCP. This causes the grains with orientations near (0001) to have a lower Taylor factor than that of others. Furthermore, grains with low Taylor factor are regarded as having low values of stored energy for deformation and are less sensitive to shearing. Thus, basal grains were able to accept the deformation only through grain boundary migration and growth consuming the grains with high values of stored energy.In contrast, even if the number of activated slip systems satisfies the von Mises criteria and can accommodate the deformation, if one of the slip systems is receiving higher stress than that of its family system, the crystal rotation would shift toward its direction . ResultsHowever, in this case, texture development was continued until the final strain, and there was no evidence that crystal rotation occurred. Eventually, basal texture was maintained and increased because of these reasons.2Ca phase, and the matrix of AZX611, with the second highest Al concentration compared to those of AZ31 and AZ61, showed the same behavior; (2) dislocation glide was the major deformation mechanism with the strain rate 5 × 10−2 s−1 and the deformation was accomplished by the <c + a> slip system and continuous DRX; (3) microstructure and texture deformation was altered by the Al2Ca. In this section, the effect of the Al2Ca phase on deformation behavior will be discussed based on these results.Until the previous section, it was concluded that: (1) Ca did not solute into the matrix, but contributed to the formation of the Al2Ca phase caused AZX611 to have higher flow stress than that those of the Ca-free alloys by precipitation hardening, and this mean precipitation can act as an obstacle to dislocation.As shown in In the previous section, it was found that continuous DRX occurred by subgrain rotation and grain boundary migration because of the grain boundary inhibiting the glide of dislocation. Similarly, the area around the precipitation can also act similar to a boundary. The misorientation would pile up to the further surface and provide the new sites and driving force for nucleation. Different types of DRX would then progress. In this study, newly formed grains developed just around the Ca precipitation, whereas in conventional DRX in the single phase, they were formed both at the grain boundaries and ingrain. The new type of DRX that occurred in this case is known as the particle stimulated nucleation (PSN) effect ,22.In the development of texture in AZX611 as shown at The relationship between texture intensities and true strain is shown in the graph in Although the dislocation glide mechanism dominates, the newly formed grains of the PSN effect had an aligned texture to a specific orientation that was not random. This mean that Ca precipitation changed the conventional [0001] stable orientation. The texture component of AZ61 at the low strain rate was demonstrated to be  and Helis et al. had reported that  and  texture occurred in AZ31 at the same strain rate and initial texture condition  during the deformation.In the solute drag mechanism, the component of deformed texture in magnesium alloy is highly related to the activation of slip systems interacting with the solute atoms. This means that a combination slip system satisfying the Von Mises criteria changes, so that the Taylor factor—which shows the stable orientation—would be changed. However, which change to the slip system would occur still remains as a problem.2Ca was discomposed and dispersed by annealing in the as-received specimens, the PSN effect occurred in specific areas. Some areas where grain refinement by the PSN effect did not appear were observed in same time. Due to the structure differences between the Mg matrix (HCP) and Al2Ca (Cubic), the lattice relationship between both phases differs from the orientation relationship. Therefore, it would be simpler to specify the change in Taylor factor if the observation of the effect of initial texture using this alloy can be conducted.To analyze the verification of the slip system combination and calculate the Taylor factor among these deformations, it could be further specified by performing compression according to the orientation conditions of the initial basal texture. Although Al2Ca precipitate at the temperature 723 K, lowering the Al concentration in the matrix. Due to the Al2Ca, AZX611 showed higher flow stress than that of AZ61.The Ca in AZX611 only contributed to the formation of Al2Ca induced the PSN effect, and because of the coarse grain size of the initial state, different types of recrystallization textures were separated from each other.Dynamic recrystallization occurred in every alloy during the deformation, AlThe dislocation glide mechanism dominated the deformation behavior in the high strain rate condition in this study and the basal texture strengthened. However, in AZX611, newly recrystallized grains formed by the PSN effect had a different texture that can weaken the basal texture.The non-basal component of PSN texture occurred during the dislocation glide mechanism, and it was similar with the texture component formed in the solute atom drag mechanism.In conclusion, the texture component developed from Ca precipitation during plane strain compression of AZ61 magnesium alloy was analyzed using SEM-EBSD and the texture caused by the PSN effect was successfully studied separately from that of the deformation behaviors. The major results are as follows: