Prader-Willi syndrome (PWS) is a developmental disorder caused by loss of maternally imprinted genes on 15q11-q13, including melanoma antigen gene family member L2 ( MAGEL2 ). The clinical phenotypes of PWS suggest impaired hypothalamic neuroendocrine function; however, the exact cellular defects are unknown. Here, we report deficits in secretory granule (SG) abundance and bioactive neuropeptide production upon loss of MAGEL2 in humans and mice. Unbiased proteomic analysis of Magel2 p Δ /m+ mice revealed a reduction in components of SG in the hypothalamus that was confirmed in 2 PWS patient–derived neuronal cell models. Mechanistically, we show that proper endosomal trafficking by the MAGEL2-regulated WASH complex is required to prevent aberrant lysosomal degradation of SG proteins and reduction of mature SG abundance. Importantly, loss of MAGEL2 in mice, NGN2-induced neurons, and human patients led to reduced neuropeptide production. Thus, MAGEL2 plays an important role in hypothalamic neuroendocrine function, and cellular defects in this pathway may contribute to PWS disease etiology. Moreover, these findings suggest unanticipated approaches for therapeutic intervention.

. Moreover, an endogenous stimulus of SGK1, insulin, increased IKK␣ and NF-B phosphorylation as well as NF-B acetylation and NF-B activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKK␣-NF-B signaling pathway, we found that transfection of the IKK␣ double mutant (IKK␣T23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity. Serum and glucocorticoid-inducible kinase 1 (SGK1)2 is a member of the serine/threonine protein kinase family that is transcriptionally induced by serum and glucocorticoids (1). SGK1 is known to regulate a variety of cellular functions, including salt homeostasis, ion channel conductance, cell proliferation, and neuronal excitability (2). In addition, SGK1 promotes cell survival and regulates cell cycle progression through phosphorylation of the forkhead transcription factor FKHRL1 (3, 4). More recently, SGK1 was found to modulate the excitatory amino acid transporter function through phosphorylation of Nedd4-2 (5). SGK1 is also known to implicate several physiological functions. For example, sgk1 mRNA expression is increased in animal models of Parkinson disease, suggesting a role of SGK1 in neuroprotection (6). SGK1 increases glutamate-induced current partly by increasing GluR6 protein level in plasma membrane of Xenopus oocytes expressing rat GluR6 (7). SGK1 was also found to increase neurite outgrowth in hippocampal neurons (8, 9). Moreover, SGK1 facilitates long term potentiation and spatial learning in rats (10).SGK1 is a downstream target of phosphatidylinositol 3-kinase signaling (11). SGK1 is first phosphorylated at Ser-422 by 3-phosphoinositide-dependent PDK2, which enables SGK1 to be further phosphorylated at Thr-256 by PDK1 (12). In addition, SGK1 also receives upstream signals from cyclic AMP (13), extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) (14), p38 MAPK (15), and big mitogen-activated protein kinase 1 (16). However, with the biological functions and upstream signals of SGK1 characterized to a certain extent, the downstream targets of SGK1 are relatively less known. Because SGK1 phosphorylates substrate proteins that contain the RXRXX(S/T) motif, where X stands for any amino acid (12), in this study we investigated the SGK1 substrate and signaling pathway by using the phospho (p)-motif antibody as a tool. We also examined the expression of genes that are regulated by this signaling pathway. Our results revealed that IB kinase ␣ (IKK␣), but not IKK␤, is a novel substrate of SGK1 and that SGK1 phosphorylation of IKK␣ increases nuclear factor B (NF-B) activity and up-regulates the expression of the N-methyl-D-aspartate (NMDA) receptor subunit NR2A and NR2B. In addition, SGK1 also phosphorylates p300 directly...

The methyl-CpG-binding protein 2 (MeCP2) gene, MECP2, is an X-linked gene encoding the MeCP2 protein, and mutations of MECP2 cause Rett syndrome (RTT). However, the molecular mechanism of MECP2-mutation-caused RTT is less known. Here we find that MeCP2 could be SUMO-modified by the E3 ligase PIAS1 at Lys-412. MeCP2 phosphorylation (at Ser-421 and Thr-308) facilitates MeCP2 SUMOylation, and MeCP2 SUMOylation is induced by NMDA, IGF-1 and CRF in the rat brain. MeCP2 SUMOylation releases CREB from the repressor complex and enhances Bdnf mRNA expression. Several MECP2 mutations identified in RTT patients show decreased MeCP2 SUMOylation. Re-expression of wild-type MeCP2 or SUMO-modified MeCP2 in Mecp2-null neurons rescues the deficits of social interaction, fear memory and LTP observed in Mecp2 conditional knockout (cKO) mice. These results together reveal an important role of MeCP2 SUMOylation in social interaction, memory and synaptic plasticity, and that abnormal MeCP2 SUMOylation is implicated in RTT.

Recurrent, reciprocal genomic disorders resulting from non-allelic homologous recombination (NAHR) between near-identical segmental duplications (SDs) are a major cause of human disease, often producing phenotypically distinct syndromes. The genomic architecture of flanking SDs presents a significant challenge for modeling these syndromes; however, the capability to efficiently generate reciprocal copy number variants (CNVs) that mimic NAHR would represent an invaluable modeling tool. We describe here a CRISPR/Cas9 genome engineering method, Single-guide-CRISPR/Cas-targeting-Of-Repetitive-Elements (SCORE), to model reciprocal genomic disorders and demonstrate its capabilities by generating reciprocal CNVs of 16p11.2 and 15q13.3, including alteration of one copy-equivalent of the SDs that mediate NAHR in vivo. The method is reproducible and RNAseq reliably clusters transcriptional signatures from human subjects with in vivo CNV and their corresponding in vitro models. This new approach will provide broad applicability for the study of genomic disorders and, with further development, may also permit efficient correction of these defects.

By using differential display PCR, we have previously identified 98 cDNA fragments from rat dorsal hippocampus, which are expressed differentially between the fast learners and slow learners from water-maze learning task. One cDNA fragment, which showed a higher expression level in fast learners, encodes the rat protein inhibitor of activated STAT1 (pias1) gene. Spatial training induced a significant increase in PIAS1 expression in rat hippocampus. Transient transfection of the wild-type (WT) PIAS1 plasmid to CA1 neurons facilitated, whereas transfection of PIAS1 siRNA impaired spatial learning in rats. Meanwhile, PIAS1WT increased STAT1 sumoylation, decreased STAT1 DNA binding and decreased STAT1 phosphorylation at Tyr-701 associated with spatial learning facilitation. But PIAS1 siRNA transfection produced an opposite effect on these measures associated with spatial learning impairment. Further, transfection of STAT1 sumoylation mutant impaired spatial acquisition, whereas transfection of STAT1 phosphorylation mutant blocked the impairing effect of PIAS1 siRNA on spatial learning. In this study, we first demonstrate the role of PIAS1 in spatial learning. Both posttranslational modifications (increased sumoylation and decreased phosphorylation) mediate the effect of PIAS1 on spatial learning facilitation.

cAMP-responsive element binding protein (CREB) phosphorylation and signaling plays an important role in long-term memory formation, but other posttranslational modifications of CREB are less known. Here, we found that CREB1⌬, the short isoform of CREB, could be sumoylated by the small ubiquitin-like modifier (SUMO) E3 ligase protein inhibitor of activated STAT1 (PIAS1) at Lys271 and Lys290 and PIAS1 SUMOylation of CREB1⌬ increased the expression level of CREB1⌬. CREB1⌬ could also be sumoylated by other PIAS family proteins, but not by the E3 ligases RanBP2 and Pc2 or by the E2 ligase Ubc9. Furthermore, water maze training increased the level of endogenous CREB SUMOylation in rat CA1 neurons determined by in vitro SUMOylation assay, but this effect was not observed in other brain areas. Moreover, transduction of Lenti-CREBWT to rat CA1 area facilitated, whereas transduction of Lenti-CREB double sumomutant (CREBK271RK290R) impaired, spatial learning and memory performance. Transduction of Lenti-CREBWT-SUMO1 fusion vector to rat CA1 area showed a more significant effect in enhancing spatial learning and memory and CREB SUMOylation. Lenti-CREBWT transduction increased, whereas Lenti-CREBK271RK290R transduction decreased, CREB DNA binding to the brain-derived neurotrophic factor (bdnf) promoter and decreased bdnf mRNA expression. Knock-down of PIAS1 expression in CA1 area by PIAS1 siRNA transfection impaired spatial learning and memory and decreased endogenous CREB SUMOylation. In addition, CREB SUMOylation was CREB phosphorylation dependent and lasted longer. Therefore, CREB phosphorylation may be responsible for signal transduction during the early phase of long-term memory formation, whereas CREB SUMOylation sustains long-term memory.

Reciprocal copy number variations (CNVs) of 16p11.2 are associated with a wide spectrum of neuropsychiatric and neurodevelopmental disorders. Here, we use human induced pluripotent stem cells (iPSCs)-derived dopaminergic (DA) neurons carrying CNVs of 16p11.2 duplication (16pdup) and 16p11.2 deletion (16pdel), engineered using CRISPR-Cas9. We show that 16pdel iPSC-derived DA neurons have increased soma size and synaptic marker expression compared to isogenic control lines, while 16pdup iPSC-derived DA neurons show deficits in neuronal differentiation and reduced synaptic marker expression. The 16pdel iPSC-derived DA neurons have impaired neurophysiological properties. The 16pdel iPSC-derived DA neuronal networks are hyperactive and have increased bursting in culture compared to controls. We also show that the expression of RHOA is increased in the 16pdel iPSC-derived DA neurons and that treatment with a specific RHOA-inhibitor, Rhosin, rescues the network activity of the 16pdel iPSC-derived DA neurons. Our data suggest that 16p11.2 deletion-associated iPSC-derived DA neuron hyperactivation can be rescued by RHOA inhibition.

Cerebral organoids can be used to gain insights into cell type specific processes perturbed by genetic variants associated with neuropsychiatric disorders. However, robust and scalable phenotyping of organoids remains challenging. Here, we perform RNA sequencing on 71 samples comprising 1,420 cerebral organoids from 25 donors, and describe a framework (Orgo-Seq) to integrate bulk RNA and single-cell RNA sequence data. We apply Orgo-Seq to 16p11.2 deletions and 15q11–13 duplications, two loci associated with autism spectrum disorder, to identify immature neurons and intermediate progenitor cells as critical cell types for 16p11.2 deletions. We further applied Orgo-Seq to identify cell type-specific driver genes. Our work presents a quantitative phenotyping framework to integrate multi-transcriptomic datasets for the identification of cell types and cell type-specific co-expressed driver genes associated with neuropsychiatric disorders.