Summary Rett Syndrome (RTT) is caused by mutations of MECP2, a methyl CpG binding protein thought to act as a global transcriptional repressor. Here we show, using an isogenic human embryonic stem cell model of RTT, that MECP2 mutant neurons display key molecular and cellular features of this disorder. Unbiased global gene expression analyses demonstrate that MECP2 functions as global gene activator in neurons but not in neural precursors. Decreased transcription in neurons was coupled with a significant reduction in nascent protein synthesis and lack of MECP2 was manifested as a severe defect in the activity of the AKT/mTOR pathway. Lack of MECP2 also leads to impaired mitochondrial function in mutant neurons. Activation of AKT/mTOR signaling by exogenous growth factors or by depleting PTEN boosted protein synthesis and ameliorated disease phenotypes in mutant neurons. Our findings indicate a vital function for MECP2 in maintaining active gene transcription in human neuronal cells.

Rett Syndrome is a neurodevelopmental disorder that arises from mutations in the X-linked gene methyl-CpG binding protein 2 (MeCP2). MeCP2 has a large number of targets and a wide range of functions, suggesting the hypothesis that functional signaling mechanisms upstream of synaptic and circuit maturation may contribute to our understanding of the disorder and provide insight into potential treatment. Here, we show that insulin-like growth factor-1 (IGF1) levels are reduced in young male Mecp2-null (Mecp2 −/y ) mice, and systemic treatment with recombinant human IGF1 (rhIGF1) improves lifespan, locomotor activity, heart rate, respiration patterns, and social and anxiety behavior. Furthermore, Mecp2-null mice treated with rhIGF1 show increased synaptic and activated signaling pathway proteins, enhanced cortical excitatory synaptic transmission, and restored dendritic spine densities. IGF1 levels are also reduced in older, fully symptomatic heterozygous (Mecp2 −/+ ) female mice, and short-term treatment with rhIGF1 in these animals improves respiratory patterns, reduces anxiety levels, and increases exploratory behavior. In addition, rhIGF1 treatment normalizes abnormally prolonged plasticity in visual cortex circuits of adult Mecp2 −/+ female mice. Our results provide characterization of the phenotypic development of Rett Syndrome in a mouse model at the molecular, circuit, and organismal levels and demonstrate a mechanism-based therapeutic role for rhIGF1 in treating Rett Syndrome. molecular therapeutic | respiration | synaptic function | male mice | female mice R ett Syndrome (RTT) is a devastating, rare neurodevelopmental disorder that primarily afflicts girls. Over 90% of individuals with RTT have sporadic mutations in the X-linked gene coding for methyl-CpG binding protein 2 (MeCP2). Affected girls are initially asymptomatic, but later develop a wide range of symptoms. Mouse models of RTT with deletion of Mecp2 recapitulate many of the key physiological, autonomic, motor, and cognitive aspects of the disorder (1, 2).MeCP2 binds widely across the genome and has complex roles that encompass activating or inhibiting gene transcription, repressing methylation, regulating chromatin remodeling, and altering noncoding RNAs (3). This wide range of functions has led to the proposal that a focus on functional signaling pathways is needed to drive an understanding of RTT and its possible therapeutics (1, 2, 4). Several lines of evidence indicate an arrested brain maturation phenotype in RTT, suggesting that loss of functional MeCP2 leads to immature synapses and circuits in the brain (5). Importantly, mouse models have suggested reversibility of specific symptoms once MeCP2 function is restored (6, 7). One well-documented target of MeCP2 is brain-derived neurotrophic factor (BDNF), which is known to be critical for neuronal and synaptic maturation and is down-regulated in Mecp2 mutant mice and RTT patients (8, 9). BDNF exerts influence on neurons and synapses mainly via the phosphoinositide 3-kinase (PI3K)/Akt pathway ...

Summary Although human induced pluripotent stem cells (hiPSCs) have enormous potential in regenerative medicine, their epigenetic variability suggests that some lines may not be suitable for human therapy. There are currently few benchmarks for assessing quality. Here we show that X-inactivation markers can be used to separate hiPSC lines into distinct epigenetic classes and that the classes are phenotypically distinct. Loss of XIST expression is strongly correlated with upregulation of X-linked oncogenes, accelerated growth rate in vitro, and poorer differentiation in vivo. Whereas differences in X-inactivation potential result in epigenetic variability of female hiPSC lines, male hiPSC lines generally resemble each other and do not overexpress the oncogenes. Neither physiological oxygen levels nor HDAC inhibitors offer advantages to culturing female hiPSC lines. We conclude that female hiPSCs may be epigenetically less stable in culture and caution that loss of XIST may result in qualitatively less desirable stem cell lines.

Rett Syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the Methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. While postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of miRNAs in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 shRNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK/MAPK) and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and 3D (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors (NPs) deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in WT mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Ca 2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino-and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.

Unbalanced visual input during development induces persistent alterations in the function and structure of visual cortical neurons. The molecular mechanisms that drive activity-dependent changes await direct visualization of underlying signals at individual synapses in vivo. By using a genetically engineered Förster resonance energy transfer (FRET) probe for the detection of CaMKII activity, and two-photon imaging of single synapses within identified functional domains, we have revealed unexpected and differential mechanisms in specific subsets of synapses in vivo. Brief monocular deprivation leads to activation of CaMKII in most synapses of layer 2/3 pyramidal cells within deprived eye domains, despite reduced visual drive, but not in nondeprived eye domains. Synapses that are eliminated in deprived eye domains have low basal CaMKII activity, implying a protective role for activated CaM-KII against synapse elimination.cortical circuits | ocular dominance plasticity | spines | excitatory synaptic transmission | serine/threonine protein kinase D uring a developmental critical period, alteration of neuronal responses in the visual cortex can occur after brief periods of monocular deprivation (MD) (1-3). These alterations are mediated by sequential mechanisms that transduce changes in the amount and pattern of visual activity from the two eyes to changes in synaptic drive (4-6). Thus, the initial loss of responses from the closed eye is known to be rapid, on the order of hours, as detailed elsewhere in PNAS (7), and mediated by mechanisms that implement synaptic depression. The gain of responses from the open eye is thought to be slower, on the order of days, and mediated by homeostatic scaling of responses as well as homosynaptic long-term potentiation (LTP)-like mechanisms (8-15), although rapid gain of responses on the order of hours also occurs (7). However, the structural and molecular basis for these changes at the level of single synapses are not fully understood (16)(17)(18)(19)(20). Two important reasons are that the precise location and distribution of synapses undergoing changes in the intact brain, and the specific molecular transformations that occur at these synapses during experience-dependent plasticity, are unknown.Excitatory neurons in layer 2/3 receive synaptic inputs from multiple sources, including feedforward, local, and long-range intracortical axons (Fig. 1A), and exhibit rapid functional changes following even brief MD (1-3). MD is known to reduce the effectiveness of deprived eye-driven synapses as well as rapidly eliminate synapses (16,18). However, significant proportions of synapses within deprived eye domains are also preserved after MD, and these synapses, depending on their origin, serve multiple functions: potentiating open eye responses (8, 12), acting as a scaffold for experience-dependent traces and accelerated shifts in response to MD, or enabling recovery of drive when the deprived eye is reopened (7, 12). Thus, understanding why some synapses are lost while others are preserve...

Human iPSC-derived organoids were co-electroporated with GFP and control vector (top) or shRNA targeting MeCP2 (bottom) and examined after 7 days. MeCP2 shRNA-targeted cells comprise an increased number of Pax6(+) neural progenitors. (Scale: 50 μm.) Highmagnification of cells is shown in each respective inset (right). The asterisks in the top right panel denote the Pax6(-) cells in the control group. Representative Pax6(+) progenitors seen after depletion of MeCP2 are denoted by arrows in the bottom right panel. (Scale: 20 μm.) For more information on this topic, please refer to the article by Mellios et al. on pages 1051-1065.

Breakthroughs in imaging techniques and optical probes in recent years have revolutionized the field of life sciences in ways that traditional methods could never match. The spatial and temporal regulation of molecular events can now be studied with great precision. There have been several key discoveries that have made this possible. Since green fluorescent protein (GFP) was cloned in 1992, it has become the dominant tracer of proteins in living cells. Then the evolution of color variants of GFP opened the door to the application of Förster resonance energy transfer (FRET), which is now widely recognized as a powerful tool to study complicated signal transduction events and interactions between molecules. Employment of fluorescent lifetime imaging microscopy (FLIM) allows the precise detection of FRET in small subcellular structures such as dendritic spines. In this review, we provide an overview of the basic and practical aspects of FRET imaging and discuss how different FRET probes have revealed insights into the molecular mechanisms of synaptic plasticity and enabled visualization of neuronal network activity both in vitro and in vivo.