Significance Human pluripotent stem (hPS) cells can self-renew indefinitely and differentiate into almost any cell type. Thus, hPS cells represent a potentially unlimited supply of cells for regenerative medicine, drug screening, and developmental studies. Realizing the full potential of hPS cells requires efficient protocols to direct their differentiation into desired cell types. Most efforts to control hPS cell differentiation have focused on soluble signaling factors, while the roles of insoluble signals, such as the mechanical properties of the ECM, have been less explored. We show that matrix mechanics alone can robustly induce neuronal differentiation of hPS cells, independent of soluble neurogenic factors. These results can guide the design of materials to influence stem cell fate.
117 Objectives: Rett syndrome is a rare neurological disorder resulting in intellectual disability caused by mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Altered neuronal morphology and reduced dendritic complexity have been observed in cell and animal models of Rett syndrome. The goal of this study was to use [11C]UCB-J, a PET radioligand binding to the pre-synaptic protein SV2A, to assess changes in synaptic density in the Mecp2 knockout (Mecp2 KO) mice, a mouse model of Rett syndrome.
Methods: Two Mecp2 KO and two wild type littermate (WT) mice underwent [11C]UCB-J scans using a Sophie G4 PET scanner. Dynamic images were acquired during 45 minutes after injection of the radiotracer (57.5±14.4μCi, 1.07±0.004 pg ) and reconstructed with 15 dynamic frames (six-1min, two-2min, seven-5min). Using FSL FLIRT the integrated PET image was normalized to the mouse MR brain template (Paxinos, 2001). The whole cortex volume of interest was created using the Paxinos mouse brain atlas. The body weight SUV was calculated using the cortical values from the last 15 minutes of the scan for the Mecp2 KO and the WT mice.
Results: SUVBW data calculated using the cerebral cortex region from 30 to 45 minutes of the scan gave values of 1.21±0.21 for the WT mice and 0.52±0.13 for the mutant mice.
Conclusions: These preliminary results reveal decreased uptake of [11C]UCB-J in the Rett syndrome mouse model are consistent with the hypothesis that the loss of Mecp2 gene may result in decreased synaptic density in the cerebral cortex. Studies are ongoing to increase the sample size that may inform future investigation into whether [11C]UCB-J imaging can serve as a valid biomarker in individuals with Rett syndrome.
Human brain organoid models have emerged as a promising tool for studying human brain development and function. These models preserve human genetics and recapitulate some aspects of human brain development, while facilitating manipulation in an in vitro setting. Despite their potential to transform biology and medicine, concerns persist about their fidelity. To fully harness their potential, it is imperative to establish reliable analytic methods, ensuring rigor and reproducibility. Here, we review current analytical platforms used to characterize human forebrain cortical organoids, highlight challenges, and propose recommendations for future studies to achieve greater precision and uniformity across laboratories.
Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)–P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.
Rett syndrome (RTT) is an autism spectrum developmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MECP2) gene. Excellent RTT mouse models have been created to study the disease mechanisms, leading to many important findings with potential therapeutic implications. These include the identification of many MeCP2 target genes, better understanding of the neurobiological consequences of the loss- or mis-function of MeCP2, and drug testing in RTT mice and clinical trials in human RTT patients. However, because of potential differences in the underlying biology between humans and common research animals, there is a need to establish cell culture-based human models for studying disease mechanisms to validate and expand the knowledge acquired in animal models. Taking advantage of the nonrandom pattern of X chromosome inactivation in female induced pluripotent stem cells (iPSC), we have generated isogenic pairs of wild type and mutant iPSC lines from several female RTT patients with common and rare RTT mutations. R294X (arginine 294 to stop codon) is a common mutation carried by 5–6% of RTT patients. iPSCs carrying the R294X mutation has not been studied. We differentiated three R294X iPSC lines and their isogenic wild type control iPSC into neurons with high efficiency and consistency, and observed characteristic RTT pathology in R294X neurons. These isogenic iPSC lines provide unique resources to the RTT research community for studying disease pathology, screening for novel drugs, and testing toxicology.
(Cell Reports 32, 107997-1–107997-15, e1–e7; August 4, 2020) In the originally published version of this article, there were multiple incorrect citations. The following citations were originally omitted but have now been included: Basu et al. (2009); Bracko et al. (2012); Brandt et al. (2003); Eisinger et al. (2013); Ge et al. (2006); Lister et al. (2013); Overall et al. (2012); Pletscher-Frankild et al. (2015); Trinchero et al. (2019); and Yun et al. (2016). Additionally, the following references were originally included but have now been deleted: Langfelder et al. (2007); Raudvere et al. (2019); Gould et al. (1999); and Zhao et al. (2008). The citations and references have now been corrected online. The authors regret this error. RGS6 Mediates Effects of Voluntary Running on Adult Hippocampal NeurogenesisGao et al.Cell ReportsAugust 04, 2020In BriefGao et al. use translational profiling to unveil genome-wide intrinsic molecular changes in adult-born hippocampal neurons that contribute to voluntary running-enhanced adult neurogenesis. The molecular mediators identified, such as RGS6, are necessary for accelerated neuronal maturation and improved learning and memory in running mice. Full-Text PDF Open Access
Our earlier work showed that stress had progressively more serious consequences in a hamster model of congestive heart failure as the magnitude of heart failure worsened. Based on that study, we hypothesized that the intensity of the stressor used might play an important part in determining this outcome as well as in influencing coronary reactivity to arginine vasopressin (AVP). Cardiomyopathic (2.5, 6.5, and 10 months) hamsters (CMHs) were stressed with a 2-hr period of supine immobilization for five consecutive days. Stressor intensity was increased by exposing the hamsters to progressively longer periods at 4°C: the low stress group was never put in the cold; the moderate stress group was exposed to cold for 1 hr, and the high stress group for 2 hr. CMHs were anesthetized and sacrificed 5 days after stress, and their hearts were perfused using a modified Langendorff system. Maximum ±dP/dt, developed pressure, ventricular relaxation time, (T), and coronary vascular resistance (CVR) were recorded, and CVR was also measured following coronary infusion of AVP. Stressor intensity had no effect on cardiac mechanics in 2.5-month CMHs. In 6.5-month CMHs, only the high-intensity stressor impaired ventricular mechanics (decreased maximum ±dP/dt and developed pressure, increased T; P <0.05), while low and moderate stress produced no effects. In 10-month CMHs, stress at all intensities exacerbated ventricular dysfunction (decreased maximum ±dP/dt and developed pressure; P < 0.05). These results support our first hypothesis that stressor intensity interacts multiplicatively with severity of the underlying disease to influence the course of heart failure. However, our second hypothesis was not supported, because stress—regardless of intensity—affected reactivity of the coronary vasculature to AVP only in 2.5-month CMHs. A further test of the relation of stressor intensity and coronary vascular reactivity requires study of additional groups of CMHs during the period of their disease characterized by coronary vasospasm.
Interneuronal gap junctional coupling is a hallmark of neural development whose functional significance is poorly understood. We have characterized the extent of electrical coupling and dye coupling and patterns of gap junction protein expression in lumbar spinal motor neurons of neonatal rats. Intracellular recordings showed that neonatal motor neurons are transiently electrically coupled and that electrical coupling is reversibly abolished by halothane, a gap junction blocker. Iontophoretic injection of Neurobiotin, a low molecular weight compound that passes across most gap junctions, into single motor neurons resulted in clusters of many labeled motor neurons at postnatal day 0 (P0)–P2, and single labeled motor neurons after P7. The compact distribution of dye-labeled motor neurons suggested that, after birth, gap junctional coupling is spatially restricted. RT-PCR, in situ hybridization, and immunostaining showed that motor neurons express five connexins, Cx36, Cx37, Cx40, Cx43, and Cx45, a repertoire distinct from that expressed by other neurons or glia. Although all five connexins are widely expressed among motor neurons in embryonic and neonatal life, Cx36, Cx37, and Cx43 continue to be expressed in many adult motor neurons, and expression of Cx45, and in particular Cx40, decreases after birth. The disappearance of electrical and dye coupling despite the persistent expression of several gap junction proteins suggests that gap junctional communication among motor neurons may be modulated by mechanisms that affect gap junction assembly, permeability, or open state.
Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain unclear. Although neuronal activity-induced phosphorylation of MeCP2 at serine 421 (S421) has been reported, the full spectrum of MeCP2 phosphorylation together with the in vivo function of such modifications are yet to be revealed. Here, we report the identification of several MeCP2 phosphorylation sites in normal and epileptic brains from multiple species. We demonstrate that serine 80 (S80) phosphorylation of MeCP2 is critical as its mutation into alanine (S80A) in transgenic knock-in mice leads to locomotor deficits. S80A mutation attenuates MeCP2 chromatin association at several gene promoters in resting neurons and leads to transcription changes of a small number of genes. Calcium influx in neurons causes dephosphorylation at S80, potentially contributing to its dissociation from the chromatin. We postulate that phosphorylation of MeCP2 modulates its dynamic function in neurons transiting between resting and active states within neural circuits that underlie behaviors.
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