Daily Rhythmic Behaviors and Thermoregulatory Patterns Are Disrupted in Adult Female MeCP2-Deficient Mice

Mutations in the X-linked gene encoding Methyl-CpG-binding protein 2 (MECP2) have been associated with neurodevelopmental and neuropsychiatric disorders including Rett Syndrome, X-linked mental retardation syndrome, severe neonatal encephalopathy, and Angelman syndrome. Although alterations in the performance of MeCP2-deficient mice in specific behavioral tasks have been documented, it remains unclear whether or not MeCP2 dysfunction affects patterns of periodic behavioral and electroencephalographic (EEG) activity. The aim of the current study was therefore to determine whether a deficiency in MeCP2 is sufficient to alter the normal daily rhythmic patterns of core body temperature, gross motor activity and cortical delta power. To address this, we monitored individual wild-type and MeCP2-deficient mice in their home cage environment via telemetric recording over 24 hour cycles. Our results show that the normal daily rhythmic behavioral patterning of cortical delta wave activity, core body temperature and mobility are disrupted in one-year old female MeCP2-deficient mice. Moreover, female MeCP2-deficient mice display diminished overall motor activity, lower average core body temperature, and significantly greater body temperature fluctuation than wild-type mice in their home-cage environment. Finally, we show that the epileptiform discharge activity in female MeCP2-deficient mice is more predominant during times of behavioral activity compared to inactivity. Collectively, these results indicate that MeCP2 deficiency is sufficient to disrupt the normal patterning of daily biological rhythmic activities

LV-pIN-KDEL: a novel lentiviral vector demonstrates the morphology, dynamics and continuity of the endoplasmic reticulum in live neurones

BACKGROUND The neuronal endoplasmic reticulum (ER) is an extensive, complex endomembrane system, containing Ca2+ pumps, and Ca2+ channels that permit it to act as a dynamic calcium store. Currently, there is controversy over the continuity of the ER in neurones, how this intersects with calcium signalling and the possibility of physical compartmentalisation. Unfortunately, available probes of ER structure such as vital dyes are limited by their membrane specificity. The introduction of ER-targeted GFP plasmids has been a considerable step forward, but these are difficult to express in neurones through conventional transfection approaches. To circumvent such problems we have engineered a novel ER-targeted GFP construct, termed pIN-KDEL, into a 3rd generation replication-defective, self-inactivating lentiviral vector system capable of mediating gene transduction in diverse dividing and post-mitotic mammalian cells, including neurones. RESULTS Following its expression in HEK293 (or COS-7) cells, LV-pIN-KDEL yielded a pattern of fluorescence that co-localised exclusively with the ER marker sec61beta but with no other major organelle. We found no evidence for cytotoxicity and only rarely inclusion body formation. To explore the utility of the probe in resolving the ER in live cells, HEK293 or COS-7 cells were transduced with LV-pIN-KDEL and, after 48 h, imaged directly at intervals from 1 min to several hours. LV-pIN-KDEL fluorescence revealed the endoplasmic reticulum as a tubular lattice structure whose morphology can change markedly within seconds. Although GFP can be phototoxic, the integrity of the cells and ER was retained for several weeks and even after light exposure for periods up to 24 h. Using LV-pIN-KDEL we have imaged the ER in diverse fixed neuronal cultures and, using real-time imaging, found evidence for extensive, dynamic remodelling of the neuronal ER in live hippocampal cultures, brain slices, explants and glia. Finally, through a Fluorescence Loss in Photobleaching (FLIP) approach, continuous irradiation at a single region of interest removed all the fluorescence of LV-pIN-KDEL-transduced nerve cells in explant cultures, thus, providing compelling evidence that in neurons the endoplasmic reticulum is not only dynamic but also continuous. CONCLUSION The lentiviral-based ER-targeted reporter, LV-pIN-KDEL, offers considerable advantages over present systems for defining the architecture of the ER, especially in primary cells such as neurones that are notoriously difficult to transfect. Images and continuous photobleaching experiments of LV-pIN-KDEL-transduced neurones demonstrate that the endoplasmic reticulum is a dynamic structure with a single continuous lumen. The introduction of LV-pIN-KDEL is anticipated to greatly facilitate a real-time visualisation of the structural plasticity and continuous nature of the neuronal ER in healthy and diseased brain tissue

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation

Altered neuronal network and rescue in a human MECP2 duplication model

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders

Altered neuronal network and rescue in a human MECP2 duplication model

Submitted by Nuzia Santos ([email protected]) on 2016-07-13T18:33:59Z No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2016-07-13T18:45:46Z (GMT) No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5)Made available in DSpace on 2016-07-13T18:45:47Z (GMT). No. of bitstreams: 1 ve_Nageshappa_Savitha_Altered_CPqRR_2016.pdf: 973558 bytes, checksum: 36ce1b9b175e435858d7c9a9c623198a (MD5) Previous issue date: 2016Center for Human Genetics. Laboratory for the Genetics of Cognition. KU Leuven, BelgiumUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversité Libre de Bruxelles. Institut de Recherches en Biologie Humaine et Moléculaire.Brussels, Belgium/ VIB Center for the Biology of Disease. Leuven, BelgiumVIB Center for the Biology of Disease. Leuven, Belgium/KU Leuven Center for Human Genetics and Leuven Institute for Neurodegenerative Diseases. KULeuven, BelgiumUniversité Libre de Bruxelles. Institut de Recherches en Biologie Humaine et Moléculaire.Brussels, Belgium/ VIB Center for the Biology of Disease. Leuven, Belgium/ WELBIO. Brussels, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, BelgiumDepartment of Development and Regeneration. Stem Cell Institute Leuven. KU Leuven Medical School. Cluster Stem Cell Biology and Embryology. Leuven, Belgium/Manipal Institute of Regenerative Medicine. Bangalore, IndiaBaylor College of Medicine. Department of Molecular and Human Genetics, and Human Genome Sequencing Center.Houston, TX, USA/Fundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Belo Horizonte, MG, BrasilVIB Center for the Biology of Disease. Leuven, Belgium/ KU Leuven Center for Human Genetics and Leuven Institute for Neurodegenerative Diseases. KULeuven, Belgium/ University of Rome Tor Vergata. Department of Biomedicine and Prevention. Rome, ItalyBaylor College of Medicine. Department of Pediatrics. Section of Pediatric Neurology and Developmental Neuroscience. Houston, TX, USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USABaylor College of Medicine. Department of Molecular and Human Genetics, and Human Genome Sequencing Center.Houston, TX, USACenter for Human Genetics. Laboratory for the Genetics of Cognition. KU Leuven, Belgium/ University Hospitals Leuven. Center for Human Genetics. Department of Clinical genetics. Leuven, BelgiumUniversity of California San Diego.School of Medicine. Department of Pediatrics/Rady Children’s Hospital San Diego. Department of Cellular & Molecular Medicine. Stem Cell Program, La Jolla, CA. USAIncreased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders