Early Postnatal Stages Research Articles

Effects of Cannabinoid Exposure during Neurodevelopment on Future Effects of Drugs of Abuse: A Preclinical Perspective.

The endocannabinoid system plays a central role in the earliest stages of embryonic, postnatal and adolescent neurodevelopment. Aberrant activity of this system at key developmental phases has been shown to affect neural development. The aim of this review is to synthesise and analyse preclinical insights within rodent populations, focusing on the effects that perinatal (embryonic, gestational and early postnatal developmental stages) and adolescent (postnatal day 21–60) cannabinoid exposure impose across time on the subsequent activity of various drugs of abuse. Results in rodents show that exposure to cannabinoids during the perinatal and adolescent period can lead to multifaceted behavioural and molecular changes. In the perinatal period, significant effects of Δ9-THC exposure on subsequent opiate and amphetamine reward-related behaviours were observed primarily in male rodents. These effects were not extended to include cocaine or alcohol. In adolescence, various cannabinoid agonists were used experimentally. This array of cannabinoids demonstrated consistent effects on opioids across sex. In contrast, no significant effects were observed regarding the future activity of amphetamines and cocaine. However, these studies focused primarily on male rodents. In conclusion, numerous gaps and limitations are apparent in the current body of research. The sparsity of studies analysing the perinatal period must be addressed. Future research within both periods must also focus on delineating sex-specific effects, moving away from a male-centric focus. Studies should also aim to utilise more clinically relevant cannabinoid treatments.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

CRISPR/Cas9-based genetic screen of SCNT-reprogramming resistant genes identifies critical genes for male germ cell development in mice

Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, although the molecular mechanisms remain largely elusive. We have previously identified somatic cell nuclear transfer-reprogramming resistant genes (SRRGs) that are highly enriched for genes essential for spermatogenesis, although many of them remain uncharacterized in knockout (KO) mice. Here, we performed a CRISPR-based genetic screen using C57BL/6N mice for five uncharacterized SRRGs (Cox8c, Cox7b2, Tuba3a/3b, Faiml, and Gm773), together with meiosis essential gene Majin as a control. RT-qPCR analysis of mouse adult tissues revealed that the five selected SRRGs were exclusively expressed in testis. Analysis of single-cell RNA-seq datasets of adult testis revealed stage-specific expression (pre-, mid-, or post-meiotic expression) in testicular germ cells. Examination of testis morphology, histology, and sperm functions in CRISPR-injected KO adult males revealed that Cox7b2, Gm773, and Tuba3a/3b are required for the production of normal spermatozoa. Specifically, Cox7b2 KO mice produced poorly motile infertile spermatozoa, Gm773 KO mice produced motile spermatozoa with limited zona penetration abilities, and Tuba3a/3b KO mice completely lost germ cells at the early postnatal stages. Our genetic screen focusing on SRRGs efficiently identified critical genes for male germ cell development in mice, which also provides insights into human reproductive medicine.

Spatial organization and transitions of spontaneous neuronal activities in the developing sensory cortex.

The sensory cortex underlies our ability to perceive and interact with the external world. Sensory perceptions are controlled by specialized neuronal circuits established through fine-tuning, which relies largely on neuronal activity during the development. Spontaneous neuronal activity is an essential driving force of neuronal circuit refinement. At early developmental stages, sensory cortices display spontaneous activities originating from the periphery and characterized by correlated firing arranged spatially according to the modality. The firing patterns are reorganized over time and become sparse, which is typical for the mature brain. This review focuses mainly on rodent sensory cortices. First, the features of the spontaneous activities during early postnatal stages are described. Then, the developmental changes in the spatial organization of the spontaneous activities and the transition mechanisms involved are discussed. The identification of the principles controlling the spatial organization of spontaneous activities in the developing sensory cortex is essential to understand the self-organization process of neuronal circuits.

LDLR expression in the cochlea suggests a role in endolymph homeostasis and cochlear amplification

There is now growing evidence that hypercholesterolemia and high serum levels of low-density lipoproteins (LDL) predispose to sensorineural hearing loss. Circulating LDL-cholesterol is delivered to peripheral tissues via LDL receptor (LDLR) -mediated endocytosis. Recently, it has been shown that LDLR gene polymorphisms are associated with higher susceptibility to sudden deafness. These findings suggested that we should investigate the expression of LDLR from the postnatal maturation of the mouse cochlea until adulthood. In the cochlea of newborn mice, we observed that LDLR is mostly expressed in the lateral wall of the cochlea, especially in a band of cells directly facing the cochlear duct. Moreover, LDLR is expressed in the inner and outer hair cells, as well as in the adjacent greater epithelial ridge. In early postnatal stages, LDLR is expressed in the marginal cells of the immature stria vascularis, in the root cells of the spiral ligament, and in the adjacent outer sulcus cells. At the same time, LDLR begins to be expressed in the pillar cells of the immature organ of Corti. From the onset of hearing, LDLR is expressed in the marginal cells of the stria vascularis, in the outer sulcus cells, and in the capillaries of the adjacent spiral ligament. In the organ of Corti, LDLR is expressed in outer pillar cells and Deiters’ cells, i.e. in the non-sensory supporting cells that directly surround the outer hair cells. These cells are believed to provide a mechanical coupling with the outer hair cells to modulate electromotility and cochlear amplification. In the stria vascularis of three-month-old mice, LDLR is further expressed in both marginal and intermediate cells. Overall, our results suggest that LDLR is mostly present in cochlear cells that are involved in endolymph homeostasis and cochlear amplification. Further functional studies will be needed to unravel how LDLR regulates extracellular and intracellular levels of cholesterol and lipoproteins in the cochlea, and how it could influence cochlear homeostasis.

Neurovascular Coupling in Development and Disease: Focus on Astrocytes.

Neurovascular coupling is a crucial mechanism that matches the high energy demand of the brain with a supply of energy substrates from the blood. Signaling within the neurovascular unit is responsible for activity-dependent changes in cerebral blood flow. The strength and reliability of neurovascular coupling form the basis of non-invasive human neuroimaging techniques, including blood oxygen level dependent (BOLD) functional magnetic resonance imaging. Interestingly, BOLD signals are negative in infants, indicating a mismatch between metabolism and blood flow upon neural activation; this response is the opposite of that observed in healthy adults where activity evokes a large oversupply of blood flow. Negative neurovascular coupling has also been observed in rodents at early postnatal stages, further implying that this is a process that matures during development. This rationale is consistent with the morphological maturation of the neurovascular unit, which occurs over a similar time frame. While neurons differentiate before birth, astrocytes differentiate postnatally in rodents and the maturation of their complex morphology during the first few weeks of life links them with synapses and the vasculature. The vascular network is also incomplete in neonates and matures in parallel with astrocytes. Here, we review the timeline of the structural maturation of the neurovascular unit with special emphasis on astrocytes and the vascular tree and what it implies for functional maturation of neurovascular coupling. We also discuss similarities between immature astrocytes during development and reactive astrocytes in disease, which are relevant to neurovascular coupling. Finally, we close by pointing out current gaps in knowledge that must be addressed to fully elucidate the mechanisms underlying neurovascular coupling maturation, with the expectation that this may also clarify astrocyte-dependent mechanisms of cerebrovascular impairment in neurodegenerative conditions in which reduced or negative neurovascular coupling is noted, such as stroke and Alzheimer’s disease.

Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains

The p75 neurotrophin receptor (p75NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.

Efnb2 haploinsufficiency induces early gap junction plaque disassembly and endocytosis in the cochlea

Chromosome 13q deletions encompassing EFNB2, which encodes the transmembrane protein ephrin-B2, are likely to cause syndromic forms of sensorineural hearing loss of unclear origin. Thus, unravelling the pathogenic mechanisms could help to improve therapeutic strategies. In the cochlea, adjacent non-sensory epithelial cells are connected via gap junction channels, the activity of which is critical to maintain cochlear homeostasis. Here we show that ephrin-B2 promotes the assembly of connexin 30 (Cx30) gap junction plaques (GJPs) between adjacent non-sensory Deiters’ cells. An in situ proximity ligation assay revealed that ephrin-B2 preferentially interacts with Cx30 in the periphery of the GJPs, i.e. where newly synthesized connexin hemichannels accrue to the GJP. Moreover, we observed that heterozygous mice encoding an Efnb2 null allele display excessive clathrin-mediated internalization of Cx30 GJPs in early postnatal stages. Finally, an in vitro organotypic assay revealed that ectopic activation of ephrin-B2 reverse signalling promotes the internalization of Cx30 GJPs. These data argue in favor of a cell-autonomous, Eph receptor-independent role of ephrin-B2 in the assembly of Cx30 GJPs. According to recent observations, early GJP degradation could certainly play a role in the pathogenic process leading to progressive sensorineural hearing loss due to Efnb2/EFNB2 haploinsufficiency.

The physiological role of Homer2a and its novel short isoform, Homer2e, in NMDA receptor-mediated apoptosis in cerebellar granule cells

Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.

Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass

ObjectiveEarly postnatal life is a critical period for the establishment of the functional β-cell mass that will sustain whole-body glucose homeostasis during the lifetime. β cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult β cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal β-cell development. MethodsTo study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from β-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. β-cell mass was studied using histomorphometric analysis and optical projection tomography. β-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted β cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation. ResultsElimination of Gsα in β cells led to reduced β-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key β-cell identity and maturation genes in postnatal β-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin. ConclusionWe conclude that Gsα is required for β-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal β cells, which may have implications for using cAMP-raising agents to promote β-cell regeneration and maturation in diabetes.

Neural cell adhesion molecule is required for ventricular conduction system development.

ABSTRACTThe most distal portion of the ventricular conduction system (VCS) contains cardiac Purkinje cells (PCs), which are essential for synchronous activation of the ventricular myocardium. Contactin-2 (CNTN2), a member of the immunoglobulin superfamily of cell adhesion molecules (IgSF-CAMs), was previously identified as a marker of the VCS. Through differential transcriptional profiling, we discovered two additional highly enriched IgSF-CAMs in the VCS: NCAM-1 and ALCAM. Immunofluorescence staining showed dynamic expression patterns for each IgSF-CAM during embryonic and early postnatal stages, but ultimately all three proteins became highly enriched in mature PCs. Mice deficient in NCAM-1, but not CNTN2 or ALCAM, exhibited defects in PC gene expression and VCS patterning, as well as cardiac conduction disease. Moreover, using ST8sia2 and ST8sia4 knockout mice, we show that inhibition of post-translational modification of NCAM-1 by polysialic acid leads to disrupted trafficking of sarcolemmal intercalated disc proteins to junctional membranes and abnormal expansion of the extracellular space between apposing PCs. Taken together, our data provide insights into the complex developmental biology of the ventricular conduction system.

Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior.

SummaryCortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers—due to a defect in precursor cell proliferation during embryogenesis—extra parvalbumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network maturation. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

Specification of oxytocinergic and vasopressinergic circuits in the developing mouse brain

Oxytocin (OXT) and arginine vasopressin (AVP) support a broad range of behaviors and homeostatic functions including sex-specific and context-appropriate social behaviors. Although the alterations of these systems have been linked with social-related disorders such as autism spectrum disorder, their formation and developmental dynamics remain largely unknown. Using novel brain clearing techniques and 3D imaging, we have reconstructed the specification of oxytocinergic and vasopressinergic circuits in the developing mouse brain with unprecedented cellular resolution. A systematic quantification indicates that OXT and AVP neurons in the hypothalamus display distinctive developmental dynamics and high cellular plasticity from embryonic to early postnatal stages. Our findings reveal new insights into the specification and consolidation of neuropeptidergic systems in the developing CNS.

Lactational Metformin Treatment in Mouse Dams Ameliorates Maternal High-Fat Diet-induced Metabolic Complications in Male Offspring

Maternal obesity/diabetes severely affects the offspring’s metabolism not only during the fetal and early life development but also later on in the childhood and adulthood stages. Recent evidence suggested that maternal metformin treatment during pregnancy and breastfeeding might provide long-term metabolic benefits on offspring’s glucose homeostasis. We employed a mouse model of maternal overnutrition induced by high fat diet (HFD) to investigate whether an early metformin treatment in mouse dams during lactation period attenuates the maternal HFD-induced metabolic complications in male offspring. The results showed that male offspring from metformin-exposed mothers during suckling period displayed lower body weight and decreased white fat contents. Furthermore, these male offspring had lower blood glucose levels, decreased hyperinsulinemia, enhanced glucose tolerance and insulin sensitivity. Moreover, we found that short-term metformin treatment during lactation period in mouse dams solely improved the glucose tolerance of male offspring exposed with high fat diet (HFD) during both in utero and early postnatal stages, which might primarily result from an increase in insulin secretion. However, this metformin treatment in mouse dams was enabled to abolish most of the metabolic complications of the male offspring exposed with HFD during lactation period. Together, these findings suggest that an early intervention in mouse dams by metformin treatment during lactation period may provide long-term metabolic benefits in regulation of male offspring’s glucose homeostasis.

Central Nervous System Developmental Regulation of Microglia via Cytokines and Chemokines

Microglia are immune cells resident in the central nervous system (CNS). It has been gradually clarified that microglia play various roles at the developmental stage of the CNS. From embryonic to early postnatal age, microglia remove apoptotic cells by phagocytosis and refine the neural circuits by synaptic pruning. In addition, microglia promote the proliferation and differentiation of neural stem cells by releasing physiologically active substances. Our group has focused on the physiological actions of microglia via cytokines and chemokines at the early postnatal developmental stage. We found that a large number of activated microglia accumulate in the early postnatal subventricular zone (SVZ). We demonstrated that the these SVZ microglia facilitate neurogenesis and oligodendrogenesis via inflammatory cytokines including IL-1β, TNFα, IL-6, IFNγ. We have also found that microglia regulate the functional maturation of the blood brain barrier (BBB) and identified the cytokines and chemokines involved in the effects of microglia. These findings indicate that microglia are physiologically more important than ever thought to reveal robust brain functions. Furthermore, the new mode of microglial action may lead to the discovery of drug targets of the incurable CNS diseases.

MiRNA and mRNA Profiling Links Connexin Deficiency to Deafness via Early Oxidative Damage in the Mouse Stria Vascularis.

Pathogenic mutations in the non-syndromic hearing loss and deafness 1 (DFNB1) locus are the primary cause of monogenic inheritance for prelingual hearing loss. To unravel molecular pathways involved in etiopathology and look for early degeneration biomarkers, we used a system biology approach to analyze Cx30−/− mice at an early cochlear post-natal developmental stage. These mice are a DFNB1 mouse model with severely reduced expression levels of two connexins in the inner ear, Cx30, and Cx26. Integrated analysis of miRNA and mRNA expression profiles in the cochleae of Cx30−/− mice at post-natal day 5 revealed the overexpression of five miRNAs (miR-34c, miR-29b, miR-29c, miR-141, and miR-181a) linked to apoptosis, oxidative stress, and cochlear degeneration, which have Sirt1 as a common target of transcriptional and/or post-transcriptional regulation. In young adult Cx30−/− mice (3 months of age), these alterations culminated with blood barrier disruption in the Stria vascularis (SV), which is known to have the highest aerobic metabolic rate of all cochlear structures and whose microvascular alterations contribute to age-related degeneration and progressive decline of auditory function. Our experimental validation of selected targets links hearing acquisition failure in Cx30−/− mice, early oxidative stress, and metabolic dysregulation to the activation of the Sirt1–p53 axis. This is the first integrated analysis of miRNA and mRNA in the cochlea of the Cx30−/− mouse model, providing evidence that connexin downregulation determines a miRNA-mediated response which leads to chronic exhaustion of cochlear antioxidant defense mechanisms and consequent SV dysfunction. Our analyses support the notion that connexin dysfunction intervenes early on during development, causing vascular damage later on in life. This study identifies also early miRNA-mediated biomarkers of hearing impairment, either inherited or age related.

BMP signaling suppresses Gemc1 expression and ependymal differentiation of mouse telencephalic progenitors

The lateral ventricles of the adult mammalian brain are lined by a single layer of multiciliated ependymal cells, which generate a flow of cerebrospinal fluid through directional beating of their cilia as well as regulate neurogenesis through interaction with adult neural stem cells. Ependymal cells are derived from a subset of embryonic neural stem-progenitor cells (NPCs, also known as radial glial cells) that becomes postmitotic during the late embryonic stage of development. Members of the Geminin family of transcriptional regulators including GemC1 and Mcidas play key roles in the differentiation of ependymal cells, but it remains largely unclear what extracellular signals regulate these factors and ependymal differentiation during embryonic and early-postnatal development. We now show that the levels of Smad1/5/8 phosphorylation and Id1/4 protein expression—both of which are downstream events of bone morphogenetic protein (BMP) signaling—decline in cells of the ventricular-subventricular zone in the mouse lateral ganglionic eminence in association with ependymal differentiation. Exposure of postnatal NPC cultures to BMP ligands or to a BMP receptor inhibitor suppressed and promoted the emergence of multiciliated ependymal cells, respectively. Moreover, treatment of embryonic NPC cultures with BMP ligands reduced the expression level of the ependymal marker Foxj1 and suppressed the emergence of ependymal-like cells. Finally, BMP ligands reduced the expression levels of Gemc1 and Mcidas in postnatal NPC cultures, whereas the BMP receptor inhibitor increased them. Our results thus implicate BMP signaling in suppression of ependymal differentiation from NPCs through regulation of Gemc1 and Mcidas expression during embryonic and early-postnatal stages of mouse telencephalic development.

Phenotypic and functional comparison of rat enteric neural crest-derived cells during fetal and early-postnatal stages.

In our previous study, we showed that with increasing time in culture, the growth characteristics of enteric neural crest-derived cells (ENCCs) change, and that the proliferation, migration and neural differentiation potential of these cells in vitro notably diminish. However, there are no studies on the developmental differences in these characteristics between fetal and early-postnatal stages in vitro or in vivo. In this study, we isolated fetal (embryonic day 14.5) and postnatal (postnatal day 2) ENCCs from the intestines of rats. Fetal ENCCs had greater maximum cross-sectional area of the neurospheres, stronger migration ability, and reduced apoptosis, compared with postnatal ENCCs. However, fetal and postnatal ENCCs had a similar differentiation ability. Fetal and postnatal ENCCs both survived after transplant into a rat model of Hirschsprung's disease. In these rats with Hirschsprung's disease, the number of ganglionic cells in the myenteric plexus was higher and the distal intestinal pressure change was greater in animals treated with fetal ENCCs compared with those treated with postnatal ENCCs. These findings suggest that, compared with postnatal ENCCs, fetal ENCCs exhibit higher survival and proliferation and migration abilities, and are therefore a more appropriate seed cell for the treatment of Hirschsprung's disease. This study was approved by the Animal Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (approval No. 2016086) on March 3, 2016.

Early postnatal oleic acid administration enhances synaptic development and cognitive abilities in the Ts65Dn mouse model of Down syndrome

ABSTRACT Objectives The brains of individuals with Down syndrome (DS) present defects in neurogenesis and synaptogenesis during prenatal and early postnatal stages that are partially responsible for their cognitive disabilities. Because oleic and linolenic fatty acids enhance neurogenesis, synaptogenesis, and cognitive abilities in rodents and humans, in this study we evaluated the ability of these compounds to restore these altered phenotypes in the Ts65Dn (TS) mouse model of DS during early postnatal stages. Methods TS and euploid mice were treated with oleic or linolenic acid from PD3 to PD15, and the short- and long- term effects of these acids on neurogenesis and synaptogenesis were evaluated. The effects of these treatments on the cognitive abilities of TS mice during early adulthood were also evaluated. Results Administration of oleic or linolenic acid did not modify cell proliferation immediately after treatment discontinuation or several weeks later. However, oleic acid increased the total number of DAPI+ cells (+ 26%), the percentage of BrdU+ cells that acquired a neural phenotype (+ 9.1%), the number of pre- (+ 29%) and post-synaptic (+ 32%) terminals and the cognitive abilities of TS mice (+ 18.1%). In contrast, linolenic acid only produced a slight cognitive improvement in TS mice. (+12.1%). Discussion These results suggest that early postnatal administration of oleic acid could palliate the cognitive deficits of DS individuals.

Abstract 17364: G-protein Signaling Regulation of the Hippo Pathway in Neonatal Heart Regeneration

Introduction: The regeneration potential in the adult mammalian heart is very limited due to the cessation of cardiomyocyte proliferation shortly after birth. Recent studies have revealed that changes after birth such as metabolic state, oxygen level, cardiomyocyte structure and maturity, immune system and polyploidy are among the factors contributing to the loss of the regenerative potential in the heart. The mechanisms that regulate the cardiac regenerative window are not well understood. Here we report that G-protein mediated signaling regulates Hippo-YAP in neonatal cardiomyocyte proliferation and heart regeneration through Rho activity. Hypothesis: Gas encoded by the Gnas gene, a downstream effector of beta-adrenergic receptor (βAR) inhibits cardiomyocyte proliferation via regulation of YAP activity. Methods: We pharmacologically inhibited the G protein coupled receptor mediated β adrenergic signaling with a β-blocker (metoprolol) at early postnatal stages, and genetically by deleting Gnas in the heart with αMHC-Cre. We accessed the cardiomyocyte proliferation, heart regeneration in these mice and elucidated molecular mechanisms. Results: We found that β-blocker enhanced cardiomyocyte proliferation and promoted cardiac regeneration post cardiac injury with improved cardiac function. Consistent with β-blocker treated mice, mice lacking Gnas in cardiomyocytes exhibited enlarged hearts with an increase in cardiomyocyte proliferation. RNA-seq analysis revealed that these cardiomyocytes maintained an immature status even at young-adult age. The genes associated with mitochondrial oxidative metabolism, the major energy source for mature cardiomyocytes, were downregulated. Moreover, YAP activity was upregulated in both cases. We also found that loss of Gαs function caused upregulation of RhoA activity, and inhibitor of Rho signaling pathway suppressed the YAP activity in cardiomyocytes. Conclusions: Our study reveals that Gαs negatively regulate cardiomyocyte proliferation and provides mechanistic insight for β-blocker treatment as a strategy for inducing cardiac dedifferentiation and proliferation in injured heart.