Timing specific requirement of microRNA function is essential for embryonic and postnatal hippocampal development

The adult hippocampus consists of the dentate gyrus (DG) and the CA1, CA2 and CA3 regions and is essential for learning and memory functions. During embryonic development, hippocampal neurons are derived from hippocampal neuroepithelial cells and dentate granular progenitors. The molecular mechanisms that control hippocampal progenitor proliferation and differentiation are not well understood. Here we show that noncoding microRNAs (miRNAs) are essential for early hippocampal development in mice. Conditionally ablating the RNAase III enzyme Dicer at different embryonic time points utilizing three Cre mouse lines causes abnormal hippocampal morphology and affects the number of hippocampal progenitors due to altered proliferation and increased apoptosis. Lack of miRNAs at earlier stages causes early differentiation of hippocampal neurons, in particular in the CA1 and DG regions. Lack of miRNAs at a later stage specifically affects neuronal production in the CA3 region. Our results reveal a timing requirement of miRNAs for the formation of specific hippocampal regions, with the CA1 and DG developmentally hindered by an early loss of miRNAs and the CA3 region to a late loss of miRNAs. Collectively, our studies indicate the importance of the Dicer-mediated miRNA pathway in hippocampal development and functions.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Subregion-specific vulnerability to endoplasmic reticulum stress-induced neurotoxicity in rat hippocampal neurons

It is well known that in certain disease states, including ischemia and Alzheimer's disease, neurodegeneration occurs in the hippocampus and that vulnerability to neuronal death is area dependent. The present study investigated the mechanism of area-dependent vulnerability to neuronal death under endoplasmic reticulum stress conditions induced by tunicamycin (TM), using rat organotypic hippocampal cultures (OHC) and hippocampal slices. Analysis of propidium iodide uptake showed that TM-induced neuronal death in a concentration-dependent manner (20-80 microg/mL) and that the rank order of vulnerability among hippocampal subregions was dentate gyrus (DG)>CA1>CA3. Results of immunohistochemistry using hippocampal slices also showed that procaspase-12-positive cells in area CA3 were significantly fewer than those in area CA1 and the DG. Moreover, procurement of neurons in areas CA1, CA3 and the DG by laser microdissection, followed by Western blot analysis, also revealed that the level of procaspase-12 in area CA3 was significantly lower than those in area CA1 and the DG. Pretreatment with z-ATAD-fmk, a cell-permeable caspase-12-selective inhibitor significantly attenuated the TM-induced increase of PI fluorescence in the CA1 and DG subregion but not in area CA3. These results suggest that TM elicits subregion-specific neuronal toxicity in OHC and that the vulnerability to TM-induced toxicity is at least partly dependent on the expression level of endogenous procaspase-12 in each area of the hippocampus.     

Role of DNMT3B in the regulation of early neural and neural crest specifiers

The de novo DNA methyltransferase DNMT3B functions in establishing DNA methylation patterns during development. DNMT3B missense mutations cause immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. The restriction of Dnmt3b expression to neural progenitor cells, as well as the mild cognitive defects observed in ICF patients, suggests that DNMT3B may play an important role in early neurogenesis. We performed RNAi knockdown of DNMT3B in human embryonic stem cells (hESCs) in order to investigate the mechanistic contribution of DNMT3B to DNA methylation and early neuronal differentiation. While DNMT3B was not required for early neuroepithelium specification, DNMT3B deficient neuroepithelium exhibited accelerated maturation with earlier expression, relative to normal hESCs, of mature neuronal markers (such as NEUROD1) and of early neuronal regional specifiers (such as those for the neural crest). Genome-wide analyses of DNA methylation by MethylC-seq identified novel regions of hypomethylation in the DNMT3B knockdowns along the X chromosome as well as pericentromeric regions, rather than changes to promoters of specific dysregulated genes. We observed a loss of H3K27me3 and the polycomb complex protein EZH2 at the promoters of early neural and neural crest specifier genes during differentiation of DNMT3B knockdown but not normal hESCs. Our results indicate that DNMT3B mediates large-scale methylation patterns in hESCs and that DNMT3B deficiency in the cells alters the timing of their neuronal differentiation and maturation.     

Enhanced hippocampal F2-isoprostane formation following kainate-induced seizures

We attempted to obtain evidence for the occurrence of oxidant injury following seizure activity by measuring hippocampal F2-isoprostanes (F2-IsoPs), a reliable marker of free radical-induced lipid peroxidation. Formation of F2-IsoPs esterified in hippocampal phospholipids was correlated with hippocampal neuronal loss and mitochondrial aconitase inactivation, a marker of superoxide production in the kainate model. F2-IsoPs were measured in microdissected hippocampal CA1, CA3 and dentate gyrus (DG) regions at various times following kainate administration. Kainate produced a large increase in F2-IsoP levels in the highly vulnerable CA3 region 16 h post injection. The CA1 region showed small, but statistically insignificant increases in F2-IsoP levels. Interestingly, the DG, a region resistant to kainate-induced neuronal death also showed marked (2.5-5-fold) increases in F2-IsoP levels 8, 16, and 24 h post injection. The increases in F2-Isop levels in CA3 and DG were accompanied by inactivation of mitochondrial aconitase in these regions. This marked subregion-specific increase in F2-Isop following kainate administration suggests that oxidative lipid damage results from seizure activity and may play an important role in seizure-induced death of vulnerable neurons.     

Cadherin-9 regulates synapse-specific differentiation in the developing hippocampus

Our understanding of mechanisms that regulate the differentiation of specific classes of synapses is limited. Here, we investigate the formation of synapses between hippocampal dentate gyrus (DG) neurons and their target CA3 neurons and find that DG neurons preferentially form synapses with CA3 rather than DG or CA1 neurons in culture, suggesting that specific interactions between DG and CA3 neurons drive synapse formation. Cadherin-9 is expressed selectively in DG and CA3 neurons, and downregulation of cadherin-9 in CA3 neurons leads to a selective decrease in the number and size of DG synapses onto CA3 neurons. In addition, loss of cadherin-9 from DG or CA3 neurons in vivo leads to striking defects in the formation and differentiation of the DG-CA3 mossy fiber synapse. These observations indicate that cadherin-9 bidirectionally regulates DG-CA3 synapse development and highlight the critical role of differentially expressed molecular cues in establishing specific connections in the mammalian brain.     

Prenatal Nicotine and Maternal Deprivation Stress De-Regulate the Development of CA1, CA3, and Dentate Gyrus Neurons in Hippocampus of Infant Rats

Adverse experiences by the developing fetus and in early childhood are associated with profound effects on learning, emotional behavior, and cognition as a whole. In this study we investigated the effects of prenatal nicotine exposure (NIC), postnatal maternal deprivation (MD) or the combination of the two (NIC+MD) to determine if hippocampal neuron development is modulated by exposure to drugs of abuse and/or stress. Growth of rat offspring exposed to MD alone or NIC+MD was repressed until after weaning. In CA1 but not CA3 of postnatal day 14 (P14) pups, MD increased pyramidal neurons, however, in dentate gyrus (DG), decreased granule neurons. NIC had no effect on neuron number in CA1, CA3 or DG. Unexpectedly, NIC plus MD combined caused a synergistic increase in the number of CA1 or CA3 neurons. Neuron density in CA regions was unaffected by treatment, but in the DG, granule neurons had a looser packing density after NIC, MD or NIC+MD exposure. When septotemporal axes were analyzed, the synergism of stress and drug exposure in CA1 and CA3 was associated with rostral, whereas MD effects were predominantly associated with caudal neurons. TUNEL labeling suggests no active apoptosis at P14, and doublecortin positive neurons and mossy fibers were diminished in NIC+MD relative to controls. The laterality of the effect of nicotine and/or maternal deprivation in right versus left hippocampus was also analyzed and found to be insiginificant. We report for the first time that early life stressors such as postnatal MD and prenatal NIC exposure, when combined, may exhibit synergistic consequences for CA1 and CA3 pyramidal neuron development, and a potential antagonistic influence on developing DG neurons. These results suggest that early stressors may modulate neurogenesis, apoptosis, or maturation of glutamatergic neurons in the hippocampus in a region-specific manner during critical periods of neurodevelopment.     

Neuropeptide Y can rescue neurons from cell death following the application of an excitotoxic insult with kainate in rat organotypic hippocampal slice cultures

In the present work we investigated the neuroprotective role of neuropeptide Y (NPY) after an excitotoxic insult in rat organotypic hippocampal slice cultures. Exposure of 2 week-old rat hippocampal slice cultures to 12muM kainate (KA) for 24h induced neuronal death in dentate gyrus (DG) granular cell layer, CA1 and CA3 pyramidal cell layers, as quantified by cellular propidium iodide (PI) uptake. The activation of Y(1) or Y(2) receptors 30min after starting the exposure to the excitotoxic insult with kainate resulted in neuroprotection by reducing the PI uptake in DG, CA1 and CA3 cell layers. The use of Y(1) or Y(2) receptors antagonists, BIBP3226 (1muM) or BIIE0246 (1muM), resulted in the loss of the neuroprotection induced by the activation of Y(1) or Y(2) receptors, respectively, in all hippocampal subfields. Taken together these results suggest that activation of NPY Y(1) or Y(2) receptors activates neuroprotective pathways that are able to rescue neurons from excitotoxic cell death.     

Impact of exercise and a complex environment on hippocampal dendritic morphology,Bdnf gene expression,and DNA methylation in male rat pups neonatally exposed to alcohol

Alcohol exposure in utero can result in Fetal Alcohol Spectrums Disorders (FASD). Measures of hippocampal neuroplasticity, including long‐term potentiation, synaptic and dendritic organization, and adult neurogenesis, are consistently disrupted in rodent models of FASD. The current study investigated whether third trimester‐equivalent binge‐like alcohol exposure (AE) [postnatal days (PD) 4–9] affects dendritic morphology of immature dentate gyrus granule cells, and brain‐derived neurotrophic factor (Bdnf ) gene expression and DNA methylation in hippocampal tissue in adult male rats. To understand immediate impact of alcohol, DNA methylation was measured in the PD10 hippocampus. In addition, two behavioral interventions, wheel running (WR) and environmental complexity (EC), were utilized as rehabilitative therapies for alcohol‐induced deficits. AE significantly decreased dendritic complexity of the immature neurons, demonstrating the long‐lasting impact of neonatal alcohol exposure on dendritic morphology of immature neurons in the hippocampus. Both housing conditions robustly enhanced dendritic complexity in the AE animals. While Bdnf exon I DNA methylation was lower in the AE and sham‐intubated animals compared with suckle controls on PD10, alterations to Bdnf DNA methylation and gene expression levels were not present at PD72. In control animals, exercise, but not exercise followed by housing in EC, resulted in higher levels of hippocampal Bdnf gene expression and lower DNA methylation. These studies demonstrate the long‐lasting negative impact of developmental alcohol exposure on hippocampal dendritic morphology and support the implementation of exercise and complex environments as therapeutic interventions for individuals with FASD. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 708–725, 2017     

Dab1 contributes differently to the morphogenesis of the hippocampal subdivisions

The hippocampal formation (HF) is morphologically and functionally distinguishable into the subdivisions, such as the dentate gyrus (DG), subiculum, and Ammon's horn. The Ammon's horn is further divided into the CA (Cornu Ammonis)1, CA2, and CA3. The Reelin‐Dab1 signal is essential for the morphogenesis of the mammalian brain. In the neocortex of Reelin‐Dab1 signal mutants the laminar pattern of the neurons is disrupted along the radial axis. Morphological abnormalities in the HF of the Reelin‐Dab1 mutants were known, but how these abnormalities appear during development had not been extensively studied. We examined the morphology of the well‐developed Dab1 deficient HF by staining with a silver impregnation method in this report, and found that disruption of lamination in the CA1, CA3, and DG was different. Abnormalities observed in the development of Dab1 deficient CA1 were similar to those reported in the neocortical development, while Dab1 deficient CA3 neuronal progenitors radially spreaded beyond presumptive pyramidal layer. The intermediate progenitor cells ectopically located in the Dab1 deficient DG, but neurogenesis was normal in the CA1 and CA3. These observations suggest that the morphogenesis in these HF subdivisions employs different developmental mechanisms involving Dab1 function.     

Glycogen Synthase Kinase-3β Regulates Equilibrium Between Neurogenesis and Gliogenesis in Rat Model of Parkinson’s Disease: a Crosstalk with Wnt and Notch Signaling

Neurogenesis involves generation of functional newborn neurons from neural stem cells (NSCs). Insufficient formation or accelerated degeneration of newborn neurons may contribute to the severity of motor/nonmotor symptoms of Parkinson’s disease (PD). However, the functional role of adult neurogenesis in PD is yet not explored and whether glycogen synthase kinase-3β (GSK-3β) affects multiple steps of adult neurogenesis in PD is still unknown. We investigated the possible underlying molecular mechanism of impaired adult neurogenesis associated with PD. Herein, we show that single intra-medial forebrain bundle (MFB) injection of 6-hydroxydopamine (6-OHDA) efficiently induced long-term activation of GSK-3β and reduced NSC self-renewal, proliferation, neuronal migration, and neuronal differentiation accompanied with increased astrogenesis in subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Indeed, 6-OHDA also delayed maturation of neuroblasts in the DG as witnessed by their reduced dendritic length and arborization. Using a pharmacological approach to inhibit GSK-3β activation by specific inhibitor SB216763, we show that GSK-3β inhibition enhances radial glial cells, NSC proliferation, self-renewal in the SVZ, and the subgranular zone (SGZ) in the rat PD model. Pharmacological inhibition of GSK-3β activity enhances neuroblast population in SVZ and SGZ and promotes migration of neuroblasts towards the rostral migratory stream and lesioned striatum from dorsal SVZ and lateral SVZ, respectively, in PD model. GSK-3β inhibition enhances dendritic arborization and survival of granular neurons and stimulates NSC differentiation towards the neuronal phenotype in DG of PD model. The aforementioned effects of GSK-3β involve a crosstalk between Wnt/β-catenin and Notch signaling pathways that are known to regulate NSC dynamics.     

Effects of Transient Cerebral Ischemia on the Expression of DNA Methyltransferase 1 in the Gerbil Hippocampal CA1 Region

DNA methylation is a key epigenetic modification of DNA that is catalyzed by DNA methyltransferases (Dnmt). Increasing evidences suggest that DNA methylation in neurons regulates synaptic plasticity as well as neuronal network activity. In the present study, we investigated the changes in DNA methyltransferases 1 (Dnmt1) immunoreactivity and its protein levels in the gerbil hippocampal CA1 region after 5 min of transient global cerebral ischemia. CA1 pyramidal neurons were well stained with NeuN (a neuron-specific soluble nuclear antigen) antibody in the sham-group, Four days after ischemia–reperfusion (I–R), NeuN-positive (⁺) cells were significantly decreased in the stratum pyramidale (SP) of the CA1 region, and many Fluro-Jade B (a marker for neuronal degeneration)⁺ cells were observed in the SP. Dnmt1 immunoreactivity was well detected in all the layers of the sham-group. Dnmt1 immunoreactivity was hardly detected only in the stratum pyramidale of the CA1 region from 4 days post-ischemia; however, at these times, Dnmt1 immunoreactivity was newly expressed in GABAergic interneurons or astrocytes in the ischemic CA1 region. In addition, the level of Dnmt1 was lowest at 4 days post-ischemia. In brief, both the Dnmt1 immunoreactivity and protein levels were distinctively decreased in the ischemic CA1 region 4 days after transient cerebral ischemia. These results indicate that the decrease of Dnmt1 expression at 4 days post-ischemia may be related to ischemia-induced delayed neuronal death.     

A Novel Suppressive Effect of Alcohol Dehydrogenase 5 in Neuronal Differentiation

Alcohol dehydrogenase 5 (ADH5) is a conserved enzyme for alcohol and aldehyde metabolism in mammals. Despite dynamic expression throughout neurogenesis, its role in neuronal development remains unknown. Here we present the first evidence that ADH5 is a negative regulator of neuronal differentiation. Gene expression analyses identify a constant reduction of ADH5 levels throughout neuronal development. Overexpression of ADH5 reduces both development and adult neuronal differentiation of mouse neurons. This effect depends on the catalytic activity of ADH5 and involves ADH5-mediated denitrosation of histone deacetylase 2 (HDAC2). Our results indicate that ADH5 counteracts neuronal differentiation of human neural stem cells and that this effect can be reversed by pharmacological inhibition of ADH5. Based on these observations, we propose that ADH5 is a novel suppressor of neuronal differentiation and maturation. Inhibition of ADH5 may improve adult neurogenesis in a physiological or pathological setting.