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1.  Neural stem cell apoptosis after low methylmercury (MeHg) exposures in postnatal hippocampus produce persistent cell loss and adolescent memory deficits 
Developmental neurobiology  2013;73(12):10.1002/dneu.22119.
The developing brain is particularly sensitive to exposures to environmental contaminants. In contrast to the adult, the developing brain contains large numbers of dividing neuronal precursors, suggesting that they may be vulnerable targets. The postnatal day 7 (P7) rat hippocampus has populations of both mature neurons in the CA1-3 region as well as neural stem cells (NSC) in the dentate gyrus (DG) hilus, that actively produce new neurons that migrate to the granule cell layer (GCL). Using this well-characterized NSC population, we examined the impact of low levels of MeHg on proliferation, neurogenesis, and subsequent adolescent learning and memory behavior. Assessing a range of exposures, we found that a single subcutaneous injection of 0.6μg/g MeHg in P7 rats induced caspase activation in proliferating NSC of the hilus and GCL. This acute NSC death had lasting impact on the DG at P21, reducing cell numbers in the hilus by 22% and the GCL by 27%, as well as reductions in neural precursor proliferation by 25%. In contrast, non-proliferative CA1-3 pyramidal neuron cell number was unchanged. Furthermore, animals exposed to P7 MeHg exhibited an adolescent spatial memory deficit as assessed by Morris water maze. These results suggest that environmentally relevant levels of MeHg exposure may decrease NSC populations and, despite ongoing neurogenesis, the brain may not restore the hippocampal cell deficits, which may contribute to hippocampal-dependent memory deficits during adolescence.
doi:10.1002/dneu.22119
PMCID: PMC3874131  PMID: 23959606
Neural stem cell; apoptosis; methylmercury; hippocampus; development
2.  Engrailed2 modulates cerebellar granule neuron precursor proliferation, differentiation and insulin-like growth factor 1 signaling during postnatal development 
Molecular Autism  2014;5:9.
Background
The homeobox transcription factor Engrailed2 (En2) has been studied extensively in neurodevelopment, particularly in the midbrain/hindbrain region and cerebellum, where it exhibits dynamic patterns of expression and regulates cell patterning and morphogenesis. Because of its roles in regulating cerebellar development and evidence of cerebellar pathology in autism spectrum disorder (ASD), we previously examined an ENGRAILED2 association and found evidence to support EN2 as a susceptibility gene, a finding replicated by several other investigators. However, its functions at the cell biological level remain undefined. In the mouse, En2 gene is expressed in granule neuron precursors (GNPs) just as they exit the cell cycle and begin to differentiate, raising the possibility that En2 may modulate these developmental processes.
Methods
To define En2 functions, we examined proliferation, differentiation and signaling pathway activation in En2 knockout (KO) and wild-type (WT) GNPs in response to a variety of extracellular growth factors and following En2 cDNA overexpression in cell culture. In vivo analyses of cerebellar GNP proliferation as well as responses to insulin-like growth factor-1 (IGF1) treatment were also conducted.
Results
Proliferation markers were increased in KO GNPs in vivo and in 24-h cultures, suggesting En2 normally serves to promote cell cycle exit. Significantly, IGF1 stimulated greater DNA synthesis in KO than WT cells in culture, a finding associated with markedly increased phospho-S6 kinase activation. Similarly, there was three-fold greater DNA synthesis in the KO cerebellum in response to IGF1 in vivo. On the other hand, KO GNPs exhibited reduced neurite outgrowth and differentiation. Conversely, En2 overexpression increased cell cycle exit and promoted neuronal differentiation.
Conclusions
In aggregate, our observations suggest that the ASD-associated gene En2 promotes GNP cell cycle exit and differentiation, and modulates IGF1 activity during postnatal cerebellar development. Thus, genetic/epigenetic alterations of EN2 expression may impact proliferation, differentiation and IGF1 signaling as possible mechanisms that may contribute to ASD pathogenesis.
doi:10.1186/2040-2392-5-9
PMCID: PMC3932947  PMID: 24507165
Autism; Engrailed2; IGF1; Cerebellum; Neurodevelopment; Cell cycle; Proliferation; Phospho-S6 kinase
3.  Pro- and Anti-Mitogenic Actions of PACAP in Developing Cerebral Cortex: Potential Mediation by Developmental Switch of PAC1 Receptor mRNA Isoforms 
During corticogenesis, pituitary adenylate cyclase-activating polypeptide (PACAP; ADCYAP1) may contribute to proliferation control by activating PAC1 receptors of neural precursors in the embryonic ventricular zone. PAC1 receptors, specifically the hop and short isoforms, couple differentially to and activate distinct pathways that produce pro- or anti-mitogenic actions. Previously we found that PACAP was an anti-mitogenic signal from embryonic day 13.5 (E13.5) onwards both in culture and in vivo, and activated cAMP signaling through the short isoform. However, we now find that mice deficient in PACAP exhibited a decrease in the BrdU labeling index in E9.5 cortex, suggesting PACAP normally promotes proliferation at this stage. To further define mechanisms, we established a novel culture model in which the viability of very early cortical precursors (E9.5 mouse and E10.5 rat) could be maintained. At this stage, we found that PACAP evoked intracellular calcium fluxes and increased phospho-PKC levels, as well as stimulated G1 cyclin mRNAs and proteins, S-phase entry and proliferation without affecting cell survival. Significantly, expression of hop receptor isoform was 24-fold greater than the short isoform at E10.5, a ratio that was reversed at E14.5 when short expression was 15-fold greater and PACAP inhibited mitogenesis. Enhanced hop isoform expression, elicited by in vitro treatment of E10.5 precursors with retinoic acid, correlated with sustained pro-mitogenic action of PACAP beyond the developmental switch. Conversely, depletion of hop receptor using shRNA abolished PACAP mitogenic stimulation at E10.5. These observations suggest PACAP elicits temporally specific effects on cortical proliferation via developmentally-regulated expression of specific receptor isoforms.
doi:10.1523/JNEUROSCI.1062-12.2013
PMCID: PMC3652328  PMID: 23447598
4.  N-acetyl cysteine (NAC) treatment reduces mercury-induced neurotoxicity in the developing rat hippocampus 
Journal of Neuroscience Research  2012;90(4):743-750.
Mercury is an environmental toxicant that can disrupt brain development. However, while progress has been made in defining its neurotoxic effects, we know far less about available therapies that can effectively protect brain in exposed individuals. We previously developed an animal model in which we defined the sequence of events underlying neurotoxicity: Methylmercury (MeHg) injection in postnatal rat acutely induced inhibition of mitosis and stimulated apoptosis in the hippocampus, that later resulted in intermediate term deficits in structure size and cell number. NAC is the N-acetyl derivative of L-cysteine used clinically for treatment of drug intoxication. Here, based on its known efficacy in promoting MeHg urinary excretion, we evaluated NAC for protective effects in the developing brain. In immature neurons and precursors MeHg (3µM) induced a >50% decrease in DNA synthesis at 24hr, an effect that was completely blocked by NAC co-incubation. In vivo, injection of MeHg (5µg/gbw) into 7 day-old rats induced a 22% decrease in DNA synthesis in whole hippocampus and a 4-fold increase in activated caspase-3 immunoreactive cells at 24hr, and reduced total cell numbers by 13% at 3 weeks. Treatment of MeHg exposed rats with repeated injections of NAC abolished MeHg toxicity. NAC prevented the reduction in DNA synthesis and the marked increase in caspase-3 immunoreactivity. Moreover, the intermediate term decrease in hippocampal cell number provoked by MeHg was fully blocked by NAC. Altogether, these results suggest that MeHg toxicity in the perinatal brain can be ameliorated by using NAC, opening potential avenues for therapeutic intervention.
PMCID: PMC3306130  PMID: 22420031
mercury; hippocampus; N-acetyl cysteine; neurogenesis; programmed cell death
5.  Methylmercury (MeHg) elicits mitochondrial-dependent apoptosis in developing hippocampus and acts at low exposures 
Neurotoxicology  2011;32(5):535-544.
The developing brain is particularly sensitive to environmental teratogens, such as methylmercury (MeHg), which may induce cell death. Although several mechanisms of MeHg-induced apoptosis have been defined in culture models, pathways mediating caspase-3 activation in vivo remain unclear, especially in the developing hippocampus. To explore apoptotic mechanisms, Sprague-Dawley rats were exposed to 5 μg/g MeHg or PBS vehicle on postnatal day 7 (P7) and the hippocampus was assessed at various times for levels of apoptotic proteins. MeHg induced a 38% increase in Bax protein and an increase in cytosolic cytochrome c at 4 h, followed by later increases in caspase-9 (40% at 12 h; 33% at 24 h) and caspase-8 (33% at 24 h), compared to controls. MeHg also induced an increase in executioner caspase-3, a protease activated by both mitochondrial-dependent caspase-9 and mitochondrial-independent caspase-8. To further define pathways, we used a forebrain culture model and found that the MeHg-induced increases in caspase-3 and caspase-8 were completely blocked by a caspase-9-specific inhibitor, while caspase-9 induction was unperturbed by the caspase-8 inhibitor. These observations suggest that MeHg acts primarily through the mitochondrial-dependent cascade to activate caspase-3 in forebrain precursors, a pathway that may contribute to previously documented neurotoxicity in developing hippocampus. In turn, using the endpoint protein, caspase-3, as a sensitive marker for neural injury, we were able to detect hippocampal cell death in vivo at ten-fold lower levels of MeHg exposure (0.6 μg/g) than previously reported. Thus mitochondrial-dependent cell death in the hippocampus may serve as a sensitive index for teratogenic insults to the developing brain.
doi:10.1016/j.neuro.2011.06.003
PMCID: PMC3256128  PMID: 21741406
Methylmercury; hippocampus; apoptosis; caspase-3; mitochondria
6.  The Cyclin-Dependent Kinase Inhibitor p57Kip2 Regulates Cell Cycle Exit, Differentiation, and Migration of Embryonic Cerebral Cortical Precursors 
Cerebral Cortex (New York, NY)  2011;21(8):1840-1856.
Mounting evidence indicates cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family, including p57Kip2 and p27Kip1, control not only cell cycle exit but also corticogenesis. Nevertheless, distinct activities of p57Kip2 remain poorly defined. Using in vivo and culture approaches, we show p57Kip2 overexpression at E14.5–15.5 elicits precursor cell cycle exit, promotes transition from proliferation to neuronal differentiation, and enhances process outgrowth, while opposite effects occur in p57Kip2-deficient precursors. Studies at later ages indicate p57Kip2 overexpression also induces precocious glial differentiation, suggesting stage-dependent effects. In embryonic cortex, p57Kip2 overexpression advances cell radial migration and alters postnatal laminar positioning. While both CKIs induce differentiation, p57Kip2 was twice as effective as p27Kip1 in inducing neuronal differentiation and was not permissive to astrogliogenic effects of ciliary neurotrophic factor, suggesting that the CKIs differentially modulate cell fate decisions. At molecular levels, although highly conserved N-terminal regions of both CKIs elicit cycle withdrawal and differentiation, the C-terminal region of p57Kip2 alone inhibits in vivo migration. Furthermore, p57Kip2 effects on neurogenesis and gliogenesis require the N-terminal cyclin/CDK binding/inhibitory domains, while previous p27Kip1 studies report cell cycle-independent functions. These observations suggest p57Kip2 coordinates multiple stages of corticogenesis and exhibits distinct and common activities compared with related family member p27Kip1.
doi:10.1093/cercor/bhq254
PMCID: PMC3138513  PMID: 21245411
gliogenesis; in utero electroporation; neurite outgrowth; neurogenesis; transfection
7.  Autism-Relevant Social Abnormalities and Cognitive Deficits in Engrailed-2 Knockout Mice 
PLoS ONE  2012;7(7):e40914.
ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.
doi:10.1371/journal.pone.0040914
PMCID: PMC3400671  PMID: 22829897
8.  Developmental mercury exposure elicits acute hippocampal cell death, reductions in neurogenesis, and severe learning deficits during puberty 
Journal of Neurochemistry  2007;103(5):1968-1981.
Normal brain development requires coordinated regulation of several processes including proliferation, differentiation, and cell death. Multiple factors from endogenous and exogenous sources interact to elicit positive as well as negative regulation of these processes. In particular, the perinatal rat brain is highly vulnerable to specific developmental insults that produce later cognitive abnormalities. We used this model to examine the developmental effects of an exogenous factor of great concern, methylmercury (MeHg). Seven-day-old rats received a single injection of MeHg (5 μg/gbw). MeHg inhibited DNA synthesis by 44% and reduced levels of cyclins D1, D3, and E at 24 h in the hippocampus, but not the cerebellum. Toxicity was associated acutely with caspase-dependent programmed cell death. MeHg exposure led to reductions in hippocampal size (21%) and cell numbers 2 weeks later, especially in the granule cell layer (16%) and hilus (50%) of the dentate gyrus defined stereologically, suggesting that neurons might be particularly vulnerable. Consistent with this, perinatal exposure led to profound deficits in juvenile hippocampal-dependent learning during training on a spatial navigation task. In aggregate, these studies indicate that exposure to one dose of MeHg during the perinatal period acutely induces apoptotic cell death, which results in later deficits in hippocampal structure and function.
doi:10.1111/j.1471-4159.2007.04882.x
PMCID: PMC3363963  PMID: 17760861
cell cycle; hippocampus; learning; methylmercury; neurogenesis; programmed cell death
9.  IGF-1 promotes G1/S cell cycle progression through bidirectional regulation of cyclins and CDK inhibitors via the PI3K/Akt pathway in developing rat cerebral cortex 
The Journal of Neuroscience  2009;29(3):775-788.
While survival promoting effects of insulin-like growth factor-1 (IGF-1) during neurogenesis are well characterized, mitogenic effects remain less well substantiated. Here, we characterize cell cycle regulators and signaling pathways underlying IGF-1 effects on embryonic cortical precursor proliferation in vitro and in vivo. In vitro, IGF-1 stimulated cell cycle progression and increased cell number without promoting cell survival. IGF-1 induced rapid increases in cyclin D1 and D3 protein levels at 4h and cyclin E at 8h. Moreover, p27KIP1 and p57KIP2 expression were reduced, suggesting downregulation of negative regulators contributes to mitogenesis. Further, the PI3K/Akt pathway specifically underlies IGF-1 activity, as blocking this pathway, but not MEK/ERK, prevented mitogenesis. To determine whether mechanisms defined in culture relate to corticogenesis in vivo, we performed transuterine intracerebroventricular injections. While blockade of endogenous factor with anti-IGF-1 antibody decreased DNA synthesis, IGF-1 injection stimulated DNA synthesis and increased the number of S-phase cells in the VZ. IGF-1 treatment increased phospho-Akt 4 fold at 30 min, cyclins D1 and E by 6h, and decreased p27KIP1 and p57KIP2 expression. Moreover, blockade of the PI3K/Akt pathway in vivo decreased DNA synthesis and cyclin E, increased p27KIP1 and p57KIP2 expression, and prevented IGF-1 induced cyclin E mRNA upregulation. Finally, IGF-1 injection in embryos increased P10 brain DNA content by 28%, suggesting a role for IGF-1 in brain growth control. These results demonstrate a mitogenic role for IGF-1 which tightly controls both positive and negative cell cycle regulators, and indicate that the PI3K/Akt pathway mediates IGF-1 mitogenic signaling during corticogenesis.
doi:10.1523/JNEUROSCI.1700-08.2009
PMCID: PMC3256126  PMID: 19158303
corticogenesis; IGF-1; p27KIP1; p57KIP2; PI3K/Akt; proliferation
10.  Animal models of autism spectrum disorders: Information for neurotoxicologists 
Neurotoxicology  2009;30(5):811-821.
Recent findings derived from large-scale datasets and biobanks link multiple genes to autism spectrum disorders. Consequently, novel rodent mutants with deletions, truncations and in some cases, overexpression of these candidate genes have been developed and studied both behaviorally and biologically. At the Annual Neurotoxicology Meeting in Rochester, NY in October of 2008, a symposium of clinicians and basic scientists gathered to present the behavioral features of autism, as well as strategies to model those behavioral features in mice and primates. The aim of the symposium was to provide researchers with up-to-date information on both the genetics of autism and how they are used in differing in vivo and in vitro animal models as well as to provide a background on the environmental exposures being tested on several animal models. In addition, researchers utilizing complementary approaches, presented on cell culture, in vitro or more basic models, which target neurobiological mechanisms, including Drosophila. Following the presentation, a panel convened to explore the opportunities and challenges of using model systems to investigate genetic and environment interactions in autism spectrum disorders. The following paper represents a summary of each presentation, as well as the discussion that followed at the end of the symposium.
doi:10.1016/j.neuro.2009.07.002
PMCID: PMC3014989  PMID: 19596370
Animal models; Autism; Neurotoxicology; Symposium
11.  Patterns of p57Kip2 Expression in Embryonic Rat Brain Suggest Roles in Progenitor Cell Cycle Exit and Neuronal Differentiation 
Developmental neurobiology  2009;69(1):1-21.
In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic day (E) 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular (VZ) and/or mantle zones of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. p57Kip2 expression was also compared to regional transcription factors, including Ngn2, Nkx2.1 and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity co-localized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas co-localization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently co-localized with neuronal marker, β-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: In proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins co-localized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.
doi:10.1002/dneu.20680
PMCID: PMC2967216  PMID: 18814313
Cyclin-Dependent Kinase Inhibitor p57Kip2; Embryonic Development/physiology; Nervous System/cytology/*embryology; Brain/embryology; Neuronal Differentiation
12.  Methylmercury elicits rapid inhibition of cell proliferation in the developing brain and decreases cell cycle regulator, cyclin E 
Neurotoxicology  2006;27(6):970-981.
The developing brain is highly sensitive to methylmercury (MeHg). Still, the initial changes in cell proliferation that may contribute to long-term MeHg effects are largely undefined. Our previous studies with growth factors indicate that acute alterations of G1/S phase transition can permanently affect cell numbers and organ size. Therefore, we determined whether an environmental toxicant could also impact brain development with rapid (6-7h) effects on DNA synthesis and cell cycle machinery in neuronal precursors. In vivo studies in newborn rat hippocampus and cerebellum, two regions of postnatal neurogenesis, were followed by in vitro analysis of two precursor models, cortical and cerebellar cells, focusing on the proteins that regulate G1/S transition. In postnatal day 7 (P7) pups, a single subcutaneous injection of MeHg (3μg/g) acutely (7h) decreased DNA synthesis in the hippocampus by 40% and produced long-term (2 weeks) reductions in total cell number, estimated by DNA quantification. Surprisingly, cerebellar granule cells were resistant to MeHg effects in vivo at comparable tissue concentrations, suggesting region-specific differences in precursor populations. In vitro, MeHg altered proliferation and cell viability, with DNA synthesis selectively inhibited at an early timepoint (6h) corresponding to our in vivo observations. Considering that G1/S regulators are targets of exogenous signals, we used a well-defined cortical cell model to examine MeHg effects on relevant cyclin dependent kinases (CDK) and CDK inhibitors. At 6h, MeHg decreased by 75% levels of cyclin E, a cell cycle regulator with roles in proliferation and apoptosis, without altering p57, p27, or CDK2 nor levels of activated caspase 3. In aggregate, our observations identify the G1/S transition as an early target of MeHg toxicity and raise the possibility that cyclin E degradation contributes to both decreased proliferation and eventual cell death.
doi:10.1016/j.neuro.2006.09.001
PMCID: PMC2013736  PMID: 17056119
Mercury; neurogenesis; neural stem cell; cell cycle; cyclin E; cell survival; cerebral cortex; cerebellar granule precursors; hippocampus; proliferation

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