Translational control plays a key role in regulation of neuronal activity and behavior. Deletion of the translational repressor 4E-BP2 in mice alters excitatory and inhibitory synaptic functions, engendering autistic-like behaviors. The contribution of 4E-BP2-dependent translational control in excitatory and inhibitory neurons and astrocytic cells to these behaviors remains unknown. To investigate this, we generated cell-type-specific conditional 4E-BP2 knockout mice and tested them for the salient features of autism, including repetitive stereotyped behaviors (self-grooming and marble burying), sociability (3-chamber social and direct social interaction tests), and communication (ultrasonic vocalizations in pups). We found that deletion of 4E-BP2 in GABAergic inhibitory neurons, defined by Gad2, resulted in impairments in social interaction and vocal communication. In contrast, deletion of 4E-BP2 in forebrain glutamatergic excitatory neurons, defined by Camk2a, or in astrocytes, defined by Gfap, failed to cause autistic-like behavioral abnormalities. Taken together, we provide evidence for an inhibitory-cell-specific role of 4E-BP2 in engendering autism-related behaviors.

OBJECTIVE

The uroguanylin-GUCY2C gut-brain axis has emerged as one component regulating feeding, energy homeostasis, body mass and metabolism. Here, we explore a role for this axis in mechanisms underlying diet-induced obesity (DIO).

METHODS

Intestinal uroguanylin expression and secretion, and hypothalamic GUCY2C expression and anorexigenic signaling, were quantified in mice on high-calorie diets for 14 weeks. The role of endoplasmic reticulum (ER) stress in suppressing uroguanylin in DIO was explored using tunicamycin, an inducer of ER stress, and tauroursodeoxycholic acid (TUDCA), a chemical chaperone that inhibits ER stress. The impact of consumed calories on uroguanylin expression was explored by dietary manipulation. The role of uroguanylin in mechanisms underlying obesity was examined using Camk2a-Cre-ER(T2)-Rosa-STOP(loxP/loxP)-Guca2b mice in which tamoxifen induces transgenic hormone expression in brain.

RESULTS

DIO suppressed intestinal uroguanylin expression and eliminated its postprandial secretion into the circulation. DIO suppressed uroguanylin through ER stress, an effect mimicked by tunicamycin and blocked by TUDCA. Hormone suppression by DIO reflected consumed calories, rather than the pathophysiological milieu of obesity, as a diet high in calories from carbohydrates suppressed uroguanylin in lean mice, whereas calorie restriction restored uroguanylin in obese mice. However, hypothalamic GUCY2C, enriched in the arcuate nucleus, produced anorexigenic signals mediating satiety upon exogenous agonist administration, and DIO did not impair these responses. Uroguanylin replacement by transgenic expression in brain repaired the hormone insufficiency and reconstituted satiety responses opposing DIO and its associated comorbidities, including visceral adiposity, glucose intolerance and hepatic steatosis.

CONCLUSIONS

These studies reveal a novel pathophysiological mechanism contributing to obesity in which calorie-induced suppression of intestinal uroguanylin impairs hypothalamic mechanisms regulating food consumption through loss of anorexigenic endocrine signaling. The correlative therapeutic paradigm suggests that, in the context of hormone insufficiency with preservation of receptor sensitivity, obesity may be prevented or treated by GUCY2C hormone replacement.

Mammalian SWI/SNF-like BAF chromatin remodeling complexes are essential for many aspects of neural development. Mutations in the genes encoding the core subunit Brg1/SmarcA4 or other complex components cause neurodevelopmental diseases and are associated with autism. Congenital hydrocephalus is a serious brain disorder often experienced by these patients. We report a role of Brg1 in the pathogenesis of hydrocephalus disorder. We discovered an unexpected early activity of mouse Camk2a-Cre transgene, which mediates Brg1 deletion in a subset of forebrain neurons beginning in the late embryonic stage. Brg1 deletion in these neurons led to severe congenital hydrocephalus with enlargement of the lateral ventricles and attenuation of the cerebral cortex. The Brg1-deficient mice had significantly smaller subcommissural organs and narrower Sylvian aqueducts than mice that express normal levels of Brg1. Effects were non-cell autonomous and may be responsible for the development of the congenital hydrocephalus phenotype. Our study provides evidence indicating that abnormalities in Brg1 function result in defects associated with neurodevelopmental disorders and autism.

Calcium/Calmodulin-dependent kinase type II (CaMKII) is essential for various forms of synaptic plasticity. The predominant α- and βCaMKII isoforms have both been shown to contribute to specific forms of plasticity at excitatory synapses, but little is known about their functions at inhibitory synapses. Here we investigated the role of both isoforms in long-term potentiation of the inhibitory molecular layer interneuron to Purkinje cell synapse (MLI-PC iLTP) upon climbing fiber (CF) stimulation. We demonstrate that deleting either the α- or βCaMKII isoform affected MLI-PC iLTP. In the presence of the PP2B blocker cyclosporin A, CF stimulation elicited iLTP in Camk2b (-) (/) (-) mice, but not in Camk2a (-) (/) (-) mice. Moreover, co-activation of the MLIs and CF suppressed iLTP in wild-type mice through activation of GABAB-receptors, whereas it evoked iLTP in Camk2b (-) (/) (-). This reversal of the effect of αCaMKII activity in Camk2b (-) (/) (-) mutants upon co-activation did not critically involve protein kinase A, but depended on calcium release from internal stores. Our results indicate that α- and βCaMKII isoforms in Purkinje cells can be differentially activated and serve distinct roles in controlling iLTP. We propose that the CaMKII holo-enzyme may be selectively activated by various GABAB-mediated pathways and that the presence of the βCaMKII isoform determines their impact on inhibitory plasticity.

Excessive copper can cause neurotoxicity and contribute to the development of some neurological diseases; however, copper neurotoxicity and the potential mechanisms remain poorly understood. We used proteomics and phosphoproteomics to quantify protein changes in the hippocampus of wild-type and 3xTg-AD mice, both of which were treated at 6 months of age with 2 months of drinking water with or without added copper chloride (0.13 ppm concentration). A total of 3960 unique phosphopeptides (5290 phosphorylation sites) from 1406 phosphoproteins was identified. Differentially expressed phosphoproteins involved neuronal and synaptic function, transcriptional regulation, energy metabolism and mitochondrial function. In addition, low-dose copper treatment of wild-type mice decreased hippocampal mitochondrial copy number, mitochondrial biogenesis and disrupted mitochondrial dynamics; these changes were associated with increased hydrogen peroxide production (H₂O₂), reduced cytochrome oxidase activity and decreased ATP content. In 3xTg-AD mice, identical low-dose oral copper treatment increased axonal degeneration, which was associated with altered phosphorylation of Camk2α at T286 and phosphorylation of mitogen-activated protein kinase (ERK1/2), which involved long-term potentiation (LTP) signaling. Mitochondrial dysfunction was mainly related to changes in phosphorylation levels of glycogen synthase kinase-3 beta (GSK3β) and serine/threonine-protein phosphatase 2B catalytic subunit alpha isoform (Ppp3ca), which involved mitochondrial biogenesis signaling. In sum, low-dose oral copper treatment changes the phosphorylation of key hippocampal proteins involved in mitochondrial, synaptic and axonal integrity. These data showing that excess of copper speeds some early events of AD changes observed suggest that excess circulating copper has the potential to perturb brain function of wild-type mice and exacerbate neurodegenerative changes in a mouse model of AD.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression and are implicated in the etiology of several neuropsychiatric disorders, including substance use disorders (SUDs). Using in silico genome-wide sequence analyses, we identified miR-495 as a miRNA whose predicted targets are significantly enriched in the Knowledgebase for Addiction Related Genes (ARG) database (KARG; http://karg.cbi.pku.edu.cn). This small non-coding RNA is also highly expressed within the nucleus accumbens (NAc), a pivotal brain region underlying reward and motivation. Using luciferase reporter assays, we found that miR-495 directly targeted the 3'UTRs of Bdnf, Camk2a and Arc. Furthermore, we measured miR-495 expression in response to acute cocaine in mice and found that it is downregulated rapidly and selectively in the NAc, along with concomitant increases in ARG expression. Lentiviral-mediated miR-495 overexpression in the NAc shell (NAcsh) not only reversed these cocaine-induced effects but also downregulated multiple ARG mRNAs in specific SUD-related biological pathways, including those that regulate synaptic plasticity. miR-495 expression was also downregulated in the NAcsh of rats following cocaine self-administration. Most importantly, we found that NAcsh miR-495 overexpression suppressed the motivation to self-administer and seek cocaine across progressive ratio, extinction and reinstatement testing, but had no effect on food reinforcement, suggesting that miR-495 selectively affects addiction-related behaviors. Overall, our in silico search for post-transcriptional regulators identified miR-495 as a novel regulator of multiple ARGs that have a role in modulating motivation for cocaine.

Although early-life stress is a significant risk factor for developing anxiety disorders, including posttraumatic stress disorder (PTSD), the underlying mechanisms are unclear. Corticotropin releasing hormone (CRH) is disrupted in individuals with PTSD and early-life stress and hence may mediate the effects of early-life stress on PTSD risk. We hypothesized that CRH hyper-signaling in the forebrain during early development is sufficient to increase response to trauma in adulthood. To test this hypothesis, we induced transient, forebrain-specific, CRH overexpression during early-life (pre-puberty, CRHOEdev) in double-mutant mice (Camk2a-rtta2 × tetO-Crh) and tested their behavioral and gene expression responses to the predator stress model of PTSD in adulthood. In one cohort of CRHOEdev exposed and unexposed mice, avoidance and arousal behaviors were examined 7-15 days after exposure to predator stress. In another cohort, gene expression changes in Crhr1, Crhr2, and Fkbp51 in forebrain of CRHOEdev exposed and unexposed mice were examined 7 days after predator stress. CRHOEdev induced robust increases in startle reactivity and reductions in startle inhibition independently of predator stress in both male and female mice. Avoidance behaviors after predator stress were highly dependent on sex and CRHOEdev exposure. Whereas stressed females exhibited robust avoidance responses that were not altered by CRHOEdev, males developed significant avoidance only when exposed to both CRHOEdev and stress. Quantitative real-time-PCR analysis indicated that CRHOEdev unexposed males exhibit significant changes in Crhr2 expression in the amygdala and bed nucleus stria terminalis in response to stress, whereas males exposed to CRHOEdev did not. Similar to CRHOEdev males, females exhibited no significant Crhr2 gene expression changes in response to stress. Cortical Fkbp51 expression was also significantly reduced by stress and CRHOEdev exposure in males, but not in females. These findings indicate that forebrain CRH hyper-signaling in early-life is sufficient to increase enduring effects of adult trauma and attenuate Crhr2 expression changes in response to stress in males. These data support growing evidence for significant sex differences in response to trauma, and support further study of CRHR2 as a candidate mechanism for PTSD risk.

BACKGROUND

Several reports suggest that the reelin protein could play a role in Alzheimer pathophysiology. This led us to ask whether genetic variability in the reelin pathway may increase the risk of developing Alzheimer disease (AD) or mild cognitive impairment (MCI).

METHODS

This was a case-control study in which neuropsychological tests were administered and peripheral blood samples taken. The study included 121 patients with AD, 94 with MCI, and 198 controls. Forty biallelic variants single nucleotide polimorphisms were genotyped in 8 genes related to reelin signaling pathway using a SNPlex genotyping system, and allele frequencies were compared between patients and controls using χ tests and obtaining odds ratios (OR).

RESULTS

A total of 413 subjects with complete neuropsychological data were analyzed. A significant association between the genotypes RELN (rs528528 and rs2299356), PLK2 (rs15009 and rs702723), and CAMK2A (rs3756577 and rs3822606) and AD or MCI was found. A significant association also was found between the GG genotype at the RELN-rs2299356 and the risk of AD (OR=2.68, P=0.003) and between the AG genotype at the CAMK2A-rs3822606 (OR=2.13, P=0.004). We found a protective effect of the RELN-rs528528 CT genotype and MCI (OR=0.36, P=0.002), and the PLK2-rs15009 CC and GG genotypes and CC genotype at PLK2-rs702723 with OR ranging from 0.40 to 0.57 on AD. These data suggest that TT or CT genotypes at CAMK2A-rs3756577 is associated with risk reduction for AD and MCI ranging from 2 to nearly 8 times.

CONCLUSIONS

Our data suggest a possible relation between certain reelin signaling pathway genotypes and cognitive impairment related to AD.

Conventional antidepressants have limited efficacy and many side effects, highlighting the need for fast-acting and specific medications. Induction of the synaptic protein Homer1a mediates the effects of different antidepressant treatments, including the rapid action of ketamine and sleep deprivation (SD). We show here that mimicking Homer1a upregulation via intravenous injection of cell-membrane-permeable TAT-Homer1a elicits rapid antidepressant effects in various tests. Similar to ketamine and SD, in vitro and in vivo application of TAT-Homer1a enhances mGlu5 signaling, resulting in increased mTOR pathway phosphorylation, and upregulates synaptic AMPA receptor expression and activity. The antidepressant action of SD and Homer1a induction depends on mGlu5 activation specifically in excitatory CaMK2a neurons and requires enhanced AMPA receptor activity, translation, and trafficking. Moreover, our data demonstrate a pronounced therapeutic potential of different TAT-fused peptides that directly modulate mGlu5 and AMPA receptor activity and thus might provide a novel strategy for rapid and effective antidepressant treatment.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Calcium/calmodulin-dependent protein kinase II (CAMK2) plays fundamental roles in synaptic plasticity that underlies learning and memory. Here, we describe a new recessive neurodevelopmental syndrome with global developmental delay, seizures and intellectual disability. Using linkage analysis and exome sequencing, we found that this disease maps to chromosome 5q31.1-q34 and is caused by a biallelic germline mutation in CAMK2A. The missense mutation, p.His477Tyr is located in the CAMK2A association domain that is critical for its function and localization. Biochemically, the p.His477Tyr mutant is defective in self-oligomerization and unable to assemble into the multimeric holoenzyme.In vivo, CAMK2AH477Y failed to rescue neuronal defects in C. elegans lacking unc-43, the ortholog of human CAMK2A. In vitro, neurons derived from patient iPSCs displayed profound synaptic defects. Together, our data demonstrate that a recessive germline mutation in CAMK2A leads to neurodevelopmental defects in humans and suggest that dysfunctional CAMK2 paralogs may contribute to other neurological disorders.

TET3 is a member of the ten-eleven translocation (TET) family of enzymes which oxidize 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Tet3 is highly expressed in the brain, where 5hmC levels are most abundant. In adult mice, we observed that TET3 is present in mature neurons and oligodendrocytes but is absent in astrocytes. To investigate the function of TET3 in adult postmitotic neurons, we crossed Tet3 floxed mice with a neuronal Cre-expressing mouse line, Camk2a-CreERT2, obtaining a Tet3 conditional KO (cKO) mouse line. Ablation of Tet3 in adult mature neurons resulted in increased anxiety-like behavior with concomitant hypercorticalism, and impaired hippocampal-dependent spatial orientation. Transcriptome and gene-specific expression analysis of the hippocampus showed dysregulation of genes involved in glucocorticoid signaling pathway (HPA axis) in the ventral hippocampus, whereas upregulation of immediate early genes was observed in both dorsal and ventral hippocampal areas. In addition, Tet3 cKO mice exhibit increased dendritic spine maturation in the ventral CA1 hippocampal subregion. Based on these observations, we suggest that TET3 is involved in molecular alterations that govern hippocampal-dependent functions. These results reveal a critical role for epigenetic modifications in modulating brain functions, opening new insights into the molecular basis of neurological disorders.

Glyphosate-containing herbicides are the most used agrochemicals in the world. Their indiscriminate application raises some concerns regarding the possible health and environmental hazards. In this study, we investigated in human neuroblastoma cell line SH-SY5Y if oxidative stress, altered neurodevelopment and cell death pathways are involved in response to glyphosate and its metabolite aminomethylphosphonic acid (AMPA) exposures. MTT and LDH assays were carried out to assess the glyphosate and AMPA cytotoxicity. Lipid peroxides measured as malondialdehyde (MDA), nitric oxide (NO) and reactive oxygen species (ROS) production, and caspase-Glo 3/7 activity were evaluated. The neuroprotective role of melatonin (MEL), Trolox, N-acetylcysteine (NAC) and Sylibin against glyphosate- and AMPA-induced oxidative stress was examined. Glyphosate and AMPA effects on neuronal development related gene transcriptions, and gene expression profiling of cell death pathways by Real-Time PCR array were also investigated. Glyphosate (5 mM) and AMPA (10 mM) induced a significant increase in MDA levels, NO and ROS production and caspase 3/7 activity. Glyphosate exposure induced up-regulation of Wnt3a, Wnt5a, Wnt7a, CAMK2A, CAMK2B and down-regulation of GAP43 and TUBB3 mRNA expression involved in normal neural cell development. In relation to gene expression profiling of cell death pathways, of the 84 genes examined in cells a greater than 2-fold change was observed for APAF1, BAX, BCL2, CASP3, CASP7, CASP9, SYCP2, TNF, TP53, CTSB, NFκB1, PIK3C3, SNCA, SQSTMT, HSPBAP1 and KCNIPI mRNA expression for glyphosate and AMPA exposures. These gene expression data can help to define neurotoxic mechanisms of glyphosate and AMPA. Our results demonstrated that glyphosate and AMPA induced cytotoxic effects on neuronal development, oxidative stress and cell death via apoptotic, autophagy and necrotic pathways and confirmed that glyphosate environmental exposure becomes a concern. This study demonstrates that SH-SY5Y cell line could be considered an in vitro system for pesticide screening.

Bipolar disorder (BPD) is genetically heterogeneous with a growing list of BPD associated genes reported in recent years resulting from increased genetic testing using advanced genetic technology, expanded genomic databases, and better awareness of the disorder. We compiled a master list of recognized susceptibility and genes associated with BPD identified from peer-reviewed medical literature sources using PubMed and by searching online databases, such as OMIM. Searched keywords were related to bipolar disorder and genetics. Our compiled list consisted of 290 genes with gene names arranged in alphabetical order in tabular form with source documents and their chromosome location and gene symbols plotted on high-resolution human chromosome ideograms. The identified genes impacted a broad range of biological pathways and processes including cellular signaling pathways particularly cAMP and calcium (e.g., CACNA1C, CAMK2A, CAMK2D, ADCY1, ADCY2); glutamatergic (e.g., GRIK1, GRM3, GRM7), dopaminergic (e.g., DRD2, DRD4, COMT, MAOA) and serotonergic (e.g., HTR1A, HTR2A, HTR3B) neurotransmission; molecular transporters (e.g., SLC39A3, SLC6A3, SLC8A1); and neuronal growth (e.g., BDNF, IGFBP1, NRG1, NRG3). The increasing prevalence of BPD calls for better understanding of the genetic etiology of this disorder and associations between the observed BPD phenotype and genes. Visual representation of genes for bipolar disorder becomes a tool enabling clinical and laboratory geneticists, genetic counselors, and other health care providers and researchers easy access to the location and distribution of currently recognized BPD associated genes. Our study may also help inform diagnosis and advance treatment developments for those affected with this disorder and improve genetic counseling for families.

The present study aimed to explore possible prognostic marker genes in glioblastoma (GBM). Differentially expressed genes (DEGs) were screened by comparing microarray data of tumor and normal tissue samples from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) dataset GSE22866. Subsequently, the prognosis‑associated DEGs were screened via Cox regression analysis, followed by construction of gene/protein/pathway interaction networks of these DEGs by calculating the correlation coefficient between the DEGs. Next, a prognostic prediction system was constructed using Bayes discriminant analysis, which was validated by the microarray data of samples from patients with good and bad prognosis from the TCGA and Chinese Glioma Genome Atlas (CGGA), as well as the GEO dataset. Finally, a co‑expression network of the signature genes in the prediction system was constructed in combination with the significant pathways. A total of 288 overlapping DEGs (false discovery rate <0.5 and |log2 of fold change|>1) were screened, 123 of which were identified to be associated with the prognosis of GBM patients. The co‑expression network of these prognosis‑associated DEGs included 1405 interactions and 112 DEGs, and 6 functional modules were identified in the network. The prognostic prediction system was comprised of 63 signature genes with a specificity value of 0.929 and a sensitivity value of 0.948. GBM samples with good and bad prognosis in the TCGA, CGGA and GEO datasets were distinguishable by these signature genes (P=1.33x10‑6, 1.63x10‑4 and 0.00534, respectively). The co‑expression network of signature genes with significant pathways was comprised of 56 genes and 361 interactions. Protein kinase Cγ (PRKCG), protein kinase Cβ (PRKCB) and calcium/calmodulin‑dependent protein kinase IIα (CAMK2A) were important genes in the network, and based on the expression of these genes, it was possible to distinguish between samples with significantly different survival risks. In the present study, an effective prognostic prediction system for GBM patients was constructed and validated. PRKCG, PRKCB and CAMK2A may be potential prognostic factors for GBM.

Ca²⁺/calmodulin-dependent protein kinase II (CAMK2) is a key player in synaptic plasticity and memory formation. Mutations in Camk2a or Camk2b cause intellectual disability in humans, and severe plasticity and learning deficits in mice, indicating unique functions for each isoform. However, considering the high homology between CAMK2A and CAMK2B, it is conceivable that for critical functions, one isoform compensates for the absence of the other, and that the full functional spectrum of neuronal CAMK2 remains to be revealed.Here we show that germline as well as adult deletion of both CAMK2 isoforms in male or female mice is lethal. Moreover, Ca²⁺-dependent activity as well as autonomous activity of CAMK2 is essential for survival. Loss of both CAMK2 isoforms abolished LTP, whereas synaptic transmission remained intact. The double-mutants showed no gross morphological changes of the brain, and in contrast to the long-considered role for CAMK2 in the structural organization of the postsynaptic density (PSD), deletion of both CAMK2 isoforms did not affect the biochemical composition of the PSD. Together, these results reveal an essential role for CAMK2 signaling in early postnatal development as well as the mature brain, and indicate that the full spectrum of CAMK2 requirements cannot be revealed in the single mutants because of partial overlapping functions of CAMK2A and CAMK2B.SIGNIFICANCE STATEMENT CAMK2A and CAMK2B have been studied for over 30 years for their role in neuronal functioning. However, most studies were performed using single knock-out mice. Because the two isoforms show high homology with respect to structure and function, it is likely that some redundancy exists between the two isoforms, meaning that for critical functions CAMK2B compensates for the absence of CAMK2A and vice versa, leaving these functions to uncover. In this study, we generated Camk2a/Camk2b double-mutant mice, and observed that loss of CAMK2, as well as the loss of Ca²⁺-dependent and Ca²⁺-independent activity of CAMK2 is lethal. These results indicate that despite 30 years of research the full spectrum of CAMK2 functioning in neurons remains to be unraveled.

Essentials Potential neurodevelopmental side effects of thrombopoietin mimetics need to be considered. The effects of eltrombopag (ELT) on neuronal iron status and dendrite development were assessed. ELT crosses the blood-brain barrier and causes iron deficiency in developing neurons. ELT blunts dendrite maturation, indicating a need for more safety studies before neonatal use.

Background Thrombocytopenia is common in sick neonates. Thrombopoietin mimetics (e.g. eltrombopag [ELT]) might provide an alternative therapy for selected neonates with severe and prolonged thrombocytopenia, and for infants and young children with different varieties of thrombocytopenia. However, ELT chelates intracellular iron, which may adversely affect developing organs with high metabolic requirements. Iron deficiency (ID) is particularly deleterious during brain development, impairing neuronal myelination, dopamine signaling and dendritic maturation and ultimately impairing long-term neurological function (e.g. hippocampal-dependent learning and memory). Objective To determine whether ELT crosses the blood-brain barrier (BBB), causes neuronal ID and impairs hippocampal neuron dendrite maturation. Methods ELT transport across the BBB was assessed using primary bovine brain microvascular endothelial cells. Embryonic mouse primary hippocampal neuron cultures were treated with ELT or deferoxamine (DFO, an iron chelator) from 7 days in vitro (DIV) through 14 DIV and assessed for gene expression and neuronal dendrite complexity. Results ELT crossed the BBB in a time-dependent manner. 2 and 6 μm ELT increased Tfr1 and Slc11a2 (iron-responsive genes involved in neuronal iron uptake) mRNA levels, indicating neuronal ID. 6 μm ELT, but not 2 μm ELT, decreased BdnfVI, Camk2a and Vamp1 mRNA levels, suggesting impaired neuronal development and synaptic function. Dendrite branch number and length were reduced in 6 μm ELT-treated neurons, resulting in blunted dendritic arbor complexity that was similar to DFO-treated neurons. Conclusions Eltrombopag treatment during development may impair neuronal structure as a result of neuronal ID. Preclinical in vivo studies are warranted to assess ELT safety during periods of rapid brain development.

Autoantibody profiling with a systems medicine approach can help identify critical dysregulated signaling pathways (SPs) in cancers. In this way, immunoglobulins G (IgG) purified from the serum samples of 92 healthy controls, 10 pre-treated (PR) non-Hodgkin lymphoma (NHL) patients, and 20 NHL patients who underwent chemotherapy (PS) were screened with a phage-displayed random peptide library. Protein-protein interaction networks of the PR and PS groups were analyzed and visualized by Gephi. The results indicated AXIN2, SENP2, TOP2A, FZD6, NLK, HDAC2, HDAC1, and EHMT2, in addition to CAMK2A, PLCG1, PLCG2, GRM5, GRIN2B, GRIN2D, CACNA2D3, and SPTAN1 as hubs in 11 and 7 modules of PR and PS networks, respectively. PR- and PS-specific hubs were evaluated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Reactome databases. The PR-specific hubs were involved in Wnt SP, signaling by Notch1 in cancer, telomere maintenance, and transcriptional misregulation. In contrast, glutamate receptor SP, Fc receptor-related pathways, growth factors-related SPs, and Wnt SP were statistically significant enriched pathways, based on the pathway analysis of PS hubs. The results revealed that the most PR-specific proteins were associated with events involved in tumor development, while chemotherapy in the PS group was associated with side effects of drugs and/or cancer recurrence. As the findings demonstrated, PR- and PS-specific proteins in this study can be promising therapeutic targets in future studies.

This study examined the neurobehavioral changes and alteration in gene expression in the brains of metallothionein (MT)-I/II null mice exposed to low-levels of mercury vapor (Hg(0)) during postnatal development. MT-I/II null and wild-type mice were repeatedly exposed to Hg(0) at 0.030 mg/m(3) (range: 0.023-0.043 mg/m(3)), which was similar to the current threshold value (TLV), for 6 hr per day until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. Hg(0)-exposed MT-I/II null mice showed a significant decrease in total locomotor activity in females, though learning ability and spatial learning ability were not affected. Immediately after Hg(0) exposure, mercury concentrations in the brain did not exceed 0.5 µg/g in any animals. Hg(0) exposure resulted in significant alterations in gene expression in the brains of both strains using DNA microarray analysis. The number of altered genes in MT-I/II null mice was higher than that in wild-type mice and calcium-calmodulin kinase II (Camk2a) involved in learning and memory in down-regulated genes was detected. These results provide useful information to elucidate the development of behavioral toxicity following low-level exposure to Hg(0).

Twin and family studies have shown that genetic factors play a role in the development of conduct disorder (CD). The purpose of this study was to identify genetic variants associated with CD using a family-based association study. We used 4,720 single nucleotide polymorphisms (SNPs) from the Illumina Panel and 11,120 SNPs from the Affymetrix 10K GeneChips genotyped in 155 Caucasian nuclear families from Genetic Analysis Workshop (GAW) 14, a subset from the Collaborative Study on the Genetics of Alcoholism (COGA). 20 SNPs had suggestive associations with CD (p<10(-3)), nine of which were located in known genes, including ADAM10 (rs383902, p=0.00036) and CAMK2A (rs2053053, p=0.00098). Our results were verified using the International Multi-Center ADHD Genetics Project (IMAGE) dataset. In conclusion, we identified several loci associated with CD. Especially, the two genes (ADAM10 and CAMK2A) have been reported to be associated with Alzheimer's disease, bipolar disorder and depression. These findings may serve as a resource for replication in other populations.

OBJECTIVE

Although inhibition of histone deacetylases (HDACs) has been shown to protect against cisplatin-induced hearing loss, the underlying mechanism is still poorly understood. In the present study, we aim to investigate the protective effect of trichostatin A (TSA), a specific inhibitor of HDACs, on cisplatin-induced ototoxicity and to determine the differentially expressed genes involved in this process.

METHODS

The basilar membrane of the cochlea was isolated from 3-day newborn Wistar rats. Organotypic cultures were treated with 150 μM cisplatin or 200 nM TSA. For combination treatment, cells were pre-incubated with TSA for 1h, followed by TSA plus cisplatin treatment. Rhodamine-phalloidin staining was used to label hair cells, and immunocytochemistry with an anti-neurofilament-200 antibody was applied to label spiral ganglion neurons (SGNs). Global expression profile microarray analysis was used to identify differentially expressed genes. Molecular function and signal pathway analysis were performed using a protein analysis through evolutionary relationships (PANTHER) classification system. Real-time quantitative PCR (qPCR) was carried out for data validation.

RESULTS

Severe loss of hair cells and SGNs occurred after 48 h of cisplatin incubation, while TSA significantly increased the number of hair cells and SGNs in the combination treatment group (P<0.05). Compared with control, expression of 71 genes were up-regulated and 383 genes were down-regulated upon cisplatin treatment. Addition of TSA induced the up-regulation of 1387 genes and down-regulation of 1226 genes as compared with cisplatin administration alone. After cisplatin treatment, we observed significant down-regulation of mRNA for several genes related to synaptic function genes, including Camk2a, Camk2b, Vglut1, Snap25 and Rab3b, whereas pretreatment with TSA elevated mRNA levels of these genes. TSA greatly decreased expression of genes related to the calcium signaling pathway (Capn1 and Capn2) and apoptosis signaling pathway (Tnfrsf1a and Tp53), while addition of TSA significantly reduced levels of Tnfrsf1a and Tp53 compared with cisplatin alone (P<0.01).

CONCLUSIONS

Our results suggested that TSA might protect against cisplatin-induced ototoxicity via mediating expression of genes responsible for regulating apoptosis, intracellular calcium homeostasis, neurotransmitter synthesis and release, and synaptic plasticity.

Arteriovenous malformations (AVMs) of the central nervous system are considered as congenital disorders. They are composed of abnormally developed dilated arteries and veins and are characterized microscopically by the absence of a capillary network. We previously reported DNA fragmentation and increased expression of apoptosis-related factors in AVM lesions. In this article, we used microarray analysis to examine differential gene expression in relation to clinical manifestations in 11 AVM samples from Japanese patients. We categorized the genes with altered expression into four groups: death-related, neuron-related, inflammation-related, and other. The death-related differentially expressed genes were MMP9, LIF, SOD2, BCL2A1, MMP12, and HSPA6. The neuron-related genes were NPY, S100A9, NeuroD2, S100Abeta, CAMK2A, SYNPR, CHRM2, and CAMKV. The inflammation-related genes were PTX3, IL8, IL6, CXCL10, GBP1, CHRM3, CXCL1, IL1R2, CCL18, and CCL13. In addition, we compared gene expression in those with or without clinical characteristics including deep drainer, embolization, and high-flow nidus. We identified a small number of genes. Using these microarray data we are able to generate and test new hypotheses to explore AVM pathophysiology. Microarray analysis is a useful technique to study clinical specimens from patients with brain vascular malformations.

Mandibulofacial dysostosis is part of a clinically and genetically heterogeneous group of disorders of craniofacial development, which lead to malar and mandibular hypoplasia. Treacher Collins syndrome is the major cause of mandibulofacial dysostosis and is due to mutations in the TCOF1 gene. Usually patients with Treacher Collins syndrome do not present with intellectual disability. Recently, the EFTUD2 gene was identified in patients with mandibulofacial dysostosis associated with microcephaly, intellectual disability and esophageal atresia. We report on two patients presenting with mandibulofacial dysostosis characteristic of Treacher Collins syndrome, but associated with unexpected intellectual disability, due to a large deletion encompassing several genes including the TCOF1 gene. We discuss the involvement of the other deleted genes such as CAMK2A or SLC6A7 in the cognitive development delay of the patients reported, and we propose the systematic investigation for 5q32 deletion when intellectual disability is associated with Treacher Collins syndrome.