Upregulation Of Immediate Early Genes Research Articles

CSF1R inhibitors induce a sex-specific resilient microglial phenotype and functional rescue in a tauopathy mouse model

Microglia are central to pathogenesis in many neurological conditions. Drugs targeting colony-stimulating factor-1 receptor (CSF1R) to block microglial proliferation in preclinical disease models have shown mixed outcomes, thus the therapeutic potential of this approach remains unclear. Here, we show that CSF1R inhibitors given by multiple dosing paradigms in the Tg2541 tauopathy mouse model cause a sex-independent reduction in pathogenic tau and reversion of non-microglial gene expression patterns toward a normal wild type signature. Despite greater drug exposure in male mice, only female mice have functional rescue and extended survival. A dose-dependent upregulation of immediate early genes and neurotransmitter dysregulation are observed in the brains of male mice only, indicating that excitotoxicity may preclude functional benefits. Drug-resilient microglia in male mice exhibit morphological and gene expression patterns consistent with increased neuroinflammatory signaling, suggesting a mechanistic basis for sex-specific excitotoxicity. Complete microglial ablation is neither required nor desirable for neuroprotection and therapeutics targeting microglia must consider sex-dependent effects.

An Adaptive Role for DNA Double-Strand Breaks in Hippocampus-Dependent Learning and Memory.

DNA double-strand breaks (DSBs), classified as the most harmful type of DNA damage based on the complexity of repair, lead to apoptosis or tumorigenesis. In aging, DNA damage increases and DNA repair decreases. This is exacerbated in disease, as post-mortem tissue from patients diagnosed with mild cognitive impairment (MCI) or Alzheimer’s disease (AD) show increased DSBs. A novel role for DSBs in immediate early gene (IEG) expression, learning, and memory has been suggested. Inducing neuronal activity leads to increases in DSBs and upregulation of IEGs, while increasing DSBs and inhibiting DSB repair impairs long-term memory and alters IEG expression. Consistent with this pattern, mice carrying dominant AD mutations have increased baseline DSBs, and impaired DSB repair is observed. These data suggest an adaptive role for DSBs in the central nervous system and dysregulation of DSBs and/or repair might drive age-related cognitive decline (ACD), MCI, and AD. In this review, we discuss the adaptive role of DSBs in hippocampus-dependent learning, memory, and IEG expression. We summarize IEGs, the history of DSBs, and DSBs in synaptic plasticity, aging, and AD. DSBs likely have adaptive functions in the brain, and even subtle alterations in their formation and repair could alter IEGs, learning, and memory.

Synaptotagmin 2 is ectopically overexpressed in excitatory presynapses of a widely used CaMKΙΙα-Cre mouse line

SummaryThe CaMKΙΙα-Cre mouse lines, possibly the most used Cre lines in neuroscience, have resulted in over 800 articles to date. Here, we demonstrate that the second most widely used CaMKΙΙα-Cre line, Tg(Camk2a-cre)2Gsc (or CamiCre), shows ectopic overexpression of synaptotagmin 2, the most efficient Ca2+ sensor for fast synchronous neurotransmitter release, in excitatory presynapses of Cre+ brains. Moreover, the upregulation of immediate-early genes and genes incorporated in bacterial artificial chromosome (BAC) transgenes, such as L-proline transporter Slc6a7, was found in Cre+ hippocampus. The copy number and integration site of the transgene are suggested to have caused the aberrant gene expression in Cre+ brains. Most importantly, CamiCre+ mice showed functional phenotypes, such as hyperactivity and enhanced associative learning, suggesting that neural activities are affected. These unexpected results suggest difficulties in interpreting results from studies using the CamiCre line and raise a warning of potential pitfalls in using Cre driver lines in general.

N-methyl-d-aspartate Receptor-mediated Preconditioning Mitigates Excitotoxicity in Human Induced Pluripotent Stem Cell-derived Brain Organoids

Studies in rodent models of acute and chronic neurodegenerative disorders have uncovered that glutamate-induced excitotoxic cell death is mediated primarily by extrasynaptic N-methyl-d-aspartate receptors (NMDARs). Rodent neurons can also build up in an activity-dependent manner a protective shield against excitotoxicity. This form of acquired neuroprotection is induced by preconditioning with low doses of NMDA or by activation of synaptic NMDARs triggered by bursts of action potentials. Whether NMDARs in human neurons have similar dichotomous actions in cell death and survival is unknown. To investigate this, we established an induced pluripotent stem cell (iPSC)-derived forebrain organoid model for excitotoxic cell death and explored conditions of NMDAR activation that promote neuronal survival when applied prior to a toxic insult. We found that glutamate-induced excitotoxicity in human iPSC-derived neurons is mediated by NMDARs. Treatment of organoids with high concentrations of glutamate or NMDA caused the typical excitotoxicity pathology, comprising structural disintegration, neurite blebbing, shut-off of the transcription factor CRE binding protein (CREB), and cell death. In contrast, bath-applied low doses of NMDA elicited synaptic activity, a robust and sustained increase in CREB phosphorylation as well as function, and upregulation of immediate-early genes, including neuroprotective genes. Moreover, we found that conditions of enhanced synaptic activity increased survival of human iPSC-derived neurons if applied as pre-treatment before toxic NMDA application. These results revealed that both toxic and protective actions of NMDARs are preserved in human neurons. The experimental platform described in this study may prove useful for the validation of neuroprotective gene products and drugs in human neurons.

Repressed Chromatin Drives Leukaemogenesis in Mutant IDH2 Acute Myeloid Leukaemia Via Inhibition of Granulocyte Differentiation and Cell Cycle Progression

Differentiation arrest in acute myeloid leukaemia (AML) results in accumulation of leukaemic progenitors (L-Prog) and bone marrow failure. Mutant isocitrate dehydrogenase enzyme produces d-2-hydroxyglutarate (2HG), which inhibits α-ketoglutarate-dependent dioxygenases, including Jumonji histone demethylases (JKDM) and TET2, but how this causes AML is unclear. Inhibitors of mutant IDH enzyme (mIDHi) restore differentiation in IDH-mutant (mIDH) AML (Amatangelo et al., 2018). Here, we studied transcriptional networks involved using single-cell (SC) gene expression (GEX) and transcription factor (TF) motif accessibility in primary AML treated with the mIDH2 inhibitor enasidenib (ENA) and found that ENA activates cell cycle (CC) and pro-differentiation programmes through increased promoter accessibility of granulocyte-monocyte (GM)-TF targets.We treated patient L-Prog in vitro with ENA or vehicle, and performed SC RNA-seq (Chromium 10x) in 4 responsive (R), and one non-responsive (NR) patient samples in early, mid and late timepoints. GEX signatures were used to annotate cells according to function (undifferentiated [U], early and late GM [EGM and LGM]) and CC states. In R samples, ENA yielded more dividing late-GM at mid-late timepoints than DMSO (18% vs 6.5%), and more terminally differentiated neutrophils at late timepoints (46% vs 16%). Using SCENIC (Aibar et al., 2017) to assign highly differentially-expressed genes to TF motifs, we computed regulatory networks (regulons, ‘R‘). Expression of the SP1 R was strongly correlated with active proliferation and ENA conditions led to generation of more cells that co-expressed CEBPA R or CEBPE R with SP1 R, emphasising simultaneous engagement of CC and GM programmes. SP1 function is associated with CC and GM differentiation, and silencing of its binding to its targets contributes to AML pathogenesis (Maiques-Diaz et al., 2012). Control and NR samples failed to produce neutrophils, had reduced co-expression of CEBPE/SP1 R and yielded more poorly differentiated cells expressing GATA2 R.At the individual gene level, ENA stimulated downregulation of GATA2, GFI1B, IKZF1/2, and RUNX3 together with upregulation of immediate early genes which respond to cytokine and mitogenic stimuli (EGR1, IER2, AP-1) in early-mid phase. Later there is upregulation of CEBP TFs and effector genes FUT4, ELANE, AZU1 and PRTN3. Interestingly, expression of some GM-TFs (RUNX1, SPI1/PU.1, GFI1) was similar between ENA and DMSO, indicating that gene expression alone was insufficient for GM differentiation.Given the effects of 2-HG on JKDM, we assessed chromatin accessibility and TF binding using SC ATAC-seq. Overall, we had 25% of differentially accessible (DA) peaks, from which 75% were more accessible in ENA than in DMSO. ENA DA peaks were highly enriched in promoters. Using ArchR (Granja et al., 2021), we clustered cells and used ELANE expression levels to compute trajectories in parallel with SC RNA-seq data. ENA peaks were sequentially enriched for CBF/RUNX and GATA families, followed by AP-1 (JUN/FOS) and EGR/CEBP/KLF motifs. Footprinting analysis showed sequential decrease and increase of TF binding for GATA2 and CEBPA/E respectively during ENA-induced differentiation. Although it did not cause higher expression of SPI1/PU.1, ENA induced increased accessibility of its target binding sites at promoters, which included CEBPA/E and GM effectors (MPO, FUT4, PRTN3). This provides a novel mechanism by which ENA induces differentiation of L-prog.Regulatory network analysis around active, differentially expressed TFs at different phases of ENA-induced differentiation showed a switch from a repressive transcriptional landscape driven by stem-progenitor TFs, to one where AP-1 and GM-TFs activate expression of GM-effector genes. We postulate a model where MYC, E2F8 and EGR1 upregulate the CEBP family in early-mid differentiation. In addition to stimulation of promoter accessibility of TFBS, we find that ENA increases accessibility of cis-regulatory elements of CEBP TFs, adding another mechanism by which differentiation of L-Prog occurs.Our data on the mechanism of action of ENA suggest that differentiation arrest in IDHm AML involves suppression of CC and GM differentiation programs in a repressive chromatin landscape, likely via inhibition of KDM6A and demethylation of repressive H3K27me3 marks. DisclosuresSilveira: Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Servier/Agios: Research Funding; BMS/Celgene: Research Funding. Hasan: Bristol Myers Squibb: Current Employment. Thakurta: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Vyas: Gilead: Honoraria; Astellas: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Takeda: Honoraria; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Daiichi Sankyo: Honoraria; Jazz: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding.

Chronic treatment with the antipsychotic drug blonanserin modulates the responsiveness to acute stress with anatomical selectivity.

Patients diagnosed with schizophrenia typically receive life-long treatments with antipsychotic drugs (APDs). However, the impact of chronic APDs treatment on neuroplastic mechanisms in the brain remains largely elusive. Here, we focused on blonanserin, a second-generation antipsychotic (SGA) that acts as an antagonist at dopamine D2, D3, and serotonin 5-HT2A receptors, and represents an important tool for the treatment of schizophrenia. We used rats to investigate the ability of chronic treatment blonanserin to modulate the activity of brain structures relevant for schizophrenia, under baseline conditions or in response to an acute forced swim session (FSS). We measured the expression of different immediate early genes (IEGs), including c-Fos, Arc/Arg 3.1, Zif268 and Npas4. Blonanserin per se produced limited changes in the expression of these genes under basal conditions, while, as expected, FSS produced a significant elevation of IEGs transcription in different brain regions. The response of blonanserin-treated rats to FSS show anatomical and gene-selective differences. Indeed, the upregulation of IEGs was greatly reduced in the striatum, a brain structure enriched in dopamine receptors, whereas the upregulation of some genes (Zif268, Npas4) was largely preserved in other regions, such as the prefrontal cortex and the ventral hippocampus. Taken together, our findings show that chronic exposure to blonanserin modulates selective IEGs with a specific anatomical profile. Moreover, the differential activation of specific brain regions under challenging conditions may contribute to specific clinical features of the drug.

Urethane attenuates early neuropathology of diisopropylfluorophosphate-induced status epilepticus in rats

Seizures can be evident within minutes of exposure to an organophosphorus (OP) agent and often progress to status epilepticus (SE) resulting in a high mortality if left untreated. Effective medical countermeasures are necessary to sustain patients suffering from OP poisoning and to mitigate the ensuing brain injury. Here, the hypothesis was tested that a single subanesthetic dose of urethane prevents neuropathology measured 24 h following diisopropylfluorophosphate (DFP)-induced SE. Adult Sprague-Dawley rats were injected with DFP to induce SE. During SE rats displayed increased neuronal activity in the hippocampus and an upregulation of immediate early genes as well as pro-inflammatory mediators. In additional experiments rats were administered diazepam (10 mg/kg, ip) or urethane (0.8 g/kg, sc) 1 h after DFP-induced SE and compared to rats that experienced uninterrupted SE. Cortical electroencephalography (EEG) and power analysis strengthen the conclusion that urethane effectively terminates SE and prevents the overnight return of seizure activity. Neurodegeneration in limbic brain regions and the seizure-induced upregulation of key inflammatory mediators present 24 h after DFP-induced SE were strongly attenuated by administration of urethane. A trivial explanation for these beneficial effects, that urethane simply reactivates acetylcholinesterase, has been ruled out. These findings indicate that, by contrast to rats administered diazepam or rats that experience uninterrupted SE, the early neuropathology after SE is prevented by subanesthetic urethane, which terminates rather than interrupts, SE.

Tet3 ablation in adult brain neurons increases anxiety-like behavior and regulates cognitive function in mice.

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.

Glial cell diversity and methamphetamine-induced neuroinflammation in human cerebral organoids

Methamphetamine (METH) is a potent stimulant that induces a euphoric state but also causes cognitive impairment, neurotoxicity and neurodevelopmental deficits. Yet, the molecular mechanisms by which METH causes neurodevelopmental defects have remained elusive. Here we utilized human cerebral organoids and single-cell RNA sequencing (scRNA-seq) to study the effects of prenatal METH exposure on fetal brain development. We analyzed 20,758 cells from eight untreated and six METH-treated cerebral organoids and found that the organoids developed from embryonic stem cells contained a diverse array of glial and neuronal cell types. We further identified transcriptionally distinct populations of astrocytes and oligodendrocytes within cerebral organoids. Treatment of organoids with METH-induced marked changes in transcription in multiple cell types, including astrocytes and neural progenitor cells. METH also elicited novel astrocyte-specific gene expression networks regulating responses to cytokines, and inflammasome. Moreover, upregulation of immediate early genes, complement factors, apoptosis, and immune response genes suggests a neuroinflammatory program induced by METH regulating neural stem cell proliferation, differentiation, and cell death. Finally, we observed marked METH-induced changes in neuroinflammatory and cytokine gene expression at the RNA and protein levels. Our data suggest that human cerebral organoids represent a model system to study drug-induced neuroinflammation at single-cell resolution.

Modification of gene expression in rat cardiomyocytes by linoleic and docosahexaenoic acids 1.

Regulation of cardiac fatty acid metabolism is central to the development of cardiac hypertrophy and heart failure. We investigated the effects of select fatty acids on the expression of genes involved in immediate early as well as inflammatory and hypertrophic responses in adult rat cardiomyocytes. Cardiac remodeling begins with upregulation of immediate early genes for c-fos and c-jun, followed by upregulation of inflammatory genes for nuclear factor kappa B (NF-κB) and nuclear factor of activated T-cells (NFAT). At later stages, genes involved in hypertrophic responses, such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are upregulated. Adult rat cardiomyocytes were treated with palmitic acid, a saturated fatty acid; oleic acid, a monounsaturated fatty acid; linoleic acid, a polyunsaturated fatty acid belonging to the n-6 class; and docosahexaenoic acid, a polyunsaturated fatty acid belonging to the n-3 class. Linoleic acid produced a greater increase in the mRNA expression of c-fos, c-jun, NF-κB, NFAT3, ANP, and BNP relative to palmitic acid and oleic acid. In contrast, docosahexaenoic acid caused a decrease in the expression of genes involved in cardiac hypertrophy. Our findings suggest that linoleic acid may be a potent inducer of genes involved in cardiac hypertrophy, whereas docosahexaenoic acid may be protective against the cardiomyocyte hypertrophic response.

Suppression of the ERK\u2013SRF axis facilitates somatic cell reprogramming

The molecular mechanism underlying the initiation of somatic cell reprogramming into induced pluripotent stem cells (iPSCs) has not been well described. Thus, we generated single-cell-derived clones by using a combination of drug-inducible vectors encoding transcription factors (Oct4, Sox2, Klf4 and Myc) and a single-cell expansion strategy. This system achieved a high reprogramming efficiency after metabolic and epigenetic remodeling. Functional analyses of the cloned cells revealed that extracellular signal-regulated kinase (ERK) signaling was downregulated at an early stage of reprogramming and that its inhibition was a driving force for iPSC formation. Among the reprogramming factors, Myc predominantly induced ERK suppression. ERK inhibition upregulated the conversion of somatic cells into iPSCs through concomitant suppression of serum response factor (SRF). Conversely, SRF activation suppressed the reprogramming induced by ERK inhibition and negatively regulated embryonic pluripotency by inducing differentiation via upregulation of immediate early genes, such as c-Jun, c-Fos and EGR1. These data reveal that suppression of the ERK-SRF axis is an initial molecular event that facilitates iPSC formation and may be a useful surrogate marker for cellular reprogramming.

Response to Pretreatment With Low‐Dose β‐Adrenergic Antagonist Therapy Does Not Affect Severity of Takotsubo Cardiomyopathy

Palla AR et al. Clin Cardiol. In press. To the Editor: Palla et al have recently reported that pretreatment with low-dose β-adrenergic antagonist therapy does not affect the severity of takotsubo cardiomyopathy.1. Their study result provides new insights into the prevention of takotsubo cardiomyopathy because β-adrenoceptor blocker has been considered to prevent or at least attenuate the intensity of the detrimental cardiac effects in this syndrome. Takotsubo cardiomyopathy has been regarded in states of catecholamine excess due to stress. One study reported more than 2 times higher norepinephrine levels in patients with takotsubo cardiomyopathy than those with myocardial infarction. However, elevated catecholamine levels are not uniformly found in patients with this syndrome. Our previous study demonstrated that only half of the study patients had elevated catecholamine levels,2. and another study also reported no elevated catecholamine levels even in the acute phase. Sharkey et al conducted a study on 136 patients with takotsubo cardiomyopathy.3. Of these, 25 patients had this syndrome while β-blockers were administered. They thus concluded that β-blockers administered in traditional dosage did not absolutely prevent either the first or recurrent episodes of takotsubo cardiomyopathy. In rat models, we have reported that blocking of both α- and β-adrenoceptors eliminates the upregulation of immediate early genes induced by stress.2. A recent study has investigated cardiac and vascular gene profiles in response to immobilization stress in rats, and reported that altered expression of cardiac genes is blunted by the pretreatment with β-1 adrenoceptor blocker or α-1 + β-1 adrenoceptor blockers.4. We previously suggested that combined α- and β-blockers would be rational for the prevention of this syndrome.2. Accordingly, further investigation in patients administered with β-1 adrenoreceptor blocker and those administered with both α- and β-adrenoreceptor blockers will provide different aspects and more valuable knowledge of takotsubo cardiomyopathy. The study result presented by Palla et al is thus considered appropriate for more detailed investigation of possible takotsubo cardiomyopathy prevention.

Poly(ADP-ribosyl)ation is implicated in the G0–G1 transition of resting cells

Poly(ADP-ribosyl)ation, catalysed by a family of poly(ADP-ribose) polymerases (PARPs), plays an important role in a large variety of physiological processes, including cell proliferation, but its role in cell cycle progression is not yet completely defined. As reported here, the examination of early times following serum stimulation of quiescent fibroblasts suggests that poly(ADP-ribosyl)ation is necessary for the transition from the G0 phase to the G1 phase. We show that PARP activity is involved in this step through the regulation of immediate-early response genes, such as c-Fos and c-Myc. This is supported by the finding that exogenous Myc expression substantially restores cell cycle reactivation in the absence of polymer synthesis. Furthermore, using RNA interference, we show that PARP-1 is the PARP family member playing the most prominent role in the upregulation of c-Fos and c-Myc during G0-G1 transition. We report that even in lectin-stimulated peripheral blood mononucleated cells, the inhibition of PARP activity interferes with the upregulation of immediate-early genes and delays the induction of proliferation, suggesting a general role for PARP-1 in linking growth factor signaling with cell cycle entry.

Gene expression profiling in a mouse model of infantile neuronal ceroid lipofuscinosis reveals upregulation of immediate early genes and mediators of the inflammatory response

BackgroundThe infantile form of neuronal ceroid lipofuscinosis (also known as infantile Batten disease) is caused by hereditary deficiency of a lysosomal enzyme, palmitoyl-protein thioesterase-1 (PPT1), and is characterized by severe cortical degeneration with blindness and cognitive and motor dysfunction. The PPT1-deficient knockout mouse recapitulates the key features of the disorder, including seizures and death by 7–9 months of age. In the current study, we compared gene expression profiles of whole brain from PPT1 knockout and normal mice at 3, 5 and 8 months of age to identify temporal changes in molecular pathways implicated in disease pathogenesis.ResultsA total of 267 genes were significantly (approximately 2-fold) up- or downregulated over the course of the disease. Immediate early genes (Arc, Cyr61, c-fos, jun-b, btg2, NR4A1) were among the first genes upregulated during the presymptomatic period whereas immune response genes dominated at later time points. Chemokine ligands and protease inhibitors were among the most transcriptionally responsive genes. Neuronal survival factors (IGF-1 and CNTF) and a negative regulator of neuronal apoptosis (DAP kinase-1) were upregulated late in the course of the disease. Few genes were downregulated; these included the α2 subunit of the GABA-A receptor, a component of cortical and hippocampal neurons, and Hes5, a transcription factor important in neuronal differentiation.ConclusionA molecular description of gene expression changes occurring in the brain throughout the course of neuronal ceroid lipofuscinosis suggests distinct phases of disease progression, provides clues to potential markers of disease activity, and points to new targets for therapy.

Activated microglia stimulate transcriptional changes in primary oligodendrocytes via IL-1β

No therapy currently exists to repair demyelinated lesions in multiple sclerosis. However, the use of IgM antibodies may provide a valuable therapeutic avenue for evoking such repair. Unfortunately, the mechanism of immunoglobulin action in CNS repair is currently unknown but may depend upon complex interactions between multiple cell types rather than upon direct activation of a single cell type. Using rat mixed glial cultures containing oligodendrocytes, microglia, and astrocytes, we found that the Fc portion of human IgM shifts microglia to an activated phenotype, reduces glial proliferation, upregulates a variety of immediate early genes, including JunB, Egr-1, and c-Fos, and stimulates microglial production and release of IL-1beta. Microglia-derived IL-1beta consequently triggers transcriptional upregulation of immediate early genes such as c-Jun, Egr-1, and c-Fos in the mixed glial cultures, and stimulates the upregulation of late response genes such as lipocalin in purified oligodendrocytes. Treatment with an IL-1beta receptor antagonist abrogates the effects of Fcmu on glial proliferation and prevents the upregulation of lipocalin in response to Fcmu, but does not prevent Fcmu-mediated upregulation of IL-1beta, suggesting that IL-1beta mediates at least some of the downstream effects of Fcmu in mixed glial cultures. We hypothesize that Fcmu-stimulated IL-1beta-induced upregulation of immediate early and late response genes in oligodendrocytes may promote CNS repair.

Differential gene expression in the rat cochlea after exposure to impulse noise

Understanding the molecular biology of noise trauma is vital to developing effective and timely interventions. In a model of explosion-mediated impulse noise injury, differential gene expression was studied in whole rat cochlea preparations at 3 and 24 h following the exposure. We developed a technique using mRNA from a single cochlea on each oligonucleotide microarray to avoid pooling of mRNA samples. Application of a conservative statistical analysis approach resulted in the identification of 61 differentially expressed genes. Within 3 h after the exposure, there was an up-regulation of immediate early genes, mainly transcription factors and genes involved in the tissue’s response to oxidative stress. No genes were found to be significantly down-regulated. At 24 h following the exposure, up-regulated genes included members of inflammatory and antioxidant pathways and one gene involved in glutathione metabolism was down-regulated. A subset of genes was confirmed by real-time reverse transcriptase–polymerase chain reaction (RT-PCR). The present study demonstrates the power of the microarray technique in providing a global view of the gene regulation following noise exposure, and in identifying genes that may be mechanistically important in hearing loss, and thereby serve as a basis for the development of therapeutic interventions.

Gene expression is differentially regulated by neurotransmitters in embryonic neuronal cortical culture

Neurotransmitters and their receptors have been involved in both proper brain development and neurodevelopmental disorders. The role that nicotinic receptors play in immature cortical neurons was initially investigated by gene profiling using Affymetrix DNA arrays. Both short (15 min) and prolonged (18 h) treatments with nicotine did not induce modification in gene expression, whereas a significant down-regulation of c-fos protein levels was observed after 18 h treatment. Conversely, a brief treatment with the glutamatergic agonist NMDA triggered up-regulation of immediate early genes and transcription factors, which remained unaffected by pre-treatment for 18 h with nicotine. Calcium imaging studies revealed that NMDA activated a sustained increase in intracellular calcium concentration in the majority of neurons, whereas nicotine evoked only a transient calcium increase in a smaller percentage of neurons, suggesting that the calcium signalling response was correlated with activation of gene expression. Nicotine effects on immature cortical neurons perhaps do not require gene regulation but may be still acting on signalling pathways.

Early left ventricular gene expression profile in response to increase in blood pressure

The heart adapts to increased pressure overload by hypertrophic growth of terminally differentiated cardiomyocytes. At the genetic level, the hypertrophic response is characterized by the reprogramming of gene expression, i.e. upregulation of immediate early genes, natriuretic peptide genes and genes encoding structural proteins. In the present study, we characterized the early changes in gene expression with cDNA expression arrays in response to increase in blood pressure produced by arginine8–vasopressin infusion (0.05 µg/kg/min, i.v.) for 30 min and 4 h in conscious normotensive rats. Expression profiling revealed differential expression of 14 genes in the left ventricle, and several novel factors of immediate early genetic response to pressure overload were identified, such as growth arrest and DNA damage inducible protein 45 (GADD45α), epidermal fatty acid‐binding protein (E‐FABP) and Bcl‐X. Administration of angiotensin II (Ang II) for 6 h by osmotic minipumps also increased left ventricular GADD45α, E‐FABP and Bcl‐X gene expression. Furthermore, the induction of GADD45α and Bcl‐X gene expression by Ang II was blocked by angiotensin II type 1 receptor antagonist losartan. In summary, our analysis provided new insights into the pathogenesis of pressure overload‐induced hypertrophy by suggesting the existence of novel regulators of the immediate early gene expression program.

β-Carotene Interferes with Ultraviolet Light A-Induced Gene Expression by Multiple Pathways

Ultraviolet light A (UVA) exposure is thought to cause skin aging mainly by singlet oxygen ((1)O(2))-dependent pathways. Using microarrays, we assessed whether pre-treatment with the (1)O(2) quencher beta-carotene (betaC; 1.5 microM) prevents UVA-induced gene regulation in HaCaT human keratinocytes. Downregulation of growth factor signaling, moderate induction of proinflammatory genes, upregulation of immediate early genes including apoptotic regulators and suppression of cell cycle genes were hallmarks of the UVA effect. Of the 568 UVA-regulated genes, betaC reduced the UVA effect for 143, enhanced it for 180, and did not interact with UVA for 245 genes. The different interaction modes imply that betaC/UVA interaction involved multiple mechanisms. In unirradiated keratinocytes, gene regulations suggest that betaC reduced stress signals and extracellular matrix (ECM) degradation, and promoted keratinocyte differentiation. In irradiated cells, expression profiles indicate that betaC inhibited UVA-induced ECM degradation, and enhanced UVA induction of tanning-associated protease-activated receptor 2. Combination of betaC-promoted keratinocyte differentiation with the cellular "UV response" caused synergistic induction of cell cycle arrest and apoptosis. In conclusion, betaC at physiological concentrations interacted with UVA effects in keratinocytes by mechanisms that included, but were not restricted to (1)O(2) quenching. The retinoid effect of betaC was minor, indicating that the betaC effects reported here were predominantly mediated through vitamin A-independent pathways.

Differential modulation of gene expression among rat tissues with warm ischemia

The aim of this study is to determine if warm ischemia after surgical extirpation impacts gene expression in tissue samples which will be used for cDNA array analysis. We investigated effects of warm ischemia on gene expression in lung, liver, kidney, and spleen of rats, chronologically, using an original cDNA array, real-time quantitative RT-PCR and immunohistochemistry. Although no visible alteration was found in RNA quality, cDNA array showed that expression of many genes was modulated by warm ischemia within 60 min in these tissues, 19.1% of the tested genes in lung, 11.0% in liver, 5.1% in kidney, and 16.2% in spleen. Quantitative RT-PCR revealed that warm ischemia significantly induced up-regulation of immediate early genes, c- fos, Egr-1, and c- jun, in lung, but not in liver. These findings suggest that genes may show tissue-dependent differential transcriptional response against warm ischemia. Tissue samples obtained from patients during surgery cannot completely escape effects of ischemia. In case of examination by cDNA array analysis, biologists should keep in mind that tissue samples come equipped with particular footprints.