Eukaryotic mRNAs are 5' end capped with a 7-methylguanosine, which is important for processing and translation of mRNAs. Cap methyltransferase 1 (CMTR1) catalyzes 2'-O-ribose methylation of the first transcribed nucleotide (N1 2'-O-Me) to mask mRNAs from innate immune surveillance by retinoic-acid-inducible gene-I (RIG-I). Nevertheless, whether this modification regulates gene expression for neuronal functions remains unexplored. Here, we find that knockdown of CMTR1 impairs dendrite development independent of secretory cytokines and RIG-I signaling. Using transcriptomic analyses, we identify altered gene expression related to dendrite morphogenesis instead of RIG-I-activated interferon signaling, such as decreased calcium/calmodulin-dependent protein kinase 2α (Camk2α). In line with these molecular changes, dendritic complexity in CMTR1-insufficient neurons is rescued by ectopic expression of CaMK2α but not by inactivation of RIG-I signaling. We further generate brain-specific CMTR1-knockout mice to validate these findings in vivo. Our study reveals the indispensable role of CMTR1-catalyzed N1 2'-O-Me in gene regulation for brain development.

Keywords: 2′-O-ribose methylation; CMTR; CaMK2; RIG-I; cap1 modification; dendrite development; innate immunity.

The neuronal RNA-binding protein HuD is involved in synaptic plasticity and learning and memory mechanisms. These effects are thought to be due to HuD-mediated stabilization and translation of target mRNAs associated with plasticity. To investigate the potential role of HuD in drug addiction, we first used bioinformatics prediction algorithms together with microarray analyses to search for specific genes and functional networks upregulated within the forebrain of HuD overexpressing mice (HuDOE ). When this set was further limited to genes in the knowledgebase of addiction-related genes database (KARG) that contains predicted HuD-binding sites in their 3' untranslated regions (3'UTRs), we found that HuD regulates networks that have been associated with addiction-like behavior. These genes included Bdnf and Camk2a, 2 previously validated HuD targets. Since addiction is hypothesized to be a disorder stemming from altered gene expression causing aberrant plasticity, we sought to test the role of HuD in cocaine conditioned placed preference (CPP), a model of addiction-related behaviors. HuD mRNA and protein were upregulated by CPP within the nucleus accumbens of wild-type C57BL/6J mice. These changes were associated with increased expression of Bdnf and Camk2a mRNA and protein. To test this further, we trained HuDOE and wild-type mice in CPP and found that HuDOE mice showed increased cocaine CPP compared with controls. This was also associated with elevated expression of HuD target mRNAs and proteins, CaMKIIα and BDNF. These findings suggest HuD involvement in addiction-related behaviors such as cocaine conditioning and seeking, through increased plasticity-related gene expression.

The neuritogenic cAMP sensor (NCS), encoded by the Rapgef2 gene, links cAMP elevation to activation of extracellular signal-regulated kinase (ERK) in neurons and neuroendocrine cells. Transducing human embryonic kidney (HEK)293 cells, which do not express Rapgef2 protein or respond to cAMP with ERK phosphorylation, with a vector encoding a Rapgef2 cDNA reconstituted cAMP-dependent ERK activation. Mutation of a single residue in the cyclic nucleotide-binding domain (CNBD) conserved across cAMP-binding proteins abrogated cAMP-ERK coupling, while deletion of the CNBD altogether resulted in constitutive ERK activation. Two types of mRNA are transcribed from Rapgef2 in vivo. Rapgef2 protein expression was limited to tissues, i.e., neuronal and endocrine, expressing the second type of mRNA, initiated exclusively from an alternative first exon called here exon 1', and an alternative 5' protein sequence leader fused to a common remaining open reading frame, which is termed here NCS-Rapgef2. In the male mouse brain, NCS-Rapgef2 is prominently expressed in corticolimbic excitatory neurons, and striatal medium spiny neurons (MSNs). Rapgef2-dependent ERK activation by the dopamine D1 agonist SKF81297 occurred in neuroendocrine neuroscreen-1 (NS-1) cells expressing the human D1 receptor and was abolished by deletion of Rapgef2. Corticolimbic [e.g., dentate gyrus (DG), basolateral amygdala (BLA)] ERK phosphorylation induced by SKF81297 was significantly attenuated in CamK2α-Cre+/- ; Rapgef2cko/cko male mice. ERK phosphorylation in nucleus accumbens (NAc) MSNs induced by treatment with SKF81297, or the psychostimulants cocaine or amphetamine, was abolished in male Rapgef2cko/cko mice with NAc NCS-Rapgef2-depleting AAV-Synapsin-Cre injections. We conclude that D1-dependent ERK phosphorylation in mouse brain requires NCS-Rapgef2 expression.

WDR13 expresses from the X chromosome and has a highly conserved coding sequence. There have been multiple associations of WDR13 with memory. However, its detailed function in context of brain and behavior remains unknown. We characterized the behavioral phenotype of 2 month old male mice lacking the homolog of WDR13 gene (Wdr13 (-/0)). Taking cue from analysis of its expression in the brain, we chose hippocampus for molecular studies to delineate its function. Wdr13 (-/0) mice spent less time in the central area of the open field test (OFT) and with the novel object in novel object recognition test (NOR) as compared to the wild-type. However, these mice didn't show any significant changes in total time spent in arms or in frequency of arm entries in elevated plus maze (EPM). In the absence of Wdr13, there was a significant upregulation of synaptic proteins, viz., SYN1, RAB3A, CAMK2A etc. accompanied with increased spine density of hippocampal CA1 neurons and better spatial memory in mice as measured by increased time spent in the target quadrant of Morris water maze (MWM) during probe test. Parallel study from our lab has established c-JUN, ER α/β, and HDAC 1,3,7 as interacting partners of WDR13. WDR13 represses transcription from AP1 (c-JUN responsive) and Estrogen Receptor Element (ERE) promoters. We hypothesized that absence of Wdr13 would result in de-regulated expression of a number of genes including multiple synaptic genes leading to the observed phenotype. Knocking down Wdr13 in Neuro2a cell lines led to increased transcripts of Camk2a and Nrxn2 consistent with in-vivo results. Summarily, our data provides functional evidence for the role of Wdr13 in brain.

Hypopharyngeal carcinoma (HPC) is a subtype of head and neck squamous cell carcinoma, and prognosis has improved significantly over the past three decades. Induction docetaxel/cisplatin/5 fluorouracil (TPF) chemotherapy is regarded as the standard of treatment for locoregionally advanced HPC. However, patients who do not respond to cisplatin suffer, rather than benefit, from chemotherapy treatment. The goal of this study was to identify molecules involved in TPF resistance and to clarify their molecular mechanisms. Using the FaDu cell line as the cell model, the TPF IC50 was identified, and c-Jun, IL6, Camk2a, c-fos knockdown using siRNAs resulted in a significant declined TPF IC50. Retrospective analysis of the expression status of c-Jun, IL6, Camk2a, and c-fos by immunohistochemistry staining in sectioned HPC tissues from TPF-sensitive and TPF-insensitive patients shows that Camk2a and c-Jun were associated with the clinical pathogenesic features in HPC. The in vitro experiments also indicate that both Camk2a and c-Jun were responsive to TPF treatment. This study identified Camk2a and c-Jun as candidate genes that confer induction TPF resistance, which would help in the discovery of potential therapeutic markers and in developing a personalized and precise treatment approach for HPC patients.

Ca²⁺/calmodulin dependent protein kinase2α (CaMK2α) is a serine/threonine protein kinase in neurons and leads to neuronal injury when it is activated abnormally. Bupivacaine, a local anesthetic commonly used in regional nerve block, could induce neurotoxicity via apoptotic injury. Whether or not CaMK2α is involved in bupivacaine-induced neurotoxicity and it is regulated remains unclear. In this study, bupivacaine was administered for intrathecal injection in C57BL/6 mice for building vivo injury model and was used to culture human neuroblastoma (SH-SY5Y) cells for building vitro injury model. The results showed that bupivacaine induced mitochondrial oxidative stress and neurons apoptotic injury, promoted phosphorylation of CaMK2α and cAMP-response element binding protein (CREB), and elevated mitochondrial Ca²⁺ uniporter (MCU) expression. Furthermore, it induced CaMK2α phosphorylation at Thr286 which phosphorylated CREB at Ser133 and up-regulated MCU transcriptional expression. Inhibition of CaMK2α-MCU signaling with knock-down of CaMK2α and MCU or with inhibitors (KN93 and Ru360) significantly mitigated bupivacaine-induced neurotoxic injury. Over-expression of CaMK2α significantly enhanced above oxidative injury. Activated MCU with agonist (spermine) reversed protective effect of siCaMK2α on bupivacaine-induced mitochondrial oxidative stress. Our data revealed that CaMK2α-MCU-mitochondrial oxidative stress pathway is a major mechanism whereby bupivacaine induces neurotoxicity and inhibition of above signaling could be a therapeutic strategy in the treatment of bupivacaine-induced neurotoxicity.

Vilse/Arhgap39 is a Rho GTPase activating protein (RhoGAP) and utilizes its WW domain to regulate Rac/Cdc42-dependent morphogenesis in Drosophila and murine hippocampal neurons. However, the function of Vilse in mammalian dendrite architecture and synaptic plasticity remained unclear. In the present study, we aimed to explore the possible role of Vilse in dendritic structure and synaptic function in the brain. Homozygous knockout of Vilse resulted in premature embryonic lethality in mice. Changes in dendritic complexity and spine density were noticed in hippocampal neurons of Camk2a-Cre mediated forebrain-specific Vilse knockout (VilseΔ/Δ) mice. VilseΔ/Δ mice displayed impaired spatial memory in water maze and Y-maze tests. Electrical stimulation in hippocampal CA1 region revealed that the synaptic transmission and plasticity were defected in VilseΔ/Δ mice. Collectively, our results demonstrate that Vilse is essential for embryonic development and required for spatial memory.

PHRF1 is involved in transforming growth factor β (TGF-β) signaling to constrain the formation of acute promyelocytic leukemia (APL) in mouse APL models. PHRF1 also participates in modulating non-homologous end-joining. However, the role of PHRF1 in mammalian dendrite architecture and synaptic plasticity is unclear. Here, we investigated the role of PHRF1 in dendritic formation in the murine hippocampus using Camk2a promoter driven-iCre recombinase to conduct a PHRF1 conditional knockout, namely PHRF1^Δ/Δ, in the forebrain region. PHRF1^Δ/Δ mice developed normally, but exhibited anxiety-like behaviors and displayed defective spatial memory. Alterations of dendritic complexity in apical and basal dendrites of pyramidal neurons were noticed in PHRF1^Δ/Δ mutants. Furthermore, electrical stimulation in the hippocampal CA1 region after the TGF-β1 treatment showed a reduced synaptic plasticity in PHRF1^Δ/Δ mice. Immunoblotting analysis indicated that PHRF1 ablation affected the TGF-β signaling. Collectively, our results demonstrate that PHRF1 is important for the dendritic architecture and required for spatial memory formation in the hippocampus.

Spinocerebellar ataxia type 2 (SCA2) is caused by polyglutamine expansion in Ataxin-2 (ATXN2). This factor binds RNA/proteins to modify metabolism after stress, and to control calcium (Ca²⁺) homeostasis after stimuli. Cerebellar ataxias and corticospinal motor neuron degeneration are determined by gain/loss in ATXN2 function, so we aimed to identify key molecules in this atrophic process, as potential disease progression markers. Our Atxn2-CAG100-Knock-In mouse faithfully models features observed in patients at pre-onset, early and terminal stages. Here, its cerebellar global RNA profiling revealed downregulation of signaling cascades to precede motor deficits. Validation work at mRNA/protein level defined alterations that were independent of constant physiological ATXN2 functions, but specific for RNA/aggregation toxicity, and progressive across the short lifespan. The earliest changes were detected at three months among Ca²⁺ channels/transporters (Itpr1, Ryr3, Atp2a2, Atp2a3, Trpc3), IP₃ metabolism (Plcg1, Inpp5a, Itpka), and Ca²⁺-Calmodulin dependent kinases (Camk2a, Camk4). CaMKIV-Sam68 control over alternative splicing of Nrxn1, an adhesion component of glutamatergic synapses between granule and Purkinje neurons, was found to be affected. Systematic screening of pre/post-synapse components, with dendrite morphology assessment, suggested early impairment of CamKIIα abundance together with the weakening of parallel fiber connectivity. These data reveal molecular changes due to ATXN2 pathology, primarily impacting excitability and communication.

Keywords: K-homology RNA-binding domain; amyotrophic lateral sclerosis (ALS); fragile-X-associated tremor-ataxia syndrome; fronto-temporal-lobar-dementia; inositol signaling; long-term potentiation; neurexin; spatial learning; synaptic plasticity; tauopathies.

Exposure to early life stress can interfere with neurodevelopmental trajectories to increase the vulnerability for psychiatric disorders later in life. With this respect, epigenetic mechanisms play a key role for the long-lasting changes in brain functions that may elicit and sustain psychopathologic outcomes. Here, we investigated DNA methylation changes as possible epigenetic mechanism mediating the effect of prenatal stress (PNS), an experimental paradigm associated with behavioral and molecular alterations relevant for psychiatric disorders. We identified 138 genes as being differentially methylated in the prefrontal cortex (PFC) and in the hippocampus (HIP) of male and female adult rats exposed to PNS. Among these genes, miR-30a and Neurod1 emerged as potential players for the negative outcomes associated with PNS exposure. Indeed, in addition to showing consistent methylation differences in both brain regions and in both sexes, and interacting with each other, they are both involved in Axon guidance and Neurotrophin signaling, which are important to neurodevelopmental disorders. We also found a significant reduction in the expression of a panel of genes (CAMK2A, c-JUN, LIMK1, MAP2K1, MAP2K2, PIK3CA and PLCG1) that belong to these two biological pathways and are also validated targets of miR-30a, pointing to a down-regulation of these pathways as a consequence of PNS exposure. Interestingly, we also found that miR-30a levels were significantly upregulated in depressed patients exposed to childhood trauma, as compared to control individuals. Importantly, we also found that a sub-chronic treatment with the atypical antipsychotic drug, lurasidone, during adolescence was able to prevent the up-regulation of miR-30a and normalized the expression of its target genes in response to PNS exposure. Our results demonstrate that miR-30a undergoes epigenetic changes following early life stress exposure and suggest that this miRNA could play a key role in producing broad and long-lasting alterations in neuroplasticity-related pathways, contributing to the etiology of psychiatric disorders.

Keywords: Axon guidance signaling; DNA methylation; Neurotrophin signaling; Prenatal stress; miR-30a.

This study aimed to screen the target miRNAs and to investigate the differential miR-3557/324-targeted signal mechanisms in the rats' model of Parkinson's disease (PD) with regular aerobic exercise. Rats were divided into sedentary control PD group (SED-PD, n = 18) and aerobic exercise PD group (EX-PD, n = 22). After 8 weeks of regular aerobic exercise, a 6-hydroxydopamine- (6-OHDA-) induced PD lesion model was constructed. Preregular aerobic exercises enhanced the injury resistance of rats with 6-OHDA-induced PD. The rotational behavior after injection of apomorphine hydrochloride was alleviated. Under the scanning electron microscopy, we found the neurons, axons, and villi of the striatum were clearly and tightly arranged, and neurons and axons significantly becoming larger. Tyrosine hydroxylase (TH) was increased significantly and α-synuclein protein expression was reduced in the EX-PD group compared to the SED-PD group. Screening from miRNA microarray chip, we further found upregulation of miR-3557 and downregulation of miR-324 were closely related to the calcium-modulating signaling pathway, remitting the progress of Parkinson's disease on aerobic exercise. Compared to the SED-PD group, Ca²⁺/calmodulin dependent protein kinase II (CaMK2α) was upregulated, but CaMKV and voltage-dependent anion-selective channel protein 1 (Vdac1) were significantly downregulated in the EX-PD group. Additionally, phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) expression were activated, and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) expression was upregulated in the EX-PD group. Conclusions: the adaptive mechanism of regular aerobic exercise delaying neurodegenerative diseases and lesions was that miR-3557/324 was activated to regulate one of its targets CaMKs signaling pathways. CaMKs, coordinated with mTOR pathway-related gene expression, improved UCH-L1 level to favor for delaying neurodegeneration or improving the pathogenesis of PD lesions.

To observe the regulation of electroacupuncture on gene expression at calcium signaling pathways in mice with cerebral ischemia reperfusion.

Sixty male, inbred Kunming mice were randomly assigned to three groups: repeated cerebral ischemia reperfusion group (RG, n = 24), sham-operated group (SG, n = 12), and electroacupuncture group (EG, n = 24). Mice in RG and EG groups were modeled by repeated cerebral ischemia reperfusion surgery, and EG mice were treated with electroacupuncture for 30 min after recovery from anesthesia. Changes in gene expression profile of mice hippocampi were analyzed by global expression profile microarray. Genes that were up-regulated or down-regulated greater than 1.5 folds were considered to be biologically meaningful. Real-time quantitative polymerase chain reaction (q-PCR) method was used to verify the expression of selected genes based on the algorithm [2^ (ΔΔCt)].

Compared with SG mice, 242 genes showed different in expressions in RG mice: 107 down-regulated and 135 up-regulated. Compared with RG mice, 609 genes showed a difference of expression in EG mice: 315 down-regulated and 375 up-regulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated two pathways: calcium signaling and long-term potentiation in which 11 differentially expressed genes selected. Six of the 11 genes in the calcium signaling pathway were verified after real-time q-PCR testing.

Electroacupuncture treatment of cerebral ischemia reperfusion appears to regulate Atp2a2, Cacna1e, Camk2a, Gnas, Grm1, Rapgef3 genes in the calcium signaling pathway.

Hippocampal pyramidal neurons play an essential role in processing spatial information as implicated with its place-dependent firing. Although, previous slice physiology studies have reported that voltage gated calcium channels contribute to spike shapes and corresponding firing rate in the hippocampus, the roles of P/Q type calcium channels (Cav2.1) underlying neural activity in behaving mice have not been well-investigated. To determine physiological and behavioral roles of Cav2.1, we conducted place cell recordings in CA1 and hippocampus dependent learning/memory tasks using mice lacking Cav2.1 in hippocampal pyramidal neurons under CamK2α-Cre recombinase expression. Results suggested that impairments shown in behavioral tasks requiring spatial and contextual information processing were statistically significant while general neurological behaviors did not differ between groups. In particular, deficits were more profound in recognition than in acquisition. Furthermore, place cell recordings also revealed that the ability to recollect spatial representation on re-visit in the conditional knockout was also altered in terms of the cue recognition while the capability of a place cell to encode a place was intact compared to the control group. Interestingly, CA1 pyramidal neurons of conditional knockout mice showed reduced burst frequency as well as abnormal temporal patterns of burst spiking. These results provide potential evidence that Cav2.1 in hippocampal pyramidal cells modulates temporal integration of bursts, which, in turn, might influence the recognition of place field and consequently disrupt spatial recognition ability.

Acute or chronic effects of consuming or skipping breakfast on cognitive performance in humans are controversial. To evaluate the effects of chronically skipping breakfast (SB) on hippocampus-dependent long-term memory formation, we examined hippocampal gene expression and applied the novel object recognition test (NORT) after two weeks of repeated fasting for six hours from lights off to mimic SB in mice. We also examined the effects of SB on circadian rhythms of locomotor activity, food intake, core body temperature (CBT) and sleep-wake cycles. Skipping breakfast slightly but significantly decreased total daily food intake without affecting body weight gain. Locomotor activity and CBT significantly decreased during the fasting period under SB. The degree of fasting-dependent CBT reduction gradually increased and then became stabilized after four days of SB. Electroencephalographic data revealed that repeated SB significantly decreased the duration of wakefulness and increased that of rapid eye movement (REM) and of non-REM (NREM) sleep during the period of SB. Furthermore, total daily amounts of wakefulness and NREM sleep were significantly decreased and increased, respectively, under SB, suggesting that SB disrupts sleep homeostasis. Skipping breakfast significantly suppressed mRNA expression of the memory-related genes, Camk2a, Fkbp5, Gadd45b, Gria1, Sirt1 and Tet1 in the hippocampus. Recognition memory assessed by NORT was impaired by SB in accordance with the gene expression profiles. These findings suggested that chronic SB causes dysregulated CBT, sleep-wake cycles and hippocampal gene expression, which results in impaired long-term memory formation.

Late-phase long-term potentiation (L-LTP) in hippocampus, thought to be the cellular basis of long-term memory, requires new protein synthesis. Neural activity enhances local protein synthesis in dendrites, which in turn mediates long-lasting synaptic plasticity. Ca²⁺/calmodulin-dependent protein kinase IIα (CaMKIIα) is a locally synthesized protein crucial for this plasticity, as L-LTP is impaired when its local synthesis is eliminated. However, the distribution of Camk2a mRNA during L-LTP induction remains unclear. In this study, we investigated the dendritic targeting of Camk2a mRNA after high-frequency stimulation, which induces L-LTP in synapses of perforant path and granule cells in the dentate gyrus in vivoIn situ hybridization studies revealed that Camk2a mRNA was immediately but transiently targeted to the site receiving high-frequency stimulation. This was associated with an increase in de novo protein synthesis of CaMKIIα. These results suggest that dendritic translation of CaMKIIα is locally mediated where L-LTP is induced. This phenomenon may be one of the essential processes for memory establishment.

The present study aimed to investigate the gene functions and expression profiles in perihematomal (PH) brain regions following spontaneous intracerebral hemorrhage. The gene expression profiles were downloaded from the Gene Expression Omnibus database under accession number GSE24265, which includes 11 brain samples from different regions, including four samples from PH areas, four from contralateral grey matter (CG) and three from contralateral white matter (CW). The gene expression profiles were pre-processed and the differentially expressed genes (DEGs) between PH and CG tissue, and PH and CW tissue were identified using R packages. The expression of genes in different tissues was analyzed by hierarchical clustering. Then, the interaction network between the DEGs was constructed using String software. Finally, Gene Ontology was performed and pathway analysis was conducted using FuncAssociate and Expression Analysis Systematic Explorer to identify the gene function. As a result, 399 DEGs were obtained between PH and CG, and 756 DEGs were identified between PH and CW. There were 35 common DEGs between the two groups. These DEGs may be involved in PH edema by regulating the calcium signaling pathway [calcium channel, voltage‑dependent, T-type, α1I subunit, Ca2+/calmodulin‑dependent protein kinase II α (CAMK2A), ryanodine receptor 2 (RYR2) and inositol 1,4,5-trisphosphate receptor, type 1 (ITPR1)], cell proliferation (sphingosine kinase 1), neuron differentiation (Ephrin-A5) or extracellular matrix-receptor interaction [collagen, type I, α 2, laminin B1 (LAMB1), syndecan 2, fibronectin 1 and integrin α5 (ITGA5)]. A number of genes may cooperate to participate in the same pathway, such as ITPR1-RYR2, CAMK2A-RYR2 and ITGA5-LAMB1 interaction pairs. The present study provides several potential targets to decrease hematoma expansion and alleviate neuronal cell death following spontaneous intracerebral hemorrhage.

The brain contains a large variety of projection neurons with different functional properties. The functional properties of projection neurons arise from their connectivity with other neurons and their molecular composition. We describe a novel tool for obtaining the gene expression profiles of projection neurons that are anatomically defined by the location of their soma and axon terminals. Our tool utilizes adeno-associated virus serotype 9 (AAV9), which we found to retrogradely transduce projection neurons after injection at the site of the axon terminals. We used AAV9 to express Enhanced Green Fluorescent Protein (EGFP)-tagged ribosomal protein L10a (EGFP-L10a), which enables the immunoprecipitation of EGFP-tagged ribosomes and associated mRNA with a method known as Translating Ribosome Affinity Purification (TRAP). To achieve high expression of the EGFP-L10a protein in projection neurons, we placed its expression under control of a 1.3 kb alpha-calcium/calmodulin-dependent protein kinase II (Camk2a) promoter. We injected the AAV9-Camk2a-TRAP virus in either the hippocampus or the bed nucleus of the stria terminalis (BNST) of the mouse brain. In both brain regions the 1.3 kb Camk2a promoter did not confer complete cell-type specificity around the site of injection, as EGFP-L10a expression was observed in Camk2a-expressing neurons as well as in neuronal and non-neuronal cells that did not express Camk2a. In contrast, cell-type specific expression was observed in Camk2a-positive projection neurons that were retrogradely transduced by AAV9-Camk2a-TRAP. Injection of AAV9-Camk2a-TRAP into the BNST enabled the use of TRAP to collect ribosome-bound mRNA from basal amygdala projection neurons that innervate the BNST. AAV9-Camk2a-TRAP provides a single-virus system that can be used for the molecular profiling of anatomically defined projection neurons in mice and other mammalian model organisms. In addition, AAV9-Camk2a-TRAP may enable the discovery of protein synthesis events that support information storage in projection neurons.

Several psychiatric and neurological diseases are associated with altered hippocampal neurogenesis, suggesting differing neural stem cell (NSC) function may play a critical role in these diseases. To investigate the role of resident NSCs in a murine model of psychiatric disease, we sought to isolate and characterize NSCs from alpha-calcium-/calmodulin-dependent protein kinase II heterozygous knockout (CaMK2α-hKO) mice, a model of schizophrenia/bipolar disorder. These mice display altered neurogenesis, impaired neuronal development and are part of a larger family possessing phenotypic and behavioral correlates of schizophrenia/bipolar disorder and a shared pathology referred to as the immature dentate gyrus (iDG). The extent to which NSCs contribute to iDG pathophysiology remains unclear. To address this, we established heterogeneous cultures of NSCs isolated from the hippocampal neuropoietic niche. When induced to differentiate, CaMK2α-hKO-derived NSCs recapitulate organotypic hippocampal neurogenesis, but generate larger numbers of immature neurons than wild-type (WT) littermates. Furthermore, mutant neurons fail to assume mature phenotypes (including morphology and MAP2/calbindin expression) at the same rate observed in WT counterparts. The increased production of immature neurons which fail to mature indicates that this reductionist model retains key animal- and iDG-specific maturational deficits observed in animal models and human patients. This is doubly significant, as these stem cells lack several developmental inputs present in vivo. Interestingly, NSCs were isolated from animals prior to the emergence of overt iDG pathophysiology, suggesting mutant NSCs may possess lasting intrinsic alterations and that altered NSC function may contribute to iDG pathophysiology in adult animals.

Calcium/calmodulin dependent protein kinase 2 (CaMKII) is a multifunctional protein that is highly enriched in the synapse. It plays important roles in neuronal functions such as synaptic plasticity, synaptogenesis, and neural development. Gene duplication in zebrafish has resulted in the occurrence of seven CaMKII genes (camk2a, camk2b1, camk2b2, camk2g1, camk2g2, camk2d1, and camk2d2) that are developmentally expressed. In this study, we used single cell, real-time quantitative PCR to investigate the expression of CaMKII genes in individual Mauthner cells (M-cells) of 2 days post fertilization (dpf) zebrafish embryos. We found that out of seven different CaMKII genes, only the mRNA for CaMKII-α was expressed in the M-cell at detectable levels, while all other isoforms were undetectable. Morpholino knockdown of CaMKII-α had no significant effect on AMPA synaptic currents (mEPSCs) but decreased the amplitude of NMDA mEPSCs. NMDA events exhibited a biexponential decay with τfast ≈ 30 ms and τslow ≈ 300 ms. Knockdown of CaMKII-α specifically reduced the amplitude of the slow component of the NMDA-mediated currents (mEPSCs), without affecting the fast component, the frequency, or the kinetics of the mEPSCs. Immunolabelling of the M-cell showed increased dendritic arborizations in the morphants compared with controls, and knockdown of CaMKII-α altered locomotor behaviors of touch responses. These results suggest that CaMKII-α is present in embryonic M-cells and that it plays a role in the normal development of excitatory synapses. Our findings pave the way for determining the function of specific CaMKII isoforms during the early stages of M-cell development.

OBJECTIVE

This study aimed to better understand gene expression profiles of human hepatic stellate cell (HSC) activation and the relationship with the Wnt signaling pathway.

METHODS

The global transcript levels in platelet derived growth factor-BB (PDGF-BB)-stimulated hTERT HSCs were analyzed using oligonucleotide microarrays. Oligonucleotide microarrays with 19K human oligo chips were performed to obtain gene expression profiles associated with proliferation in human hTERT HSCs. The microarray data was verified by real time quantitative PCR and expression of the components of Wnt signaling was analyzed by Western blot.

RESULTS

Microarray data showed 243 up-regulated and 265 down-regulated genes in PDGF-BB-treated HSCs. The changes in expression of glypican3 and BH3 interacting domain death agonist (BID) mRNA in real time quantitative PCR, especially among the highly up- or down-regulated genes, were statistically consistent with the microarray data. The Wnt signaling pathway components, frizzled10 (FZD10) and calcium/calmodulindependent protein kinase II alpha (CAMK2A), showed increased expression in the short time course microarray and the up-regulation of FZD10 also occurred at the protein level. Our data showed various gene expression profiles during activation of human HSC.

CONCLUSIONS

The up-regulated expression of FZD10 and CAMK2A suggests that the Wnt/Ca(2+) signaling pathway is active in hTERT HSCs and may participate in HSC activation and proliferation.

The nervous system of higher eukaryotes is composed of numerous types of neurons and glia that together orchestrate complex neuronal responses. However, this complex pool of cells typically poses analytical challenges in investigating gene expression profiles and their epigenetic basis for specific cell types. Here, we developed a novel method that enables cell type-specific analyses of epigenetic modifications using tandem chromatin immunoprecipitation sequencing (tChIP-Seq). FLAG-tagged histone H2B, a constitutive chromatin component, was first expressed in Camk2a-positive pyramidal cortical neurons and used to purify chromatin in a cell type-specific manner. Subsequent chromatin immunoprecipitation using antibodies against H3K4me3-a chromatin modification mainly associated with active promoters-allowed us to survey the histone modifications in Camk2a-positive neurons. Indeed, tChIP-Seq identified hundreds of H3K4me3 modifications in promoter regions located upstream of genes associated with neuronal functions and genes with unknown functions in cortical neurons. tChIP-Seq provides a versatile approach to investigating the epigenetic modifications of particular cell types in vivo.

BACKGROUND

Here we describe a detailed, reliable protocol for isolation of polysomal fractions from mouse brain synaptoneurosomes. This method is an important tool to study local protein synthesis in neurons.

UNASSIGNED

We combined rapid preparation of synaptoneurosomes by filtration with polysome profiling. We provide a detailed protocol highlighting difficulties and critical steps of: i) preparation of synaptoneurosomes; ii) polyribosome fractionation from synaptoneurosomes; iii) extraction of proteins and RNA from sucrose gradient fractions.

RESULTS

and Comparison with Existing Methods We fractionated polyribosomes from synaptoneurosomes and detected the association of Mmp9, Camk2a and Stx1B mRNA with polysomes in the unstimulated conditions. Synaptic stimulation led to increased levels of Mmp9 and Camk2a mRNA in the heavy polysomal fractions. We compared our protocol with existing methods CONCLUSIONS: We have developed a reliable, effective method to prepare polyribosomal fractions from synaptoneurosomes to study polyribosomal binding of mRNAs as an aspect of synaptic translation in vitro.

UNASSIGNED

Inhibitors of fibroblast growth factor receptors (FGFRs) have recently arisen as a promising treatment option for patients with FGFR alterations. Gene fusions involving FGFR3 and transforming acidic coiled-coil protein 3 (TACC3) have been detected in diffuse gliomas and other malignancies, and fusion-positive cases have responded well to FGFR inhibition. As high FGFR3 expression has been detected in fusion-positive tumors, we sought to determine the clinical significance of FGFR3 protein expression level as well as its potential for indicating FGFR3 fusions.

UNASSIGNED

We performed FGFR3 immunohistochemistry on tissue microarrays containing 676 grades II-IV astrocytomas and 116 grades II-III oligodendroglial tumor specimens. Fifty-one cases were further analyzed using targeted sequencing.

UNASSIGNED

Moderate to strong FGFR3 staining was detected in gliomas of all grades, was more common in females, and was associated with poor survival in diffuse astrocytomas. Targeted sequencing identified FGFR3-TACC3 fusions and an FGFR3-CAMK2A fusion in 10 of 15 strongly stained cases, whereas no fusions were found in 36 negatively to moderately stained cases. Fusion-positive cases were predominantly female and negative for IDH and EGFR/PDGFRA/MET alterations. These and moderately stained cases show lower MIB-1 proliferation index than negatively to weakly stained cases. Furthermore, stronger FGFR3 expression was commonly observed in malignant tissue regions of lower cellularity in fusion-negative cases. Importantly, subregional negative FGFR3 staining was also observed in a few fusion-positive cases.

UNASSIGNED

Strong FGFR3 protein expression is indicative of FGFR3 fusions and may serve as a clinically applicable predictive marker for treatment regimens based on FGFR inhibitors.