Small G-Protein Rheb Gates Mammalian Target of Rapamycin Signaling to Regulate Morphine Tolerance in Mice.

BACKGROUND: Analgesic tolerance due to long-term use of morphine remains a challenge for pain management. Morphine acts on µ-opioid receptors and downstream of the phosphatidylinositol 3-kinase signaling pathway to activate the mammalian target of rapamycin (mTOR) pathway. Rheb is an important regulator of growth and cell-cycle progression in the central nervous system owing to its critical role in the activation of mTOR. The hypothesis was that signaling via the GTP-binding protein Rheb in the dorsal horn of the spinal cord is involved in morphine-induced tolerance. METHODS: Male and female wild-type C57BL/6J mice or transgenic mice (6 to 8 weeks old) were injected intrathecally with saline or morphine twice daily at 12-h intervals for 5 consecutive days to establish a tolerance model. Analgesia was assessed 60 min later using the tail-flick assay. After 5 days, the spine was harvested for Western blot or immunofluorescence analysis. RESULTS: Chronic morphine administration resulted in the upregulation of spinal Rheb by 4.27 ± 0.195-fold (P = 0.0036, n = 6), in turn activating mTOR by targeting rapamycin complex 1 (mTORC1). Genetic overexpression of Rheb impaired morphine analgesia, resulting in a tail-flick latency of 4.65 ± 1.10 s (P < 0.0001, n = 7) in Rheb knock-in mice compared to 10 s in control mice (10 ± 0 s). Additionally, Rheb overexpression in spinal excitatory neurons led to mTORC1 signaling overactivation. Genetic knockout of Rheb or inhibition of mTORC1 signaling by rapamycin potentiated morphine-induced tolerance (maximum possible effect, 52.60 ± 9.56% in the morphine + rapamycin group vs. 16.60 ± 8.54% in the morphine group; P < 0.0001). Moreover, activation of endogenous adenosine 5'-monophosphate-activated protein kinase inhibited Rheb upregulation and retarded the development of morphine-dependent tolerance (maximum possible effect, 39.51 ± 7.40% in morphine + metformin group vs. 15.58 ± 5.79% in morphine group; P < 0.0001). CONCLUSIONS: This study suggests spinal Rheb as a key molecular factor for regulating mammalian target of rapamycin signaling.

The calcium channel Orai1 is required for osteoblast development: Studies in a chimeric mouse with variable in vivo Runx-cre deletion of Orai-1.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

The neuronal pentraxin Nptx2 regulates complement activity and restrains microglia-mediated synapse loss in neurodegeneration.

Complement overactivation mediates microglial synapse elimination in neurological diseases such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), but how complement activity is regulated in the brain remains largely unknown. We identified that the secreted neuronal pentraxin Nptx2 binds complement C1q and thereby regulates its activity in the brain. Nptx2-deficient mice show increased complement activity, C1q-dependent microglial synapse engulfment, and loss of excitatory synapses. In a neuroinflammation culture model and in aged TauP301S mice, adeno-associated virus (AAV)-mediated neuronal overexpression of Nptx2 was sufficient to restrain complement activity and ameliorate microglia-mediated synapse loss. Analysis of human cerebrospinal fluid (CSF) samples from a genetic FTD cohort revealed reduced concentrations of Nptx2 and Nptx2-C1q protein complexes in symptomatic patients, which correlated with elevated C1q and activated C3. Together, these results show that Nptx2 regulates complement activity and microglial synapse elimination in the brain and that diminished Nptx2 concentrations might exacerbate complement-mediated neurodegeneration in patients with FTD.

NMDA Receptor-Arc Signaling Is Required for Memory Updating and Is Disrupted in Alzheimer's Disease.

BACKGROUND: Memory deficits are central to many neuropsychiatric diseases. During acquisition of new information, memories can become vulnerable to interference, yet mechanisms that underlie interference are unknown. METHODS: We describe a novel transduction pathway that links the NMDA receptor (NMDAR) to AKT signaling via the immediate early gene Arc and evaluate its role in memory. The signaling pathway is validated using biochemical tools and transgenic mice, and function is evaluated in assays of synaptic plasticity and behavior. The translational relevance is evaluated in human postmortem brain. RESULTS: Arc is dynamically phosphorylated by CaMKII (calcium/calmodulin-dependent protein kinase II) and binds the NMDAR subunits NR2A/NR2B and a previously unstudied PI3K (phosphoinositide 3-kinase) adapter p55PIK (PIK3R3) in vivo in response to novelty or tetanic stimulation in acute slices. NMDAR-Arc-p55PIK recruits p110α PI3K and mTORC2 (mechanistic target of rapamycin complex 2) to activate AKT. NMDAR-Arc-p55PIK-PI3K-mTORC2-AKT assembly occurs within minutes of exploratory behavior and localizes to sparse synapses throughout hippocampal and cortical regions. Studies using conditional (Nestin-Cre) p55PIK deletion mice indicate that NMDAR-Arc-p55PIK-PI3K-mTORC2-AKT functions to inhibit GSK3 and mediates input-specific metaplasticity that protects potentiated synapses from subsequent depotentiation. p55PIK conditional knockout mice perform normally in multiple behaviors including working memory and long-term memory tasks but exhibit deficits indicative of increased vulnerability to interference in both short-term and long-term paradigms. The NMDAR-AKT transduction complex is reduced in postmortem brain of individuals with early Alzheimer's disease. CONCLUSIONS: A novel function of Arc mediates synapse-specific NMDAR-AKT signaling and metaplasticity that contributes to memory updating and is disrupted in human cognitive disease.

Upregulation of the long non-coding RNA, LIPCAR promotes proliferation, migration, and metastasis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) early diagnosis remains a challenge to date. Alpha-feto protein, though less sensitive remains widely used for both diagnosis and prognosis. Recently however, a number of molecular biomarkers have been suggested as alternatives to Alpha feto protein, especially for early diagnosis. OBJECTIVE: To determine the role of the long non-coding RNA, LIPCAR in the pathogenesis and early diagnosis of hepatocellular carcinoma. METHODS: Quantitative real-time PCR, and Fluorescence in situ hybridization assays were conducted to determine LIPCAR expression in HCC vs normal blood samples, and HCC cell lines vs normal liver cell lines. Transfection was done to upregulate LIPCAR in one HCC cell line, and used to study cell proliferation, migration, apoptosis and epithelial-mesenchymal transformation. Animal experiment was finally done to determine its effect on metastasis. RESULTS: LIPCAR was significantly upregulated in HCC blood samples and HCC cell lines compared to their respective normal ones. Its overexpression promoted hepatocellular carcinoma cell proliferation, and migration, while inhibiting apoptosis. Its overexpression also promoted epithelial-mesenchymal transformation in hepatocellular carcinoma cells, and metastasis in vivo. CONCLUSION: The study demonstrated that the lncRNA, LIPCAR is significantly upregulated in hepatocellular carcinoma patients and that its upregulation promotes HCC proliferation, migration, and metastases.

RHEB is a potential therapeutic target in T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of immature T lymphocytes. Although various therapeutic approaches have been developed, refractoriness of chemotherapy and relapse cause a poor prognosis of the disease and further therapeutic strategies are required. Here, we report that Ras homolog enriched in brain (RHEB), a critical regulator of mTOR complex 1 activity, is a potential target for T-ALL therapy. In this study, we established an sgRNA library that comprehensively targeted mTOR upstream and downstream pathways, including autophagy. CRISPR/Cas9 dropout screening revealed critical roles of mTOR-related molecules in T-ALL cell survival. Among the regulators, we focused on RHEB because we previously found that it is dispensable for normal hematopoiesis in mice. Transcriptome and metabolic analyses revealed that RHEB deficiency suppressed de novo nucleotide biosynthesis, leading to human T-ALL cell death. Importantly, RHEB deficiency suppressed tumor growth in both mouse and xenograft models. Our data provide a potential strategy for efficient therapy of T-ALL by RHEB-specific inhibition.

A Farnesyltransferase Inhibitor Restores Cognitive Deficits in Tsc2+/- Mice through Inhibition of Rheb1.

Tuberous sclerosis complex (TSC) is caused by mutations in Tsc1 or Tsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability, and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in cultured Tsc2+/- neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in cultured Tsc2+/- neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1 and MAP (mitogen-activated protein (MAP)) kinase signaling, and restored dendritic spine morphology in the hippocampi of male Tsc2+/- mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in male Tsc2+/- mice in vivo Heterozygous Rheb1 knockout in male Tsc2+/- mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments in Tsc2+/- mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID has been reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found that Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used the farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1 and recovered proper synapse formation and plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in TSC neurons. Furthermore, in vivo lonafarnib treatment restored contextual memory and Arc induction in TSC mice. Together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.

Homer2 and Homer3 Act as Novel Biomarkers in Diagnosis of hepatitis B virus-induced Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related mortality worldwide. Early detection of HCC can significantly improve patients' outcomes. An increasing number of studies have validated that Homer is dysregulated in cancers and may serve as diagnostic markers. In the present study, we investigated the expression profile and diagnostic significance of Homer2 and Homer3 in hepatitis B virus-induced HCC (HBV-HCC). Methods: Quantitative real-time PCR (QRT-PCR), western blot analysis and immunohistochemistry analysis. Results: Homer2 and Homer3 were downregulated in HCC. The expression of Homer2 was associated with tumor differentiation grade (P= 0.012) and total protein (TP) level (P= 0.032). Homer3 was related to tumor size (P= 0.010), tumor nodes (P= 0.026) and γ-glutamyl transferase (GGT) level (P= 0.001). The receiver operating characteristic curve analyses indicated that the combination of Homer2, Homer3 and AFP possessed a high accuracy (AUC=0.900) to diagnose HCC cases from healthy controls. Conclusion: Our data indicated that Homer2 and Homer3 were downregulated in HCC and might be potential diagnostic marker for HCC.

Persistently Elevated mTOR Complex 1-S6 Kinase 1 Disrupts DARPP-32-Dependent D1 Dopamine Receptor Signaling and Behaviors.

BACKGROUND: The serine-threonine kinase mTORC1 (mechanistic target of rapamycin complex 1) is essential for normal cell function but is aberrantly activated in the brain in both genetic-developmental and sporadic diseases and is associated with a spectrum of neuropsychiatric symptoms. The underlying molecular mechanisms of cognitive and neuropsychiatric symptoms remain controversial. METHODS: The present study examines behaviors in transgenic models that express Rheb, the most proximal known activator of mTORC1, and profiles striatal phosphoproteomics in a model with persistently elevated mTORC1 signaling. Biochemistry, immunohistochemistry, electrophysiology, and behavior approaches are used to examine the impact of persistently elevated mTORC1 on D1 dopamine receptor (D1R) signaling. The effect of persistently elevated mTORC1 was confirmed using D1-Cre to elevate mTORC1 activity in D1R neurons. RESULTS: We report that persistently elevated mTORC1 signaling blocks canonical D1R signaling that is dependent on DARPP-32 (dopamine- and cAMP-regulated neuronal phosphoprotein). The immediate downstream effector of mTORC1, ribosomal S6 kinase 1 (S6K1), phosphorylates and activates DARPP-32. Persistent elevation of mTORC1-S6K1 occludes dynamic D1R signaling downstream of DARPP-32 and blocks multiple D1R responses, including dynamic gene expression, D1R-dependent corticostriatal plasticity, and D1R behavioral responses including sociability. Candidate biomarkers of mTORC1-DARPP-32 occlusion are increased in the brain of human disease subjects in association with elevated mTORC1-S6K1, supporting a role for this mechanism in cognitive disease. CONCLUSIONS: The mTORC1-S6K1 intersection with D1R signaling provides a molecular framework to understand the effects of pathological mTORC1 activation on behavioral symptoms in neuropsychiatric disease.

LanCL1 promotes motor neuron survival and extends the lifespan of amyotrophic lateral sclerosis mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Improving neuronal survival in ALS remains a significant challenge. Previously, we identified Lanthionine synthetase C-like protein 1 (LanCL1) as a neuronal antioxidant defense gene, the genetic deletion of which causes apoptotic neurodegeneration in the brain. Here, we report in vivo data using the transgenic SOD1G93A mouse model of ALS indicating that CNS-specific expression of LanCL1 transgene extends lifespan, delays disease onset, decelerates symptomatic progression, and improves motor performance of SOD1G93A mice. Conversely, CNS-specific deletion of LanCL1 leads to neurodegenerative phenotypes, including motor neuron loss, neuroinflammation, and oxidative damage. Analysis reveals that LanCL1 is a positive regulator of AKT activity, and LanCL1 overexpression restores the impaired AKT activity in ALS model mice. These findings indicate that LanCL1 regulates neuronal survival through an alternative mechanism, and suggest a new therapeutic target in ALS.

Clinical and Diagnostic Significance of Homer1 in hepatitis B virus-induced Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a malignant tumor worldwide. Attributed to the lack of early diagnosis index, most patients are diagnosed in their late stage. Homer1, as a member of scaffold protein family, is made up of two different isoforms: Homer1a and Homer1b/c. More and more evidences show that Homer1 is dysregulated in cancers. Here, in this study, we investigated the expression profile, clinical, diagnostic and prognostic significance of Homer1 in hepatitis B virus-induced HCC (HBV-HCC). Methods: We first tested the expression of Homer1 in HCC cell lines by quantitative real-time PCR (qRT-PCR), western blot. Then, 86 pairs of tumorous and adjacent normal tissues from HCC together with a total number of 245 peripheral blood samples were enrolled to check the expression levels of Homer1 by quantitative real-time PCR (qRT-PCR). Results: The results revealed that the levels of Homer1 were both downregulated in HCC cell line and tissue and were associated with tumor size, but were not related to the prognosis of HBV-HCC. Receiver-operating characteristic curve analyses indicated that the sensitivity of Homer1 to differentiate HCC patients from the controls was high to 100.0% and the combination of Homer1 and AFP got a higher prediction value of HCC (AUC=0.890). Conclusion: Our data highlighted that Homer1 played a critical role in HCC tumorigenesis and might be a potential diagnostic marker for HCC.

Distinct roles of Rheb and Raptor in activating mTOR complex 1 for the self-renewal of hematopoietic stem cells.

The mammalian target of rapamycin (mTOR) complex 1 (mTORC1) senses a cell's energy status and environmental levels of nutrients and growth factors. In response, mTORC1 mediates signaling that controls protein translation and cellular metabolism. Although mTORC1 plays a critical role in hematopoiesis, it remains unclear which upstream stimuli regulate mTORC1 activity in the context of hematopoietic stem cells (HSC) maintenance in vivo. In this study, we investigated the function of Rheb, a critical regulator of mTORC1 activity controlled by the PI3K-AKT-TSC axis, both in HSC maintenance in mice at steady-state and in HSC-derived hematopoiesis post-transplantation. In contrast to the severe hematopoietic dysfunction caused by Raptor deletion, which completely inactivates mTORC1, Rheb deficiency in adult mice did not show remarkable hematopoietic failure. Lack of Rheb caused abnormalities in myeloid cells but did not have impact on hematopoietic regeneration in mice subjected to injury by irradiation. As previously reported, Rheb deficiency resulted in defective HSC-derived hematopoiesis post-transplantation. However, while Raptor is essential for HSC competitiveness in vivo, Rheb is dispensable for HSC maintenance under physiological conditions, indicating that the PI3K-AKT-TSC pathway does not contribute to mTORC1 activity for sustaining HSC self-renewal activity at steady-state. Thus, the various regulatory elements that impinge upstream of mTORC1 activation pathways are differentially required for HSC homeostasis in vivo.

NPTX2 and cognitive dysfunction in Alzheimer's Disease.

Memory loss in Alzheimer's disease (AD) is attributed to pervasive weakening and loss of synapses. Here, we present findings supporting a special role for excitatory synapses connecting pyramidal neurons of the hippocampus and cortex with fast-spiking parvalbumin (PV) interneurons that control network excitability and rhythmicity. Excitatory synapses on PV interneurons are dependent on the AMPA receptor subunit GluA4, which is regulated by presynaptic expression of the synaptogenic immediate early gene NPTX2 by pyramidal neurons. In a mouse model of AD amyloidosis, Nptx2-/- results in reduced GluA4 expression, disrupted rhythmicity, and increased pyramidal neuron excitability. Postmortem human AD cortex shows profound reductions of NPTX2 and coordinate reductions of GluA4. NPTX2 in human CSF is reduced in subjects with AD and shows robust correlations with cognitive performance and hippocampal volume. These findings implicate failure of adaptive control of pyramidal neuron-PV circuits as a pathophysiological mechanism contributing to cognitive failure in AD.

Homer1a drives homeostatic scaling-down of excitatory synapses during sleep.

Sleep is an essential process that supports learning and memory by acting on synapses through poorly understood molecular mechanisms. Using biochemistry, proteomics, and imaging in mice, we find that during sleep, synapses undergo widespread alterations in composition and signaling, including weakening of synapses through removal and dephosphorylation of synaptic AMPA-type glutamate receptors. These changes are driven by the immediate early gene Homer1a and signaling from group I metabotropic glutamate receptors mGluR1/5. Homer1a serves as a molecular integrator of arousal and sleep need via the wake- and sleep-promoting neuromodulators, noradrenaline and adenosine, respectively. Our data suggest that homeostatic scaling-down, a global form of synaptic plasticity, is active during sleep to remodel synapses and participates in the consolidation of contextual memory.

Disrupted Homer scaffolds mediate abnormal mGluR5 function in a mouse model of fragile X syndrome.

Enhanced metabotropic glutamate receptor subunit 5 (mGluR5) function is causally associated with the pathophysiology of fragile X syndrome, a leading inherited cause of intellectual disability and autism. Here we provide evidence that altered mGluR5-Homer scaffolds contribute to mGluR5 dysfunction and phenotypes in the fragile X syndrome mouse model, Fmr1 knockout (Fmr1(-/y)). In Fmr1(-/y) mice, mGluR5 was less associated with long Homer isoforms but more associated with the short Homer1a. Genetic deletion of Homer1a restored mGluR5-long Homer scaffolds and corrected several phenotypes in Fmr1(-/y) mice, including altered mGluR5 signaling, neocortical circuit dysfunction and behavior. Acute, peptide-mediated disruption of mGluR5-Homer scaffolds in wild-type mice mimicked many Fmr1(-/y) phenotypes. In contrast, Homer1a deletion did not rescue altered mGluR-dependent long-term synaptic depression or translational control of target mRNAs of fragile X mental retardation protein, the gene product of Fmr1. Our findings reveal new functions for mGluR5-Homer interactions in the brain and delineate distinct mechanisms of mGluR5 dysfunction in a mouse model of cognitive dysfunction and autism.

STIM1-dependent and STIM1-independent function of transient receptor potential canonical (TRPC) channels tunes their store-operated mode.

Ca(2+) influx by store-operated Ca(2+) channels is a key component of the receptor-evoked Ca(2+) signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca(2+) influx. Recent studies have revealed how STIM1 activates TRPC1 in response to store depletion; however, the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. Here, we established mutants of TRPC channels that could not be activated by STIM1 but were activated by the "charge-swap" mutant STIM1(K684E,K685E). Significantly, WT but not mutant TRPC channels were inhibited by scavenging STIM1 with Orai1(R91W), indicating the STIM1 dependence and independence of WT and mutant TRPC channels, respectively. Importantly, mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca(2+) influx.

Gene expression analysis identifies potential biomarkers of neurofibromatosis type 1 including adrenomedullin.

PURPOSE: Plexiform neurofibromas (pNF) are Schwann cell tumors found in a third of individuals with neurofibromatosis type 1 (NF1). pNF can undergo transformation to malignant peripheral nerve sheath tumors (MPNST). There are no identified serum biomarkers of pNF tumor burden or transformation to MPNST. Serum biomarkers would be useful to verify NF1 diagnosis, monitor tumor burden, and/or detect transformation. EXPERIMENTAL DESIGN: We used microarray gene expression analysis to define 92 genes that encode putative secreted proteins in neurofibroma Schwann cells, neurofibromas, and MPNST. We validated differential expression by quantitative reverse transcription-PCR, Western blotting, and ELISA assays in cell conditioned medium and control and NF1 patient sera. RESULTS: Of 13 candidate genes evaluated, only adrenomedullin (ADM) was confirmed as differentially expressed and elevated in serum of NF1 patients. ADM protein concentrati on was further elevated in serum of a small sampling of NF1 patients with MPNST. MPNST cell conditioned medium, containing ADM and hepatocyte growth factor, stimulated MPNST migration and endothelial cell proliferation. CONCLUSIONS: Thus, microarray analysis identifies potential serum biomarkers for disease, and ADM is a serum biomarker of NF1. ADM serum levels do not seem to correlate with the presence of pNFs but may be a biomarker of transformation to MPNST.

Widespread transcription at neuronal activity-regulated enhancers.

We used genome-wide sequencing methods to study stimulus-dependent enhancer function in mouse cortical neurons. We identified approximately 12,000 neuronal activity-regulated enhancers that are bound by the general transcriptional co-activator CBP in an activity-dependent manner. A function of CBP at enhancers may be to recruit RNA polymerase II (RNAPII), as we also observed activity-regulated RNAPII binding to thousands of enhancers. Notably, RNAPII at enhancers transcribes bi-directionally a novel class of enhancer RNAs (eRNAs) within enhancer domains defined by the presence of histone H3 monomethylated at lysine 4. The level of eRNA expression at neuronal enhancers positively correlates with the level of messenger RNA synthesis at nearby genes, suggesting that eRNA synthesis occurs specifically at enhancers that are actively engaged in promoting mRNA synthesis. These findings reveal that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.

Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels.

Ca(2+) influx by store-operated Ca(2+) influx channels (SOCs) mediates many cellular functions regulated by Ca(2+), and excessive SOC-mediated Ca(2+) influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca(2+)-dependent inactivation, which limits Ca(2+) influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca(2+)-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca(2+)-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca(2+)-dependent inactivation. Fast Ca(2+)-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca(2+)-dependent inactivation that has implications for Ca(2+) influx by SOC in physiological and pathological states.

TRPC channels as STIM1-regulated SOCs.

Store-operated Ca(2+) channels (SOCs) are Ca(2+) influx channels at the plasma membrane whose opening is determined by the level of Ca(2+) stored in the endoplasmic reticulum lumen. SOCs are activated in response to receptor-mediated or passive depletion of ER Ca(2+) to regulate many Ca(2+)-dependent cellular functions. Early work implicated the TRPC channels as SOCs. More recently, it was found that the Orai channels mediate the CRAC current and that the Ca(2+) binding protein STIM1 functions as the ER Ca(2+) sensor that mediates activation of the SOCs in response to depletion of ER Ca(2+). Key questions are whether both TRPC and Orai channels are opened by STIM1 and the molecular mechanism by which STIM1 opens the SOCs. Ample biochemical and functional evidence indicate interaction of the TRPC channels with STIM1. Furthermore, it was found that STIM1 gates TRPC channels by electrostatic interaction of STIM1(K684,K685) in the polybasic domain of STIM1 with two negative charges (aspartates or glutamates) that are conserved in all TRPC channels. Charge mutants of STIM1(K684,K685) and TRPC1(D639,D640) and TRPC3(D697,D698) were used to develop further direct evidence for the function of TRPC channels as SOCs. The evidence in favor of TRPC channels as SOCs are discussed.