Exposure to high levels of organophosphorus compounds (OP) can induce status epilepticus (SE) in humans and rodents via acute cholinergic toxicity, leading to neurodegeneration and brain inflammation. Currently there is no treatment to combat the neuropathologies associated with OP exposure. We recently demonstrated that inhibition of the EP2 receptor for PGE2 reduces neuronal injury in mice following pilocarpine-induced SE. Here, we investigated the therapeutic effects of an EP2 inhibitor (TG6-10-1) in a rat model of SE using diisopropyl fluorophosphate (DFP). We tested the hypothesis that EP2 receptor inhibition initiated well after the onset of DFP-induced SE reduces the associated neuropathologies. Adult male Sprague-Dawley rats were injected with pyridostigmine bromide (0.1 mg/kg, sc) and atropine methylbromide (20 mg/kg, sc) followed by DFP (9.5 mg/kg, ip) to induce SE. DFP administration resulted in prolonged upregulation of COX-2. The rats were administered TG6-10-1 or vehicle (ip) at various time points relative to DFP exposure. Treatment with TG6-10-1 or vehicle did not alter the observed behavioral seizures, however six doses of TG6-10-1 starting 80-150 min after the onset of DFP-induced SE significantly reduced neurodegeneration in the hippocampus, blunted the inflammatory cytokine burst, reduced microglial activation and decreased weight loss in the days after status epilepticus. By contrast, astrogliosis was unaffected by EP2 inhibition 4 d after DFP. Transient treatments with the EP2 antagonist 1 h before DFP, or beginning 4 h after DFP, were ineffective. Delayed mortality, which was low (10%) after DFP, was unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor within a time window that coincides with the induction of cyclooxygenase-2 by DFP is neuroprotective and accelerates functional recovery of rats.
DFP; PGE2; EP2; COX-2; organophosphorus; Hippocampus; Neurodegeneration; Inflammation; Status Epilepticus
As a prominent inflammatory effector of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) mediates brain inflammation and injury in many chronic central nervous system (CNS) conditions including seizures and epilepsy, largely through its receptor subtype EP2. However, EP2 receptor activation might also be neuroprotective in models of excitotoxicity and ischemia. These seemingly incongruent observations expose the delicacy of immune and inflammatory signaling in the brain, thus the therapeutic window for quelling neuroinflammation might vary with injury type and target molecule. Here, we identify a therapeutic window for EP2 antagonism to reduce delayed mortality and functional morbidity after status epilepticus (SE) in mice. Importantly, treatment must be delayed relative to SE onset to be effective, a finding that could be explained by the time-course of COX-2 induction after SE and compound pharmacokinetics. A large number of inflammatory mediators were upregulated in hippocampus after SE with COX-2 and IL-1β temporally leading many others. Thus, EP2 antagonism represents a novel anti-inflammatory strategy to treat SE with a tightly-regulated therapeutic window.
Epilepsy; Seizure; Status epilepticus; Neuroinflammation; Cyclooxygenase; Prostaglandin; Interleukin-1β; EP2; Cytokine; Chemokine; Gliosis
Status epilepticus (SE) is a life-threatening neurological emergency often refractory to available treatment options. It is a very heterogeneous condition in terms of clinical presentation and causes, which besides genetic, vascular and other structural causes also include CNS or severe systemic infections, sudden withdrawal from benzodiazepines or anticonvulsants and rare autoimmune etiologies. Treatment of SE is essentially based on expert opinions and antiepileptic drug treatment per se seems to have no major impact on prognosis. There is, therefore, urgent need of novel therapies that rely upon a better understanding of the basic mechanisms underlying this clinical condition. Accumulating evidence in animal models highlights that inflammation ensuing in the brain during SE may play a determinant role in ongoing seizures and their long-term detrimental consequences, independent of an infection or auto-immune cause; this evidence encourages reconsideration of the treatment flow in SE patients.
animal models; anti-inflammatory treatments; blood–brain barrier; comorbidities; COX-2; cytokines; neurodegeneration; outcome; prognosis; refractory status epilepticus; seizures
Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response.
EP1; EP2; GluK2; GluK4; GluK5; Kainate receptor; AMPA; Protein Kinase C; status epilepticus
A variety of medicinal chemistry approaches can be used for the identification of hits, generation of leads and to accelerate the development of drug candidates. The Emory Chemical and Biology Discovery Center (ECBDC) has been an active participant in the NIH’s high-throughput screening (HTS) endeavor to identify potent small molecule probes for poorly studied proteins. Several of Emory’s projects relate to cancer or virus infection. We have chosen three successful examples including discovery of potent measles virus RNA-dependent RNA polymerase inhibitors, development of Heat Shock Protein 90 (Hsp90) blockers and identification of angiogenesis inhibitors using transgenic Zebrafish as a HTS model. In parallel with HTS, a unique component of the Emory virtual screening (VS) effort, namely, substructure enrichment analysis (SEA) program has been utilized in several cases.
Epilepsy is a prevalent neurological disorder afflicting nearly 50 million people worldwide. The disorder is characterized clinically by recurrent spontaneous seizures attributed to abnormal synchrony of brain neurons. Despite advances in the treatment of epilepsy, nearly one-third of patients are resistant to current therapies, and the underlying mechanisms whereby a healthy brain becomes epileptic remain unresolved. Therefore, researchers have a major impetus to identify and exploit new drug targets. Here we distinguish between epileptic effectors, or proteins that set the seizure threshold, and epileptogenic mediators, which control the expression or functional state of the effector proteins. Under this framework, we then discuss attempts to regulate the mediators to control epilepsy. Further insights into the complex processes that render the brain susceptible to seizures and the identification of novel mediators of these processes will lead the way to the development of drugs to modify disease outcome and, potentially, to prevent epileptogenesis.
epileptogenesis; anticonvulsant; neuroinflammation; cytokines; neuroprotection; disease modification
Epilepsy is one of the more prevalent neurological disorders in the world, affecting approximately 50 million people of different ages and backgrounds. Epileptic seizures propagating through both lobes of the forebrain can have permanent debilitating effects on a patient's cognitive and somatosensory brain functions. Epilepsy, defined by the sporadic occurrence of recurrent seizures (SRS), is often accompanied by inflammation of the brain. Pronounced increases in the expression of key inflammatory mediators (e.g. IL-1β, TNFα, cyclooxygenase-2, CXCL10) after seizures may cause secondary damage in the brain and increase the likelihood of repetitive seizures. The cyclooxygenase-2 (COX-2) enzyme is induced rapidly during seizures. The increased level of COX-2 in specific areas of the epileptic brain can help to identify regions of seizure-induced brain inflammation. A good deal of effort has been expended to determine whether COX-2 inhibition might be neuroprotective and represent an adjunct therapeutic strategy along with antiepileptic drugs to treat epilepsy. However, the effectiveness of COX-2 inhibitors on epilepsy animal models appears to depend on the timing of administration. With all of the effort placed on making use of COX-2 inhibitors as therapeutic agents for the treatment of epilepsy, inflammation, and neurodegenerative diseases there has yet to be a selective and potent COX-2 inhibitor that has shown a clear therapeutic outcome with acceptable side effects.
Seizure; Neurodegeneration; Anti-convulsant; Prostaglandin; Blood-brain Barrier; cognitive deficit; EP2; EP1
The knowledge that excitatory synapses on aspiny hippocampal interneurons can develop genuine forms of activity-dependent remodeling, independently from the surrounding network of principal cells, is a relatively new concept. Cumulative evidence has now unequivocally demonstrated that, despite the absence of specialized postsynaptic spines that serve as compartmentalized structure for intracellular signaling in principal cell plasticity, excitatory inputs onto interneurons can undergo forms of synaptic plasticity that are induced and expressed autonomously from principal cells. Yet, the rules for induction and expression of interneuron plasticity are much more heterogeneous than in principal neurons. Long-term plasticity in interneurons is not necessarily dependent upon postsynaptic activation of NMDA receptors nor relies on the same postsynaptic membrane potential requirements as principal cells. Plasticity in interneurons rather requires activation of Ca2+-permeable AMPA receptors and/or metabotropic glutamate receptors and is triggered by postsynaptic hyperpolarization. In this review we will outline these distinct features of interneuron plasticity and identify potential critical candidate molecules that might be important for sustaining long-lasting changes in synaptic strength at excitatory inputs onto interneurons.
This article is part of a Special Issue entitled ‘Synaptic Plasticity & Interneurons’.
Interneurons; Synapses; GABA; Plasticity; Hippocampus
Kainate receptors contribute to synaptic plasticity and rhythmic oscillatory firing of neurons in corticolimbic circuits including hippocampal area CA3. We use zinc chelators and mice deficient in zinc transporters to show that synaptically released zinc inhibits postsynaptic kainate receptors at mossy fiber synapses and limits frequency facilitation of kainate, but not AMPA EPSCs during thetapattern stimulation. Exogenous zinc also inhibits the facilitatory modulation of mossy fiber axon excitability by kainate but does not suppress the depressive effect of kainate on CA3 axons. Recombinant kainate receptors are inhibited in a subunit-dependent manner by physiologically relevant concentrations of zinc, with receptors containing the KA1 subunit being sensitive to submicromolar concentrations of zinc. Zinc inhibition does not alter receptor desensitization nor apparent agonist affinity and is only weakly voltage dependent, which points to an allosteric mechanism. Zinc inhibition is reduced at acidic pH. Thus, in the presence of zinc, a fall in pH potentiates kainate receptors by relieving zinc inhibition. Acidification of the extracellular space, as occurs during repetitive activity, may therefore serve to unmask kainate receptor neurotransmission. We conclude that zinc modulation of kainate receptors serves an important role in shaping kainate neurotransmission in the CA3 region.
zinc; kainate receptor; CA3; glutamate receptor; mossy fiber; hippocampus; pH
Osteopontin is a cytokine found in many tissues and plays a role in tissue injury and repair. This study had two goals: to characterize osteopontin expression after status epilepticus (SE), and to test the hypotheses that osteopontin affects the susceptibility to seizures or alters cell death and inflammation after SE.
Pilocarpine was used to induce SE in OPN−/− and OPN+/+ mice to compare seizure susceptibility, neuropathological markers including real time PCR for inflammatory genes, and osteopontin immunohistochemistry. The effect of added osteopontin on excitotoxicity by N-methyl-d-aspartate in neuronal cultures of ONP−/− mice was determined.
Neurons undergoing degeneration showed osteopontin immunoreactivity 2–3 days after SE. After 10 to 31 days degenerating axons in the thalamus were osteopontin-positive. The susceptibility to seizures of OPN−/− and OPN+/+ mice in the pilocarpine, fluorothyl, and maximal electroshock models was similar. There were no significant differences in the extent of neuronal damage after pilocarpine-induced SE, the expression of several neuropathological markers or the RNA levels of selected inflammatory genes. Recombinant and natural bovine osteopontin did not affect the extent of NMDA-induced cell death in OPN−/− mouse neuronal cultures.
We demonstrated that osteopontin is up-regulated in response to SE in distinct temporal sequences in the hippocampus, specifically in degenerating neurons and axons. However, osteopontin did not appear to regulate neurodegeneration or inflammation within the first 3 days after SE.
Seizure; Pilocarpine; Inflammation; Axonal degeneration; Neuronal degeneration
Astrocytes are relatively resistant to injury compared to neurons and oligodendrocytes. Here, we report transient region-specific loss of astrocytes in mice early after pilocarpine-induced status epilepticus (SE). In the dentate hilus, immunoreactivity for glial acidic fibrillary protein (GFAP) was decreased, and the number of healthy appearing GFAP- or S100β-positive cells was significantly reduced (≥ 65%) 1 and 3 days after pilocarpine-induced SE. Many remaining GFAP-positive cells were shrunken, and 1 day after SE electron microscopy revealed numerous electron-dense degenerating astrocyte processes and degenerating glial somata in the hilus. Degeneration of GFAP-expressing cells may be linked to hilar neuronal death, because we did not observe loss of astrocytes after kainate-induced SE, after which hilar neurons remained intact. Ten days after SE, hilar GFAP immunoreactivity had returned, partially from GFAP-positive cells in the hilus. Unlike control mice, many GFAP-positive hilar processes originated from cell bodies located in the subgranular zone (SGZ). To investigate whether proliferation contributes to hilar repopulation, we injected 5-bromo-2′-deoxyuridine (BrdU) 3 days after SE. Five hours later and up to 31 days after SE, many BrdU/GFAP colabeled cells were found in the hilus and the SGZ, some with hilar processes, indicating that proliferation in both areas contributes to generation of hilar astrocytes and astrocyte processes. In contrast to pilocarpine-induced SE in mice, astrocyte degeneration was not found after pilocarpine-induced SE in rats. These findings demonstrate astrocyte degeneration in the mouse dentate hilus specifically in the mouse pilocarpine epilepsy model, followed by astrogenesis leading to hilar repopulation.
Stem cell; Seizure; Hilus; Kainate; Glial fibrillary acidic protein; Adult progenitor cell; Mouse strain
One compelling challenge in the therapy of epilepsy is to develop anti-epileptogenic drugs with an impact on the disease progression. The search for novel targets has focused recently on brain inflammation since this phenomenon appears to be an integral part of the diseased hyperexcitable brain tissue from which spontaneous and recurrent seizures originate. Although the contribution of specific proinflammatory pathways to the mechanism of ictogenesis in epileptic tissue has been demonstrated in experimental models, the role of these pathways in epileptogenesis is still under evaluation. We review the evidence conceptually supporting the involvement of brain inflammation and the associated blood-brain barrier damage in epileptogenesis, and describe the available pharmacological evidence where post-injury intervention with anti-inflammatory drugs has been attempted. Our review will focus on three main inflammatory pathways, namely the IL-1 receptor/Toll-like receptor signalling, COX-2 and the TGF-β signalling. The mechanisms underlying neuronal-glia network dysfunctions induced by brain inflammation are also discussed, highlighting novel neuromodulatory effects of classical inflammatory mediators such as cytokines and prostaglandins.
The increase in knowledge about a role of inflammation in disease progression, may prompt the use of specific anti-inflammatory drugs for developing disease-modifying treatments.
Seizures can give rise to enduring changes that reflect alterations in gene expression patterns, intra and inter cellular signaling and ultimately network alterations that are a hallmark of epilepsy. A growing body of literature suggests that long-term changes in gene transcription associated with epilepsy are mediated via modulation of chromatin structure. One transcription factor in particular, REST (repressor element 1-silencing transcription factor), has received a lot of attention due to the possibility that it may control fundamental transcription patterns that drive circuit excitability, seizures and epilepsy. REST represses a suite of genes in the nervous system by utilizing nuclear protein complexes that were originally identified as mediators of epigenetic inheritance. Epigenetics has traditionally referred to mechanisms that allow a heritable change in gene expression in the absence of DNA mutation. However a more contemporaneous definition acknowledges that many of the mechanisms used to perpetuate epigenetic traits in dividing cells are utilized by neurons to control activity dependent gene expression.
This review will survey what is currently understood about the role of epigenetic mechanisms in epilepsy. We discuss how REST controls gene expression to effect circuit excitability and neurogenesis in epilepsy. We also discuss how the repressor MeCP2 and activator CREB regulate neuronal activity and are themselves controlled by activity. Finally we highlight possible future directions in the field of epigenetics and epilepsy.
REST; RE1; NRSF; epilepsy; epigenetics; chromatin; histone; neurogenesis; MeCP2; CREB; seizure; excitability; G9a; CoREST; SIN3; HDAC
Pilocarpine induces prolonged status epilepticus (SE) in rodents that results in neurodegeneration and cognitive deficits, both commonly observed to be associated with human temporal lobe epilepsy. The multifunctional neuronal modulator, cyclooxygenase-2 (PTGS2 or COX-2), is rapidly induced after SE, mainly in principal neurons of the hippocampal formation and cortex. We used mice in which COX-2 is conditionally ablated in principal forebrain neurons to investigate the involvement of neuron-derived COX-2 in delayed mortality and performance in the Barnes maze.
Using the COX-2 conditional knockout mouse (nCOX-2 cKO) and their littermate wildtype controls, we compared motor behavior and performance in the Barnes maze before and three weeks after the induction of SE by pilocarpine. Mortality rate was also measured during SE and in the week following SE.
nCOX-2 cKO mice showed less delayed mortality than wildtype mice in the week after SE. Although motor behavior and most cognitive measures were not different in the nCOX-2 cKO, upon re-exposure to the maze three weeks after pilocarpine the latency to find the previously-learned target hole was significantly shorter in the nCOX-2 cKO than their wildtype littermate controls. By this measure pilocarpine-treated nCOX-2 cKO mice were identical to mice that had not experienced SE.
Results point to a role for neuronal COX-2 in delayed mortality in mice during the week following SE and suggest that neuronal COX-2 contributes to selected cognitive deficits observed after SE. Taking into consideration our previous findings that neurodegeneration and neuroinflammation after SE are reduced in the nCOX-2 cKO, and opening of the blood-brain barrier after pilocarpine is prevented, we conclude that neuronal COX-2 induction is an early step in many of the deleterious consequences of SE.
Seizure-induced neuronal COX-2; pilocarpine; retrograde memory; anterograde memory; Barnes maze
Functional MRI (fMRI) indirectly measures neural activity by detecting the signal change associated with the hemodynamic response following brain activation. In order to alleviate the temporal and spatial specificity problems associated with fMRI, a number of attempts have been made to detect neural magnetic fields (NMFs) with MRI directly, but have thus far provided conflicting results. In the present study, we used magnetic resonance to detect axonal NMFs in the median giant fiber of the earthworm, Lumbricus terrestris, by examining the free-induction decay (FID) with a sampling interval of 0.32 ms. The earthworm nerve cords were isolated from the vasculature and stimulated at the threshold of action potential generation. FIDs were acquired shortly after the stimulation and simultaneous field potential recordings identified the presence or absence of single evoked action potentials. FIDs acquired when the stimulus did not evoke an action potential were summed as background. The phase of the background-subtracted FID exhibited a systematic change, with a peak phase difference of [-1.2 ± 0.3] ×10-5 radians occurring at a time corresponding to the timing of the action potential. In addition, we calculated the possible changes in the FID magnitude and phase due to a simulated action potential using a volume conductor model. The measured phase difference matched the theoretical prediction well in both amplitude and temporal characteristics. This study provides the first evidence for the direct detection of a magnetic field from an evoked action potential using magnetic resonance.
Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed “Dual-Readout F2 assay” with F2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.
Coronaviruses are a family of enveloped single-stranded positive-sense RNA viruses causing respiratory, enteric, and neurologic diseases in mammals and fowl. Human coronaviruses are recognized to cause up to a third of common colds and are suspected to be involved in enteric and neurologic diseases. Coronavirus replication involves the generation of nested subgenomic mRNAs (sgmRNAs) with a common capped 5′ leader sequence. The translation of most of the sgmRNAs is thought to be cap dependent and displays a requirement for eukaryotic initiation factor 4F (eIF4F), a heterotrimeric complex needed for the recruitment of 40S ribosomes. We recently reported on an ultrahigh-throughput screen to discover compounds that inhibit eIF4F activity by blocking the interaction of two of its subunits (R. Cencic et al., Proc. Natl. Acad. Sci. U. S. A. 108:1046–1051, 2011). Herein we describe a molecule from this screen that prevents the interaction between eIF4E (the cap-binding protein) and eIF4G (a large scaffolding protein), inhibiting cap-dependent translation. This inhibitor significantly decreased human coronavirus 229E (HCoV-229E) replication, reducing the percentage of infected cells and intra- and extracellular infectious virus titers. Our results support the strategy of targeting the eIF4F complex to block coronavirus infection.
Interactions between cholinergic and glutamatergic neurotransmitter systems influence synaptic transmission and plasticity. While previous studies have examined cross-talk between acetylcholine (ACh) and NMDA or AMPA receptors, little is known about the effect of ACh on kainate receptors (KARs). We show that stimulation of m1 or m3 muscarinic ACh receptors (mAChRs) for 2 min potentiates recombinant KAR currents in a long lasting fashion. Muscarinic AChR activation potentiates heteromeric GluK2/GluK4 and GluK2/GluK5 receptors, but not homomeric GluK2 receptors. In hippocampal slices kainate potentiates mossy fiber axon excitability. Transient mAChR activation enhances this action of kainate, suggesting a novel mechanism through which acetylcholine could modulate synaptic transmission in the hippocampus. KAR over-activation has been implicated in excitotoxic cell death. To establish the functional significance of the interaction between mAChRs and KARs we examined the effect of mAChR activation on KAR-mediated excitotoxicity. We find that during pharmacological blockade of NMDA and AMPA receptors, KAR activation with AMPA produces significant cell death in primary cortical culture. Concanavalin A (Con A), which selectively blocks KAR desensitization, markedly increases this KAR-mediated neurotoxicity. Brief activation of mAChRs with pilocarpine significantly enhances KAR-mediated excitotoxicity both in the presence and absence of Con A. We conclude that KARs are modulated in a subunit dependent manner by mAChRs. We suggest that ACh may induce long lasting alterations in neuronal excitability and enhance excitotoxicity in part by potentiating KAR function.
Excitotoxicity; GluK2; GluK5; Hippocampus; Glutamate; Acetylcholine
Enantiomeric propanolamines have been identified as a new class of NR2B-selective NMDA receptor antagonists. The most effective agents are biaryl structures, synthesized in six steps with overall yields ranging from 11–64%. The compounds are potent and selective inhibitors of NR2B-containing recombinant NMDA receptors with IC50 values between 30–100 nM. Potency is strongly controlled by substitution on both rings and the centrally located amine nitrogen. SAR analysis suggests that well-balanced polarity and chain-length factors provide the greatest inhibitory potency. Structural comparisons based on 3D shape analysis and electrostatic complementarity support this conclusion. The antagonists are neuroprotective in both in vitro and in vivo models of ischemic cell death. In addition, some compounds exhibit anticonvulsant properties. Unlike earlier generation NMDA receptor antagonists and some NR2B-selective antagonists, the present series of propanolamines does not cause increased locomotion in rodents. Thus, the NR2B-selective antagonists exhibit a range of therapeutically interesting properties.
Dopamine (DA) neurons in the substantia nigra (SNDA neurons) are among the most severely affected in Parkinson’s disease (PD). Mitochondrial complex I inhibition by rotenone or MPTP can induce SNDA neurodegeneration and recapitulate motor disability in rodents. We performed a transcriptional analysis of the midbrain response to complex I inhibition focused on selected metabolic transcripts using quantitative real-time RT-PCR in conjunction with laser-capture microdissection (LCM) of immunofluorescently-targeted SNDA and ventral tegmental area (VTA) DA neurons. There were DA neuron-selective alterations in metabolic transcripts in response to generalized complex I inhibition dependent on the behavioral response of the animal, and vulnerable SNDA neurons were more dynamic in their metabolic transcriptional response than less vulnerable VTADA neurons. The metabolic transcriptional response of DA neurons may contribute significantly to the ultimate toxicity associated with mitochondrial inhibition, and better understanding of this response may provide insight into potential targets for neuroprotection in PD.
Parkinson’s disease; mitochondria; gene expression; rotenone; MPTP; dopamine; substantia nigra; ventral tegmental area
We have used laser-capture microdissection and microarray hybridization to characterize gene expression in the three principal neuron layers of rat hippocampus. Correlative and clustering analyses revealed all three layers to be easily differentiated from one another based on gene expression profile alone. A greater disparity in gene expression exists between dentate granule and pyramidal cell layers, reflecting phenotypic and ontological differences between those cell populations. Remarkably, the level of more than 45% of expressed transcripts was significantly different among the three neuron populations, with more than a third of those (>1,000 transcripts) being at least 2-fold different between layers. Even CA1 and CA3 pyramidal cell layers were dramatically different on a transcriptional level with a separate analysis indicating that nearly 20% of transcripts are differentially expressed between them. Only a small number of transcripts were specific for a given hippocampal cell layer, suggesting that functional differences are more likely secondary to wide-ranging expression differences of modest magnitude rather than very large disparities in a few genes. Categorical analysis of transcript abundance revealed concerted differences in gene expression among the three cell layers referable to specific cellular pathways. For instance, transcripts encoding proteins involved in glucose metabolism are most highly expressed in the CA3 pyramidal layer, which may reflect an underlying greater metabolic rate of these neurons and partially explain their exquisite vulnerability to seizure-induced damage. Conversely, transcripts related to MAP kinase signaling pathways and transcriptional regulator activity are prominent in the dentate granule cell layer, which could contribute to its resistance to damage following seizure activity by positioning these neurons to respond to external stimuli by altering transcription. Taken together, these data suggest that unique physiological characteristics of major cell layers, such as neuronal activity, neuronal plasticity, and vulnerability to neurodegeneration, are reflected in substantial transcriptional heterogeneity within the hippocampus.
laser capture microdissection; pyramidal neuron; dentate gyrus; granule cell; microarray
Therapeutic block of estrogen action is typically achieved with conventional antagonists (CAs), compounds that displace estradiol from the estrogen receptor (ER) and induce formation of an ER conformation that cannot bind to coactivator proteins, such as the steroid receptor coactivators (SRCs). As an alternative mode for blocking estrogen action, we are seeking small molecules that act as coactivator binding inhibitors (CBIs), i.e., they compete directly with SRC3 for interaction with estradiol-bound ER. CBIs would be interesting mechanistic probes of estrogen action and might also provide an alternative, more durable endocrine therapy for hormone-responsive breast cancer, where cellular adaptations lead to resistance to CAs. We have designed and optimized a set of time-resolved fluorescence resonance energy transfer (TR-FRET) assays to monitor the interaction of ER with SRC3 and ligands, and we have used them in high-throughput screens to discover small molecule CBIs that are able to disrupt this interaction. These assays also distinguish CBIs from CAs. These robust and sensitive “mix and measure” assays use low concentrations of ER labeled with a europium chelate as FRET donor and a Cy5-labeled SRC as acceptor. This multiplexed protocol produces excellent signal to noise ratios (> 100) and Z' values (> 0.8).
The GluR2 subunit determines many of the functional properties of the AMPA subtype of glutamate receptor. The roles of untranslated regions (UTRs) in mRNA stability, transport or translation are increasingly recognized. The 3′end of the GluR2 transcripts are alternatively processed to form a short and long 3′UTR, giving rise to two pools of GluR2 mRNA of 4 and 6 kb in length respectively in the mammalian brain. However, the role of these alternative 3′UTRs in GluR2 expression has not been reported. We demonstrate that in the cytoplasm of rat hippocampus, native GluR2 mRNAs bearing the long 3′UTR are mostly retained in translationally dormant complexes of ribosome-free messenger ribonucleoprotein (mRNP), whereas GluR2 transcripts bearing the short 3′UTR are predominantly associated with actively translating ribosomes. One day after pilocarpine-induced status epilepticus (SE), the levels of both long and short GluR2 transcripts were markedly decreased in rat hippocampus. However, after SE GluR2 mRNAs bearing long 3′UTRs were shifted from untranslating mRNP complexes to ribosome-containing complexes, pointing to a selective translational derepression of GluR2 mRNA mediated by the long 3′UTR. In Xenopus oocytes, expression of firefly luciferase reporters bearing alternative GluR2 3′UTRs confirmed that the long 3′UTR is sufficient to suppress translation. The stability of reporter mRNAs in oocytes was not significantly influenced by alternative 5′ or 3′UTRs of GluR2 over the time period examined. Overall our findings that the long 3′UTR of GluR2 mRNA alone is sufficient to suppress translation, and the evidence for seizure-induced derepression of translation of GluR2 via the long 3′UTR, strongly suggests that a regulatory signaling mechanism exists that differentially targets GluR2 transcripts with alternative 3′UTRs.
The molecular chaperone Hsp90 plays important roles in maintaining the malignant phenotypes. Recent studies suggest that Hsp90 exerts high affinity interactions with multiple oncoproteins, which are essential for the growth of tumor cells. As a result, research has been focused on finding Hsp90 probes as potential and selective anticancer agents. In a high-throughput screening exercise, we identified quinoline 7 as a moderate inhibitor of Hsp90. Further hit identification, SAR studies and biological investigation revealed several synthetic analogs in this series with micromolar activities in both fluorescent polarization (FP) assay and a cell-based western-blot (WB) assay. These compounds represent a new class of Hsp90 inhibitors with simple chemical structures.
Heat Shock Protein 90 (Hsp90); high-throughput screening (HTS); aminoquinoline
Many mechanisms of neurodegeneration have been implicated in Parkinson’s disease, but which ones are most important and potential interactions among them are unclear. To provide a broader perspective on the parkinsonian neurodegenerative process, we have performed a global analysis of gene expression changes caused by chronic, low-level exposure of neuroblastoma cells to the mitochondrial complex I inhibitor and parkinsonian neurotoxin rotenone. Undifferentiated SK-N-MC neuroblastoma cells were grown in the presence of rotenone (5 nM), and RNA was extracted at three different time points (baseline, 1 week, and 4 weeks) for labeling and hybridization to Affymetrix Human U133 Plus 2.0 GeneChips. Our results show that rotenone induces concerted alterations in gene expression that change over time. Particularly, alterations in transcripts related to DNA damage, energy metabolism, and protein metabolism are prominent during chronic complex I inhibition. These data suggest that early augmentation of capacity for energy production in response to mitochondrial inhibition might be deleterious to cellular function and survival. These experiments provide the first transcriptional analysis of a rotenone model of Parkinson’s disease and insight into which mechanisms of neurodegeneration may be targeted for therapeutic intervention.
Parkinson’s disease; rotenone; mitochondria; microarray; oxidative stress; neurodegeneration