News Article | July 14, 2017
Scientists in the Vollum Institute at OHSU have identified an enzyme that plays a crucial role in the degeneration of axons, the threadlike portions of a nerve cell that transmit signals within the nervous system. Axon loss occurs in all neurodegenerative diseases, so this discovery could open new pathways to treating or preventing a wide array of brain diseases. The research team discovered a new role of the enzyme Axundead - or Axed - in promoting the self-destruction of axons. They found that when Axed function was blocked, injured axons not only maintained their integrity but remained capable of transmitting signals within the brain's complex circuitry for weeks. Their research was published July 5 in the journal Neuron. "If you target this pathway, you have a really good chance of preserving the functional aspects of neurons after a variety of types of trauma or injury," said senior author Marc Freeman, Ph.D., director of the Vollum Institute at OHSU. "It's a very attractive therapeutic target." Freeman conducted the work in the Department of Neurobiology at the University of Massachusetts Medical School. He has since been recruited to head the Vollum Institute, which conducts cutting-edge basic research into how the nervous system works at a molecular level. Severing axons, or axotomy, is a simple way to study the molecular basis of neurodegeneration as it leads to the activation of explosive axonal degeneration. In the laboratory, researchers using this technique can identify pro-degenerative genes with great specificity, especially when using sophisticated genetic approaches in the fruit fly Drosophila, Freeman's primary research model organism. Drosophila shares these same pathways with humans. Previous work by Freeman's lab identified another enzyme, a gene called SARM, which was the first shown to activate a process that causes axons to disintegrate when damaged. In the current study, Freeman and colleagues identified Axed, showed that it functions downstream of SARM to execute axonal degeneration, and, surprisingly, that the protection afforded by blocking Axed was even stronger than SARM. "There was really nothing we could do to kill axons where Axed function was blocked," Freeman said. From an evolutionary perspective, Freeman said SARM and Axed function are likely important in the peripheral nervous system after injury because programmed axon death allows for efficient packaging of damaged cellular materials for removal by immune cells. This process thereby clears the pathway for new neuronal processes to regrow, reinnervate tissues, and recover function. From a therapeutic perspective, the goal of the work is to understand at the molecular level how axons degenerate, and block those pathways in patients to preserve nervous system function. In many nervous system injuries axons are not severed but become stretched or crushed, which activates the SARM-dependent death program and drives axon loss. In those cases, it's imperative to block SARM and Axed signaling to preserve axon integrity, and in turn neuronal function. At the same time, Freeman and others have shown that SARM-dependent signaling pathways also drive axon loss in neurodegenerative conditions including glaucoma, traumatic brain injury and peripheral neuropathy. This suggests the notion of an ancient and conserved axon death signaling pathway that is widely activated to drive axon loss. Since axon loss is a universal feature of neurodegenerative diseases, it seems likely that blocking this pathway could have enormous therapeutic benefit. "If we can find ways to block it, maybe we can preserve function in a wide array of patients who have lost axons through neurodegenerative diseases or other neural trauma," Freeman said. Lead author Lukas J. Neukomm, Ph.D., was supported by the Charles A. King Trust Postdoctoral Fellowship, supported by the Harold Whitworth Pierce Charitable Trust. Freeman's work on the study was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health, grant R01 NS053538. During the period of the study, Freeman was an investigator with the Howard Hughes Medical Institute.
Zhang X.-D.,University of California at Davis |
Timofeyev V.,University of California at Davis |
Li N.,University of California at Davis |
Myers R.E.,University of California at Davis |
And 8 more authors.
Cardiovascular Research | Year: 2014
AimsSmall conductance Ca2+-activated K+ channels (KCa2 or SK channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+ () with membrane potentials. We have recently reported the functional expression of SK channels in human and mouse cardiac myocytes. Additionally, we have found that the channel is highly expressed in atria compared with the ventricular myocytes. We demonstrated that human cardiac myocytes expressed all three members of SK channels (SK1, 2, and 3); moreover, the different members are capable of forming heteromultimers. Here, we directly tested the contribution of SK3 to the overall repolarization of atrial action potentials.Methods and resultsWe took advantage of a mouse model with site-specific insertion of a tetracycline-based genetic switch in the 5′ untranslated region of the KCNN3 (SK3 channel) gene (SK3T/T). The gene-targeted animals overexpress the SK3 channel without interfering with the normal profile of SK3 expression. Whole-cell, patch-clamp techniques show a significant shortening of the action potential duration mainly at 90% repolarization (APD90) in atrial myocytes from the homozygous SK3T/T animals. Conversely, treatment with dietary doxycycline results in a significant prolongation of APD90 in atrial myocytes from SK3T/T animals. We further demonstrate that the shortening of APDs in SK3 overexpression mice predisposes the animals to inducible atrial arrhythmias.ConclusionSK3 channel contributes importantly towards atrial action potential repolarization. Our data suggest the important role of the SK3 isoform in atrial myocytes. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2013.
PubMed | Chonnam National University, Seoul National University, Baylor College of Medicine, Pape Family Pediatric Research Institute and 2 more.
Type: | Journal: Nature communications | Year: 2015
While microRNAs have emerged as an important component of gene regulatory networks, it remains unclear how microRNAs collaborate with transcription factors in the gene networks that determines neuronal cell fate. Here we show that in the developing spinal cord, the expression of miR-218 is directly upregulated by the Isl1-Lhx3 complex, which drives motor neuron fate. Inhibition of miR-218 suppresses the generation of motor neurons in both chick neural tube and mouse embryonic stem cells, suggesting that miR-218 plays a crucial role in motor neuron differentiation. Results from unbiased RISC-trap screens, in vivo reporter assays and overexpression studies indicated that miR-218 directly represses transcripts that promote developmental programs for interneurons. In addition, we found that miR-218 activity is required for Isl1-Lhx3 to effectively induce motor neurons and suppress interneuron fates. Together our results reveal an essential role of miR-218 as a downstream effector of the Isl1-Lhx3 complex in establishing motor neuron identity.
Ahuja S.,Genentech |
Ahuja S.,Vollum Institute |
Mukund S.,Genentech |
Deng L.,Genentech |
And 33 more authors.
Science | Year: 2015
Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies.Targeting the human Nav1.7 channel involved in pain perception,we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4),where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate themembrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.
Vaaga C.E.,Oregon Health And Science University |
Westbrook G.L.,Vollum Institute
Journal of Physiology | Year: 2016
Key points: The functional synaptic connectivity between olfactory receptor neurons and principal cells within the olfactory bulb is not well understood. One view suggests that mitral cells, the primary output neuron of the olfactory bulb, are solely activated by feedforward excitation. Using focal, single glomerular stimulation, we demonstrate that mitral cells receive direct, monosynaptic input from olfactory receptor neurons. Compared to external tufted cells, mitral cells have a prolonged afferent-evoked EPSC, which serves to amplify the synaptic input. The properties of presynaptic glutamate release from olfactory receptor neurons are similar between mitral and external tufted cells. Our data suggest that afferent input enters the olfactory bulb in a parallel fashion. Primary olfactory receptor neurons terminate in anatomically and functionally discrete cortical modules known as olfactory bulb glomeruli. The synaptic connectivity and postsynaptic responses of mitral and external tufted cells within the glomerulus may involve both direct and indirect components. For example, it has been suggested that sensory input to mitral cells is indirect through feedforward excitation from external tufted cells. We also observed feedforward excitation of mitral cells with weak stimulation of the olfactory nerve layer; however, focal stimulation of an axon bundle entering an individual glomerulus revealed that mitral cells receive monosynaptic afferent inputs. Although external tufted cells had a 4.1-fold larger peak EPSC amplitude, integration of the evoked currents showed that the synaptic charge was 5-fold larger in mitral cells, reflecting the prolonged response in mitral cells. Presynaptic afferents onto mitral and external tufted cells had similar quantal amplitude and release probability, suggesting that the larger peak EPSC in external tufted cells was the result of more synaptic contacts. The results of the present study indicate that the monosynaptic afferent input to mitral cells depends on the strength of odorant stimulation. The enhanced spiking that we observed in response to brief afferent input provides a mechanism for amplifying sensory information and contrasts with the transient response in external tufted cells. These parallel input paths may have discrete functions in processing olfactory sensory input. © 2016 The Physiological Society.
Lu J.,Vollum Institute |
Lu J.,Chinese National Institute for the Control of Pharmaceutical and Biological Products |
Maddison L.A.,Vanderbilt University |
Chen W.,Vanderbilt University
Transgenic Research | Year: 2011
Site-specific recombinases catalyze recombination between specific targeting sites to delete, insert, invert, or exchange DNA with high fidelity. In addition to the widely used Cre and Flp recombinases, the phiC31 integrase system from Streptomyces phage may also be used for these genetic manipulations in eukaryotic cells. Unlike Cre and Flp, phiC31 recognizes two heterotypic sites, attB and attP, for recombination. While the phiC31 system has been recently applied in mouse and human cell lines and in Drosophila, it has not been demonstrated whether it can also catalyze efficient DNA recombination in zebrafish. Here we show that phiC31 integrase efficiently induces site-specific deletion of episomal targets as well as chromosomal targets in zebrafish embryos. Thus, the phiC31 system can be used in zebrafish for genetic manipulations, expanding the repertoire of available tools for genetic manipulation in this vertebrate model. © 2010 Springer Science+Business Media B.V.
Parent A.-S.,University of Liège |
Naveau E.,University of Liège |
Gerard A.,University of Liège |
Bourguignon J.-P.,University of Liège |
Westbrook G.L.,Vollum Institute
Journal of Toxicology and Environmental Health - Part B: Critical Reviews | Year: 2011
Sex steroids and thyroid hormones play a key role in the development of the central nervous system. The critical role of these hormonal systems may explain the sensitivity of the hypothalamus, the cerebral cortex, and the hippocampus to endocrine-disrupting chemicals (EDC). This review examines the evidence for endocrine disruption of glial-neuronal functions in the hypothalamus, hippocampus, and cerebral cortex. Focus was placed on two well-studied EDC, the insecticide dichlorodiphenyltrichloroethane (DDT) and polychlorinated biphenyls (PCB). DDT is involved in neuroendocrine disruption of the reproductive axis, whereas polychlorinated biphenyls (PCB) interact with both the thyroid hormone-and sex steroid-dependent systems and disturb the neuroendocrine control of reproduction and development of hippocampus and cortex. These results highlight the impact of EDC on the developing nervous system and the need for more research in this area. Copyright © Taylor & Francis Group, LLC.
PubMed | Vollum Institute and University of Liège
Type: | Journal: The European journal of neuroscience | Year: 2016
Neurogenesis in the dentate gyrus is sensitive to endogenous and exogenous factors that influence hippocampal function. Ongoing neurogenesis and the integration of these new neurons throughout life thus may provide a sensitive indicator of environmental stress. We examined the effects of Aroclor 1254 (A1254), a mixture of polychlorinated biphenyls (PCBs), on the development and function of newly generated dentate granule cells. Early exposure to A1254 has been associated with learning impairment in children, suggesting potential impact on the development of hippocampus and/or cortical circuits. Oral A1254 (from the 6th day of gestation to postnatal day 21) produced the expected increase in PCB levels in brain at postnatal day 21, which persisted at lower levels into adulthood. A1254 did not affect the proliferation or survival of newborn neurons in immature animals nor did it cause overt changes in neuronal morphology. However, A1254 occluded the normal developmental increase in sEPSC frequency in the third post-mitotic week without altering the average sEPSC amplitude. Our results suggest that early exposure to PCBs can disrupt excitatory synaptic function during a period of active synaptogenesis, and thus could contribute to the cognitive effects noted in children exposed to PCBs.
Simmen T.,University of Alberta |
Lynes E.M.,University of Alberta |
Gesson K.,University of Alberta |
Thomas G.,Vollum Institute
Biochimica et Biophysica Acta - Biomembranes | Year: 2010
The production of secretory proteins at the ER (endoplasmic reticulum) depends on a ready supply of energy and metabolites as well as the close monitoring of the chemical conditions that favor oxidative protein folding. ER oxidoreductases and chaperones fold nascent proteins into their export-competent three-dimensional structure. Interference with these protein folding enzymes leads to the accumulation of unfolded proteins within the ER lumen, causing an acute organellar stress that triggers the UPR (unfolded protein response). The UPR increases the transcription of ER chaperones commensurate with the load of newly synthesized proteins and can protect the cell from ER stress. Persistant stress, however, can force the UPR to commit cells to undergo apoptotic cell death, which requires the emptying of ER calcium stores. Conversely, a continuous ebb and flow of calcium occurs between the ER and mitochondria during resting conditions on a domain of the ER that forms close contacts with mitochondria, the MAM (mitochondria-associated membrane). On the MAM, ER folding chaperones such as calnexin and calreticulin and oxidoreductases such as ERp44, ERp57 and Ero1α regulate calcium flux from the ER through reversible, calcium and redox-dependent interactions with IP3Rs (inositol 1,4,5-trisphophate receptors) and with SERCAs (sarcoplasmic/endoplasmic reticulum calcium ATPases). During apoptosis progression and depending on the identity of the ER chaperone and oxidoreductase, these interactions increase or decrease, suggesting that the extent of MAM targeting of ER chaperones and oxidoreductases could shift the readout of ER-mitochondria calcium exchange from housekeeping to apoptotic. However, little is known about the cytosolic factors that mediate the on/off interactions between ER chaperones and oxidoreductases with ER calcium channels and pumps. One candidate regulator is the multi-functional molecule PACS-2 (phosphofurin acidic cluster sorting protein-2). Recent studies suggest that PACS-2 mediates localization of a mobile pool of calnexin to the MAM in addition to regulating homeostatic ER calcium signaling as well as MAM integrity. Together, these findings suggest that cytosolic, membrane and lumenal proteins combine to form a two-way switch that determines the rate of protein secretion by providing ions and metabolites and that appears to participate in the pro-apoptotic ER-mitochondria calcium transfer. © 2010 Elsevier B.V.
PubMed | Vollum Institute
Type: Journal Article | Journal: eNeuro | Year: 2016
Despite representing only a small fraction of hippocampal granule cells, adult-generated newborn granule cells have been implicated in learning and memory (Aimone et al., 2011). Newborn granule cells undergo functional maturation and circuit integration over a period of weeks. However, it is difficult to assess the accompanying gene expression profiles in vivo with high spatial and temporal resolution using traditional methods. Here we used a novel method [thiouracil (TU) tagging] to map the profiles of nascent mRNAs in mouse immature newborn granule cells compared with mature granule cells. We targeted a nonmammalian uracil salvage enzyme, uracil phosphoribosyltransferase, to newborn neurons and mature granule cells using retroviral and lentiviral constructs, respectively. Subsequent injection of 4-TU tagged nascent RNAs for analysis by RNA sequencing. Several hundred genes were significantly enhanced in the retroviral dataset compared with the lentiviral dataset. We compared a selection of the enriched genes with steady-state levels of mRNAs using quantitative PCR. Ontology analysis revealed distinct patterns of nascent mRNA expression, with newly generated immature neurons showing enhanced expression for genes involved in synaptic function, and neural differentiation and development, as well as genes not previously associated with granule cell maturation. Surprisingly, the nascent mRNAs enriched in mature cells were related to energy homeostasis and metabolism, presumably indicative of the increased energy demands of synaptic transmission and their complex dendritic architecture. The high spatial and temporal resolution of our modified TU-tagging method provides a foundation for comparison with steady-state RNA analyses by traditional transcriptomic approaches in defining the functional roles of newborn neurons.