Blockade of prostaglandin (PG) production by COX inhibitors is the treatment of choice for inflammatory pain but is also prone to severe side effects. Identification of signaling elements downstream of COX inhibition, particularly of PG receptor subtypes responsible for pain sensitization (hyperalgesia), provides a strategy for better-tolerated analgesics. Here, we have identified PGE2 receptors of the EP2 receptor subtype as key signaling elements in spinal inflammatory hyperalgesia. Mice deficient in EP2 receptors (EP2–/– mice) completely lack spinal PGE2-evoked hyperalgesia. After a peripheral inflammatory stimulus, EP2–/– mice exhibit only short-lasting peripheral hyperalgesia but lack a second sustained hyperalgesic phase of spinal origin. Electrophysiological recordings identify diminished synaptic inhibition of excitatory dorsal horn neurons as the dominant source of EP2 receptor–dependent hyperalgesia. Our results thus demonstrate that inflammatory hyperalgesia can be treated by targeting of a single PG receptor subtype and provide a rational basis for new analgesic strategies going beyond COX inhibition.
Heiko Reinold, Seifollah Ahmadi, Ulrike B. Depner, Beate Layh, Cornelia Heindl, May Hamza, Andreas Pahl, Kay Brune, Shuh Narumiya, Ulrike Müller, Hanns Ulrich Zeilhofer
Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that γ-aminobutyric acidA (GABAA) receptor α1–/– mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABAA receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABAA receptor α1–/– mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.
Jason E. Kralic, Hugh E. Criswell, Jessica L. Osterman, Todd K. O’Buckley, Mary E. Wilkie, Douglas B. Matthews, Kristin Hamre, George R. Breese, Gregg E. Homanics, A. Leslie Morrow
Mutations in genes encoding chromatin-remodeling proteins, such as the ATRX gene, underlie a number of genetic disorders including several X-linked mental retardation syndromes; however, the role of these proteins in normal CNS development is unknown. Here, we used a conditional gene-targeting approach to inactivate Atrx, specifically in the forebrain of mice. Loss of ATRX protein caused widespread hypocellularity in the neocortex and hippocampus and a pronounced reduction in forebrain size. Neuronal “birthdating” confirmed that fewer neurons reached the superficial cortical layers, despite normal progenitor cell proliferation. The loss of cortical mass resulted from a 12-fold increase in neuronal apoptosis during early stages of corticogenesis in the mutant animals. Moreover, cortical progenitors isolated from Atrx-null mice undergo enhanced apoptosis upon differentiation. Taken together, our results indicate that ATRX is a critical mediator of cell survival during early neuronal differentiation. Thus, increased neuronal loss may contribute to the severe mental retardation observed in human patients.
Nathalie G. Bérubé, Marie Mangelsdorf, Magdalena Jagla, Jackie Vanderluit, David Garrick, Richard J. Gibbons, Douglas R. Higgs, Ruth S. Slack, David J. Picketts
Neuritic plaques are a defining feature of Alzheimer disease (AD) pathology. These structures are composed of extracellular accumulations of amyloid-β peptide (Aβ) and other plaque-associated proteins, surrounded by large, swollen axons and dendrites (dystrophic neurites) and activated glia. Dystrophic neurites are thought to disrupt neuronal function, but whether this damage is static, dynamic, or reversible is unknown. To address this, we monitored neuritic plaques in the brains of living PDAPP;Thy-1:YFP transgenic mice, a model that develops AD-like pathology and also stably expresses yellow fluorescent protein (YFP) in a subset of neurons in the brain. Using multiphoton microscopy, we observed and monitored amyloid through cranial windows in PDAPP;Thy-1:YFP double-transgenic mice using the in vivo amyloid-imaging fluorophore methoxy-X04, and individual YFP-labeled dystrophic neurites by their inherent fluorescence. In vivo studies using this system suggest that amyloid-associated dystrophic neurites are relatively stable structures in PDAPP;Thy-1:YFP transgenic mice over several days. However, a significant reduction in the number and size of dystrophic neurites was seen 3 days after Aβ deposits were cleared by anti-Aβ antibody treatment. This analysis suggests that ongoing axonal and dendritic damage is secondary to Aβ and is, in part, rapidly reversible.
Robert P. Brendza, Brian J. Bacskai, John R. Cirrito, Kelly A. Simmons, Jesse M. Skoch, William E. Klunk, Chester A. Mathis, Kelly R. Bales, Steven M. Paul, Bradley T. Hyman, David M. Holtzman
Evidence suggests that Alzheimer disease (AD) begins as a disorder of synaptic function, caused in part by increased levels of amyloid β-peptide 1–42 (Aβ42). Both synaptic and cognitive deficits are reproduced in mice double transgenic for amyloid precursor protein (AA substitution K670N,M671L) and presenilin-1 (AA substitution M146V). Here we demonstrate that brief treatment with the phosphodiesterase 4 inhibitor rolipram ameliorates deficits in both long-term potentiation (LTP) and contextual learning in the double-transgenic mice. Most importantly, this beneficial effect can be extended beyond the duration of the administration. One course of long-term systemic treatment with rolipram improves LTP and basal synaptic transmission as well as working, reference, and associative memory deficits for at least 2 months after the end of the treatment. This protective effect is possibly due to stabilization of synaptic circuitry via alterations in gene expression by activation of the cAMP-dependent protein kinase (PKA)/cAMP regulatory element–binding protein (CREB) signaling pathway that make the synapses more resistant to the insult inflicted by Aβ. Thus, agents that enhance the cAMP/PKA/CREB pathway have potential for the treatment of AD and other diseases associated with elevated Aβ42 levels.
Bing Gong, Ottavio V. Vitolo, Fabrizio Trinchese, Shumin Liu, Michael Shelanski, Ottavio Arancio
The most common form of human congenital muscular dystrophy (CMD) is caused by mutations in the laminin-α2 gene. Loss of laminin-α2 function in this autosomal recessive type 1A form of CMD results in neuromuscular dysfunction and, often, early death. Laminin-α2–deficient skeletal muscles in both humans and mice show signs of muscle cell death by apoptosis. To examine the significance of apoptosis in CMD1A pathogenesis, we determined whether pathogenesis in laminin-α2–deficient (Lama2–/–) mice could be ameliorated by inhibiting apoptosis through either (a) inactivation of the proapoptosis protein Bax or (b) overexpression of the antiapoptosis protein Bcl-2 from a muscle-specific transgene. We found that both of these genetic interventions produced a several-fold increase in the lifespan of Lama2–/– mice. Bax inactivation also improved postnatal growth rate and myofiber histology and decreased fixed contractures of Lama2–/– mice. Thus, Bcl-2 family–mediated apoptosis contributes significantly to pathogenesis in the mouse model of CMD1A, and antiapoptosis therapy may be a possible route to amelioration of neuromuscular dysfunction due to laminin-α2 deficiency in humans.
Mahasweta Girgenrath, Janice A. Dominov, Christine A. Kostek, Jeffrey Boone Miller
Neurofibrillary tangles (NFTs) are composed of abnormal aggregates of the cytoskeletal protein tau. Together with amyloid β (Aβ) plaques and neuronal and synaptic loss, NFTs constitute the primary pathological hallmarks of Alzheimer disease (AD). Recent evidence also suggests that caspases are activated early in the progression of AD and may play a role in neuronal loss and NFT pathology. Here we demonstrate that tau is cleaved at D421 (ΔTau) by executioner caspases. Following caspase-cleavage, ΔTau facilitates nucleation-dependent filament formation and readily adopts a conformational change recognized by the early pathological tau marker MC1. ΔTau can be phosphorylated by glycogen synthase kinase-3β and subsequently recognized by the NFT antibody PHF-1. In transgenic mice and AD brains, ΔTau associates with both early and late markers of NFTs and is correlated with cognitive decline. Additionally, ΔTau colocalizes with Aβ1–42 and is induced by Aβ1–42 in vitro. Collectively, our data imply that Aβ accumulation triggers caspase activation, leading to caspase-cleavage of tau, and that this is an early event that may precede hyperphosphorylation in the evolution of AD tangle pathology. These results suggest that therapeutics aimed at inhibiting tau caspase-cleavage may prove beneficial not only in preventing NFT formation, but also in slowing cognitive decline.
Robert A. Rissman, Wayne W. Poon, Mathew Blurton-Jones, Salvatore Oddo, Reidun Torp, Michael P. Vitek, Frank M. LaFerla, Troy T. Rohn, Carl W. Cotman
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, we propose that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations.
Jen-Zen Chuang, Carrie Vega, Wenjin Jun, Ching-Hwa Sung
Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cδ (PKCδ) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCδ-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCδ-null mice. These results indicate an important role for neutrophil PKCδ in reperfusion injury and strongly suggest that PKCδ inhibitors could prove useful in the treatment of stroke.
Wen-Hai Chou, Doo-Sup Choi, Hong Zhang, Dezhi Mu, Tom McMahon, Viktor N. Kharazia, Clifford A. Lowell, Donna M. Ferriero, Robert O. Messing
Uracil-DNA glycosylase (UNG) is involved in base excision repair of aberrant uracil residues in nuclear and mitochondrial DNA. Ung knockout mice generated by gene targeting are viable, fertile, and phenotypically normal and have regular mutation rates. However, when exposed to a nitric oxide donor, Ung–/– fibroblasts show an increase in the uracil/cytosine ratio in the genome and augmented cell death. After combined oxygen-glucose deprivation, Ung–/– primary cortical neurons have increased vulnerability to cell death, which is associated with early mitochondrial dysfunction. In vivo, UNG expression and activity are low in brains of naive WT mice but increase significantly after reversible middle cerebral artery occlusion and reperfusion. Moreover, major increases in infarct size are observed in Ung–/– mice compared with littermate control mice. In conclusion, our results provide compelling evidence that UNG is of major importance for tissue repair after brain ischemia.
Matthias Endres, Detlev Biniszkiewicz, Robert W. Sobol, Christoph Harms, Michael Ahmadi, Andreas Lipski, Juri Katchanov, Philipp Mergenthaler, Ulrich Dirnagl, Samuel H. Wilson, Andreas Meisel, Rudolf Jaenisch
Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line–derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase–positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.
Mari Dezawa, Hiroshi Kanno, Mikio Hoshino, Hirotomi Cho, Naoya Matsumoto, Yutaka Itokazu, Nobuyoshi Tajima, Hitoshi Yamada, Hajime Sawada, Hiroto Ishikawa, Toshirou Mimura, Masaaki Kitada, Yoshihisa Suzuki, Chizuka Ide
Alzheimer disease (AD) is characterized by excessive deposition of amyloid β-peptides (Aβ peptides) in the brain. In the nonamyloidogenic pathway, the amyloid precursor protein (APP) is cleaved by the α-secretase within the Aβ peptide sequence. Proteinases of the ADAM family (adisintegrin and metalloproteinase) are the main candidates as physiologically relevant α-secretases, but early lethality of knockout animals prevented a detailed analysis in neuronal cells. To overcome this restriction, we have generated transgenic mice that overexpress either ADAM10 or a catalytically inactive ADAM10 mutant. In this report we show that a moderate neuronal overexpression of ADAM10 in mice transgenic for human APP[V717I] increased the secretion of the neurotrophic soluble α-secretase–released N-terminal APP domain (APPsα), reduced the formation of Aβ peptides, and prevented their deposition in plaques. Functionally, impaired long-term potentiation and cognitive deficits were alleviated. Expression of mutant catalytically inactive ADAM10 led to an enhancement of the number and size of amyloid plaques in the brains of double-transgenic mice. The results provide the first in vivo evidence for a proteinase of the ADAM family as an α-secretase of APP, reveal activation of ADAM10 as a promising therapeutic target, and support the hypothesis that a decrease in α-secretase activity contributes to the development of AD.
Rolf Postina, Anja Schroeder, Ilse Dewachter, Juergen Bohl, Ulrich Schmitt, Elzbieta Kojro, Claudia Prinzen, Kristina Endres, Christoph Hiemke, Manfred Blessing, Pascaline Flamez, Antoine Dequenne, Emile Godaux, Fred van Leuven, Falk Fahrenholz
Recently, pathological prion protein PrPSc, the putative key constituent of infectious agents causing transmissible spongiform encephalopathies (TSEs), was found in muscles of rodents experimentally infected with scrapie and in patients with Creutzfeldt-Jakob disease (CJD). For the assessment of risk scenarios originating from these findings (e.g., alimentary transmission of pathogens associated with bovine spongiform encephalopathy [BSE] and chronic wasting disease [CWD] via tainted beef and game or iatrogenic dissemination of CJD agent through contaminated surgical instruments) more detailed information about the time course of PrPSc accumulation in muscles at preclinical and clinical stages of incubation is needed. Here we show that PrPSc in muscles of hamsters fed with scrapie can be detected prior to the onset of clinical symptoms, but that the bulk of PrPSc was deposited late in clinical disease. Additionally, regarding the question of how muscles become invaded, we report on the intramuscular location of PrPSc and substantial indications for centrifugal spread of infection from spinal motor neurons to myofibers. Our findings in a well-established animal model for TSEs contribute to a better assessment of the risks for public health emanating from “Prions in skeletal muscle” and provide new insights into the pathophysiological spread of TSE agents through the body.
Achim Thomzig, Walter Schulz-Schaeffer, Christine Kratzel, Jessica Mai, Michael Beekes
Cortical spreading depression (CSD) is a propagating wave of neuronal and glial depolarization and has been implicated in disorders of neurovascular regulation such as stroke, head trauma, and migraine. In this study, we found that CSD alters blood-brain barrier (BBB) permeability by activating brain MMPs. Beginning at 3–6 hours, MMP-9 levels increased within cortex ipsilateral to the CSD, reaching a maximum at 24 hours and persisting for at least 48 hours. Gelatinolytic activity was detected earliest within the matrix of cortical blood vessels and later within neurons and pia arachnoid (≥3 hours), particularly within piriform cortex; this activity was suppressed by injection of the metalloprotease inhibitor GM6001 or in vitro by the addition of a zinc chelator (1,10-phenanthroline). At 3–24 hours, immunoreactive laminin, endothelial barrier antigen, and zona occludens-1 diminished in the ipsilateral cortex, suggesting that CSD altered proteins critical to the integrity of the BBB. At 3 hours after CSD, plasma protein leakage and brain edema developed contemporaneously. Albumin leakage was suppressed by the administration of GM6001. Protein leakage was not detected in MMP-9–null mice, implicating the MMP-9 isoform in barrier disruption. We conclude that intense neuronal and glial depolarization initiates a cascade that disrupts the BBB via an MMP-9–dependent mechanism.
Yasemin Gursoy-Ozdemir, Jianhua Qiu, Norihiro Matsuoka, Hayrunnisa Bolay, Daniela Bermpohl, Hongwei Jin, Xiaoying Wang, Gary A. Rosenberg, Eng H. Lo, Michael A. Moskowitz
Neural stem/progenitor cell (NSPC) migration toward sites of damaged central nervous system (CNS) tissue may represent an adaptive response for the purpose of limiting and/or repairing damage. Little is known of the mechanisms responsible for this migratory response. We constructed a cDNA library of injured mouse forebrain using subtractive suppression hybridization (SSH) to identify genes that were selectively upregulated in the injured hemisphere. We demonstrate that stem cell factor (SCF) mRNA and protein are highly induced in neurons within the zone of injured brain. Additionally, the SCF receptor c-kit is expressed on NSPCs in vitro and in vivo. Finally, we demonstrate that recombinant SCF induces potent NSPC migration in vitro and in vivo through the activation of c-kit on NSPCs. These data suggest that the SCF/c-kit pathway is involved in the migration of NSPCs to sites of brain injury and that SCF may prove useful for inducing progenitor cell recruitment to specific areas of the CNS for cell-based therapeutic strategies.
Lixin Sun, Jeongwu Lee, Howard A. Fine
Eleven studies now report significant associations between schizophrenia and certain haplotypes of single-nucleotide polymorphisms in the gene encoding dysbindin-1 at 6p22.3. Dysbindin-1 is best known as dystrobrevin-binding protein 1 (DTNBP1) and may thus be associated with the dystrophin glycoprotein complex found at certain postsynaptic sites in the brain. Contrary to expectations, however, we found that when compared to matched, nonpsychiatric controls, 73–93% of cases in two schizophrenia populations displayed presynaptic dysbindin-1 reductions averaging 18–42% (P = 0.027–0.0001) at hippocampal formation sites lacking neuronal dystrobrevin (i.e., β-dystrobrevin). The reductions, which were not observed in the anterior cingulate of the same schizophrenia cases, occurred specifically in terminal fields of intrinsic, glutamatergic afferents of the subiculum, the hippocampus proper, and especially the inner molecular layer of the dentate gyrus (DGiml). An inversely correlated increase in vesicular glutamate transporter-1 (VGluT-1) occurred in DGiml of the same schizophrenia cases. Those changes occurred without evidence of axon terminal loss or neuroleptic effects on dysbindin-1 or VGluT-1. Our findings indicate that presynaptic dysbindin-1 reductions independent of the dystrophin glycoprotein complex are frequent in schizophrenia and are related to glutamatergic alterations in intrinsic hippocampal formation connections. Such changes may contribute to the cognitive deficits common in schizophrenia.
Konrad Talbot, Wess L. Eidem, Caroline L. Tinsley, Matthew A. Benson, Edward W. Thompson, Rachel J. Smith, Chang-Gyu Hahn, Steven J. Siegel, John Q. Trojanowski, Raquel E. Gur, Derek J. Blake, Steven E. Arnold
Control of cancer, neuropathic, and postoperative pain is frequently inadequate or compromised by debilitating side effects. Inhibition or removal of certain nociceptive neurons, while retaining all other sensory modalities and motor function, would represent a new therapeutic approach to control severe pain. The enriched expression of transient receptor potential cation channel, subfamily V, member 1 (TRPV1; also known as the vanilloid receptor, VR1) in nociceptive neurons of the dorsal root and trigeminal ganglia allowed us to test this concept. Administration of the potent TRPV1 agonist resiniferatoxin (RTX) to neuronal perikarya induces calcium cytotoxicity by opening the TRPV1 ion channel and selectively ablates nociceptive neurons. This treatment blocks experimental inflammatory hyperalgesia and neurogenic inflammation in rats and naturally occurring cancer and debilitating arthritic pain in dogs. Sensations of touch, proprioception, and high-threshold mechanosensitive nociception, as well as locomotor function, remained intact in both species. In separate experiments directed at postoperative pain control, subcutaneous administration of RTX transiently disrupted nociceptive nerve endings, yielding reversible analgesia. In human dorsal root ganglion cultures, RTX induced a prolonged increase in intracellular calcium in vanilloid-sensitive neurons, while leaving other, adjacent neurons unaffected. The results suggest that nociceptive neuronal or nerve terminal deletion will be effective and broadly applicable as strategies for pain management.
Laszlo Karai, Dorothy C. Brown, Andrew J. Mannes, Stephen T. Connelly, Jacob Brown, Michael Gandal, Ofer M. Wellisch, John K. Neubert, Zoltan Olah, Michael J. Iadarola
Programmed cell death pathways have been implicated in the mechanism by which neurons die following brief and prolonged seizures, but the significance of proapoptotic Bcl-2 family proteins in the process remains poorly defined. Expression of the death agonist Bcl-2–interacting mediator of cell death (Bim) is under the control of the forkhead in rhabdomyosarcoma (FKHR) transcription factors. This prompted us to examine the response of this pathway to experimental seizures and in hippocampi from patients with intractable temporal lobe epilepsy. A short period of status epilepticus in rats that damaged the hippocampus activated FKHR/FKHRL-1 and induced a significant increase in expression of Bim. Blocking of FKHR/FKHRL-1 dephosphorylation after seizures improved hippocampal neuronal survival in vivo, and Bim antisense oligonucleotides were neuroprotective against seizures in vitro. Inhibition of Akt increased the FKHR/Bim response and DNA fragmentation within the normally resistant cortex. Analysis of hippocampi from patients with intractable epilepsy revealed that Bim levels were significantly lower than in controls and FKHR was inhibited; we were able to reproduce these results experimentally in rats by evoking multiple brief, noninjurious electroshock seizures. We conclude that Bim expression may be a critical determinant of whether seizures damage the brain, and that its control may be neuroprotective in status epilepticus and epilepsy.
Sachiko Shinoda, Clara K. Schindler, Robert Meller, Norman K. So, Tomohiro Araki, Akitaka Yamamoto, Jing-Quan Lan, Waro Taki, Roger P. Simon, David C. Henshall
Cerebellar ataxia, a devastating neurological disease, may be initiated by hyperexcitability of deep cerebellar nuclei (DCN) secondary to loss of inhibitory input from Purkinje neurons that frequently degenerate in this disease. This mechanism predicts that intrinsic DCN hyperexcitability would cause ataxia in the absence of upstream Purkinje degeneration. We report the generation of a transgenic (Tg) model that supports this mechanism of disease initiation. Small-conductance calcium-activated potassium (SK) channels, regulators of firing frequency, were silenced in the CNS of Tg mice with the dominant-inhibitory construct SK3-1B-GFP. Transgene expression was restricted to the DCN within the cerebellum and was detectable beginning on postnatal day 10, concomitant with the onset of cerebellar ataxia. Neurodegeneration was not evident up to the sixth month of age. Recordings from Tg DCN neurons revealed loss of the apamin-sensitive after-hyperpolarization current (IAHP) and increased spontaneous firing through SK channel suppression, indicative of DCN hyperexcitability. Spike duration and other electrogenic conductance were unaffected. Thus, a purely electrical alteration is sufficient to cause cerebellar ataxia, and SK openers such as the neuroprotective agent riluzole may reduce neuronal hyperexcitability and have therapeutic value. This dominant-inhibitory strategy may help define the in vivo role of SK channels in other neuronal pathways.
Vikram G. Shakkottai, Chin-hua Chou, Salvatore Oddo, Claudia A. Sailer, Hans-Günther Knaus, George A. Gutman, Michael E. Barish, Frank M. LaFerla, K. George Chandy
We produced and analyzed mice deficient for Na/Ca exchanger 3 (NCX3), a protein that mediates cellular Ca2+ efflux (forward mode) or Ca2+ influx (reverse mode) and thus controls intracellular Ca2+ concentration. NCX3-deficient mice (Ncx3–/–) present a skeletal muscle fiber necrosis and a defective neuromuscular transmission, reflecting the absence of NCX3 in the sarcolemma of the muscle fibers and at the neuromuscular junction. The defective neuromuscular transmission is characterized by the presence of electromyographic abnormalities, including low compound muscle action potential amplitude, a decremental response at low-frequency nerve stimulation, an incremental response, and a prominent postexercise facilitation at high-frequency nerve stimulation, as well as neuromuscular blocks. The analysis of quantal transmitter release in Ncx3–/– neuromuscular junctions revealed an important facilitation superimposed on the depression of synaptic responses and an elevated delayed release during high-frequency nerve stimulation. It is suggested that Ca2+ entering nerve terminals is cleared relatively slowly in the absence of NCX3, thereby enhancing residual Ca2+ and evoked and delayed quantal transmitter release during repetitive nerve stimulation. Our findings indicate that NCX3 plays an important role in vivo in the control of Ca2+ concentrations in the skeletal muscle fibers and at the neuromuscular junction.
Sophie Sokolow, Mario Manto, Philippe Gailly, Jordi Molgó, Clarisse Vandebrouck, Jean-Marie Vanderwinden, Andre Herchuelz, Stéphane Schurmans