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
Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb–/– mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb–/– mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor γ gene (FcRγ) was additionally disrupted in Hexb–/– mice, as it plays a key role in immune complex–mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb–/–FcRγ–/– mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies.
Akira Yamaguchi, Kayoko Katsuyama, Kiyotaka Nagahama, Toshiyuki Takai, Ichiro Aoki, Shoji Yamanaka
In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients’ disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons.
Fatima Ferreirinha, Angelo Quattrini, Marinella Pirozzi, Valentina Valsecchi, Giorgia Dina, Vania Broccoli, Alberto Auricchio, Fiorella Piemonte, Giulia Tozzi, Laura Gaeta, Giorgio Casari, Andrea Ballabio, Elena I. Rugarli
The regulation of cerebrovascular permeability is critical for normal brain homeostasis, and the “breakdown” of the blood-brain barrier (BBB) is associated with the development of vasogenic edema and intracranial hypertension in a number of neurological disorders. In this study we demonstrate that an increase in endogenous tissue-type plasminogen activator (tPA) activity in the perivascular tissue following cerebral ischemia induces opening of the BBB via a mechanism that is independent of both plasminogen (Plg) and MMP-9. We also show that injection of tPA into the cerebrospinal fluid in the absence of ischemia results in a rapid dose-dependent increase in vascular permeability. This activity is not seen with urokinase-type Plg activator (uPA) but is induced in Plg–/– mice, confirming that the effect is Plg-independent. However, the activity is blocked by antibodies to the LDL receptor–related protein (LRP) and by the LRP antagonist, receptor-associated protein (RAP), suggesting a receptor-mediated process. Together these studies demonstrate that tPA is both necessary and sufficient to directly increase vascular permeability in the early stages of BBB opening, and suggest that this occurs through a receptor-mediated cell signaling event and not through generalized degradation of the vascular basement membrane.
Manuel Yepes, Maria Sandkvist, Elizabeth G. Moore, Thomas H. Bugge, Dudley K. Strickland, Daniel A. Lawrence
CNS-resident cells, in particular microglia and macrophages, are a source of inflammatory cytokines during inflammation within the CNS. Expression of IL-23, a recently discovered cytokine, has been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE) in mice. Expression of the p40 subunit of IL-12 and IL-23 by microglia has been shown in situ and in vitro, but direct evidence for a functional significance of p40 expression by CNS cells during an immune response in vivo is still lacking. Here we report that p40 plays a critical role in maintaining encephalitogenicity during the disease course. By using irradiation bone marrow chimeras, we have generated mice in which p40 is deleted from the CNS parenchyma but not the systemic immune compartment. Our studies show that p40 expressed by CNS-endogenous cells is critical for the development of myelin oligodendrocyte glycoprotein–induced EAE. In spite of the reduced clinical disease, the absence of p40 from the CNS has little impact on the degree of inflammation. Expression profiles of the CNS lesions show an increase in Th2 cytokines when compared with mice that develop EAE in the presence of CNS IL-12 and/or IL-23. Taken together, our data demonstrate that p40 expression by CNS-resident cells forms the basis for the Th1 bias of the CNS.
Burkhard Becher, Brigit G. Durell, Randolph J. Noelle
Parkinson disease (PD) is a neurodegenerative disorder characterized by a loss of the nigrostriatal dopaminergic neurons accompanied by a deficit in mitochondrial respiration. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that causes dopaminergic neurodegeneration and a mitochondrial deficit reminiscent of PD. Here we show that the infusion of the ketone body D-β-hydroxybutyrate (DβHB) in mice confers partial protection against dopaminergic neurodegeneration and motor deficits induced by MPTP. These effects appear to be mediated by a complex II–dependent mechanism that leads to improved mitochondrial respiration and ATP production. Because of the safety record of ketone bodies in the treatment of epilepsy and their ability to penetrate the blood-brain barrier, DβHB may be a novel neuroprotective therapy for PD.
Kim Tieu, Celine Perier, Casper Caspersen, Peter Teismann, Du-Chu Wu, Shi-Du Yan, Ali Naini, Miquel Vila, Vernice Jackson-Lewis, Ravichandran Ramasamy, Serge Przedborski
Subsets of parasympathetic and enteric neurons require neurturin signaling via glial cell line–derived neurotrophic factor family receptor α2 (GFRα2) for development and target innervation. Why GFRα2-deficient (Gfra2–/–) mice grow poorly has remained unclear. Here, we analyzed several factors that could contribute to the growth retardation. Neurturin mRNA was localized in the gut circular muscle. GFRα2 protein was expressed in most substance P–containing myenteric neurons, in most intrapancreatic neurons, and in surrounding glial cells. In the Gfra2–/– mice, density of substance P–containing myenteric ganglion cells and nerve bundles in the myenteric ganglion cell layer was significantly reduced, and transit of test material through small intestine was 25% slower compared to wild-type mice. Importantly, the knockout mice had approximately 80% fewer intrapancreatic neurons, severely impaired cholinergic innervation of the exocrine but not the endocrine pancreas, and increased fecal fat content. Vagally mediated stimulation of pancreatic secretion by 2-deoxy-glucose in vivo was virtually abolished. Retarded growth of the Gfra2–/– mice was accompanied by reduced fat mass and elevated basal metabolic rate. Moreover, the knockout mice drank more water than wild-type controls, and wet-mash feeding resulted in partial growth rescue. Taken together, the results suggest that the growth retardation in mice lacking GFRα2 is largely due to impaired salivary and pancreatic secretion and intestinal dysmotility.
Jari Rossi, Karl-Heinz Herzig, Vootele Võikar, Päivi H. Hiltunen, Mikael Segerstråle, Matti S. Airaksinen
The type I IFNs (IFN-α and IFN-β), which are crucial in antiviral defense and immune regulation, signal via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway with activation of STAT1 and STAT2. Here, the function of STAT2 was studied in transgenic mice (termed GIFN/STAT2–/–) with CNS production of IFN-α. Surprisingly, GIFN/STAT2–/–, but not GIFN/STAT1-null, transgenic mice, with CNS production of IFN-α, died prematurely with medulloblastoma. An immune response also induced in the brain of the GIFN/STAT2–/– mice was associated with IFN-γ gene expression by CD3+ T cells and the activation of the STAT1, STAT3, STAT4, and STAT5 molecules. Expression of the Sonic hedgehog (Shh) and the downstream transcriptional factor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, was found to be significantly increased and cotranscribed in cerebellar granule neurons of the GIFN/STAT2–/– mice. IFN-γ, but not IFN-α, induced STAT1-dependent expression of the Shh gene in cultured cerebellar granule neurons. Thus, there is an unexpected and extraordinarily adverse biological potency of IFN-α in the CNS when the primary signal transduction molecule STAT2 is absent. Moreover, a hitherto unknown role is indicated for the immune system in the pathogenesis of developmental disorders and tumorigenesis of the CNS via dysregulated Shh signaling mediated by IFN-γ.
Jianping Wang, Ngan Pham-Mitchell, Christian Schindler, Iain L. Campbell
Epidemiologic studies demonstrate that long-term use of NSAIDs is associated with a reduced risk for the development of Alzheimer disease (AD). In this study, 20 commonly used NSAIDs, dapsone, and enantiomers of flurbiprofen were analyzed for their ability to lower the level of the 42-amino-acid form of amyloid β protein (Aβ42) in a human H4 cell line. Thirteen of the NSAIDs and the enantiomers of flurbiprofen were then tested in acute dosing studies in amyloid β protein precursor (APP) transgenic mice, and plasma and brain levels of Aβ and the drug were evaluated. These studies show that (a) eight FDA-approved NSAIDs lower Aβ42 in vivo, (b) the ability of an NSAID to lower Aβ42 levels in cell culture is highly predicative of its in vivo activity, (c) in vivo Aβ42 lowering in mice occurs at drug levels achievable in humans, and (d) there is a significant correlation between Aβ42 lowering and levels of ibuprofen. Importantly, flurbiprofen and its enantiomers selectively lower Aβ42 levels in broken cell γ-secretase assays, indicating that these compounds directly target the γ-secretase complex that generates Aβ from APP. Of the compounds tested, meclofenamic acid, racemic flurbiprofen, and the purified R and S enantiomers of flurbiprofen lowered Aβ42 levels to the greatest extent. Because R-flurbiprofen reduces Aβ42 levels by targeting γ-secretase and has reduced side effects related to inhibition of cyclooxygenase (COX), it is an excellent candidate for clinical testing as an Aβ42 lowering agent.
Jason L. Eriksen, Sarah A. Sagi, Tawnya E. Smith, Sascha Weggen, Pritam Das, D.C. McLendon, Victor V. Ozols, Kevin W. Jessing, Kenton H. Zavitz, Edward H. Koo, Todd E. Golde
Vascular endothelial growth factor (VEGF) is an angiogenic protein with therapeutic potential in ischemic disorders, including stroke. VEGF confers neuroprotection and promotes neurogenesis and cerebral angiogenesis, but the manner in which these effects may interact in the ischemic brain is poorly understood. We produced focal cerebral ischemia by middle cerebral artery occlusion for 90 minutes in the adult rat brain and measured infarct size, neurological function, BrdU labeling of neuroproliferative zones, and vWF-immunoreactive vascular profiles, without and with intracerebroventricular administration of VEGF on days 1–3 of reperfusion. VEGF reduced infarct size, improved neurological performance, enhanced the delayed survival of newborn neurons in the dentate gyrus and subventricular zone, and stimulated angiogenesis in the striatal ischemic penumbra, but not the dentate gyrus. We conclude that in the ischemic brain VEGF exerts an acute neuroprotective effect, as well as longer latency effects on survival of new neurons and on angiogenesis, and that these effects appear to operate independently. VEGF may, therefore, improve histological and functional outcome from stroke through multiple mechanisms.
Yunjuan Sun, Kunlin Jin, Lin Xie, Jocelyn Childs, Xiao Ou Mao, Anna Logvinova, David A. Greenberg
The physiologic role of the μ opioid receptor (MOR) in gut nociception, motility, and secretion is well established. To evaluate whether MOR may also be involved in controlling gut inflammation, we first showed that subcutaneous administration of selective peripheral MOR agonists, named DALDA and DAMGO, significantly reduces inflammation in two experimental models of colitis induced by administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) or peripheral expansion of CD4+ T cells in mice. This therapeutic effect was almost completely abolished by concomitant administration of the opioid antagonist naloxone. Evidence of a genetic role for MOR in the control of gut inflammation was provided by showing that MOR-deficient mice were highly susceptible to colon inflammation, with a 50% mortality rate occurring 3 days after TNBS administration. The mechanistic basis of these observations suggests that the anti-inflammatory effects of MOR in the colon are mediated through the regulation of cytokine production and T cell proliferation, two important immunologic events required for the development of colon inflammation in mice and patients with inflammatory bowel disease (IBD). These data provide evidence that MOR plays a role in the control of gut inflammation and suggest that MOR agonists might be new therapeutic molecules in IBD.
David Philippe, Laurent Dubuquoy, Hervé Groux, Valérie Brun, Myriam Tran Van Chuoï-Mariot, Claire Gaveriaux-Ruff, Jean-Frédéric Colombel, Brigitte L. Kieffer, Pierre Desreumaux
EGF promotes proliferation and migration of stem/progenitor cells in the normal adult brain. The effect of epidermal growth factor on neurogenesis in ischemic brain is unknown, however. Here we show that intraventricular administration of EGF and albumin augments 100-fold neuronal replacement in the injured adult mouse striatum after cerebral ischemia. Newly born immature neurons migrate into the ischemic lesion and differentiate into mature parvalbumin-expressing neurons, replacing more than 20% of the interneurons lost by 13 weeks after ischemia and representing 2% of the total BrdU-labeled cells. These data suggest that administration of EGF and albumin could be used to manipulate endogenous neurogenesis in the injured brain and to promote brain self-repair.
Tetsuyuki Teramoto, Jianhua Qiu, Jean-Christophe Plumier, Michael A. Moskowitz
Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12–/–) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12–/– bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12–/– mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia.
Tomonori Kaifu, Jin Nakahara, Masanori Inui, Kenichi Mishima, Toshihiko Momiyama, Mitsuji Kaji, Akiko Sugahara, Hisami Koito, Azusa Ujike-Asai, Akira Nakamura, Kiyoshi Kanazawa, Kyoko Tan-Takeuchi, Katsunori Iwasaki, Wayne M. Yokoyama, Akira Kudo, Michihiro Fujiwara, Hiroaki Asou, Toshiyuki Takai
Manuela Neumann, Philipp J. Kahle, Benoit I. Giasson, Laurence Ozmen, Edilio Borroni, Will Spooren, Veronika Müller, Sabine Odoy, Hideo Fujiwara, Masato Hasegawa, Takeshi Iwatsubo, John Q. Trojanowski, Hans A. Kretzschmar, Christian Haass
Felix Kreier, Eric Fliers, Peter J. Voshol, Corbert G. Van Eden, Louis M. Havekes, Andries Kalsbeek, Caroline L. Van Heijningen, Arja A. Sluiter, Thomas C. Mettenleiter, Johannes A. Romijn, Hans P. Sauerwein, Ruud M. Buijs
Clyde W. Hodge, Jacob Raber, Thomas McMahon, Helen Walter, Ana Maria Sanchez-Perez, M. Foster Olive, Kristin Mehmert, A. Leslie Morrow, Robert O. Messing