- Email: [email protected]
P2-012
Poster P2:: Monday Posters beta-protein precursor (APP) and stabilizes its metabolism (J. Biol. Chem. [1999] 274, 2243). We reported the cellular functions ( J. Biol. Chem. [2000] 275, 13056; J. Biol. Chem. [2003] 278, 49448; J. Biol. Chem. [2004] 279, 21628; J. Biol. Chem. [2004] 279, 24343). To analyze the physiological function of X11L and X11 in vivo is important for understanding comprehensive neural activity and the mechanism of neurodegenerative disease. Previous analysis reported that X11 gene-deficient [X11-/-] mice showed no defects in excitatory synaptic transmission and only a subtle defect in dopamine and GABA transmission (Neurosci. Res. [2002] 43, 251, Proc. Natl. Acad. Sci. USA [2003] 100, 1409). Here we generate X11L gene-deficient [X11L-/-] mice and X11/X11L gene-deficient [X11-/-, X11L-/-] mice and analyze their phenotypes. Methods: Genotype analysis of offspring and survival rate of each genotype were examined. We monitored behavior of [X11-/-, X11L] mice and examined presence or absence of abnormal behavior. Brain morphology was explored by producing brain tissue-sections, which were analyzed by chemical- and immuno-staining. Results: We found that each of the genotype mice are viable and fertile. Both [X11-/-] and [X11L-/-] mice do not show remarkable behavioral and brain morphological abnormality, while [X11-/-, X11L-/-] mice showed obvious abnormality in behavior and neural activity. After this abnormal behavior, we observed alternation of c-Fos immunoreactivity in several regions. Conclusions: Our observations suggest that X11 and X11L play complementary in part to preserve neural function and that deficiency of both genes induces crisis of neural function, leading to neuropathology. P2-011
TRANSMISSIBILITY OF PRION DISEASES IN A LEMURIAN PRIMATE, MICROCEBUS MURINUS
Nadine Mestre-Frances1, Carole Crozet2, Sylvain Lehmann2, Sylvie Rouland1, Armand Perret-Liaudet3, Jean-Michel Verdier1, 1 INSERM U710, Montpellier, France; 2CNRS UPR 1142, Montpellier, France; 3Hospices Civils de Lyon, Lyon, France. Contact e-mail: [email protected] Background: The transmission of prion agent to animal models is an essential tool to better understand human prion diseases. Objective(s): Here, we report on the investigation of transmission of various strains to a non-human primate, Microcebus murinus: BSE, macaque-adaptated BSE (MBSE), CJD and vCJD (new variant of Creutzfeldt-Jakob). Methods: Ten microcebes were inoculated by intracerebral and intraperitoneal route (3 MBSE, 3 CJD, 4 vCJD), and by oral, intracerebral and intraperitoneal route for BSE (3 animals). Results: Following BSE inoculation, none of the microcebes presented the symptoms of prion disease, even after 5 years of incubation. By contrast, all microcebes that received BSE-macaca brain and vCJD brain developed a neurological disease after 16 and 20 months, respectively. For CJD, only one microcebe developed disease after 37 months. The duration of clinical stage was 2 months in all diseased animals. The symptoms begin by nervousness evolving rapidly to agressivity, visual troubles, imbalance, then incoordination of movements, myoclonic jerks, and later on, theybecame ataxic, turning round on themselves. The spongiform extent was proportional to the duration of illness. It ranged from small discrete vacuoles randomly dispersed throughout the neuropil to large confluent cystic spaces. Spongiform changes were most pronounced in the thalamus, the basal ganglia, the hypothalamus and the brainstem. The neocortex was relatively spared by spongiosis: small sparse vacuoles were seen in the frontal and occipital lobes whereas the hippocampus showed larger vacuoles. Spongiform changes were accompanied by important astrocytic gliosis. The prion immunostaining was more intense in thalamus, basal ganglia and brainstem. Focal deposits or plaques were seen in the cortex. In the cerebellum, some plaques were evidenced in the molecular layer. Finally, proteinase K resistant-prion protein was evidenced by Western-blot and immunocytochemistry. PrPres accumulation was observed in all microcebes with clinical signs. Although, BSE agent does not seem to be directly pathogenic to microcebe, we found resistantprion protein in spleen by Western blot in one microcebe after 70 months of incubation.These results suggest that BSE may require a longer incu-
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bation period to adapt to microcebes. Conclusions: In conclusion, the microcebe is a good model for studying the transmission of prion strains into primate. P2-012
HUMANIN IS UPREGULATED FOLLOWING IN VIVO BRAIN INJURY
Jill H. Fowler, Hsiang-Yun Tang, University of Edinburgh, Edinburgh, United Kingdom. Contact e-mail: [email protected] Background: Humanin (HN), a 24 amino acid polypeptide, was initially identified by functional screening of a cDNA library from an Alzheimer’s Disease (AD) brain. HN is a novel survival protein that protects against cell death induced by AD-related insults (APP, PS1 and PS2 mutations and by A toxicity), although recent studies suggest that HN might be a more promiscuous survival factor in the brain, protecting against various proapoptotic insults, serum deprivation and impaired metabolic activity in vitro. Brain injuries, including cerebral ischemia, have been identified as major risk factors for developing AD, however the response of Humanin to brain injury in vivo is unknown. Objective: To examine the temporal and spatial profile of the endogenous Humanin protein response to entorhinal cortex lesion and focal cerebral ischemia in the mouse. Methods: Adult C57 mice received a stereotaxic injection of 0.5l ibotenic acid (10mg/ml) into the entorhinal cortex and survived for 2 or 24 hours, 3, 7, 28 or 90 days (n⫽6 per group). For focal cerebral ischemia, a monofilament blocked the origin of the middle cerebral artery for 1 hour in adult C57 mice. Following reperfusion, animals survived for 1, 5, 23 or 71 hours (n⫽5-7 per group; Sham procedure n⫽2/3 per group). Neuronal damage was detected with H&E staining, and Humanin was detected using immunohistochemistry. Results: Humanin expression was rapidly up-regulated at 2 hours throughout the entorhinal cortex lesion, with minimal staining detected on the contralateral control hemisphere. Between 24 hours and 90 days, Humanin expression was sustained at the boundary of the excitotoxic lesion. Humanin was also up-regulated 2 hours following the onset of ischemia in the caudate nucleus and cortex. However, Humanin expression declined between 6 and 72 hours, particularly within the caudate nucleus, whereas the amount of neuronal damage increased. Minimal Humanin immunostaining was detected in sham operated mice. Conclusions: Similar to AD, Humanin is rapidly up-regulated in the injured rodent brain and may therefore serve a general survival role following brain injury. P2-013
LOCALIZED APP OVEREXPRESSION USING AN AAV VECTOR CAUSES AMYLOID DEPOSITION IN C57BL/6 MICE
Inga Kadish1, Ronald L. Klein2, Edwin M. Meyer3, Yan Gong4, Michael A. King3, James M. Wyss1, Thomas van Groen1, 1University of Alabama at Birmingham, Birmingham, AL, USA; 2Louisiana State University, Shreveport, LA, USA; 3University of Florida Coll. Med., Gainesville, FL, USA; 4University of Florida Coll. Pharmacy, Gainesville, FL, USA. Contact e-mail: [email protected] Background: Transgenic AD model mice overexpressing APPswe develop plaques (with a thioflavine S positive core) and diffuse deposits. We have shown that these amyloid  deposits have a specific time course of development with plaques appearing before diffuse deposits. Objective(s): We hypothesized that local overexpression of APP would cause APP overproduction leading to A pathology, i.e., the mice would show locally increased amyloid deposition. Methods: Young adult, C57BL/6 animals were injected in the entorhinal cortex with an adeno-associated virus (AAV) that expresses human APP (with the swedish mutation), and sacrificed 1 and 3 months later. The animals were transcardially perfused, the brains were cut and stained for APP, A and inflammatory markers. Results: In the animals with 1 month of survival following the injection there is a high number of cells expression APP at the injection site, and APP and a few amyloid deposits are present. Mice with 3 months survival demonstrated significantly increased numbers of amyloid deposits, further, some diffuse deposits were present. Conclusions: These results support the
TRANSMISSIBILITY OF PRION DISEASES IN A LEMURIAN PRIMATE, MICROCEBUS MURINUS
Nadine Mestre-Frances1, Carole Crozet2, Sylvain Lehmann2, Sylvie Rouland1, Armand Perret-Liaudet3, Jean-Michel Verdier1, 1 INSERM U710, Montpellier, France; 2CNRS UPR 1142, Montpellier, France; 3Hospices Civils de Lyon, Lyon, France. Contact e-mail: [email protected] Background: The transmission of prion agent to animal models is an essential tool to better understand human prion diseases. Objective(s): Here, we report on the investigation of transmission of various strains to a non-human primate, Microcebus murinus: BSE, macaque-adaptated BSE (MBSE), CJD and vCJD (new variant of Creutzfeldt-Jakob). Methods: Ten microcebes were inoculated by intracerebral and intraperitoneal route (3 MBSE, 3 CJD, 4 vCJD), and by oral, intracerebral and intraperitoneal route for BSE (3 animals). Results: Following BSE inoculation, none of the microcebes presented the symptoms of prion disease, even after 5 years of incubation. By contrast, all microcebes that received BSE-macaca brain and vCJD brain developed a neurological disease after 16 and 20 months, respectively. For CJD, only one microcebe developed disease after 37 months. The duration of clinical stage was 2 months in all diseased animals. The symptoms begin by nervousness evolving rapidly to agressivity, visual troubles, imbalance, then incoordination of movements, myoclonic jerks, and later on, theybecame ataxic, turning round on themselves. The spongiform extent was proportional to the duration of illness. It ranged from small discrete vacuoles randomly dispersed throughout the neuropil to large confluent cystic spaces. Spongiform changes were most pronounced in the thalamus, the basal ganglia, the hypothalamus and the brainstem. The neocortex was relatively spared by spongiosis: small sparse vacuoles were seen in the frontal and occipital lobes whereas the hippocampus showed larger vacuoles. Spongiform changes were accompanied by important astrocytic gliosis. The prion immunostaining was more intense in thalamus, basal ganglia and brainstem. Focal deposits or plaques were seen in the cortex. In the cerebellum, some plaques were evidenced in the molecular layer. Finally, proteinase K resistant-prion protein was evidenced by Western-blot and immunocytochemistry. PrPres accumulation was observed in all microcebes with clinical signs. Although, BSE agent does not seem to be directly pathogenic to microcebe, we found resistantprion protein in spleen by Western blot in one microcebe after 70 months of incubation.These results suggest that BSE may require a longer incu-
S235
bation period to adapt to microcebes. Conclusions: In conclusion, the microcebe is a good model for studying the transmission of prion strains into primate. P2-012
HUMANIN IS UPREGULATED FOLLOWING IN VIVO BRAIN INJURY
Jill H. Fowler, Hsiang-Yun Tang, University of Edinburgh, Edinburgh, United Kingdom. Contact e-mail: [email protected] Background: Humanin (HN), a 24 amino acid polypeptide, was initially identified by functional screening of a cDNA library from an Alzheimer’s Disease (AD) brain. HN is a novel survival protein that protects against cell death induced by AD-related insults (APP, PS1 and PS2 mutations and by A toxicity), although recent studies suggest that HN might be a more promiscuous survival factor in the brain, protecting against various proapoptotic insults, serum deprivation and impaired metabolic activity in vitro. Brain injuries, including cerebral ischemia, have been identified as major risk factors for developing AD, however the response of Humanin to brain injury in vivo is unknown. Objective: To examine the temporal and spatial profile of the endogenous Humanin protein response to entorhinal cortex lesion and focal cerebral ischemia in the mouse. Methods: Adult C57 mice received a stereotaxic injection of 0.5l ibotenic acid (10mg/ml) into the entorhinal cortex and survived for 2 or 24 hours, 3, 7, 28 or 90 days (n⫽6 per group). For focal cerebral ischemia, a monofilament blocked the origin of the middle cerebral artery for 1 hour in adult C57 mice. Following reperfusion, animals survived for 1, 5, 23 or 71 hours (n⫽5-7 per group; Sham procedure n⫽2/3 per group). Neuronal damage was detected with H&E staining, and Humanin was detected using immunohistochemistry. Results: Humanin expression was rapidly up-regulated at 2 hours throughout the entorhinal cortex lesion, with minimal staining detected on the contralateral control hemisphere. Between 24 hours and 90 days, Humanin expression was sustained at the boundary of the excitotoxic lesion. Humanin was also up-regulated 2 hours following the onset of ischemia in the caudate nucleus and cortex. However, Humanin expression declined between 6 and 72 hours, particularly within the caudate nucleus, whereas the amount of neuronal damage increased. Minimal Humanin immunostaining was detected in sham operated mice. Conclusions: Similar to AD, Humanin is rapidly up-regulated in the injured rodent brain and may therefore serve a general survival role following brain injury. P2-013
LOCALIZED APP OVEREXPRESSION USING AN AAV VECTOR CAUSES AMYLOID DEPOSITION IN C57BL/6 MICE
Inga Kadish1, Ronald L. Klein2, Edwin M. Meyer3, Yan Gong4, Michael A. King3, James M. Wyss1, Thomas van Groen1, 1University of Alabama at Birmingham, Birmingham, AL, USA; 2Louisiana State University, Shreveport, LA, USA; 3University of Florida Coll. Med., Gainesville, FL, USA; 4University of Florida Coll. Pharmacy, Gainesville, FL, USA. Contact e-mail: [email protected] Background: Transgenic AD model mice overexpressing APPswe develop plaques (with a thioflavine S positive core) and diffuse deposits. We have shown that these amyloid  deposits have a specific time course of development with plaques appearing before diffuse deposits. Objective(s): We hypothesized that local overexpression of APP would cause APP overproduction leading to A pathology, i.e., the mice would show locally increased amyloid deposition. Methods: Young adult, C57BL/6 animals were injected in the entorhinal cortex with an adeno-associated virus (AAV) that expresses human APP (with the swedish mutation), and sacrificed 1 and 3 months later. The animals were transcardially perfused, the brains were cut and stained for APP, A and inflammatory markers. Results: In the animals with 1 month of survival following the injection there is a high number of cells expression APP at the injection site, and APP and a few amyloid deposits are present. Mice with 3 months survival demonstrated significantly increased numbers of amyloid deposits, further, some diffuse deposits were present. Conclusions: These results support the