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LAMP-2 deficient mice brain as a model for lysosomal storage disease
S128
Abstracts
P1-n13 Establishment of animal model of olfactory hypersensitivity to volatile organic compounds Yoshika Kurokawa 1 , Rieko Hojo 2 , Hidekazu Fujimaki 1
P1-n16 Accumulation of GAPDH enhances delayed neuronal cell death in LAMP-2 deficient mice Yohei Fujimoto 1,2 , Akiko Furuta 2 , Keiji Wada 1,2
1
1 Dept. of Elect. Engineer and Biosci., Grad. Sch. of Adv. Sci. and Engineer, Waseda University, Tokyo, Japan; 2 Dept. of Degen. Neurol. Dis., Natl. Inst. Neurosci., NCNP, Tokyo, Japan
National Institute for Environmental Studies, Japan; Occupational Safety and Health, Japan
2
National Institute of
The aim of our study is to establish an animal model of olfactory hypersensitivity to volatile organic compounds (VOCs) and to apply the model for pathological understanding of VOC-related human disorders such as sick house syndrome. We developed a system for evaluating olfactory detection of VOCs in mice, which is composed of a standard operant chamber, an odor-sniffing glassy port, solenoid valves, diaphragm pumps, two polyethylene gas bags, and a personal computer. Male mice were trained for an operant learning task of pressing the levers coresponding to sniffed gas, and then they performed a session of tasks to discriminate between VOC gas with various concentrations and pure nitrogen gas. We found that the detection thresholds of the two VOC gas (toluene and dichloromethane (DCM)) in mice were below the limit of finding with our system but that the accuracy for detecting DCM with fixed concentration (100 ppb) may be a useful index in mice for monitoring olfactory sensitivity within or after the period with repeated exposure to VOC gas.
LAMP-2A, a subtype of lysosome-associated membrane protein-2 (LAMP-2), functions in chaperone-mediated autophagy (CMA) that is a selective pathway of protein degradation in lysosome. We have examined the expression and regulation of GAPDH, one of the target proteins of CMA, and evaluated the cell death in the cerebral cold injury. Robust GAPDH immunoreactivity was seen in the cytoplasm of astrocytes around the lesion of both LAMP-2 deficient and wild type mice on days 3 and 7 after cold injury. However, on day 14, GAPDH was detected in the nuclei of large neurons in LAMP-2 deficient mice. Besides, TUNEL-positive cells in the LAMP-2 deficient mice were abundant on the day 14. Since the translocation of GAPDH to the nucleus are known to cause cell death, our result suggests GAPDHmediated delayed neuronal cell death is enhanced in the brain lesion of LAMP-2 deficient mice. Thus, CMA via LAMP-2 may protect neurons under the pathological conditions through degradation of excessive GAPDH.
doi:10.1016/j.neures.2009.09.615
doi:10.1016/j.neures.2009.09.618
P1-n14 Anomalous properties of cortical inhibitory neural mechanism in behavioral dysfunction of the Bronx waltzer mouse Yoshiki Matsuda 1 , Hitomi Izumi 1 , Yuki Inoue 1 , Masumi Inagaki 1 , Makiko Kaga 1 , Yu-ichi Goto 2
P1-n17 Depolarizing high-[K+ ] load model of rat brain energy metabolism: ␣ 31 P-NMR study Osamu Tokumaru 1 , Chihiro Kuroki 1 , Kazue Ogata 1 , Takaaki Kitano 2 , Isao Yokoi 1
1
1
Natl. Inst. Ment. Health, NCNP, Tokyo, Japan; Tokyo, Japan
2
Natl. Inst. Neurosci., NCNP,
2
Department Neurophysiol, Oita University Faculty of Medicine, Oita, Japan; Med. Edu. Ctr., Oita University Faculty of Medicine, Oita, Japan
The homozygous Bronx waltzer (bv) mouse, which is known as a model of congenital hearing and vestibular dysfunction, exhibits obvious anxiety-like behavior. The present study investigated whether the anxiety-like behavior of bv mouse is caused by abnormalities in cortical GABAergic interneurons. Double immunofluorescent staining revealed that the number of parvalbumin-expressing GAD67 positive cells were significantly decreased in cingulate, somatosensory and visual cortices compared to the control group (p < 0.01 or 0.05). In contrast, no difference was observed in hippocampal CA1, CA3 area and dentate gyri. Electrocorticography from parietal region of bv mouse showed bilaterally synchronous interictal discharges. The latency to incidence of first tonic extension induced by the intraperitoneal administration of pentylenetetrazol was remarkably decreased in bv mice. These results suggest that the cortical inhibitory neuronal dysfunctions contribute to the behavioral abnormality including anxiety-like behavior in bv mouse.
We study energy metabolism of rat brain slices superfused with artificial cerebrospinal fluid (ACSF) by measuring high energy phosphates using 31 P-NMR. When [K+ ] in ACSF is increased from 5 mM to 60 mM, NMR peaks corresponding to phosphocreatine (PCr), a buffering compound for ATP in Lohmann’s reaction, are reduced, indicating increased energy expenditure of brain slices.The present study investigated the effects of [K+ ] on energy demand using 31 P-NMR. Logarithm of [K+ ] and decrease in PCr were linearly correlated (p < 0.0001). Such decrease in PCr was reduced by 44% with tetrodotoxin (TTX). If action potentials (AP) play a major role in increase in energy expenditure, it should be all-or-none in character. Thus the observed linearity between decrease in PCr and log[K+ ] did not suggest a significant contribution of AP to the increased energy demand due to high [K+ ]. On the contrary, partial blockage of such decrease in PCr by TTX might indicate that 44% of the increased energy expenditure attribute to AP.
doi:10.1016/j.neures.2009.09.616
doi:10.1016/j.neures.2009.09.619
P1-n15 LAMP-2 deficient mice brain as a model for lysosomal storage disease Akiko Furuta, Hisae Kikuchi, Keiji Wada
P1-n18 Novel ataxia mouse with heavy iron deposition in the liver and kidneys, but not in the heart or central nervous system Moriaki Kusakabe 1,2,3 , Toshiaki Tachibana 6 , Miki Tanzawa 4 , Yukari Wakana 4 , Tomonori Kawabe 4 , Jorge Zavaleta-Ahane 4 , Takahiro Fukuda 5 , Hisashi Hashimoto 6
Department Degenerat. Neurol. Dis., Natl. Inst. Neurosci., NCNP, Tokyo, Japan Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein localized to lysosomal membrane. Mutations in LAMP-2 gene cause Danon disease that is characterized by cardiomyopathy, myopathy and variable mental retardation; however, the mechanism of mental disorder in Danon disease is not elucidated. We have investigated possible neuropathological changes of LAMP-2 deficient mouse. Increased immunoreactivity of cathepsin D in large neurons and GFAP-positive astrocytosis were seen in the hippocampus and the cerebral cortex from LAMP-2 deficient mice. Among several lectin staining sections, concanavalin A positive aggregation was found in the neurons from LAMP-2 deficient mice. Whereas previous studies on electron microscopy reported the accumulation of autophagic vacuoles in the visceral organs from LAMP-2 deficient mice, here we showed that various lysosomal changes in the neurons, astrocytes and pericytes, which were similar to changes in lysosomal storage disease. These results indicate that LAMP-2 is crucial for lysosomal protein degradation and regulated with cell-specific manner in the brain. doi:10.1016/j.neures.2009.09.617
1 Res. Ctr. for Food Safety, Grad Sch. Agri. Life Sci., University of Tokyo, Tokyo, Japan; 2 Inst. Anim. Rep., Japan; 3 Matrix Cell Res. Inst., Inc., Japan; 4 Tokyo Col. Medico-Pharmaco Tec., Japan; 5 Department Neuropath, Jikei Univ Sch Med, Japan; 6 Department Anat., Jikei. University Sch. Med., Japan
Many neurodegenerative diseases, including Friedreich ataxia, are associated with dysregulation of iron metabolism. We have investigated the iron deposition in various organs of our novel mutant mouse which is characterized by heavy ataxia with severe gait disorder of the hind legs. Heavy Fe3+ deposition was found in the liver and kidneys, but not in the heart, the skeletal muscle or the central nervous system. Fe2+ was not detected in any organs. In the kidneys, iron was deposited in each cell of selected proximal tubules but no deposition was found in other proximal tubules. Ultrastructurally, electron-dense depositions were found in lysosomes in the apical side of the cell and not in mitochondria. These data suggest that this mutant mouse is a very useful model to reveal a relationship between neurodegeneration and iron metabolism. doi:10.1016/j.neures.2009.09.620
Abstracts
P1-n13 Establishment of animal model of olfactory hypersensitivity to volatile organic compounds Yoshika Kurokawa 1 , Rieko Hojo 2 , Hidekazu Fujimaki 1
P1-n16 Accumulation of GAPDH enhances delayed neuronal cell death in LAMP-2 deficient mice Yohei Fujimoto 1,2 , Akiko Furuta 2 , Keiji Wada 1,2
1
1 Dept. of Elect. Engineer and Biosci., Grad. Sch. of Adv. Sci. and Engineer, Waseda University, Tokyo, Japan; 2 Dept. of Degen. Neurol. Dis., Natl. Inst. Neurosci., NCNP, Tokyo, Japan
National Institute for Environmental Studies, Japan; Occupational Safety and Health, Japan
2
National Institute of
The aim of our study is to establish an animal model of olfactory hypersensitivity to volatile organic compounds (VOCs) and to apply the model for pathological understanding of VOC-related human disorders such as sick house syndrome. We developed a system for evaluating olfactory detection of VOCs in mice, which is composed of a standard operant chamber, an odor-sniffing glassy port, solenoid valves, diaphragm pumps, two polyethylene gas bags, and a personal computer. Male mice were trained for an operant learning task of pressing the levers coresponding to sniffed gas, and then they performed a session of tasks to discriminate between VOC gas with various concentrations and pure nitrogen gas. We found that the detection thresholds of the two VOC gas (toluene and dichloromethane (DCM)) in mice were below the limit of finding with our system but that the accuracy for detecting DCM with fixed concentration (100 ppb) may be a useful index in mice for monitoring olfactory sensitivity within or after the period with repeated exposure to VOC gas.
LAMP-2A, a subtype of lysosome-associated membrane protein-2 (LAMP-2), functions in chaperone-mediated autophagy (CMA) that is a selective pathway of protein degradation in lysosome. We have examined the expression and regulation of GAPDH, one of the target proteins of CMA, and evaluated the cell death in the cerebral cold injury. Robust GAPDH immunoreactivity was seen in the cytoplasm of astrocytes around the lesion of both LAMP-2 deficient and wild type mice on days 3 and 7 after cold injury. However, on day 14, GAPDH was detected in the nuclei of large neurons in LAMP-2 deficient mice. Besides, TUNEL-positive cells in the LAMP-2 deficient mice were abundant on the day 14. Since the translocation of GAPDH to the nucleus are known to cause cell death, our result suggests GAPDHmediated delayed neuronal cell death is enhanced in the brain lesion of LAMP-2 deficient mice. Thus, CMA via LAMP-2 may protect neurons under the pathological conditions through degradation of excessive GAPDH.
doi:10.1016/j.neures.2009.09.615
doi:10.1016/j.neures.2009.09.618
P1-n14 Anomalous properties of cortical inhibitory neural mechanism in behavioral dysfunction of the Bronx waltzer mouse Yoshiki Matsuda 1 , Hitomi Izumi 1 , Yuki Inoue 1 , Masumi Inagaki 1 , Makiko Kaga 1 , Yu-ichi Goto 2
P1-n17 Depolarizing high-[K+ ] load model of rat brain energy metabolism: ␣ 31 P-NMR study Osamu Tokumaru 1 , Chihiro Kuroki 1 , Kazue Ogata 1 , Takaaki Kitano 2 , Isao Yokoi 1
1
1
Natl. Inst. Ment. Health, NCNP, Tokyo, Japan; Tokyo, Japan
2
Natl. Inst. Neurosci., NCNP,
2
Department Neurophysiol, Oita University Faculty of Medicine, Oita, Japan; Med. Edu. Ctr., Oita University Faculty of Medicine, Oita, Japan
The homozygous Bronx waltzer (bv) mouse, which is known as a model of congenital hearing and vestibular dysfunction, exhibits obvious anxiety-like behavior. The present study investigated whether the anxiety-like behavior of bv mouse is caused by abnormalities in cortical GABAergic interneurons. Double immunofluorescent staining revealed that the number of parvalbumin-expressing GAD67 positive cells were significantly decreased in cingulate, somatosensory and visual cortices compared to the control group (p < 0.01 or 0.05). In contrast, no difference was observed in hippocampal CA1, CA3 area and dentate gyri. Electrocorticography from parietal region of bv mouse showed bilaterally synchronous interictal discharges. The latency to incidence of first tonic extension induced by the intraperitoneal administration of pentylenetetrazol was remarkably decreased in bv mice. These results suggest that the cortical inhibitory neuronal dysfunctions contribute to the behavioral abnormality including anxiety-like behavior in bv mouse.
We study energy metabolism of rat brain slices superfused with artificial cerebrospinal fluid (ACSF) by measuring high energy phosphates using 31 P-NMR. When [K+ ] in ACSF is increased from 5 mM to 60 mM, NMR peaks corresponding to phosphocreatine (PCr), a buffering compound for ATP in Lohmann’s reaction, are reduced, indicating increased energy expenditure of brain slices.The present study investigated the effects of [K+ ] on energy demand using 31 P-NMR. Logarithm of [K+ ] and decrease in PCr were linearly correlated (p < 0.0001). Such decrease in PCr was reduced by 44% with tetrodotoxin (TTX). If action potentials (AP) play a major role in increase in energy expenditure, it should be all-or-none in character. Thus the observed linearity between decrease in PCr and log[K+ ] did not suggest a significant contribution of AP to the increased energy demand due to high [K+ ]. On the contrary, partial blockage of such decrease in PCr by TTX might indicate that 44% of the increased energy expenditure attribute to AP.
doi:10.1016/j.neures.2009.09.616
doi:10.1016/j.neures.2009.09.619
P1-n15 LAMP-2 deficient mice brain as a model for lysosomal storage disease Akiko Furuta, Hisae Kikuchi, Keiji Wada
P1-n18 Novel ataxia mouse with heavy iron deposition in the liver and kidneys, but not in the heart or central nervous system Moriaki Kusakabe 1,2,3 , Toshiaki Tachibana 6 , Miki Tanzawa 4 , Yukari Wakana 4 , Tomonori Kawabe 4 , Jorge Zavaleta-Ahane 4 , Takahiro Fukuda 5 , Hisashi Hashimoto 6
Department Degenerat. Neurol. Dis., Natl. Inst. Neurosci., NCNP, Tokyo, Japan Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein localized to lysosomal membrane. Mutations in LAMP-2 gene cause Danon disease that is characterized by cardiomyopathy, myopathy and variable mental retardation; however, the mechanism of mental disorder in Danon disease is not elucidated. We have investigated possible neuropathological changes of LAMP-2 deficient mouse. Increased immunoreactivity of cathepsin D in large neurons and GFAP-positive astrocytosis were seen in the hippocampus and the cerebral cortex from LAMP-2 deficient mice. Among several lectin staining sections, concanavalin A positive aggregation was found in the neurons from LAMP-2 deficient mice. Whereas previous studies on electron microscopy reported the accumulation of autophagic vacuoles in the visceral organs from LAMP-2 deficient mice, here we showed that various lysosomal changes in the neurons, astrocytes and pericytes, which were similar to changes in lysosomal storage disease. These results indicate that LAMP-2 is crucial for lysosomal protein degradation and regulated with cell-specific manner in the brain. doi:10.1016/j.neures.2009.09.617
1 Res. Ctr. for Food Safety, Grad Sch. Agri. Life Sci., University of Tokyo, Tokyo, Japan; 2 Inst. Anim. Rep., Japan; 3 Matrix Cell Res. Inst., Inc., Japan; 4 Tokyo Col. Medico-Pharmaco Tec., Japan; 5 Department Neuropath, Jikei Univ Sch Med, Japan; 6 Department Anat., Jikei. University Sch. Med., Japan
Many neurodegenerative diseases, including Friedreich ataxia, are associated with dysregulation of iron metabolism. We have investigated the iron deposition in various organs of our novel mutant mouse which is characterized by heavy ataxia with severe gait disorder of the hind legs. Heavy Fe3+ deposition was found in the liver and kidneys, but not in the heart, the skeletal muscle or the central nervous system. Fe2+ was not detected in any organs. In the kidneys, iron was deposited in each cell of selected proximal tubules but no deposition was found in other proximal tubules. Ultrastructurally, electron-dense depositions were found in lysosomes in the apical side of the cell and not in mitochondria. These data suggest that this mutant mouse is a very useful model to reveal a relationship between neurodegeneration and iron metabolism. doi:10.1016/j.neures.2009.09.620