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Axonal degeneration in mice doubly deficient in cathepsin D or B and L
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Abstracts / Neuroscience Research 68S (2010) e109–e222
of Purkinje cells around neurons in the deep cerebellar nuclei. Our present data showing the specific localization of Atg9Ap in synaptic regions suggest that Atg9Ap may play an important role in the maintenance of metabolic turnover in pre- and postsynaptic regions. doi:10.1016/j.neures.2010.07.2436
P1-o11 Shifted migration profile of synaptic structural components observed in familial amyotrophic lateral sclerosis patient brain may reflect a misfolded SOD1 toxicity
P1-o13 Mutant ␥PKC causing spinocerebellar ataxia type 14 (SCA14) preferably interacts with heat shock cognate protein 70 (Hsc70) and affects its translocation to lysosome Takahiro Seki 1 , Ken-ichi Yoshino 2 , Kouta Ogawa 1 , Tomoya Onji 1 , Shigeru Tanaka 1 , Izumi Hide 1 , Naoaki Saito 2 , Norio Sakai 1 1 Dept Mol Pharmacol Neurosci, Grad Sch Biomed Sci, Hiroshima Univ, Hiroshima 2 Lab Mol Pharmacol, Biosig Res Ctr, Kobe Univ, Japan
Toxicity of misfolded mutant superoxide dismutase (SOD)1 protein is thought to be responsible for the selective loss of motoneurons in SOD1related familial amyotrophic lateral sclerosis (FALS), although the underlying molecular mechanism has been unclear. We previously showed that mutant SOD1 aggregates in motoneuronal axons in G93A SOD1-Tg mice specifically associate with a Kinesin II motor subunit and thereby inhibit axonal transport of choline acetyltransferase, an enzyme essential for acetylcholine generation at motor terminals (Hum Mol Genet. 2009). Here we analyzed postmortem human FALS brain tissue lysates with a method similar to what we have used in the previous report. We found that misfolded SOD1 is abundant in precentral gyrus of SOD1-related FALS patient cerebral cortex, while it is undetectable in control or synaptic ALS cases. We also found by densitygradient centrifugation analysis that several synaptic structural component molecules co-migrate with misfolded SOD1 to high density fractions, which is also a finding specific to FALS cases. These results may suggest that reorganization of synaptic structure that results in shifted mobility of synaptic components is a part of a previously unknown mechanism of misfolded SOD1 toxicity.
Missense mutations have been identified in ␥PKC gene in several spinocereballar ataxia type 14 (SCA14) families. We have previously demonstrated that SCA14 mutant ␥PKC is susceptible to aggregation and causes apoptotic cell death. However, it remains unclear how mutant ␥PKC causes apoptosis. To clarify the mechanism, we attempted to identify the protein that preferably interacts with SCA14 mutant ␥PKC. Pull-down analysis using HaloTag system revealed that heat shock cognate protein 70 (Hsc70) was strongly bound to mutant ␥PKC, compared with wild type ␥PKC, in SHSY5Y cells and primary cultured cerebellar Purkinje cells (PCs). Since Hsc70 is known to be involved in chaperone-mediated autophagy (CMA), one of the lysosomal protein degradation pathways, we examined whether mutant ␥PKC affects the translocation of Hsc70 from cytoplasm to lysosome, which reflects the intracellular CMA activity, in primary cultured PCs. The translocation of Hsc70 was visualized by using HT system. We confirmed that 6-aminonicotinamide treatment and oxidative stress, which are reported to activate CMA, enhanced lysosomal accumulation of Hsc70-HT in PCs. In PCs expressing wild type ␥PKC-GFP, co-expressed Hsc70-HT spontaneously translocated into lysosome, while this translocation of Hsc70-HT was significantly repressed in PCs expressing mutant ␥PKC-GFP. These results suggest that mutant ␥PKC inhibits translocation of Hsc70 to lysosome by its strong interaction with Hsc70, leading to repression of intracellular CMA activity in PCs. Since CMA is activated in various stress conditions, mutant ␥PKC might impair the cellular adaptation to stress, resulting in neurodegeneration of PCs.
doi:10.1016/j.neures.2010.07.2437
doi:10.1016/j.neures.2010.07.2439
P1-o12 Cytoplasmic aggregation of C-terminal ATN1 fragment in Dentatorubral-pallidoluysian atrophy (DRPLA)
P1-o14 Axonal degeneration in mice doubly deficient in cathepsin D or B and L
Toshiyuki Araki , Minako Tateno Department of PNS Res, National Institute of Neuroscience, National Center of Neurology and Psychiatry
Yasuyo Suzuki , Ikuru Yazawa Laboratory of Research Resources, National Center for Geriatics and Gerontology (NCGG) Dentatorubral-pallidoluysian atrophy (DRPLA) is a hereditary neurodegenerative disorder. DRPLA is caused by expansion of CAG trinucleotide repeats encoding a polyglutamine tract in the responsible genes. The responsible gene product (DRPLA protein), also known as atrophin-1 (ATN1), carries polyglutamine tract. Nuclear accumulation of ATN1 might be a primary disease process in DRPLA as well as other polyglutamine diseases, but the mechanism of neurodegeneration is still unclear. Here, using COS-7 and Neuro2a cells expressing the ATN1 gene, we identified a novel C-terminal fragment containing a polyglutamine tract, and demonstrated that ATN1 accumulated with increasing number of polyglutamines. Accumulation of the C-terminal fragment was enhanced by inhibition of caspase activities in the COS-7 cells. Western blot analysis of subcellular fractions of both the brain tissues of patient with DRPLA and COS-7 cells expressed ATN1 showed full-length ATN1 and C-terminal fragment localized nuclear and cytoskeletal matrix fractions, and the mutant C-terminal fragment was preferentially found in the membrane/organella fraction. Immunocytochemical analysis revealed that the accumulation of ATN1 occurred in both the cytoplasm and nucleus of the brain tissues of patient with DRPLA and mammalian cells expressed ATN1. In COS-7 cells, accelerated aggregations of the C-terminal fragment in cytoplasm were observed in inhibition of caspase activities which enhanced of accumulation of C-terminal fragment. In addition, formation of perinuclear aggregates was caused by selective accumulation of the C-terminal fragment. We suggest that the C-terminal fragment plays a role in DRPLA neurodegeneration. doi:10.1016/j.neures.2010.07.2438
Masato Koike , Yasuo Uchiyama Dept of Cell Biology and Neurosci, Juntendo Univ We have noticed that granular staining for LC3 is distinct not only in neuronal perikarya but also in axons of mouse brains deficient in cathepsin D (CD−/−) or cathepsins B and L (CB−/−CL−/−) (Koike et al., 2005). The accumulation of autophagosomes in the axons corresponded well with meganeurites/spheroids, which were found to be one of the pathological findings in various neurodegenerative disorders. When such neuropathological phenotypes were compared between CD−/− and CB−/−CL−/− mice, the most striking difference was detected in constituents stacked in axons of the corpus callosum. In coronal sections of the corpus callosum from CB−/−CL−/− mice, massive accumulation of spheroids was distinct in axons, whereas those from CD−/− mice possessed only a few spheroids in them. We next examined the pathological features in the corpus callosum between CD(/(, CD(/(CL(/(, and CD(/(CB(/( mouse brains, and found that abundant spheroids were detected in that of CD(/(CL(/( and CD(/(CB(/( brains. It is well known that Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E (CPE) are localized to neuronal biosynthetic, recycling/endocytic compartments. Such molecules highly increase in lysosomes of CB(/(CL(/( mouse brain neurons, when examined by a proteomic analysis in combination with subcellular fractionation and LC-MS/MS (Stahl et al., 2007). Immunohistochemical staining of Rab14, DNER, and CPE using brain tissues of CD(/(, CD(/(CL(/( and CD(/(CB(/( mice found that these three molecules were specifically accumulated in the spheroids of these mutant animals. These results indicate that accumulated vacuoles contain not only nascent autophagosomes but also transporting vesicles that contain Rab14, DNER, and CPE. Moreover, it is also interesting that such pathologically accumulating materials in vacuoles of the axons appears significantly more abundant in brains doubly deficient in cathepsins than in those deficient in CD. doi:10.1016/j.neures.2010.07.2440
Abstracts / Neuroscience Research 68S (2010) e109–e222
of Purkinje cells around neurons in the deep cerebellar nuclei. Our present data showing the specific localization of Atg9Ap in synaptic regions suggest that Atg9Ap may play an important role in the maintenance of metabolic turnover in pre- and postsynaptic regions. doi:10.1016/j.neures.2010.07.2436
P1-o11 Shifted migration profile of synaptic structural components observed in familial amyotrophic lateral sclerosis patient brain may reflect a misfolded SOD1 toxicity
P1-o13 Mutant ␥PKC causing spinocerebellar ataxia type 14 (SCA14) preferably interacts with heat shock cognate protein 70 (Hsc70) and affects its translocation to lysosome Takahiro Seki 1 , Ken-ichi Yoshino 2 , Kouta Ogawa 1 , Tomoya Onji 1 , Shigeru Tanaka 1 , Izumi Hide 1 , Naoaki Saito 2 , Norio Sakai 1 1 Dept Mol Pharmacol Neurosci, Grad Sch Biomed Sci, Hiroshima Univ, Hiroshima 2 Lab Mol Pharmacol, Biosig Res Ctr, Kobe Univ, Japan
Toxicity of misfolded mutant superoxide dismutase (SOD)1 protein is thought to be responsible for the selective loss of motoneurons in SOD1related familial amyotrophic lateral sclerosis (FALS), although the underlying molecular mechanism has been unclear. We previously showed that mutant SOD1 aggregates in motoneuronal axons in G93A SOD1-Tg mice specifically associate with a Kinesin II motor subunit and thereby inhibit axonal transport of choline acetyltransferase, an enzyme essential for acetylcholine generation at motor terminals (Hum Mol Genet. 2009). Here we analyzed postmortem human FALS brain tissue lysates with a method similar to what we have used in the previous report. We found that misfolded SOD1 is abundant in precentral gyrus of SOD1-related FALS patient cerebral cortex, while it is undetectable in control or synaptic ALS cases. We also found by densitygradient centrifugation analysis that several synaptic structural component molecules co-migrate with misfolded SOD1 to high density fractions, which is also a finding specific to FALS cases. These results may suggest that reorganization of synaptic structure that results in shifted mobility of synaptic components is a part of a previously unknown mechanism of misfolded SOD1 toxicity.
Missense mutations have been identified in ␥PKC gene in several spinocereballar ataxia type 14 (SCA14) families. We have previously demonstrated that SCA14 mutant ␥PKC is susceptible to aggregation and causes apoptotic cell death. However, it remains unclear how mutant ␥PKC causes apoptosis. To clarify the mechanism, we attempted to identify the protein that preferably interacts with SCA14 mutant ␥PKC. Pull-down analysis using HaloTag system revealed that heat shock cognate protein 70 (Hsc70) was strongly bound to mutant ␥PKC, compared with wild type ␥PKC, in SHSY5Y cells and primary cultured cerebellar Purkinje cells (PCs). Since Hsc70 is known to be involved in chaperone-mediated autophagy (CMA), one of the lysosomal protein degradation pathways, we examined whether mutant ␥PKC affects the translocation of Hsc70 from cytoplasm to lysosome, which reflects the intracellular CMA activity, in primary cultured PCs. The translocation of Hsc70 was visualized by using HT system. We confirmed that 6-aminonicotinamide treatment and oxidative stress, which are reported to activate CMA, enhanced lysosomal accumulation of Hsc70-HT in PCs. In PCs expressing wild type ␥PKC-GFP, co-expressed Hsc70-HT spontaneously translocated into lysosome, while this translocation of Hsc70-HT was significantly repressed in PCs expressing mutant ␥PKC-GFP. These results suggest that mutant ␥PKC inhibits translocation of Hsc70 to lysosome by its strong interaction with Hsc70, leading to repression of intracellular CMA activity in PCs. Since CMA is activated in various stress conditions, mutant ␥PKC might impair the cellular adaptation to stress, resulting in neurodegeneration of PCs.
doi:10.1016/j.neures.2010.07.2437
doi:10.1016/j.neures.2010.07.2439
P1-o12 Cytoplasmic aggregation of C-terminal ATN1 fragment in Dentatorubral-pallidoluysian atrophy (DRPLA)
P1-o14 Axonal degeneration in mice doubly deficient in cathepsin D or B and L
Toshiyuki Araki , Minako Tateno Department of PNS Res, National Institute of Neuroscience, National Center of Neurology and Psychiatry
Yasuyo Suzuki , Ikuru Yazawa Laboratory of Research Resources, National Center for Geriatics and Gerontology (NCGG) Dentatorubral-pallidoluysian atrophy (DRPLA) is a hereditary neurodegenerative disorder. DRPLA is caused by expansion of CAG trinucleotide repeats encoding a polyglutamine tract in the responsible genes. The responsible gene product (DRPLA protein), also known as atrophin-1 (ATN1), carries polyglutamine tract. Nuclear accumulation of ATN1 might be a primary disease process in DRPLA as well as other polyglutamine diseases, but the mechanism of neurodegeneration is still unclear. Here, using COS-7 and Neuro2a cells expressing the ATN1 gene, we identified a novel C-terminal fragment containing a polyglutamine tract, and demonstrated that ATN1 accumulated with increasing number of polyglutamines. Accumulation of the C-terminal fragment was enhanced by inhibition of caspase activities in the COS-7 cells. Western blot analysis of subcellular fractions of both the brain tissues of patient with DRPLA and COS-7 cells expressed ATN1 showed full-length ATN1 and C-terminal fragment localized nuclear and cytoskeletal matrix fractions, and the mutant C-terminal fragment was preferentially found in the membrane/organella fraction. Immunocytochemical analysis revealed that the accumulation of ATN1 occurred in both the cytoplasm and nucleus of the brain tissues of patient with DRPLA and mammalian cells expressed ATN1. In COS-7 cells, accelerated aggregations of the C-terminal fragment in cytoplasm were observed in inhibition of caspase activities which enhanced of accumulation of C-terminal fragment. In addition, formation of perinuclear aggregates was caused by selective accumulation of the C-terminal fragment. We suggest that the C-terminal fragment plays a role in DRPLA neurodegeneration. doi:10.1016/j.neures.2010.07.2438
Masato Koike , Yasuo Uchiyama Dept of Cell Biology and Neurosci, Juntendo Univ We have noticed that granular staining for LC3 is distinct not only in neuronal perikarya but also in axons of mouse brains deficient in cathepsin D (CD−/−) or cathepsins B and L (CB−/−CL−/−) (Koike et al., 2005). The accumulation of autophagosomes in the axons corresponded well with meganeurites/spheroids, which were found to be one of the pathological findings in various neurodegenerative disorders. When such neuropathological phenotypes were compared between CD−/− and CB−/−CL−/− mice, the most striking difference was detected in constituents stacked in axons of the corpus callosum. In coronal sections of the corpus callosum from CB−/−CL−/− mice, massive accumulation of spheroids was distinct in axons, whereas those from CD−/− mice possessed only a few spheroids in them. We next examined the pathological features in the corpus callosum between CD(/(, CD(/(CL(/(, and CD(/(CB(/( mouse brains, and found that abundant spheroids were detected in that of CD(/(CL(/( and CD(/(CB(/( brains. It is well known that Rab14, the Delta/Notch-like epidermal growth factor-related receptor (DNER), calcyon, and carboxypeptidase E (CPE) are localized to neuronal biosynthetic, recycling/endocytic compartments. Such molecules highly increase in lysosomes of CB(/(CL(/( mouse brain neurons, when examined by a proteomic analysis in combination with subcellular fractionation and LC-MS/MS (Stahl et al., 2007). Immunohistochemical staining of Rab14, DNER, and CPE using brain tissues of CD(/(, CD(/(CL(/( and CD(/(CB(/( mice found that these three molecules were specifically accumulated in the spheroids of these mutant animals. These results indicate that accumulated vacuoles contain not only nascent autophagosomes but also transporting vesicles that contain Rab14, DNER, and CPE. Moreover, it is also interesting that such pathologically accumulating materials in vacuoles of the axons appears significantly more abundant in brains doubly deficient in cathepsins than in those deficient in CD. doi:10.1016/j.neures.2010.07.2440