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Characterization of Tau splicing factors in Alzheimer's disease
Poster Presentations P3 by the BFCNs. Cholinergic innervation of the hippocampus is important for BDNF expression. BDNF and TrkB were significantly reduced while TrkC andNT-3 were not altered. Moreover, BDNF was negatively related to AD-like tau phosphorylation. To proof the role of NGF and cholinergic innervation in this model, transgenic mice and controls were treated with intraventricular infusion of NGF or galantamine i.p. over two weeks. Treated animals displayed significantly increased numbers of BFCN and improved cognition. NGF treatment was superior over galantamine in this short-term treatment. Conclusions: Tau pathology plays an important role as it impairs axonal transport apparently involved in cholinergic degeneration. Here, we demonstrated the pivotal role of NTF in AD.
P3-141
CHARACTERIZATION OF TAU SPLICING FACTORS IN ALZHEIMER’S DISEASE
Aderonke Adelodun, Michael Niblock, Tibor Hortobagyi, JeanMarc Gallo, King’s College London, London, United Kingdom. Background: Pathological aggregates of tau, a microtubule associated protein, is a characteristic hallmark of tauopathies including Alzheimer’s disease (AD). Transcripts of tau undergo aternative splicing; inclusion or exclusion of tau exon 10 (E10) in the MAPT gene, encoding tau generates tau protein isoforms with four (4R) or three (3R) tubulin binding repeats respectively. In the normal adult human brain, the 4R/3R ratio is approximately one. An increase in tauE 10 inclusion in some forms of Front temporal Dementia with Parkinsonism linked to chromosome 17 (FTDP17). The H1 (unlike H2) haplotype of the MAPT gene produces higher levels of E10 mRNA and predisposes to AD. An increase in 4R/3R ratio has also been associated with sporadic AD. Hence, misregulation of tau RNA splicing may contribute to the pathogenesis of sporadic AD. Specific RNA binding proteins including serine and arginine (SR) rich proteins and CELF proteins regulate E10 splicing and may have an abnormal activity in AD. Methods: In this study, we investigated the levels of expression of specific CELF proteins transcripts (CELF1, CELF3 and CELF4) and SR proteins transcripts (SRp55, SRp40 and SC35) in five brain regions (frontal cortex, temporal cortex, amygdala, hippocampus and cerebellum) using human post-mortem brain tissue from AD patients. About 30% of AD cases have inclusion of the RNA binding protein, TDP-43. We have investigated AD cases with (+) and without () TDP-43 inclusions compared to agedmatched controls. Results: We found, by quantitative RT-PCR, that the expression levels of CELF1 and CELF3 transcripts was significantly decreased in the hippocampus of sporadic AD-TDP-43-and AD-TDP-43+ compared to controls. In contrast, CELF1 and CELF3 transcripts expression was significantly increased in the amygdala of sporadic AD-TDP-43 and AD-TDP-43+when compared to controls. Interestingly, we found higher expression levels of CELF1 in the hippocampus of AD-TDP-43+ when compared to AD-TDP-43-. Conclusions: Hence, TDP-43 may exacerbate the pathology of AD by affecting the RNA processing machinery. These results show that the splicing regulatory factors, CELF1 and CELF3 undergo altered expression in sporadic AD and may contribute to pathogenesis of AD.
P3-142
SECRETION AND AUTOPHAGY-DEPENDENT LYSOSOMAL SEQUESTRATION OF Ab ARE UNSUCCESSFUL RESCUE RESPONSES TO PROTEASOME INHIBITION
Lotta Agholme1, Martin Hallbeck2, Eirikur Benedikz3, Jan Marcusson1, Katarina K agedal1, 1Link€ oping University, Link€oping, Sweden; 2Link€oping University Hospital, Linkoping, Sweden; 3Karolinska Institutet, Stockholm, Sweden. Background: The proteasome, being important for degradation of aggregated and misfolded protein, and vital for cellular function, has been shown to be dysfunctional in Alzheimer’s disease (AD). Proteasome inhibition has been shown to induce autophagy, but it is still debated whether autophagy is beneficial or deleterious in neurons. The aim of this study
S561
was to investigate the effects of proteasomal inhibition on intracellular ß-amyloid (Aß) accumulation in lysosomes, secretion of Aß and APP processing. We furthermore investigated the involvement of autophagy in these events. Methods: Proteasome inhibition of SH-SY5Y neuroblastoma cells transfected with APP harbouring the Swedish mutation was carried out using MG-115. Autophagy was inhibited using 3MA, and fusion of autophagosomes with lysosomes was inhibited using E64d/Pepstatin A. Investigation of autophagic flux was investigated by Western blot using antibodies against LC3 and p70s6 kinase. Intracellular Aß accumulation was investigated by immunocytochemistry and ELISA, whereas secretion was investigated by the Mesoscale system. APP processing was investigated by western blot. Results: Proteasome inhibition resulted in up regulation of the lysosomal system, as well as autophagy-dependent lysosomal sequestration of Aß. Moreover, proteasome inhibition increased the secretion of Aß, not being affected by autophagy inhibition. Proteasome inhibition increased the levels of C-terminal fragment (C99), but not full length APP. Inhibition of autophagy tended to further increase C99 levels, implicating involvement of both proteasomes and autophagosomes in C99 processing. Proteasome inhibition also resulted in reduced cellular viability, which could be rescued by autophagy inhibition. Conclusions: Decreased proteasome activity led to both lysosomal sequestration and increased secretion of Aß. These pathways appear to be at least partly facilitated by autophagy. We also detected a decrease in cellular viability, suggesting that these unsuccessful rescue mechanisms may contribute to AD pathogenesis. P3-143
INFECTION OF HUMAN MONOCYTES, OLFACTORY NEUROEPITHELIA, AND NEURONAL CELLS WITH CHLAMYDIA PNEUMONIAE ALTERS EXPRESSION OF GENES ASSOCIATED WITH ALZHEIMER’S DISEASE
Brian Balin1, Morgan Devins1, Corey Caruthers1, Juliana Zoga1, Chris Cappellini2, Christine Hammond1, Susan Hingley1, Denah Appelt1, 1 Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, United States; 2Washington & Jefferson College, Washington, Pennsylvania, United States. Background: Our laboratory has been studying the role of infection with Chlamydia pneumoniae in sporadic late-onset Alzheimer disease (LOAD). This infection may be a trigger for the pathology observed in LOAD as a function of initiating changes in gene regulation following entry of the organism into the brain. We have shown that entry and infection in the brain may result from infection of both blood-borne monocytes and olfactory neuroepithelial cells. Methods: Our current studies focus on infection of human THP1 monocytes, olfactory neuroepithelial cells, and SKNMC neuronal cells in vitro followed by analysis of infection using immunofluorescence and Alzheimer pathway-specific Real-Time PCR microarrays. The different cell types were infected for 48hrs with the laboratory strain of Chlamydia pneumoniae, AR39, at a multiplicity of infection ¼ 0.5-1. Following infection, cells were analyzed by immunocytochemistry using immunofluorescent tagged antibodies. To determine gene regulation changes, RNA extraction was followed by cDNA first strand synthesis and Real-Time PCR. Results: C. pneumoniae prominently and stably infected all cell types at 48hr post-infection. Numerous large inclusions were labelled using anti-chlamydial monoclonal antibodies. Gene expression was altered dramatically in all three cell types. Of the genes up-regulated, only 6 were in common to all 3 cell types at 48hr pi (ACHE, BACE2, CLU, GSK3b, NCSTN, and PRKI), and of the genes down-regulated, only 3 were in common to all 3 cell types (BDNF, INSR, and CTSG). When comparing one cell type to another 33 genes were found to be up-regulated and in common to monocytes and the neuronal cells whereas 14 genes were in common to monocytes and olfactory cells; when genes from monocytes were up-regulated as compared to genes down-regulated in the other cell types, 39 genes were identified to be different in neuronal cells, whereas 52 genes were different in the olfactory cells. Conclusions: Our data suggest that C. pneumoniae-infected monocytes, olfactory neuroepithelia, and neuronal cells exhibit specific, but often disparate, changes in
P3-141
CHARACTERIZATION OF TAU SPLICING FACTORS IN ALZHEIMER’S DISEASE
Aderonke Adelodun, Michael Niblock, Tibor Hortobagyi, JeanMarc Gallo, King’s College London, London, United Kingdom. Background: Pathological aggregates of tau, a microtubule associated protein, is a characteristic hallmark of tauopathies including Alzheimer’s disease (AD). Transcripts of tau undergo aternative splicing; inclusion or exclusion of tau exon 10 (E10) in the MAPT gene, encoding tau generates tau protein isoforms with four (4R) or three (3R) tubulin binding repeats respectively. In the normal adult human brain, the 4R/3R ratio is approximately one. An increase in tauE 10 inclusion in some forms of Front temporal Dementia with Parkinsonism linked to chromosome 17 (FTDP17). The H1 (unlike H2) haplotype of the MAPT gene produces higher levels of E10 mRNA and predisposes to AD. An increase in 4R/3R ratio has also been associated with sporadic AD. Hence, misregulation of tau RNA splicing may contribute to the pathogenesis of sporadic AD. Specific RNA binding proteins including serine and arginine (SR) rich proteins and CELF proteins regulate E10 splicing and may have an abnormal activity in AD. Methods: In this study, we investigated the levels of expression of specific CELF proteins transcripts (CELF1, CELF3 and CELF4) and SR proteins transcripts (SRp55, SRp40 and SC35) in five brain regions (frontal cortex, temporal cortex, amygdala, hippocampus and cerebellum) using human post-mortem brain tissue from AD patients. About 30% of AD cases have inclusion of the RNA binding protein, TDP-43. We have investigated AD cases with (+) and without () TDP-43 inclusions compared to agedmatched controls. Results: We found, by quantitative RT-PCR, that the expression levels of CELF1 and CELF3 transcripts was significantly decreased in the hippocampus of sporadic AD-TDP-43-and AD-TDP-43+ compared to controls. In contrast, CELF1 and CELF3 transcripts expression was significantly increased in the amygdala of sporadic AD-TDP-43 and AD-TDP-43+when compared to controls. Interestingly, we found higher expression levels of CELF1 in the hippocampus of AD-TDP-43+ when compared to AD-TDP-43-. Conclusions: Hence, TDP-43 may exacerbate the pathology of AD by affecting the RNA processing machinery. These results show that the splicing regulatory factors, CELF1 and CELF3 undergo altered expression in sporadic AD and may contribute to pathogenesis of AD.
P3-142
SECRETION AND AUTOPHAGY-DEPENDENT LYSOSOMAL SEQUESTRATION OF Ab ARE UNSUCCESSFUL RESCUE RESPONSES TO PROTEASOME INHIBITION
Lotta Agholme1, Martin Hallbeck2, Eirikur Benedikz3, Jan Marcusson1, Katarina K agedal1, 1Link€ oping University, Link€oping, Sweden; 2Link€oping University Hospital, Linkoping, Sweden; 3Karolinska Institutet, Stockholm, Sweden. Background: The proteasome, being important for degradation of aggregated and misfolded protein, and vital for cellular function, has been shown to be dysfunctional in Alzheimer’s disease (AD). Proteasome inhibition has been shown to induce autophagy, but it is still debated whether autophagy is beneficial or deleterious in neurons. The aim of this study
S561
was to investigate the effects of proteasomal inhibition on intracellular ß-amyloid (Aß) accumulation in lysosomes, secretion of Aß and APP processing. We furthermore investigated the involvement of autophagy in these events. Methods: Proteasome inhibition of SH-SY5Y neuroblastoma cells transfected with APP harbouring the Swedish mutation was carried out using MG-115. Autophagy was inhibited using 3MA, and fusion of autophagosomes with lysosomes was inhibited using E64d/Pepstatin A. Investigation of autophagic flux was investigated by Western blot using antibodies against LC3 and p70s6 kinase. Intracellular Aß accumulation was investigated by immunocytochemistry and ELISA, whereas secretion was investigated by the Mesoscale system. APP processing was investigated by western blot. Results: Proteasome inhibition resulted in up regulation of the lysosomal system, as well as autophagy-dependent lysosomal sequestration of Aß. Moreover, proteasome inhibition increased the secretion of Aß, not being affected by autophagy inhibition. Proteasome inhibition increased the levels of C-terminal fragment (C99), but not full length APP. Inhibition of autophagy tended to further increase C99 levels, implicating involvement of both proteasomes and autophagosomes in C99 processing. Proteasome inhibition also resulted in reduced cellular viability, which could be rescued by autophagy inhibition. Conclusions: Decreased proteasome activity led to both lysosomal sequestration and increased secretion of Aß. These pathways appear to be at least partly facilitated by autophagy. We also detected a decrease in cellular viability, suggesting that these unsuccessful rescue mechanisms may contribute to AD pathogenesis. P3-143
INFECTION OF HUMAN MONOCYTES, OLFACTORY NEUROEPITHELIA, AND NEURONAL CELLS WITH CHLAMYDIA PNEUMONIAE ALTERS EXPRESSION OF GENES ASSOCIATED WITH ALZHEIMER’S DISEASE
Brian Balin1, Morgan Devins1, Corey Caruthers1, Juliana Zoga1, Chris Cappellini2, Christine Hammond1, Susan Hingley1, Denah Appelt1, 1 Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania, United States; 2Washington & Jefferson College, Washington, Pennsylvania, United States. Background: Our laboratory has been studying the role of infection with Chlamydia pneumoniae in sporadic late-onset Alzheimer disease (LOAD). This infection may be a trigger for the pathology observed in LOAD as a function of initiating changes in gene regulation following entry of the organism into the brain. We have shown that entry and infection in the brain may result from infection of both blood-borne monocytes and olfactory neuroepithelial cells. Methods: Our current studies focus on infection of human THP1 monocytes, olfactory neuroepithelial cells, and SKNMC neuronal cells in vitro followed by analysis of infection using immunofluorescence and Alzheimer pathway-specific Real-Time PCR microarrays. The different cell types were infected for 48hrs with the laboratory strain of Chlamydia pneumoniae, AR39, at a multiplicity of infection ¼ 0.5-1. Following infection, cells were analyzed by immunocytochemistry using immunofluorescent tagged antibodies. To determine gene regulation changes, RNA extraction was followed by cDNA first strand synthesis and Real-Time PCR. Results: C. pneumoniae prominently and stably infected all cell types at 48hr post-infection. Numerous large inclusions were labelled using anti-chlamydial monoclonal antibodies. Gene expression was altered dramatically in all three cell types. Of the genes up-regulated, only 6 were in common to all 3 cell types at 48hr pi (ACHE, BACE2, CLU, GSK3b, NCSTN, and PRKI), and of the genes down-regulated, only 3 were in common to all 3 cell types (BDNF, INSR, and CTSG). When comparing one cell type to another 33 genes were found to be up-regulated and in common to monocytes and the neuronal cells whereas 14 genes were in common to monocytes and olfactory cells; when genes from monocytes were up-regulated as compared to genes down-regulated in the other cell types, 39 genes were identified to be different in neuronal cells, whereas 52 genes were different in the olfactory cells. Conclusions: Our data suggest that C. pneumoniae-infected monocytes, olfactory neuroepithelia, and neuronal cells exhibit specific, but often disparate, changes in