- Email: [email protected]
Key role of inflammation in Alzheimer pathology
P478
Poster Presentations P4
3
The Alfred Hospital, Melbourne, Australia. Contact e-mail: gmevin@ unimelb.edu.au
Background: BACE initiates the amyloid proteolytic processing of APP that leads to the production of Ab peptides. We have previously reported that BACE levels are increased in the frontal cortex of patients with Alzheimer’s disease (AD). Because no change in BACE mRNA expression was observed, we hypothesized that an alteration of BACE cellular trafficking may be responsible for increased BACE protein and activity in AD. The cellular trafficking of BACE between TNG and endosomes is controlled by Golgi-localized g-ear containing ADP ribosylation factor-binding proteins (GGA). The aim of the present study was to analyse quantitatively the expression GGA1 and GGA3 in frontal cortex of Alzheimer’s disease and control patients in relation to BACE expression. Methods: Frontal cortical samples from AD (N¼20), healthy age-matched controls (HC; N¼18) and controls with a neurological pathology other than AD (NC; N¼8) were homogenized in Trizol to isolate RNA, DNA, and protein. Protein precipitates were analysed by western blotting. Signals were captured with a Syngene DCC imaging system. Statistical analysis was performed using SPSS software (one-way ANOVA) and the results expressed relative to the neuronal marker, b-tubulin III. Results: GGA1 was detected as a 60 kDa doublet. Quantitative analysis indicated that GGA1 levels were decreased by an average of 43% in the AD group compared to the HC (p ¼ 0.03). GGA3 was detected as a 75 kDa signal. A 45 kDa species that may represent a caspase cleavage product was also observed in some AD and CT samples. Quantitation of the 75 kDa band indicated a 74% decrease in AD samples compared to HC (p<0.01). Conclusions: This is the first report of simultaneous analysis of GGA1 and GGA3 expression in AD and control brains. A marked decreased expression of both GGA1 and GGA3 was observed and consistent with other reports. Quantitative analysis of BACE with two alternate antibodies and correlation study of GGA and BACE expression are in progress. P4-158
A LOW-DOSE OF INTRAVENOUS IMMUNOGLOBULIN PROVIDES PROTECTION IN FOCAL AND GLOBAL BRAIN ISCHEMIA MODELS AND ABOLISHES THE INCREASED ISCHEMIC VULNERABILITY OF AGED APP/PS1 MICE
Morris water maze at 14 days. Finally, delayed apoptotic death of CA1 pyramidal neurons was assessed at 15 days. Results: IVIG provided protection against focal ischemia, especially at the dose of 0.5 g/kg when the treatment was started at or one hour after the onset of ischemia. Ischemic lesions were 58% larger in APP/PS1 mice than in controls. IVIG treatment abolished this increased ischemic vulnerability in APP/PS1 mice. IVIG group outperformed the saline group in spatial memory after the global ischemia suggesting that IVIG protects the CA1 subregion against ischemic damage. Conclusions: These data indicate that IVIG may be a promising new neuroprotective treatment in acute brain ischemia, and further study of this possibility is warranted. Supported by Baxter Innovations, GmbH, Austria. P4-159
KEY ROLE OF INFLAMMATION IN ALZHEIMER PATHOLOGY
Ana M. Fernandez, Ignacio Torres-Aleman, Instituto Cajal, CSIC, Madrid, Spain. Contact e-mail: [email protected] Background: Inflammation undoubtedly plays a role in Alzheimer’s disease (AD), but its relative contribution to disease load is uncertain. Examination of inflammatory pathways potentially relevant in AD revealed a key role for astrocytes. Methods: Astrocyte cultures, animal transgenesis, qPCR, western blot, immunohistochemistry, ChIP assay, Elisa Results: Inflammatory cascades in murine and human astrocytes activated by inflammatory cytokines overrepresented in AD brains such as TNFa relied on the interaction between the transcription factor Foxo3 and the phosphatase calcineurin. Foxo/calcineurin complexes were observed in AD brains and transgenic mice (APP/PS1) and correlated with pathology. Genetic modulation of calcineurin in astrocytes at different stages of the pathological process in APP/PS1 mice indicated that inflammation is a key step in disease progression and its regulation is therapeutically significant. Full protection was achieved in the initial stages of the disease, with a protracted development of amyloidosis and preserved cognition. Accordingly, disruption of the Foxo/calcineurin association after IGF-I treatment was sufficient to reduce AD symptoms. Conclusions: The results indicate that inflammatory processes in astrocytes constitute a driving force of AD pathology. P4-160
EFFECT OF LACTOFERRIN ON Aß NEUROTOXICITY AND ITS CLEARANCE
Riikka Heikkinen1, Tarja Malm1, Susanna Boman1, Susanna Kuhmonen1, Toni Ahtoniemi2, Anu Muona2, Heikki Tanila1,3, Jari Koistinaho1,4, Milla Koistinaho2, 1Department of Neurobiology, A.I.V. Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio, Kuopio, Finland; 2 Medeia Therapeutics Ltd., Kuopio, Finland; 3Department of Neurology, Kuopio University Hospital, Kuopio, Finland; 4Department of Oncology, Kuopio University Hospital, Kuopio, Finland. Contact e-mail: riikka. [email protected]
Haruhisa Sato1,2, Li An2,3, Toshio Kawamata4, Yoshihiro Konishi5, Ikuo Tooyama2, 1Department of Pathology, Marutamachi Hospital, Kyoto, Japan; 2Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan; 3China Medical University, Shenyang, China; 4Faculty of Health Sciences, Kobe University School of Medicine, Kobe, Japan; 5Department of Clinical Research, Nishi-Tottori National Hospital, Tottori, Japan. Contact e-mail: [email protected]
Background: Stroke is a major cause of disability in the elderly. In particular, cerebrovascular lesions may contribute to the pathogenesis of Alzheimer’s disease (AD), as up to 90 % of patients show cerebrovascular pathology at autopsy. Interestingly, several transgenic mouse models of AD have shown increased vulnerability to ischemia at an early age. Because recent studies indicate that clinically safe intravenous immunoglobulin (IVIG) dose of 2 g/kg provides protection against focal brain ischemia in young wild-type (wt) mice, we investigated whether IVIG could abolish the increased vulnerability to ischemic injury in aged APP/PS1 transgenic mice. We also tested the effect of IVIG in a model of global brain ischemia affecting the brain regions important for memory functions. Methods: Permanent middle cerebral artery occlusion was induced in male Balb/c, female APP/PS1 mice and their C57Bl wt controls. The mice were given an intravenous injection of 0.5 or 1.0 g/kg of IVIG (Gammagard Liquid, Baxter) or saline 1 h before or 0, 1 or 3 h after the onset of ischemia. Lesion volume was determined by T2 weighted 9.4 T MRI 24 and 72 h after the ischemia in Balb/cA mice and by TTC-staining in APP/PS1 mice and their controls. Global forebrain ischemia was induced in C57Bl/6 wt mice by reversible compression of carotid arteries. IVIG (0.5 g/kg) or saline was given 1 h after the ischemia and effect on cognitive decline was tested in
Background: Lactoferrin (lactotransferrin; LF), an 80 kDa iron-binding protein, is greatly upregulated in Alzheimer’s disease (AD) brain. However, its function remains unclear. LF is strongly associated with diffuse and consolidated amyloid deposits in AD, LF may be involved in b-amyloid peptide (Ab) deposition. In this study, we investigated the effect of LF on Aß neurotoxicity and its clearance using cell line. Methods: Human IMR-32 cells and mouse astrocyte cells (purchased from ATCC) were used in this study. Ab42 and Ab40 as well as fluorescent Ab40 were purchased from AnaSpec, Inc. (San Jose, CA USA) or Peptide Institute (Osaka, Japan). IMR-32 cells were incubated for 24 hours with and without 10 mM of Aß42 combined with 0, 10, 50 or 100 mg/ml of LF. Cell vulnerability was examined by the LDH assay. Astrocytes were incubated for 30 min in media containing 10 mg/ml of Aß40, Aß42, fluorescent Ab40 combined with 0, 10, 40 or 160 mg/ml of LF. Fluorescent signals were observed by a confocal microscopy or fluorescent microscopy. After incubation for 30 min, cells were recovered. The uptake of Aß40 or Aß42 was analyzed by Western blots. Results: The LDH assay showed that IMR-32 cells damaged by Aß42 were not protected by LF. Image analysis clearly shows that LF inhibited the internalization of fluorescent Aß40 into astrocytes dose-dependently. Similarly, Western blot analysis also showed that LF inhibited the internalization of Aß40 and
Poster Presentations P4
3
The Alfred Hospital, Melbourne, Australia. Contact e-mail: gmevin@ unimelb.edu.au
Background: BACE initiates the amyloid proteolytic processing of APP that leads to the production of Ab peptides. We have previously reported that BACE levels are increased in the frontal cortex of patients with Alzheimer’s disease (AD). Because no change in BACE mRNA expression was observed, we hypothesized that an alteration of BACE cellular trafficking may be responsible for increased BACE protein and activity in AD. The cellular trafficking of BACE between TNG and endosomes is controlled by Golgi-localized g-ear containing ADP ribosylation factor-binding proteins (GGA). The aim of the present study was to analyse quantitatively the expression GGA1 and GGA3 in frontal cortex of Alzheimer’s disease and control patients in relation to BACE expression. Methods: Frontal cortical samples from AD (N¼20), healthy age-matched controls (HC; N¼18) and controls with a neurological pathology other than AD (NC; N¼8) were homogenized in Trizol to isolate RNA, DNA, and protein. Protein precipitates were analysed by western blotting. Signals were captured with a Syngene DCC imaging system. Statistical analysis was performed using SPSS software (one-way ANOVA) and the results expressed relative to the neuronal marker, b-tubulin III. Results: GGA1 was detected as a 60 kDa doublet. Quantitative analysis indicated that GGA1 levels were decreased by an average of 43% in the AD group compared to the HC (p ¼ 0.03). GGA3 was detected as a 75 kDa signal. A 45 kDa species that may represent a caspase cleavage product was also observed in some AD and CT samples. Quantitation of the 75 kDa band indicated a 74% decrease in AD samples compared to HC (p<0.01). Conclusions: This is the first report of simultaneous analysis of GGA1 and GGA3 expression in AD and control brains. A marked decreased expression of both GGA1 and GGA3 was observed and consistent with other reports. Quantitative analysis of BACE with two alternate antibodies and correlation study of GGA and BACE expression are in progress. P4-158
A LOW-DOSE OF INTRAVENOUS IMMUNOGLOBULIN PROVIDES PROTECTION IN FOCAL AND GLOBAL BRAIN ISCHEMIA MODELS AND ABOLISHES THE INCREASED ISCHEMIC VULNERABILITY OF AGED APP/PS1 MICE
Morris water maze at 14 days. Finally, delayed apoptotic death of CA1 pyramidal neurons was assessed at 15 days. Results: IVIG provided protection against focal ischemia, especially at the dose of 0.5 g/kg when the treatment was started at or one hour after the onset of ischemia. Ischemic lesions were 58% larger in APP/PS1 mice than in controls. IVIG treatment abolished this increased ischemic vulnerability in APP/PS1 mice. IVIG group outperformed the saline group in spatial memory after the global ischemia suggesting that IVIG protects the CA1 subregion against ischemic damage. Conclusions: These data indicate that IVIG may be a promising new neuroprotective treatment in acute brain ischemia, and further study of this possibility is warranted. Supported by Baxter Innovations, GmbH, Austria. P4-159
KEY ROLE OF INFLAMMATION IN ALZHEIMER PATHOLOGY
Ana M. Fernandez, Ignacio Torres-Aleman, Instituto Cajal, CSIC, Madrid, Spain. Contact e-mail: [email protected] Background: Inflammation undoubtedly plays a role in Alzheimer’s disease (AD), but its relative contribution to disease load is uncertain. Examination of inflammatory pathways potentially relevant in AD revealed a key role for astrocytes. Methods: Astrocyte cultures, animal transgenesis, qPCR, western blot, immunohistochemistry, ChIP assay, Elisa Results: Inflammatory cascades in murine and human astrocytes activated by inflammatory cytokines overrepresented in AD brains such as TNFa relied on the interaction between the transcription factor Foxo3 and the phosphatase calcineurin. Foxo/calcineurin complexes were observed in AD brains and transgenic mice (APP/PS1) and correlated with pathology. Genetic modulation of calcineurin in astrocytes at different stages of the pathological process in APP/PS1 mice indicated that inflammation is a key step in disease progression and its regulation is therapeutically significant. Full protection was achieved in the initial stages of the disease, with a protracted development of amyloidosis and preserved cognition. Accordingly, disruption of the Foxo/calcineurin association after IGF-I treatment was sufficient to reduce AD symptoms. Conclusions: The results indicate that inflammatory processes in astrocytes constitute a driving force of AD pathology. P4-160
EFFECT OF LACTOFERRIN ON Aß NEUROTOXICITY AND ITS CLEARANCE
Riikka Heikkinen1, Tarja Malm1, Susanna Boman1, Susanna Kuhmonen1, Toni Ahtoniemi2, Anu Muona2, Heikki Tanila1,3, Jari Koistinaho1,4, Milla Koistinaho2, 1Department of Neurobiology, A.I.V. Institute for Molecular Sciences, Biocenter Kuopio, University of Kuopio, Kuopio, Finland; 2 Medeia Therapeutics Ltd., Kuopio, Finland; 3Department of Neurology, Kuopio University Hospital, Kuopio, Finland; 4Department of Oncology, Kuopio University Hospital, Kuopio, Finland. Contact e-mail: riikka. [email protected]
Haruhisa Sato1,2, Li An2,3, Toshio Kawamata4, Yoshihiro Konishi5, Ikuo Tooyama2, 1Department of Pathology, Marutamachi Hospital, Kyoto, Japan; 2Molecular Neuroscience Research Center, Shiga University of Medical Science, Otsu, Japan; 3China Medical University, Shenyang, China; 4Faculty of Health Sciences, Kobe University School of Medicine, Kobe, Japan; 5Department of Clinical Research, Nishi-Tottori National Hospital, Tottori, Japan. Contact e-mail: [email protected]
Background: Stroke is a major cause of disability in the elderly. In particular, cerebrovascular lesions may contribute to the pathogenesis of Alzheimer’s disease (AD), as up to 90 % of patients show cerebrovascular pathology at autopsy. Interestingly, several transgenic mouse models of AD have shown increased vulnerability to ischemia at an early age. Because recent studies indicate that clinically safe intravenous immunoglobulin (IVIG) dose of 2 g/kg provides protection against focal brain ischemia in young wild-type (wt) mice, we investigated whether IVIG could abolish the increased vulnerability to ischemic injury in aged APP/PS1 transgenic mice. We also tested the effect of IVIG in a model of global brain ischemia affecting the brain regions important for memory functions. Methods: Permanent middle cerebral artery occlusion was induced in male Balb/c, female APP/PS1 mice and their C57Bl wt controls. The mice were given an intravenous injection of 0.5 or 1.0 g/kg of IVIG (Gammagard Liquid, Baxter) or saline 1 h before or 0, 1 or 3 h after the onset of ischemia. Lesion volume was determined by T2 weighted 9.4 T MRI 24 and 72 h after the ischemia in Balb/cA mice and by TTC-staining in APP/PS1 mice and their controls. Global forebrain ischemia was induced in C57Bl/6 wt mice by reversible compression of carotid arteries. IVIG (0.5 g/kg) or saline was given 1 h after the ischemia and effect on cognitive decline was tested in
Background: Lactoferrin (lactotransferrin; LF), an 80 kDa iron-binding protein, is greatly upregulated in Alzheimer’s disease (AD) brain. However, its function remains unclear. LF is strongly associated with diffuse and consolidated amyloid deposits in AD, LF may be involved in b-amyloid peptide (Ab) deposition. In this study, we investigated the effect of LF on Aß neurotoxicity and its clearance using cell line. Methods: Human IMR-32 cells and mouse astrocyte cells (purchased from ATCC) were used in this study. Ab42 and Ab40 as well as fluorescent Ab40 were purchased from AnaSpec, Inc. (San Jose, CA USA) or Peptide Institute (Osaka, Japan). IMR-32 cells were incubated for 24 hours with and without 10 mM of Aß42 combined with 0, 10, 50 or 100 mg/ml of LF. Cell vulnerability was examined by the LDH assay. Astrocytes were incubated for 30 min in media containing 10 mg/ml of Aß40, Aß42, fluorescent Ab40 combined with 0, 10, 40 or 160 mg/ml of LF. Fluorescent signals were observed by a confocal microscopy or fluorescent microscopy. After incubation for 30 min, cells were recovered. The uptake of Aß40 or Aß42 was analyzed by Western blots. Results: The LDH assay showed that IMR-32 cells damaged by Aß42 were not protected by LF. Image analysis clearly shows that LF inhibited the internalization of fluorescent Aß40 into astrocytes dose-dependently. Similarly, Western blot analysis also showed that LF inhibited the internalization of Aß40 and