- Email: [email protected]
NEUROPATHOGENIC FUNCTION OF A NOVEL TAU REGULATOR IN TAU MODELS OF ALZHEIMER’S DISEASE
Poster Presentations: Sunday, July 16, 2017 SUNDAY, JULY 16, 2017 ALZHEIMER’S ASSOCIATION INTERNATIONAL CONFERENCE POSTER PRESENTATIONS P1
P1-001
IDENTIFYING THE NEURONAL AbIMMUNOPOSITIVE POOL WITHIN THE HUMAN HIPPOCAMPUS
Lindsay A. Welikovitch, Sonia Do Carmo, A. Claudio Cuello, McGill University, Montreal, QC, Canada. Contact e-mail: lindsay. [email protected] Background: The established neuropathological features of Alzheimer’s disease (AD) include the presence of extracellular amyloid-b (Ab) plaques within the brain. Despite the fact that the AD-neuropathology is typically studied in already-diagnosed patients, it is now apparent that these neuropathological events evolve silently for decades before symptom-onset. Indeed it has been shown by our lab and others’ that Ab first accumulates intraneuronally (iAb) before the appearance of extracellular plaques both in humans and animal models of AD. Despite its apparent intraneuronal localization early in the disease-process, studying the occurrence and pathological relevance of iAb has been confounded by much controversial debate: antibodies targeting Ab peptides typically show specificity for the amyloid precursor protein (APP), which is physiologically abundant within the intraneuronal space. High-resolution fluorescent microscopy imaging of human brain material is made especially difficult by the presence of auto-fluorescent lipofuscin, which occludes the detection of true immunoreactive sites. Methods: To detect intraneuronal Ab and APP, human hippocampal tissue from control subjects was probed using highly specific Ab- and APP-directed antibodies, McSA1 and pab27576 respectively, and imaged by high-resolution confocal microscopy and super-resolution structured illumination microscopy. To measure the extent of co-colocalization between neuronal Ab- and APP-immunoreactive sites, Manders’ and Pearson’s coefficients were calculated using ImageJ-image analysis. Antibody specificity was then tested by peptide-adsorption and MALDI-mass spectrometry. Results: We show that neurons within the control human hippocampus carry appreciable amounts of detectable intraneuronal Abimmunoreactive material, and that these immunoreactive sites are distinguishable from those associated with APP; indeed, co-localization analysis revealed that over 90% of Ab-immunoreactive sites are spatially distinct from those associated with APP. The specificity of the antibodies used was verified by immunohistochemistry following peptide-adsorption as well as MALDI-mass spectrometry analysis. Conclusions: Our results definitively reveal the presence of an Ab-immunopositive pool within the neuronal space, which is spatially distinct from that associated with APP. These results validate the physiological occurrence of the intraneuronal Ab species, implicating the toxic intracellular material as an early feature of the asymptomatic phase of AD.
P1-002
NEUROPATHOGENIC FUNCTION OF A NOVEL TAU REGULATOR IN TAU MODELS OF ALZHEIMER’S DISEASE
Jisu Park, Hyunwoo Choi, Youngdoo Kim, Yong-Keun Jung, Seoul National University, Seoul, Republic of South Korea. Contact e-mail: [email protected]
P231
Background: Abnormal modification of tau and its deposition in brains is a marked characteristic of Alzheimer’s disease. Tau phosphorylation by kinases including GSK3ß and CDK5 leads to its accumulation in the somatodendritic compartment of neurons, forming aggregates and neurofibrillary tangles at last. Those aggregates and NFTs are neurotoxic and result in neuronal cell death. Methods: HT22, SH-SY5Y cells were transfected with full-length human ALK to activate downstream signal. Antagonistic/agonistic antibody (mAb30, mAb46) were treated to primary hippocampal neurons to inhibit or activate ALK. The roles of ALK in tau-mediated neurotoxicity and memory loss were investigated in vivo, using Tau fly model and 3xTg-AD mouse model. Results: ALK activation by overexpression or ALK agonistic antibody treatment causes tau phosphorylation and increased tau stability in neuronal cell lines and primary neurons. Tauopathic effect of ALK relies on its tyrosine kinase activity and downstream tau kinases, ERK and CDK5. Furthermore, ALK impedes tau degradation by inhibiting autophagy flux in a CDK5 dependent manner, resulting in tau accumulation. D. melanogaster fly model with neuronal expression of ALK shows aggravated tau rough eye phenotype and shortened lifespan,whereas expression of kinase-dead ALK fails to develop those phenotypes. In mammals, primary hippocampal neurons treated with agonistic antibody displays reduced synaptic density. In addition, ALK activation by lentivirus injection accelerates memory loss in 3x-AD mice, while administration of ALK inhibitor (Ceritinib) recovers the memory impairment. Conclusions: Aberrant regulation of ALK activity in neurons is crucial for tau pathology found in AD. References: Ittner, L.M. and J. Gotz, Amyloid-beta and tau–a toxic pas de deux in Alzheimer’s disease. Nat Rev Neurosci, 2011. 12(2): p. 65-72. Moog-Lutz, C., et al., Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin. J Biol Chem, 2005. 280(28): p. 26039-48. Loren, C.E., et al., Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo. Genes Cells, 2001. 6(6): p. 531-44.
P1-003
IDENTIFICATION OF NOVEL GENETIC RISK VARIANTS FOR ALZHEIMER’S DISEASE IN THE HMGCR GENE LOCUS
Nathalie I. V. Nilsson1,2, Cynthia Picard2, Judes Poirier3,4,5, 1 McGill University, Montreal, QC, Canada; 2Douglas Mental Health University Institute Research Centre, Montreal, QC, Canada; 3Centre for the Studies on the Prevention of Alzheimer’s Disease, Douglas Mental Health University Institute, Montreal, QC, Canada; 4Douglas Mental Health University Institute, Montreal, QC, Canada; 5Centre for Studies on Prevention of Alzheimer’s Disease (StoP-AD Centre), Douglas Mental Health Institute, Montreal, QC, Canada. Contact e-mail: nathalie. [email protected] Background: Cholesterol lowering drugs inhibiting HMG-CoA
reductase activity (statins) have been found to be protective in retrospective analyses of sporadic AD. Recently, a genetic variant in the HMGCR gene, also associated with reduced reductase activity, was found to be protective in AD (Leduc et al., 2015). This led us to systematically investigate all the genetic polymorphisms at the HMGCR locus with a population frequency greater than 5%, and their relationship to AD risk and HMGCR expression. Methods: Whole-genome sequencing, plasma, and cerebrospinal fluid (CSF) biomarker data were obtained from the Alzheimer’s Disease
P1-001
IDENTIFYING THE NEURONAL AbIMMUNOPOSITIVE POOL WITHIN THE HUMAN HIPPOCAMPUS
Lindsay A. Welikovitch, Sonia Do Carmo, A. Claudio Cuello, McGill University, Montreal, QC, Canada. Contact e-mail: lindsay. [email protected] Background: The established neuropathological features of Alzheimer’s disease (AD) include the presence of extracellular amyloid-b (Ab) plaques within the brain. Despite the fact that the AD-neuropathology is typically studied in already-diagnosed patients, it is now apparent that these neuropathological events evolve silently for decades before symptom-onset. Indeed it has been shown by our lab and others’ that Ab first accumulates intraneuronally (iAb) before the appearance of extracellular plaques both in humans and animal models of AD. Despite its apparent intraneuronal localization early in the disease-process, studying the occurrence and pathological relevance of iAb has been confounded by much controversial debate: antibodies targeting Ab peptides typically show specificity for the amyloid precursor protein (APP), which is physiologically abundant within the intraneuronal space. High-resolution fluorescent microscopy imaging of human brain material is made especially difficult by the presence of auto-fluorescent lipofuscin, which occludes the detection of true immunoreactive sites. Methods: To detect intraneuronal Ab and APP, human hippocampal tissue from control subjects was probed using highly specific Ab- and APP-directed antibodies, McSA1 and pab27576 respectively, and imaged by high-resolution confocal microscopy and super-resolution structured illumination microscopy. To measure the extent of co-colocalization between neuronal Ab- and APP-immunoreactive sites, Manders’ and Pearson’s coefficients were calculated using ImageJ-image analysis. Antibody specificity was then tested by peptide-adsorption and MALDI-mass spectrometry. Results: We show that neurons within the control human hippocampus carry appreciable amounts of detectable intraneuronal Abimmunoreactive material, and that these immunoreactive sites are distinguishable from those associated with APP; indeed, co-localization analysis revealed that over 90% of Ab-immunoreactive sites are spatially distinct from those associated with APP. The specificity of the antibodies used was verified by immunohistochemistry following peptide-adsorption as well as MALDI-mass spectrometry analysis. Conclusions: Our results definitively reveal the presence of an Ab-immunopositive pool within the neuronal space, which is spatially distinct from that associated with APP. These results validate the physiological occurrence of the intraneuronal Ab species, implicating the toxic intracellular material as an early feature of the asymptomatic phase of AD.
P1-002
NEUROPATHOGENIC FUNCTION OF A NOVEL TAU REGULATOR IN TAU MODELS OF ALZHEIMER’S DISEASE
Jisu Park, Hyunwoo Choi, Youngdoo Kim, Yong-Keun Jung, Seoul National University, Seoul, Republic of South Korea. Contact e-mail: [email protected]
P231
Background: Abnormal modification of tau and its deposition in brains is a marked characteristic of Alzheimer’s disease. Tau phosphorylation by kinases including GSK3ß and CDK5 leads to its accumulation in the somatodendritic compartment of neurons, forming aggregates and neurofibrillary tangles at last. Those aggregates and NFTs are neurotoxic and result in neuronal cell death. Methods: HT22, SH-SY5Y cells were transfected with full-length human ALK to activate downstream signal. Antagonistic/agonistic antibody (mAb30, mAb46) were treated to primary hippocampal neurons to inhibit or activate ALK. The roles of ALK in tau-mediated neurotoxicity and memory loss were investigated in vivo, using Tau fly model and 3xTg-AD mouse model. Results: ALK activation by overexpression or ALK agonistic antibody treatment causes tau phosphorylation and increased tau stability in neuronal cell lines and primary neurons. Tauopathic effect of ALK relies on its tyrosine kinase activity and downstream tau kinases, ERK and CDK5. Furthermore, ALK impedes tau degradation by inhibiting autophagy flux in a CDK5 dependent manner, resulting in tau accumulation. D. melanogaster fly model with neuronal expression of ALK shows aggravated tau rough eye phenotype and shortened lifespan,whereas expression of kinase-dead ALK fails to develop those phenotypes. In mammals, primary hippocampal neurons treated with agonistic antibody displays reduced synaptic density. In addition, ALK activation by lentivirus injection accelerates memory loss in 3x-AD mice, while administration of ALK inhibitor (Ceritinib) recovers the memory impairment. Conclusions: Aberrant regulation of ALK activity in neurons is crucial for tau pathology found in AD. References: Ittner, L.M. and J. Gotz, Amyloid-beta and tau–a toxic pas de deux in Alzheimer’s disease. Nat Rev Neurosci, 2011. 12(2): p. 65-72. Moog-Lutz, C., et al., Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin. J Biol Chem, 2005. 280(28): p. 26039-48. Loren, C.E., et al., Identification and characterization of DAlk: a novel Drosophila melanogaster RTK which drives ERK activation in vivo. Genes Cells, 2001. 6(6): p. 531-44.
P1-003
IDENTIFICATION OF NOVEL GENETIC RISK VARIANTS FOR ALZHEIMER’S DISEASE IN THE HMGCR GENE LOCUS
Nathalie I. V. Nilsson1,2, Cynthia Picard2, Judes Poirier3,4,5, 1 McGill University, Montreal, QC, Canada; 2Douglas Mental Health University Institute Research Centre, Montreal, QC, Canada; 3Centre for the Studies on the Prevention of Alzheimer’s Disease, Douglas Mental Health University Institute, Montreal, QC, Canada; 4Douglas Mental Health University Institute, Montreal, QC, Canada; 5Centre for Studies on Prevention of Alzheimer’s Disease (StoP-AD Centre), Douglas Mental Health Institute, Montreal, QC, Canada. Contact e-mail: nathalie. [email protected] Background: Cholesterol lowering drugs inhibiting HMG-CoA
reductase activity (statins) have been found to be protective in retrospective analyses of sporadic AD. Recently, a genetic variant in the HMGCR gene, also associated with reduced reductase activity, was found to be protective in AD (Leduc et al., 2015). This led us to systematically investigate all the genetic polymorphisms at the HMGCR locus with a population frequency greater than 5%, and their relationship to AD risk and HMGCR expression. Methods: Whole-genome sequencing, plasma, and cerebrospinal fluid (CSF) biomarker data were obtained from the Alzheimer’s Disease