- Email: [email protected]
Aß oligomer induced signal transduction at the post-synaptic density
Podium Presentations: Thursday, July 23, 2015
results indicate that this activity may be attributable to pre-fibrillar assemblies built from no more than eighty w7kDa Aß species. These results recommend further analysis of brain-derived w7 kDa Aß species, the mechanism by which they assemble and the structures which they form.
F5-02-02
Aß OLIGOMER INDUCED SIGNAL TRANSDUCTION AT THE POST-SYNAPTIC DENSITY
Adam Kaufman, Mikhail Kostylev, Laura Haas, Santiago Salazar, Stephen Strittmatter, Yale University, New Haven, CT, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease is characterized by Amyloid-ß (Aß) accumulation, with soluble oligomers (Aßo) being the most synaptotoxic. We sought to determine which Aß species correlated closely with disease progression. Once formed, Aßo acts to impair synpases through PrPCand mGluR5 to activate the Fyn tyrosine kinase, a Src-related enzyme. We sought to repurpose a Src family kinase inhibitor oncology compound for AD. Methods: We characterized multiple Aß forms throughout the lifespan of various Alzheimer model mice, and in post-mortem human brain by specific binding assays and size exclusion chromatography. Inhibition of Aßo signaling to Fyn, Pyk2 and Glu receptors by a Fyn kinase inhibitor was tested in brain slice assays. After Fyn kinase inhibitor or vehicle treatment of wild type and AD transgenic mice, memory was assessed by Morris water maze and novel object recognition. For these cohorts, APP metabolism, synaptic markers (SV2 and PSD-95), and targets of Fyn (Pyk2 and Tau) were studied by immunohistochemistry and by immunoblotting. Results: Aß exists in several populations, where PrPC-interacting oligomers are a high molecular weight Aß assembly present in multiple transgenic mice and human Alzheimer brain extract. Levels of PrPC-interacting Aßo match closely with mouse memory impairment. Fyn kinase inhibitor treatment potently inhibits both Aßo-induced signaling and downstream phosphorylation of the AD risk gene product, Pyk2, and of NR2B Glu receptors in brain slices. After 4 weeks of treatment, Fyn kinase inhibitor dosing of APP/PS1 transgenic mice fully rescues spatial memory deficits and synaptic depletion, without altering APP or Aß metabolism. Conclusions: PrPC-interacting Aßo species are high molecular weight from mouse and human brain and closely correlated with disease. Fyn inhibition reverses the effects of Aßo through the PrPC–mGluR5 complex at the synapse, and rescues transgenic mouse models of AD. F5-02-03
TARGETING Ab OLIGOMER FORMATION IN ALZHEIMER’S DISEASE: CHALLENGES AND ALTERNATIVES TO OLIGOMER-SPECIFIC THERAPEUTIC AVENUES
Timothy M. Ryan1, Blaine R. Roberts1, Gawain McColl1, Dominic J. Hare1, Mark G. Hinds2, Kevin J. Barnham1, Cyril C. Curtain1, Colin L. Masters1, 1The Florey Institute of Neuroscience and Mental Health, Parkville, Australia; 2The University of Melbourne, Melbourne, Australia. Contact e-mail: [email protected] Background: The field of Alzheimer’s Disease (AD) research is
focused strongly on the soluble oligomer theory, where Ab forms a neurotoxic oligomer that mediates synaptic dysfunction and neuronal loss. A range of theories on how this species obtains a toxic gain of function have been proposed, ranging from simple accumulation through to lipid or metal mediated modifications. None however have provided a defined target to isolate from hu-
P305
man brain, or viable a clinically viable therapeutic target. The lack of defined terminology in discussing these oligomeric species, incomplete characterisation and a general mystery about the role and biophysics of Ab in an intact brain have significantly hampered identifying how Ab self-association and oligomerisation are involved in the development of neurotoxicity. This raises the question of whether the approach of preventing, breaking down or clearing a specific endogenous Ab oligomer, that may or may not be present in vivo, is a viable approach to treat the disease. Are there other methods of targeting Ab oligomerisation, without trying to breakdown or modify specific toxic oligomers? An approach that does not require information on the composition and conformation of the endogenous oligomeric species in AD is to redirect Aß self-association to stable non-toxic oligomers that can be characterised. We have been investigating this approach, primarily using small molecules to induce specific oligomeric species. Methods: We investigated a range of therapeutic molecules primarily focusing on hydroxyquinolines. We used biophysical techniques, including analytical ultracentrifugation, size exclusion chromatography, NMR, and inductively coupled plasma mass spectroscopy in conjunction with multiple electrode array measurements of cell networks, and Caenorhabditis elegans transgenic models of Ab toxicity to characterise the effect of stabilising a small, specific oligomer of Ab. Results: We identified compounds that induced the formation of a dimeric Ab species that was non-toxic in cell culture, and reduced Ab toxicity in transgenic animal models of the disease. Conclusions: Our strategy to identify potential AD therapeutics may avoid the pitfalls that have so far beset therapeutic interventions based on the prevention, breakdown or clearance of a specific, endogenous oligomeric Ab species. F5-02-04
CEREBROSPINAL FLUID AMYLOID OLIGOMERS MEASURED IN A SUBSET OF THE AIBL AND OPTIMA AGE-MATCHED HEALTHY CONTROL AND ALZHEIMER’S COHORTS
Mary J. Savage1, Juliya Kalinina2, Dan Holder2, Julie Stromswold3, Michael Egan3, Omar Laterza4, Kenneth Emancipator4, David G. Darby5, Qiao-Xin Li6, Gordon Wilcock7, David Smith7, Colin L. Masters8, 1Merck, Rahway, NJ, USA; 2Merck and Company, West Point, PA, USA; 3Merck and Company, Upper Gwynedd, PA, USA; 4Merck and Company, Rahway, NJ, USA; 5University of Melbourne, Melbourne, Australia; 6The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia; 7Oxford University, Oxford, United Kingdom; 8The Florey Institute of Neuroscience and Mental Health, Parkville, Australia. Contact e-mail: mary_savage@ merck.com Background: Alzheimer’s disease (AD) is the most common neurodegenerative dementia in the elderly. The pathognomonic senile plaques found in brains of AD patients are comprised largely of aggregated amyloid-b (Ab) peptide. Several forms of soluble Ab oligomers have been identified and there is increasing evidence that these soluble oligomers are toxic Ab species. Methods: An Ab oligomer-specific assay which detects endogenous oligomers from cerebrospinal fluid (CSF) or from histologically confirmed human AD and healthy control subjects was previously developed and reported (J. Neuroscience 34:2884). Results: Using this assay, a robust 3-5 fold increase in Ab oligomers was observed in the CSF of AD patients (N¼63) vs. age-matched healthy controls (N¼54) from 2 commercial sources, as well as from SEC-fractionated aqueous supernatants of AD compared to non-AD cortex. To further confirm assay utility to measure Ab oligomers, and the correlation of these species with other AD-relevant endpoints, we
results indicate that this activity may be attributable to pre-fibrillar assemblies built from no more than eighty w7kDa Aß species. These results recommend further analysis of brain-derived w7 kDa Aß species, the mechanism by which they assemble and the structures which they form.
F5-02-02
Aß OLIGOMER INDUCED SIGNAL TRANSDUCTION AT THE POST-SYNAPTIC DENSITY
Adam Kaufman, Mikhail Kostylev, Laura Haas, Santiago Salazar, Stephen Strittmatter, Yale University, New Haven, CT, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease is characterized by Amyloid-ß (Aß) accumulation, with soluble oligomers (Aßo) being the most synaptotoxic. We sought to determine which Aß species correlated closely with disease progression. Once formed, Aßo acts to impair synpases through PrPCand mGluR5 to activate the Fyn tyrosine kinase, a Src-related enzyme. We sought to repurpose a Src family kinase inhibitor oncology compound for AD. Methods: We characterized multiple Aß forms throughout the lifespan of various Alzheimer model mice, and in post-mortem human brain by specific binding assays and size exclusion chromatography. Inhibition of Aßo signaling to Fyn, Pyk2 and Glu receptors by a Fyn kinase inhibitor was tested in brain slice assays. After Fyn kinase inhibitor or vehicle treatment of wild type and AD transgenic mice, memory was assessed by Morris water maze and novel object recognition. For these cohorts, APP metabolism, synaptic markers (SV2 and PSD-95), and targets of Fyn (Pyk2 and Tau) were studied by immunohistochemistry and by immunoblotting. Results: Aß exists in several populations, where PrPC-interacting oligomers are a high molecular weight Aß assembly present in multiple transgenic mice and human Alzheimer brain extract. Levels of PrPC-interacting Aßo match closely with mouse memory impairment. Fyn kinase inhibitor treatment potently inhibits both Aßo-induced signaling and downstream phosphorylation of the AD risk gene product, Pyk2, and of NR2B Glu receptors in brain slices. After 4 weeks of treatment, Fyn kinase inhibitor dosing of APP/PS1 transgenic mice fully rescues spatial memory deficits and synaptic depletion, without altering APP or Aß metabolism. Conclusions: PrPC-interacting Aßo species are high molecular weight from mouse and human brain and closely correlated with disease. Fyn inhibition reverses the effects of Aßo through the PrPC–mGluR5 complex at the synapse, and rescues transgenic mouse models of AD. F5-02-03
TARGETING Ab OLIGOMER FORMATION IN ALZHEIMER’S DISEASE: CHALLENGES AND ALTERNATIVES TO OLIGOMER-SPECIFIC THERAPEUTIC AVENUES
Timothy M. Ryan1, Blaine R. Roberts1, Gawain McColl1, Dominic J. Hare1, Mark G. Hinds2, Kevin J. Barnham1, Cyril C. Curtain1, Colin L. Masters1, 1The Florey Institute of Neuroscience and Mental Health, Parkville, Australia; 2The University of Melbourne, Melbourne, Australia. Contact e-mail: [email protected] Background: The field of Alzheimer’s Disease (AD) research is
focused strongly on the soluble oligomer theory, where Ab forms a neurotoxic oligomer that mediates synaptic dysfunction and neuronal loss. A range of theories on how this species obtains a toxic gain of function have been proposed, ranging from simple accumulation through to lipid or metal mediated modifications. None however have provided a defined target to isolate from hu-
P305
man brain, or viable a clinically viable therapeutic target. The lack of defined terminology in discussing these oligomeric species, incomplete characterisation and a general mystery about the role and biophysics of Ab in an intact brain have significantly hampered identifying how Ab self-association and oligomerisation are involved in the development of neurotoxicity. This raises the question of whether the approach of preventing, breaking down or clearing a specific endogenous Ab oligomer, that may or may not be present in vivo, is a viable approach to treat the disease. Are there other methods of targeting Ab oligomerisation, without trying to breakdown or modify specific toxic oligomers? An approach that does not require information on the composition and conformation of the endogenous oligomeric species in AD is to redirect Aß self-association to stable non-toxic oligomers that can be characterised. We have been investigating this approach, primarily using small molecules to induce specific oligomeric species. Methods: We investigated a range of therapeutic molecules primarily focusing on hydroxyquinolines. We used biophysical techniques, including analytical ultracentrifugation, size exclusion chromatography, NMR, and inductively coupled plasma mass spectroscopy in conjunction with multiple electrode array measurements of cell networks, and Caenorhabditis elegans transgenic models of Ab toxicity to characterise the effect of stabilising a small, specific oligomer of Ab. Results: We identified compounds that induced the formation of a dimeric Ab species that was non-toxic in cell culture, and reduced Ab toxicity in transgenic animal models of the disease. Conclusions: Our strategy to identify potential AD therapeutics may avoid the pitfalls that have so far beset therapeutic interventions based on the prevention, breakdown or clearance of a specific, endogenous oligomeric Ab species. F5-02-04
CEREBROSPINAL FLUID AMYLOID OLIGOMERS MEASURED IN A SUBSET OF THE AIBL AND OPTIMA AGE-MATCHED HEALTHY CONTROL AND ALZHEIMER’S COHORTS
Mary J. Savage1, Juliya Kalinina2, Dan Holder2, Julie Stromswold3, Michael Egan3, Omar Laterza4, Kenneth Emancipator4, David G. Darby5, Qiao-Xin Li6, Gordon Wilcock7, David Smith7, Colin L. Masters8, 1Merck, Rahway, NJ, USA; 2Merck and Company, West Point, PA, USA; 3Merck and Company, Upper Gwynedd, PA, USA; 4Merck and Company, Rahway, NJ, USA; 5University of Melbourne, Melbourne, Australia; 6The Florey Institute of Neuroscience and Mental Health, Melbourne, Australia; 7Oxford University, Oxford, United Kingdom; 8The Florey Institute of Neuroscience and Mental Health, Parkville, Australia. Contact e-mail: mary_savage@ merck.com Background: Alzheimer’s disease (AD) is the most common neurodegenerative dementia in the elderly. The pathognomonic senile plaques found in brains of AD patients are comprised largely of aggregated amyloid-b (Ab) peptide. Several forms of soluble Ab oligomers have been identified and there is increasing evidence that these soluble oligomers are toxic Ab species. Methods: An Ab oligomer-specific assay which detects endogenous oligomers from cerebrospinal fluid (CSF) or from histologically confirmed human AD and healthy control subjects was previously developed and reported (J. Neuroscience 34:2884). Results: Using this assay, a robust 3-5 fold increase in Ab oligomers was observed in the CSF of AD patients (N¼63) vs. age-matched healthy controls (N¼54) from 2 commercial sources, as well as from SEC-fractionated aqueous supernatants of AD compared to non-AD cortex. To further confirm assay utility to measure Ab oligomers, and the correlation of these species with other AD-relevant endpoints, we