- Email: [email protected]
COMPUTATIONAL APPROACHES IN PRECLINICAL DIAGNOSTICS AND PROGNOSIS FOR ALZHEIMER’S DISEASE
Poster Presentations: Tuesday, July 18, 2017
protein that is known to define the dendritic spine morphology. It is also involved in synaptic plasticity that mediates contextual fear conditioning. The primary objective of this study was to delineate the molecular mechanisms underlying spine loss and behavioral dysfunction seen early in AD. Methods: We used APPSwe/PS1DE9 mice (APP/PS1) and litter mate WT controls for our experiments. Contextual fear conditioning was used to assess associative fear learning and memory. We used an innovative method to isolate highly enriched F-Actin and G-Actin fractions from synaptosomes from cortices of 1, 2, 4 month old (pre-plaque phase) and 9 month old APP/PS1 mice and age matched controls. We also utilized AMS-derivatization approach to determine the reduced form of F-actin. Statistical comparisons were performed using unpaired t-tests or ANOVA followed by posthoc test. Results: We found significant decrease in F-actin levels in the synaptosomes from 1 month old APP/PS1 mice and this reduction was sustained until 9 months when overt symptoms are observed. While, F-actin levels were unaffected in post-nuclear supernatant across the ages examined. We observed loss of reduced F-actin but not reduced G-actin in the synaptosomes of APP/PS1 mice. Synaptosomal actin-Sglutathionylation, which is known to hinder F-actin polymerization, was significantly increased in 1 month old APP/PS1 mice. Interestingly the contextual fear conditioning behavioral deficits seen in APP/PS1 mice was reversed by a single intrathecal dose (500 ng/25 gram mouse) of Jasplakinolide, an actin polymerizing agent. Further, latrunculin A (500 ng/25 gram mouse), which promotes actin depolymerization induced contextual fear conditioning deficits in WT mice. Conclusions: We demonstrate that the cytoskeletal organization of F-actin is perturbed in synaptic compartment early in AD and leads to behavioral deficits, which can be reversed by actin polymerizing agents. Our results indicate that F-actin is an early target in the pathogenesis of AD.
P3-185
LOSS OF F-ACTIN IN SYNAPTOSOMES CORRELATES WITH COGNITIVE DYSFUNCTION (BRAAK STAGING, B-AMYLOID LOAD AND TANGLE LOAD) IN PATIENTS WITH MCI AND AD
Vijayalakshmi Ravindranath1,2, Ajit Ray3, Reddy Kommaddi3, David A. Bennett4, 1Centre for Neuroscience, Indian Institute of Science, Bengaluru, India; 2Centre for Brain Research, Indian Institute of Science, Bengaluru, India; 3Indian Institute of Science, Bangalore, India; 4Rush University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) has been described by
its biological signatures that mediate synaptic dysfunction and loss of dendritic spines. Neuronal cytoskeletal protein, such as F-actin (filamentous actin) are critical modulator of dendritic spine morphology and also regulates its function. Thus, maintaining the ratio of F-actin and G-actin within the dendritic spines is essential for optimal synaptic function. Our aim was to determine the status of synaptic F-actin levels in postmortem tissue from patients with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD patients. Methods: Autopsy human brain tissues were obtained from Rush Alzheimer’s Disease Center, Chicago, IL, USA. Total of 36 brains were examined (12 each of NCI, MCI and AD). Frontal neocortical tissue from each human brain
P1005
was thawed on ice and used for the preparation of synaptosomes. Further, we used an innovative method to isolate highly enriched F-Actin and G-Actin fractions from synaptosomes. Results: F-actin levels in synaptosomes prepared from NCI, MCI and AD postmortem tissue showed graded decrease were significantly correlated. Furthermore, significant correlation was also seen between the decrease in synaptosomal F-actin and poor performance in several cognitive tests (Global cognitive score, Episodic memory score, Working memory score) as also reflected Braak staging, b-amyloid load and tangle load. Conclusions: Our findings provide evidence that loss of F-actin at the synapse correlates with the histopathological and behavioral dysfunctions seen in patients with mild cognitive impairment (MCI) and AD.
P3-186
COMPUTATIONAL APPROACHES IN PRECLINICAL DIAGNOSTICS AND PROGNOSIS FOR ALZHEIMER’S DISEASE
Olivera Mitrasinovic1,2, Natasa Kablar3, 1PCM - Pathway Connectivity Maps, Inc., Mountain View, CA, USA; 2Stanford University School of Medicine, Stanford, CA, USA; 3PCM Pathway Connectivity Maps, Inc., Mountain View, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is referred to as “disease
with many faces”, biggest reason being that variability in the phenotypic manifestations are consequence of different molecular pathways underlying what is known as single, consensus prototype pathology. This variability represents one of the biggest challenges in developing specific diagnostic test. Alternative is identification of multiple biomarkers that in synchrony, accurately predict and track AD. Technologies that enable screening for complex disease markers (CDM) with high-throughput capabilities are of particular interest. Methods: We use MathWorks MATLAB to explore accuracy and usefulness of computational approaches in AD diagnostics. Experimentation is initiated by “sketching” broad mechanism of AD pathogenesis, and identifying most significant neuroinflammatory and neuro-apoptotic factors, then connecting with factors of interest. Comprehensive PubMed search allows accumulation of cumulative knowledge about molecular pathway, maximizing the output accuracy. Results: Figure shows schematic diagram for applying computational methods in elucidation of AD pathogenesis. It consists of: 1) Identifying the functional diagram or sub-set of intermolecular signal transaction interactions; 2) Biochemical equations for the molecular mechanism under study are created; 3) Conversion to the differential equations. The parameter values are then assigned based on quantitative literature information, or are otherwise “assumed”; 4) MAT LAB computations. We have demonstrated 100% accuracy of the computational results by comparison with Western and ELISA quantitative time-course measurements for inflammatory factors such as IL-1a, TNF-a, MAPK, JNK and neurotrophic factors, such as BDNF and NF-kB. Time-course graphic allows signaling progression to be determined when and whether two factors of interest are connected in signaling pathway. Using this approach we have identified (IIAC 2011) critical importance for small factor, MyD88 in signal transmission along IL-1a/ TRAF/TNFa/NF-kB pathway. Conclusions: We show how computational approach is useful in effective discovery of
P1006
Poster Presentations: Tuesday, July 18, 2017
nutes of supervised training three times per week. Aerobic exercise consisted of treadmill walking or jogging at an initial intensity that correlated with 50% VO2max and gradually increased to 70% VO2max. Stretch exercise consisted of a variety of dynamic stretch positions that were held for 15-30 second intervals. Fasting blood samples were taken at baseline, 3, 6, and 12 months of exercise training. Enzyme-linked photometric assay was used to measure nitrite/nitrate (NOx) levels in plasma and urine. Descriptive statistics were computed to assess patient baseline demographics for differences in exercise groups (Aerobic vs. Stretch). Significance level was set at P< 0.05 for all tests, and completed all analyses using the Statistical Analysis System (SAS) software 9.3. Results: Six months of exercise resulted in decrease plasma NOx levels in both groups. Percent change in plasma NOx was significantly different from baseline to 12 months (p¼0.05), but percent change in urine levels did not reach significance level. Plasma or urine NOx levels did not significantly correlate with age, MMSE, NO3 gene or VO2max. Conclusions: These results, suggests that exercise traininginduced changes in NOx levels may be independent of eNOS gene in older African American MCI participants. Consequent vasodilatory effects of NOx may be beneficial to brain perfusion at the prodromal stage of AD. signaling interactions in AD. Quantitative variability is the main reason for methodology not being Academically validated. However, early markers identification is warranted, thus preceding and accelerating validation studies, while its low-cost makes it exceptionally useful in translational studies. Support: Alzheimer’s Association, in part (OMM).
P3-187
EFFECTS OF EXERCISE ON PLASMA NITRITE/NITRATE LEVELS, AND NITRIC OXIDE SYNTHASE ACTIVITY IN ELDERLY AFRICAN AMERICANS WITH MILD COGNITIVE IMPAIRMENT
Oyonumo Ntekim1, Julius S. Ngwa2, Joanne S. Allard2, Thomas V. Fungwe1, Graham A. Lennox3,4, Richard F. Gillum5, Chimene Castor3, Thomas O. Obisesan2, 1Howard University, Washington, DC, USA; 2Howard University College of Medicine, Washington, DC, USA; 3 Howard University, Washington, DC, USA; 4College of Nursing and Allied Health Sciences, Washington, DC, USA; 5Howard University College of Medicine, Washington DC, USA. Contact e-mail: oyonumo.ntekim@ howard.edu Background: Studies have shown a positive correlation between exercise and vasodilatation, and this may be beneficial to brain perfusion and possibly memory. Proposed mediators for this observed effects of exercise include increases in nitric oxide (NO) via up-regulation of the rate controlling enzymes nitric oxide synthase (NOS) isoform (eNOS. Because endothelial dysfunction is an early marker of atherosclerosis, and hypoxia, impaired capacity of the vascular endothelium to release NO and NOS, and consequent vascular dysregulations have been implicated in cognitive impairment. This study seeks to establish whether mild cognitively impaired older adults exhibit NO and NOS dysregulation while on a supervised exercise program. Methods: African American older adult’s 55 year, determined to have mild cognitive impairment, were enrolled and randomized into a six-month program of either treadmill exercise or stretch exercise. Both groups engaged in 40 mi-
P3-188
MUTAGENESIS OF THE GENERAL AMYLOID INTERACTION MOTIF (GAIM) REVEALS A STRUCTURE-ACTIVITY RELATIONSHIP FOR MISFOLDED BETA-AMYLOID AND TAU AGGREGATES
Rajaraman Krishnan1, Eva Asp1, Ming Proschitsky1, Michal Lulu1, Charlotte Chung1, Cassandra Rockwell-Postel1, Edward Holgate2, Jonathan M. Levenson1, Kim McDowell1, Haim Tsubery1, Jason S. Wright1, Richard A. Fisher1, 1Proclara Biosciences, Cambridge, MA, USA; 2Abzena, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: The general amyloid interaction motif (GAIM) derived from the M13 phage tip protein g3p binds a wide variety of amyloid aggregates in a conformation-dependent manner (Krishnan 2014). Dimeric GAIM-Ig-fusions robustly bind and remodel Ab42 amyloid aggregates and inhibit tau aggregate propagation in primary neuronal cells. In transgenic models of AD and tauopathy, GAIM-fusion treatment reduces Ab plaque load, phospho-tau levels and improves cognition (Levenson 2016). In this study, we explored the mechanism of GAIM-mediated remodeling of amyloid aggregates by mutagenesis. Using this data, we designed a next generation Ig-fusion, NPT189, which in addition to showing improved binding potency to multiple aggregates, has reduced potential for immunogenicity after removal of potential Tcell epitopes. Methods: Data obtained from H/D exchange NMR studies, GAIM-peptide-based inhibition assays, TolA binding, and computational modeling was used for targeted mutagenesis of the GAIM scaffold. Stable, high expressing variants were then screened for binding to Ab42 and Tau-K18 fibers using SPR and binding ELISA assays. High binding variants were further screened for Ab42 fiber remodeling as well as tau and a-synuclein transmission inhibition activities. An independent mutagenesis study was carried out to sequentially eliminate
protein that is known to define the dendritic spine morphology. It is also involved in synaptic plasticity that mediates contextual fear conditioning. The primary objective of this study was to delineate the molecular mechanisms underlying spine loss and behavioral dysfunction seen early in AD. Methods: We used APPSwe/PS1DE9 mice (APP/PS1) and litter mate WT controls for our experiments. Contextual fear conditioning was used to assess associative fear learning and memory. We used an innovative method to isolate highly enriched F-Actin and G-Actin fractions from synaptosomes from cortices of 1, 2, 4 month old (pre-plaque phase) and 9 month old APP/PS1 mice and age matched controls. We also utilized AMS-derivatization approach to determine the reduced form of F-actin. Statistical comparisons were performed using unpaired t-tests or ANOVA followed by posthoc test. Results: We found significant decrease in F-actin levels in the synaptosomes from 1 month old APP/PS1 mice and this reduction was sustained until 9 months when overt symptoms are observed. While, F-actin levels were unaffected in post-nuclear supernatant across the ages examined. We observed loss of reduced F-actin but not reduced G-actin in the synaptosomes of APP/PS1 mice. Synaptosomal actin-Sglutathionylation, which is known to hinder F-actin polymerization, was significantly increased in 1 month old APP/PS1 mice. Interestingly the contextual fear conditioning behavioral deficits seen in APP/PS1 mice was reversed by a single intrathecal dose (500 ng/25 gram mouse) of Jasplakinolide, an actin polymerizing agent. Further, latrunculin A (500 ng/25 gram mouse), which promotes actin depolymerization induced contextual fear conditioning deficits in WT mice. Conclusions: We demonstrate that the cytoskeletal organization of F-actin is perturbed in synaptic compartment early in AD and leads to behavioral deficits, which can be reversed by actin polymerizing agents. Our results indicate that F-actin is an early target in the pathogenesis of AD.
P3-185
LOSS OF F-ACTIN IN SYNAPTOSOMES CORRELATES WITH COGNITIVE DYSFUNCTION (BRAAK STAGING, B-AMYLOID LOAD AND TANGLE LOAD) IN PATIENTS WITH MCI AND AD
Vijayalakshmi Ravindranath1,2, Ajit Ray3, Reddy Kommaddi3, David A. Bennett4, 1Centre for Neuroscience, Indian Institute of Science, Bengaluru, India; 2Centre for Brain Research, Indian Institute of Science, Bengaluru, India; 3Indian Institute of Science, Bangalore, India; 4Rush University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) has been described by
its biological signatures that mediate synaptic dysfunction and loss of dendritic spines. Neuronal cytoskeletal protein, such as F-actin (filamentous actin) are critical modulator of dendritic spine morphology and also regulates its function. Thus, maintaining the ratio of F-actin and G-actin within the dendritic spines is essential for optimal synaptic function. Our aim was to determine the status of synaptic F-actin levels in postmortem tissue from patients with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD patients. Methods: Autopsy human brain tissues were obtained from Rush Alzheimer’s Disease Center, Chicago, IL, USA. Total of 36 brains were examined (12 each of NCI, MCI and AD). Frontal neocortical tissue from each human brain
P1005
was thawed on ice and used for the preparation of synaptosomes. Further, we used an innovative method to isolate highly enriched F-Actin and G-Actin fractions from synaptosomes. Results: F-actin levels in synaptosomes prepared from NCI, MCI and AD postmortem tissue showed graded decrease were significantly correlated. Furthermore, significant correlation was also seen between the decrease in synaptosomal F-actin and poor performance in several cognitive tests (Global cognitive score, Episodic memory score, Working memory score) as also reflected Braak staging, b-amyloid load and tangle load. Conclusions: Our findings provide evidence that loss of F-actin at the synapse correlates with the histopathological and behavioral dysfunctions seen in patients with mild cognitive impairment (MCI) and AD.
P3-186
COMPUTATIONAL APPROACHES IN PRECLINICAL DIAGNOSTICS AND PROGNOSIS FOR ALZHEIMER’S DISEASE
Olivera Mitrasinovic1,2, Natasa Kablar3, 1PCM - Pathway Connectivity Maps, Inc., Mountain View, CA, USA; 2Stanford University School of Medicine, Stanford, CA, USA; 3PCM Pathway Connectivity Maps, Inc., Mountain View, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is referred to as “disease
with many faces”, biggest reason being that variability in the phenotypic manifestations are consequence of different molecular pathways underlying what is known as single, consensus prototype pathology. This variability represents one of the biggest challenges in developing specific diagnostic test. Alternative is identification of multiple biomarkers that in synchrony, accurately predict and track AD. Technologies that enable screening for complex disease markers (CDM) with high-throughput capabilities are of particular interest. Methods: We use MathWorks MATLAB to explore accuracy and usefulness of computational approaches in AD diagnostics. Experimentation is initiated by “sketching” broad mechanism of AD pathogenesis, and identifying most significant neuroinflammatory and neuro-apoptotic factors, then connecting with factors of interest. Comprehensive PubMed search allows accumulation of cumulative knowledge about molecular pathway, maximizing the output accuracy. Results: Figure shows schematic diagram for applying computational methods in elucidation of AD pathogenesis. It consists of: 1) Identifying the functional diagram or sub-set of intermolecular signal transaction interactions; 2) Biochemical equations for the molecular mechanism under study are created; 3) Conversion to the differential equations. The parameter values are then assigned based on quantitative literature information, or are otherwise “assumed”; 4) MAT LAB computations. We have demonstrated 100% accuracy of the computational results by comparison with Western and ELISA quantitative time-course measurements for inflammatory factors such as IL-1a, TNF-a, MAPK, JNK and neurotrophic factors, such as BDNF and NF-kB. Time-course graphic allows signaling progression to be determined when and whether two factors of interest are connected in signaling pathway. Using this approach we have identified (IIAC 2011) critical importance for small factor, MyD88 in signal transmission along IL-1a/ TRAF/TNFa/NF-kB pathway. Conclusions: We show how computational approach is useful in effective discovery of
P1006
Poster Presentations: Tuesday, July 18, 2017
nutes of supervised training three times per week. Aerobic exercise consisted of treadmill walking or jogging at an initial intensity that correlated with 50% VO2max and gradually increased to 70% VO2max. Stretch exercise consisted of a variety of dynamic stretch positions that were held for 15-30 second intervals. Fasting blood samples were taken at baseline, 3, 6, and 12 months of exercise training. Enzyme-linked photometric assay was used to measure nitrite/nitrate (NOx) levels in plasma and urine. Descriptive statistics were computed to assess patient baseline demographics for differences in exercise groups (Aerobic vs. Stretch). Significance level was set at P< 0.05 for all tests, and completed all analyses using the Statistical Analysis System (SAS) software 9.3. Results: Six months of exercise resulted in decrease plasma NOx levels in both groups. Percent change in plasma NOx was significantly different from baseline to 12 months (p¼0.05), but percent change in urine levels did not reach significance level. Plasma or urine NOx levels did not significantly correlate with age, MMSE, NO3 gene or VO2max. Conclusions: These results, suggests that exercise traininginduced changes in NOx levels may be independent of eNOS gene in older African American MCI participants. Consequent vasodilatory effects of NOx may be beneficial to brain perfusion at the prodromal stage of AD. signaling interactions in AD. Quantitative variability is the main reason for methodology not being Academically validated. However, early markers identification is warranted, thus preceding and accelerating validation studies, while its low-cost makes it exceptionally useful in translational studies. Support: Alzheimer’s Association, in part (OMM).
P3-187
EFFECTS OF EXERCISE ON PLASMA NITRITE/NITRATE LEVELS, AND NITRIC OXIDE SYNTHASE ACTIVITY IN ELDERLY AFRICAN AMERICANS WITH MILD COGNITIVE IMPAIRMENT
Oyonumo Ntekim1, Julius S. Ngwa2, Joanne S. Allard2, Thomas V. Fungwe1, Graham A. Lennox3,4, Richard F. Gillum5, Chimene Castor3, Thomas O. Obisesan2, 1Howard University, Washington, DC, USA; 2Howard University College of Medicine, Washington, DC, USA; 3 Howard University, Washington, DC, USA; 4College of Nursing and Allied Health Sciences, Washington, DC, USA; 5Howard University College of Medicine, Washington DC, USA. Contact e-mail: oyonumo.ntekim@ howard.edu Background: Studies have shown a positive correlation between exercise and vasodilatation, and this may be beneficial to brain perfusion and possibly memory. Proposed mediators for this observed effects of exercise include increases in nitric oxide (NO) via up-regulation of the rate controlling enzymes nitric oxide synthase (NOS) isoform (eNOS. Because endothelial dysfunction is an early marker of atherosclerosis, and hypoxia, impaired capacity of the vascular endothelium to release NO and NOS, and consequent vascular dysregulations have been implicated in cognitive impairment. This study seeks to establish whether mild cognitively impaired older adults exhibit NO and NOS dysregulation while on a supervised exercise program. Methods: African American older adult’s 55 year, determined to have mild cognitive impairment, were enrolled and randomized into a six-month program of either treadmill exercise or stretch exercise. Both groups engaged in 40 mi-
P3-188
MUTAGENESIS OF THE GENERAL AMYLOID INTERACTION MOTIF (GAIM) REVEALS A STRUCTURE-ACTIVITY RELATIONSHIP FOR MISFOLDED BETA-AMYLOID AND TAU AGGREGATES
Rajaraman Krishnan1, Eva Asp1, Ming Proschitsky1, Michal Lulu1, Charlotte Chung1, Cassandra Rockwell-Postel1, Edward Holgate2, Jonathan M. Levenson1, Kim McDowell1, Haim Tsubery1, Jason S. Wright1, Richard A. Fisher1, 1Proclara Biosciences, Cambridge, MA, USA; 2Abzena, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: The general amyloid interaction motif (GAIM) derived from the M13 phage tip protein g3p binds a wide variety of amyloid aggregates in a conformation-dependent manner (Krishnan 2014). Dimeric GAIM-Ig-fusions robustly bind and remodel Ab42 amyloid aggregates and inhibit tau aggregate propagation in primary neuronal cells. In transgenic models of AD and tauopathy, GAIM-fusion treatment reduces Ab plaque load, phospho-tau levels and improves cognition (Levenson 2016). In this study, we explored the mechanism of GAIM-mediated remodeling of amyloid aggregates by mutagenesis. Using this data, we designed a next generation Ig-fusion, NPT189, which in addition to showing improved binding potency to multiple aggregates, has reduced potential for immunogenicity after removal of potential Tcell epitopes. Methods: Data obtained from H/D exchange NMR studies, GAIM-peptide-based inhibition assays, TolA binding, and computational modeling was used for targeted mutagenesis of the GAIM scaffold. Stable, high expressing variants were then screened for binding to Ab42 and Tau-K18 fibers using SPR and binding ELISA assays. High binding variants were further screened for Ab42 fiber remodeling as well as tau and a-synuclein transmission inhibition activities. An independent mutagenesis study was carried out to sequentially eliminate