Browsing by Author "Haynes, Kathryn A."

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    Aging x Environment x genetic risk for late onset Alzheimer’s disease results in alterations in cognitive function in mice independent of amyloid and tau pathology
    (Wiley, 2025-01-03) Williams, Sean-Paul Gerard; Santos, Diogo Francisco Silva; Haynes, Kathryn A.; Heaton, Nicholas; Hart, Jason T.; Kotredes, Kevin P.; Pandey, Ravi S.; Persohn, Scott C.; Eldridge, Kierra; Ingraham, Cynthia M.; Lloyd, Christopher D.; Wang, Nian; Sasner, Michael; Carter, Gregory W.; Territo, Paul R.; Lamb, Bruce T.; Howell, Gareth R.; Oblak, Adrian L.; Sukoff Rizzo, Stacey J.; Neurology, School of Medicine
    Background: Alzheimer’s disease (AD) research has been historically dominated with studies in mouse models expressing familial AD mutations; however, the majority of AD patients have the sporadic, late‐onset form of AD (LOAD). To address this gap, the IU/JAX/PITT MODEL‐AD Consortium has focused on development of mouse models that recapitulate LOAD by combining genetic risk variants with environmental risk factors and aging to enable more precise models to evaluate potential therapeutics. The present studies were undertaken to characterize cognitive and neurophysiological phenotypes in LOAD mice. Method: Two genetic risk factors, APOE4 and Trem2*R47H, were incorporated into C57BL/6J mice with humanized amyloid‐beta to produce the LOAD2 model (JAX# 030670). Male and female LOAD2 and WT mice were exposed to ad libitum 45% high‐fat diet from 2‐months of age (LOAD2+HFD or WT+HFD, respectively) throughout their lifespan and compared to LOAD2 and WT mice on control diet (+CD). Cognitive training began at 14‐months of age using a touchscreen testing battery, similar to previously described methods (Oomen et al 2013). At the conclusion of touchscreen testing, subjects were implanted with wireless telemetry devices (DSI) for evaluation of electroencephalography (EEG) signatures. Result: All subjects met the touch‐reward association criteria. During task acquisition LOAD2+CD mice demonstrated impaired acquisition relative to WT+CD, while both LOAD2+HFD and WT+HFD failed to learn the task as indicated by accuracy less than chance (<50%); which was confirmed in a separate cohort. LOAD2+HFD mice demonstrated increased spikewave events as measured by EEG, relative to LOAD2+CD. At 18‐months of age +CD mice that met acquisition criteria were evaluated in a location discrimination task with LOAD2+CD mice demonstrating modest impairments in pattern separation relative to age‐matched WT+CD. Conclusion: These data are the first reports of cognitive deficits and neurophysiological alterations in mice with environmental x genetic risk for LOAD, independent of amyloid and tau pathology. Importantly, the present findings demonstrate the sensitivity of the translational touchscreen testing battery for detecting mild cognitive impairment in LOAD mice with corresponding neurophysiologic alterations, and extend previous characterization data for the LOAD2 model and its utility for the study of the biology of LOAD.
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    Characterizing Molecular and Synaptic Signatures in mouse models of Late-Onset Alzheimer’s Disease Independent of Amyloid and Tau Pathology
    (bioRxiv, 2023-12-20) Kotredes, Kevin P.; Pandey, Ravi S.; Persohn, Scott; Elderidge, Kierra; Burton, Charles P.; Miner, Ethan W.; Haynes, Kathryn A.; Santos, Diogo Francisco S.; Williams, Sean-Paul; Heaton, Nicholas; Ingraham, Cynthia M.; Lloyd, Christopher; Garceau, Dylan; O’Rourke, Rita; Herrick, Sarah; Rangel-Barajas, Claudia; Maharjan, Surendra; Wang, Nian; Sasner, Michael; Lamb, Bruce T.; Territo, Paul R.; Sukoff Rizzo, Stacey J.; Carter, Gregory W.; Howell, Gareth R.; Oblak, Adrian L.; Medical and Molecular Genetics, School of Medicine
    Introduction: MODEL-AD is creating and distributing novel mouse models with humanized, clinically relevant genetic risk factors to more accurately mimic LOAD than commonly used transgenic models. Methods: We created the LOAD2 model by combining APOE4, Trem2*R47H, and humanized amyloid-beta. Mice aged up to 24 months were subjected to either a control diet or a high-fat/high-sugar diet (LOAD2+HFD) from two months of age. We assessed disease-relevant outcomes, including in vivo imaging, biomarkers, multi-omics, neuropathology, and behavior. Results: By 18 months, LOAD2+HFD mice exhibited cortical neuron loss, elevated insoluble brain Aβ42, increased plasma NfL, and altered gene/protein expression related to lipid metabolism and synaptic function. In vivo imaging showed age-dependent reductions in brain region volume and neurovascular uncoupling. LOAD2+HFD mice also displayed deficits in acquiring touchscreen-based cognitive tasks. Discussion: Collectively the comprehensive characterization of LOAD2+HFD mice reveal this model as important for preclinical studies that target features of LOAD independent of amyloid and tau.
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    Fit for purpose high‐throughput absolute quantitation of chimeric aducanumab in mouse cortex and plasma
    (Wiley, 2025-01-03) Doud, Emma H.; Haynes, Kathryn A.; Silva Santos, Diogo Francisco; Mosley, Amber L.; Quinney, Sara K.; Sukoff Rizzo, Stacey J.; Territo, Paul R.; Biochemistry and Molecular Biology, School of Medicine
    Background: Although pharmacokinetics and pharmacodynamics of biotherapeutics are commonly studied through ELISAs; however, the extremely strong binding of modern antibody‐based therapeutics result in background, inability of secondary antibody binding, and nonlinear response curves. The selectivity and specificity imparted through the use of liquid chromatography‐targeted mass spectrometry (LC‐MS/MS) allows for absolute quantitation of chosen peptides. For MODEL‐AD, here we present a high‐throughput workflow for absolute quantification of chimeric aducanumab from cortex and plasma of 5XFAD mice. Methods: A targeted MS assay for quantitation of aducanumab was designed utilizing guidelines described by the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium. Proteotryptic peptides unique for chimeric aducanumab were selected, and stable isotope versions were purchased as spike in controls. Given that aducanumab was present in mouse cortex at very low levels, a high sensitivity and high throughput methodology was optimized with Protein A enrichment, reduction, alkylation, trypsin digestion, loading samples onto Evotips using an AssayMap Bravo (Agilent). Evosep LC was paired with a Lumos Tribrid orbitrap (Thermo Fisher Scientific) and data were analyzed in Skyline (MacCoss lab) with a concentration curve of pure protein in matrix normalized to spike in stable isotope labeled peptides. Results: The three tryptic peptides used for quantitation of aducanumab had lower limits of detection and quantification of 1‐500 Amol pure peptide on column and 2‐5 ng aducanumab/uL in plasma and 0.225 ng/ug brain homogenate. This assay was sensitive and linear over 1 to 500,000 Amol range with high reproducibility (CV 3‐10%). Using a protein A purification, the lower limit of quantification was decreased by 100 fold. This assay was micronized for 96 sample formats, where a single plate could be analyzed in 48‐72 hours. Conclusions: Although unique peptides will vary, we anticipate this general workflow will allow for quantitation of AD focused biotherapeutics. As part of the open science framework, this methodology will be made available to the broader research community to facilitate broad application.