Multiple pathways contribute to neurodegeneration in Alzheimer?s Disease (AD).Identification of appropriate targetseffectiveforinhibitingdiseaseprogressionremainselusive.Pathwaysassociatedwithsynapticplasticity are strongly implicated in the pathophysiology of AD and the associated cognitive decline. They therefore representattractive targets forpharmacological interventions designed to restore cognitivefunction. Here, we proposetoprofilehowplasticityandneurotransmitterreceptorpathwaysmalfunctioninearlyADatasingle-cell- specifictranscriptomiclevel,bothasafunctionofthecell?sproximitytob?-amyloiddepositsandasafunctionof itslevelofactivityrecordedinvivo. Forcell-specifictranscriptomicprofiling,weusearecentlydeveloped,high-throughput,multiplexed,error-robust fluorescenceinsituRNAhybridizationmethod(MERFISH),whichimplementsacombinatoriallabelingapproach followedbysequentialroundsofsingle-moleculefluorescenceinsituhybridization(smFISH)toimagesimultaneously hundredstothousands(upto10000;?Xiaetal.,PNAS,2019)ofdistinctRNAmoleculesat~100nmspatialresolution.
Aim 1 : Develop a pipeline for characterizing Alzheimer?s-disease associated transcriptomic pathways in a topographically-resolvedsingle-cellspecificway.Maptranscriptomicdysfunctioninpathwaysassociatedwithneural plasticity and neurotransmitter receptor expression in the 5xFAD/PS1 APP mouse model of Alzheimer?s disease. Determinehowcell-specificmalfunctionatthetranscriptomiclevelrelatestotheproximityofb?-amyloiddepositsearly inthediseasecourse. Neuronal hyperactivity and failure of circuit homeostasis (manifesting as hypersynchrony) confer higher risk of neurodegeneration,areassociatedwiththepresenceofb?-?amyloid,andoftenprecedeplaquepathology.Wewilluse chronic large-field-of-view, ?Mesoscopic,? 2-photon imaging to measure cellular response functions and functional connectivity(hypersynchrony)profilesinvivo.AligninginvitroMERFISHimageswithinvivorecordingsallowssingle- cell-specifictranscriptomicpathwaystobecharacterizedasafunctionofneuronalactivitylevelsimagedinvivo.
Aim 2 : Identify functional biomarkers of early neuronal dysfunction and use MERFISH to map the transcriptomic profile of neurons and glial cells at the earliest stage of disease expression. Link transcriptomic measurements to neuronalprofilesofabnormalactivityobtainedbyinvivo2-photonimagingandtothelocationofb?-amyloiddeposits.
Our aims will1)identifyplasticityandneurotransmitterpathwaysdifferentiallyexpressedinallneuronalandglial cell types in response to AD, 2) describe the role of b?-?amyloid proximity in these modifications, 3) correlate functional defects in individual cell types with transcriptional changes, 4) determine the evolution of these properties with diseaseprogression,and 5) provide anovel datasetand method that will substantially extend ourmolecularandcellularunderstandingofADand,asaresult,mayleadtorationallydesignedtreatments.This approachisgeneralandcanbeeasilyadoptedinthefuturetodissectadditionalpathwaysimplicatedinAlzheimer?s.
Synaptic plasticity mechanisms are causally implicated in neurodegenerative disorders, specifically in Alzheimer?s, and represent promising targets for ameliorating cognitive decline. However, a cell-specific investigation of how synaptic plasticity pathways malfunction as a function of time (disease stage) and proximity to amyloid plaques has yet to be undertaken. We combine chronic ultra-large field of view in vivo two photon imaging with a recently- developed high-throughput in situ RNA hybridization method (MERFISH), to dissect how plasticity pathways malfunction at the transcriptomic level in relation to the deterioration of neuronal circuit properties measured in vivo.
