Proper cortical network development and function is reliant on the generation, maturation, and activity of numerouscelltypesinadditiontotheircomplexcell-cellinteractions.Essentialtothisprocessistheoutputof glutamatergicpyramidalneurons(PyNs),whichishighlymodulatedbyinhibitoryGABAergicinterneurons.One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC),which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS).DuetotheuniqueconnectionsformedbetweentheterminalsofChCaxonalarborsandtheAISsoflarge populations of spiking PyNs, ChCs are physiologically poised to regulate the output of excitatory cortical networks. As a result, it is not surprising that disruptions in ChC biology have been linked to autism spectrum disorder(ASD)andschizophrenia,debilitatingmentalhealthdisordersresultingfromaberrantneuronalnetwork activity.DespitetheimportanceofChCs,virtuallynothingisknownaboutthemolecularfactorsgoverningtheir selective innervation at the AIS of neocortical PyNs. By performing a novel, in vivo RNA interference (RNAi) screenagainstPyNAIS-specificand-enrichedadhesionmolecules,thisstudyintriguinglyrevealedanessential rolefortheaxonalcelladhesionmoleculeL1CAMinChC/PyNAISinnervation.Specifically,L1CAMknockdown inneocorticalPyNswasfoundtosignificantlyreducePyNAISinnervationbyChCs,thusidentifyingL1CAMas the only known molecule to date to regulate ChC/PyN subcellular target recognition. This application aims to elucidate how L1CAM governs ChC/PyN AIS innervation and to identify the presynaptic binding partner(s) of L1CAM on ChC axon terminals. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAMandtheAIScytoskeletontodeterminewhethercytoskeleton-mediatedL1CAMclusteringisnecessary forproperChC/PyNAISinnervation.Inaddition,molecularreplacementstrategieswillbeutilizedtoinvestigate L1CAM?ssubcellulardistributionontheAISandaxonofneocorticalPyNsinvivo.Finally,Aim1willdetermine whetherPyNL1CAMisrequiredearlyonfortheestablishmentand/orlateronforthemaintenanceofChC/PyN innervation using RNAi technology in combination with in utero electroporation (IUE)- and adeno-associated viral-basedstrategies.
Aim2 focusesonidentifyingthepresynapticbindingpartner(s)ofL1CAMonChCaxon terminals.BasedonmutantL1CAMscreening,neuropilin-1(Nrp1)ishypothesizedtoserveasthepresynaptic partnerofL1CAMonChCsnecessaryforproperChC/PyNinnervation.Totestthis,experimentsutilizingChC- targetingIUE,RNAitechnology,andconditionalNrp1knockoutmicewillbeemployedtodepleteNrp1inChCs. Molecularandbiochemicalstrategieswillalsobepursuedtodefinethecriticaldomain(s)ofNrp1necessaryfor L1CAMbindingandproperChC/PyNsubcellulartargetrecognition.Together,theproposedstudieswillprovide vital information on the mechanisms governing ChC/PyN AIS innervation and, as such, shed new light on the connectivitydefectsunderlyingdebilitatingmentaldisorders.

Public Health Relevance

ThisstudyaimstoelucidatethemolecularmechanismsgoverningGABAergicchandeliercellsubcellulartarget recognition,acriticalprocessimplicatedinmentalhealthdisordersincludingschizophreniaandautismspectrum disorder.Whilechandeliercellsplayavitalroleinmaintainingproperexcitatory/inhibitorybalanceinthecortex, ourunderstandingofhowtheyselectivelyinnervatetheaxoninitialsegmentofneocorticalpyramidalneuronsis verylimited.Elucidatingthekeyfactorsgoverningthisuniqueformofsubcellularinnervationwillprovidenovel insights into the pathophysiology underlying these brain disorders and thereby aid in the development of new therapeuticstrategiestopreventandtreatthem.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31MH117871-02
Application #
9812187
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Van'T Veer, Ashlee V
Project Start
2018-11-16
Project End
2020-11-15
Budget Start
2019-11-16
Budget End
2020-11-15
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Neurosurgery
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794