Molecular signaling during development and maturation of the nervous system

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Our first objective for the current year was to establish a functional laboratory at the Bethesda campus of the National Institutes of Health (NIH). Sub-objective 1: set up the physical space. Renovations were originally scheduled to be completed by August / September 2019. This date was modified to late October, mid-December, end of January 2020 and finally mid-to-late March of 2020. We began to unpack purchases in March while the crew was finishing renovations when we went to minimal maintenance due to the COVID-19 pandemic. Renovations were completed during minimal maintenance and most of the space is nearing functional. Final equipment installation and training is scheduled for August / September 2020. No science has been done in the lab at NIH yet. Sub-objective 2: recruit key lab personnel. I have recruited: A staff scientist, 2 post-doctoral fellows and a post-bac. Sub-objective 3: Equip and supply the lab. We have largely accomplished this. As of the beginning of August 2020, all areas of the lab are set up with the exception of our slice electrophysiology rig (installation scheduled for mid-August), our imaging facilities (ordering of microscopes in final phases of the process, and space for our confocal awaits the move out of equipment of another investigator). Sub-objective 4: Establish our mouse colony. We shipped three lines of mice from Stony Brook University to the NIH for re-derivation by the NIMH transgenic core. To date, two of the three lines have been successfully rederived, the third is scheduled for rederivation in the next two weeks. The first animals from line 1 are now breeding and we have identified homozygous offspring to use for establishing our breeding colony. Animals from the second line have now been genotyped and heterozygotes transferred to our nascent colony. Our second objective was to begin experiments aimed at defining the Nrg1 interactome--the galaxy of proteins that physically interact with the Nrg1 protein in neurons. To this end I have built expression constructs to allow proximity tagging of interactors followed by affinity purification. The constructs have been validated using HEK293 cells. I have established a collaboration with Dr. Yan Li, Director of the NINDS proteomics core facility, for identifying interacting proteins. We have also performed an initial set of RNAseq analyses on the hippocampi of wild type and mutant littermates to examine the transcriptional consequences of disrupting gamma secretase dependent Nrg1 signaling. Our third objective was to foster and sustain ongoing collaborations with the Role lab (now Laboratory of Circuits, Synapses and Molecular Signaling in the NINDS), with Dr. Ari Kaufman (Department of Computer Sciences) and Drs. Parsey and De Lorenzo (Department of Psychiatry) (all at Stony Brook University) and with Dr. Marina Picciotto at Yale University Department of Psychiatry. The collaboration with Dr. Role and with (a) Dr. Kaufman yielded two manuscripts that under revision (developing unique image process algorithms that use AI approaches to predict trajectories of cholinergic terminal field loss during aging), (b) Drs Parsey and De Lorenzo the establishment of a cross species study of the cholinergic system during normal and pathological aging, and (c) Dr. Picciotto a manuscript under revision and a second that will be submitted in the coming weeks. These latter two studies identify specific populations of cholinergic neurons that participate in distinct types of memory and demonstrate for the first time that cholinergic neurons participate directly in memory engrams.