Molecular signaling during development and maturation of the nervous system

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Objective 1: define the Nrg1 interactome (the transcriptional targets of Nrg1 signaling in neurons and the galaxy of proteins that physically interact with Nrg1). Subobjective 1: Axonal isoforms of Nrg1 participate in axon-to-nucleus signaling. In order to identify the suite of genes targeted by Nrg1 nuclear signaling, mechanism we collected and analyzed bulk RNAseq data from dentate gyrus, nucleus accumbens, basal lateral amygdala, total hippocampus and frontal cortices of mice carrying a mutation in the Nrg1 transmembrane domain that impairs gamma secretase processing and nuclear signaling (and which was initial identified as a psychosis risk gene in a Costa Rican population). Our initial study focused on the dentate gyrus from young adults. Neurons in the dentate are continually generated throughout life and therefore by looking at the dentate gyrus we were able to look for effects of disrupted Nrg1 nuclear signaling in neuroprogenitor populations as well as in mature neurons. Analyses of the dentate gyrus data from these mutant animals and their non-mutant littermates has been completed (in collaboration with Dr. Kory Johnson, NINDS bioinformatics core; Rajebhosale et al., BioRxiv 2022; under review at ELife). The results predict alterations in cell cycle progression and in neuronal maturation. Both predictions have been confirmed using other approaches. The second major finding of the RNAseq analyses is highly significant convergence between the mouse mutant data and RNAseq differences between schizophrenia patients and controls. The population of genes whose expression is significantly altered in the absence of Nrg1 nuclear signaling overlaps significantly with populations of genes whose altered expression is associated with risk of being diagnosed with psychotic illness. Subobjective 2: How Nrg1 initiates signaling, either in axons or in the nucleus, remains unknown. To begin to unravel this question we are using a proximity tagging, coupled affinity purification and proteomics as an unbiased screen for proteins that transiently or stably interact with Nrg1 (collaboration with Dr. Yan Li, Director of the NINDS proteomics core facility and Dr. Ray Fields, Director of the NINDS viral core facility). Objective 2: identify the functional heterogeneity of cholinergic neurons. Subobjective 1: Determine the involvement of cholinergic neurons in two distinct BLA dependent behaviors, one in response to an appetitive stimulus, the other in response to learned threat. These two studies (carried out in collaboration with Dr. L Role, NINDS IRP and Dr. M Picciotto, Yale Univ) 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 (Crouse et al. ELife 2020 and Rajebhosale et al., bioRxiv 2021). These findings provide significantly new insight into the ways that the modulatory, cholinergic system participates in memory formation and retrieval with clear implications to understanding age related cognitive decline that accompanies degeneration of cholinergic brain nuclei. We observed that distinct populations of cholinergic neurons were required for threat responsive behaviors depending on whether the threat was learned (cue-dependent conditioning) or was innate (response to predator odor). The former required engagement of cholinergic neurons in the nucleus Basalis, the latter cholinergic neurons in the ventral pallidum two distinct basal forebrain structures. We have initiated a line of investigation focusing on the response of ventral pallidal cholinergic neurons to either innately appetitive or innately aversive olfactory stimuli. Both stimuli broadly activate cholinergic neurons in the ventral pallidum. Using intersectional genetic approaches, we have found that although both stimuli activate cholinergic neurons, the cholinergic neurons activated, represent two distinct, non-overlapping populations. We are now embarking on efforts to understand, at the molecular level, the fundamental properties of these distinct populations. Subobjective 2: Gain insight into molecular mechanisms that regulate the establishment and function of cholinergic axo-axonal synapses. Basal forebrain cholinergic neuron axons are highly branched estimates are single neuronal axonal arbors are up to 100 meters in the human brain. Very little is known about how these terminal fields are established or maintained across the lifespan. Much of the action of acetylcholine in the brain occurs by activating pre-synaptic ACh receptors. How these axo-axonic synapse-like interactions are established or regulated (or even what they look like) is unknown. Previously we focused on axonal signaling mechanisms that targeted nicotinic acetylcholine receptors (nAChRs) to axons and have demonstrated that levels of presynaptic alpha 7 containing ACh receptors are in part regulated by axonal synthesis of alpha 7 protein. In collaboration with Dr. Roles group, we have recently developed a co-culture system between glutamatergic neurons with axonal nAChRs with explants from basal forebrain cholinergic nuclei and demonstrated the establishment of functional cholinergic to glutamatergic axo-axonal transmission (Zhong et al., Frontiers in Neural Circuits, under review). We anticipate that this experimental preparation will significantly expand our ability to probe molecular mechanisms that underlie the establishment of these connections.