Molecular signaling during development and maturation of the nervous system

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications & trials

Abstract

Objective 1: Determine the molecular mechanisms by which axonal CRD-Nrg1 coordinates neuronal development. One challenging aspect of neuronal development is how neurons coordinate the timing of the elaboration of molecularly distinct and physically distant compartments such as axons and dendrites. Changes in developmental timing might also have more subtle but enduring effects; for example, generating a circuit capable of performing basic functions but lacking resilience in face of challenges (stresses). Because target innervation by an axon can occur meters away from the somatodendritic compartment, neurons require high fidelity, long-range mechanisms to inform this communication between the developing axon and dendrites. How might a neuron achieve this? A core goal of my research program is to understand how these distant compartments communicate during development and to elucidate the consequences of disruption in communication between these compartments, at the cellular, circuit and behavioral levels. Nrg1 signaling is neuroprotective, during development, and in the mature brain, possibly contributing to neural resilience and longevity. The critical role of Nrg1 signaling in nervous system development is underscored by linking rare missense variants in NRG1 to disorders as diverse as Hirschsprung’s disease, heritable peripheral neuropathy and psychosis. Indeed, the neuroprotective function of Nrg1 signaling is being exploited to investigate therapeutic uses of Nrg1 in various stroke models. Our studies focus predominantly on axonal Nrg1 signaling during neural development with an eye on contributions to neurodevelopmental disorders. My section on Genetics of Neuronal Signaling (GNSS) at NINDS, uses cutting edge methodologies to investigate Nrg1 back signaling mechanisms and their roles in regulated axon-dendrite development, neuronal fate specification, axonal mitochondrial & local protein synthesis dynamics, and synaptic transmission at cortico-amygdala synapses (collaboration with L. Role, LCSMS in NINDS). These studies have advanced our understanding of Nrg1 signaling in brain development and function and have pioneered conceptual advances in mechanisms of circuit development, terminal fate specification of neurons, and intersection of these basic cell biological processes with neurodevelopmental/ neuropsychiatric disease-related genetics. I. Genomic analyses of CRD-Nrg1 nuclear signaling (PMID: 39214704; Collaborators: K. Johnson, Bioinformatics Core, L. Role, LCSMS): Our initial transcriptomic analyses focused on the hippocampal dentate gyrus (DG). Bioinformatic analyses of differentially expressed genes led to several insights. 1. Axonal target innervation-induced nuclear signaling stimulates a switch from axonal extension to dendritic maturation and synaptogenesis. Genes downregulated by Nrg1 nuclear back-signaling are implicated in pathways involved in axon growth and axonal guidance. Conversely, the genes upregulated by Nrg1 nuclear back-signaling are implicated in regulation of cytoskeletal dynamics linked to dendritic arborization. These results implicate Nrg1 nuclear signaling in mediating the switch from early axonal development to dendritic maturation. 2. Loss of CRD-Nrg1 nuclear signaling reduced neuroprogenitor proliferation and accelerated differentiation of neurons. We examined neurogenesis in the DG and found that adult neurogenesis was reduced in mutant animals. This decrease was balanced by increased generation of immature granule cells (iGC) at the expense of retaining neural progenitors. In addition, the iGCs in the mutant had dysmorphic dendritic arbors. These data indicate that loss of Nrg1 nuclear back-signaling dysregulates the pace of neuronal development. 3. Nrg1-ICD controls functionally connected gene networks via regulation of the Polycomb Repressive Complex (PRC) and RE-1 Silencing Transcription factor (REST). We compared gene expression in the DG, amygdala, ventral striatum, and ventral hippocampus between Nrg1V321L and WT mice. There were similar numbers of differentially regulated genes (DEGs) in each region, but there was minimal overlap in the identity of the dysregulated genes between regions. There was significant overlap in (a) the predicted transcriptional regulators of the DEGs (PRC2 and REST) and (b) the affected biological processes/functions. These findings implicate PRC2 and/or REST as primary effectors of nuclear Nrg1-ICD. We propose Nrg1-ICD targeting of these complexes integrates developmental signals in diverse neuronal types. We found that DEGs in each brain are enriched for orthologs of known schizophrenia susceptibility genes. Network analyses predict that these dysregulated schizophrenia-linked genes coordinate a larger network of genes by functioning as network hubs. These networks are predicted to be involved in aspects of neuronal development. This indicates that disease-associated gene networks are “accessed” by developmentally regulated axon-to-nucleus signaling by CRD-Nrg1. By conducting comparative analyses of transcriptomic data from postmortem human DG tissue from patients with schizophrenia, we demonstrated shared molecular pathways between the Nrg1V321L mouse model and human schizophrenia DG [43]. These findings add insight into the genetic risk mechanisms in neurodevelopmental disorders: genetic risk for schizophrenia might be realized not only through inheritance but can also be affected by synaptogenic signaling to the nucleus. II. Regulation of terminal fate specification by CRD-Nrg1 nuclear back-signaling (PMID: 40280713; Collaborators: K. Johnson, Bioinformatics Core; Desai, Role (CSMS)): We conducted a follow-up study on the role of CRD-Nrg1 nuclear back-signaling on neuronal maturation in the DG using both morpho-electrophysiological and single nuclear multi-omics approaches. These studies revealed the intriguing observation that CRD-Nrg1 nuclear signaling controls a fate decision within the DG granule cell (GC) population. III. Dissociable mechanisms of local vs. nuclear CRD Nrg1-signaling (Collaborators: Hospes, Freeman, Role (CSMS)): We examined the contribution of local CRD-Nrg1 axonal signaling to axon outgrowth and to development of presynaptic specializations. Nrg1 signaling is necessary for proper axon targeting and for dendrite growth, branching and spine formation. Using in vitro preparations, we demonstrated that CRD-Nrg1 activation of Src/PI3K signaling stimulates axon outgrowth whereas the nuclear import of the Nrg1-ICD regulates dendrite growth. These represent two, independent functions of axonal CRD-Nrg1. We also demonstrated a role for targeting mitochondria to presynaptic specializations: decreased CRD-Nrg1 (heterozygotes) led to increased motility, whereas acutely stimulating CRD-Nrg1 signaling decreased mitochondria motility. Docked mitochondria were located at/near active release sites. Axonal CRD-Nrg1 signaling regulates local, axonal protein synthesis. Because CRD-Nrg1 presynaptic targeting of nAChRs requires both PtdIns3 kinase and new protein synthesis, we asked whether CRD-Nrg1 was regulating axonal mRNA translation. We demonstrated nAChR mRNA in axons and demonstrated CRD-Nrg1 signaling increased axonal nAChR translation Inhibitors of either PtdIns3k or Src kinases block the effects of axonal CRD-Nrg1 signaling. We predicted that the Nrg1-ICD formed a complex with Src upstream of PtdIns3k. This was confirmed by co-immunoprecipitation of Nrg1 and Src from cortical extracts and by proximity ligation staining of primary neurons. We have mapped the Src interaction to an SH3 ligand in the Nrg1-ICD. Point mutations in the SH3 ligand domain eliminate CRD-Nrg1 support of axon outgrowth. Objective 2: Understand the functional heterogeneity of basal forebrain cholinergic neurons. We have identified populations of cholinergic neurons that participate in distinct types of memory and demonstrated for the first time that cholinergic neurons participate directly in memory engrams (PMID: 32945260; PMID: 38363713) (collaborators: Dr. L Role, LCSMS; Dr. M Picciotto, Yale Univ). These findings provide novel insight into the participation of the cholinergic system in memory formation and retrieval with clear implications to understanding age related cognitive decline that accompanies degeneration of cholinergic brain nuclei. We have also demonstrated the existence of distinct populations of cholinergic neurons that are involved in the coding of valence: within the ventral pallidum there are two, non-overlapping populations of cholinergic neurons that respond to, and behaviorally influence responses to positive vs negative valence odors (PMID: 38536818). In a collaborative project (with Drs. L Role, LCSMS, K. Johnson, NINDS Bioinformatics Core and T Petros, NICHD), Dr. M Ananth, LCSMS, generated a single nucleus RNAseq dataset from >20,000 forebrain cholinergic neurons. This far surpasses existing data sets of cholinergic transcriptomes, expanding the number of identifiable subpopulations from 2-3 to over 20. The transcriptomic clusters to not obey anatomical boundaries, but rather are distributed across multiple classical defined BFCN nuclei. The cholinergic transcriptome changes with age. Some cluster show resilience to age, some vulnerability. Still others are major contributors to age related differentially expressed gene changes without changing in relative numbers. Early age vulnerability is conserved from rodents to primates (PMID: 39891909). Of particular interest to GNSS, BFCN populations vary widely in expression of CRD-Nrg1. In CRD Nrg1 heterozygous mutants, BFCN populations that normally express the highest level of CRD-Nrg1, show accelerated age-related declines in cholinergic neuron number, target field innervation and have alterations in the patterns of behaviorally relevant ACh release. The effect of Nrg1 genotype on basal forebrain cholinergic neurons shows complex inheritance: phenotypes are influenced by parent-of-origin of the mutant allele and show sex bias.