Spatial & temporal regulation of beta-actin mRNA local translation in neurons

Abstract

The physical manifestation of learning and memory formation in the brain can be expressed by strengthening or weakening of synaptic connections through morphological changes. Compartmentalization of protein synthesis in neurons is an essential component of synaptic plasticity and brain development. Local actin remodeling underlies some forms of plasticity and may be facilitated by local beta-actin synthesis, but dynamic information is lacking. We use single-molecule in situ hybridization to demonstrate that endogenous, dendritic beta-actin mRNA and ribosomes are in a masked, neuron-specific form. Chemically induced long-term potentiation prompts transient mRNA unmasking, which depends on factors active during synaptic activity. The motility of ribosomes and single beta-actin mRNAs increases after stimulation, indicative of release from complexes. By measuring activity induced single mRNA and granule dynamics, we have identified and characterized a mechanism whereby the translation of beta-actin mRNA can be precisely regulated in response to synaptic activity with temporal and spatial precision.;We expand our investigations of how beta-actin mRNA localization and translational regulation contribute to neuronal function in a mouse model lacking the mRNA binding protein Zipcode Binding Protein I (ZBP1). ZBP1 has been shown to regulate the transport and asymmetric distribution of beta-actin mRNA into growth cones and dendrites, as well as repress its translation. Live imaging of single beta-actin mRNAs revealed that actively transported mRNAs in ZBP1-/- neurons exhibited decreased net travel lengths in dendrites compared to wildtype cells. Although mRNA transport is altered, we did not find a significant loss of mRNA localization to dendrites. ZBP1 -/- neurons exhibited increased transcription rates, which may compensate for decreased transport. We also find diminished beta-actin mRNA masking in dendrites and increased actin protein, indicative of reduced translational repression. Despite an abundance of actin protein, ZBP1-/- neurons have a paucity of synapses and aberrant dendritic arborization, emphasizing the significance of correctly compartmentalized translational activity.;Hence, the single-molecule assays we developed demonstrate that beta-actin mRNA and ribosomes are in a masked state and allow for the quantification of activity-induced mRNA availability for translation. Further, single molecule data in ZBP1-/- neurons begin to shed light on the regulation of beta-actin mRNA distribution and local translation and its possible role in the development of the brain.