RNP components condense into repressive RNP granules in the aging brain.

Cytoplasmic RNP condensates enriched in mRNAs and proteins are found in various cell types and associated with both buffering and regulatory functions. While a clear link has been established between accumulation of aberrant RNP aggregates and progression of aging-related neurodegenerative diseases, the impact of physiological aging on neuronal RNP condensates has never been explored. Through high-resolution imaging, we uncover that RNP components progressively cluster into large yet dynamic granules in the aging Drosophila brain. We further show that age-dependent clustering is caused by an increase in the stoichiometry of the conserved helicase Me31B/DDX6, and requires PKA kinase activity. Finally, our functional analysis reveals that mRNA species recruited to RNP condensates upon aging exhibit age-dependent translational repression, indicating that co-clustering of selected mRNAs and translation regulators into repressive condensates may contribute to the specific post-transcriptional changes in gene expression observed in the course of aging.

Detecting Stress Granules in Drosophila Neurons.

Stress granules (SGs) are cytoplasmic ribonucleoprotein condensates that dynamically and reversibly assemble in response to stress. They are thought to contribute to the adaptive stress response by storing translationally inactive mRNAs as well as signaling molecules. Recent work has shown that SG composition and properties depend on both stress and cell types, and that neurons exhibit a complex SG proteome and a strong vulnerability to mutations in SG proteins. Drosophila has emerged as a powerful genetically tractable organism where to study the physiological regulation and functions of SGs in normal and pathological contexts. In this chapter, we describe a protocol enabling quantitative analysis of SG properties in both larval and adult Drosophila CNS samples. In this protocol, fluorescently tagged SGs are induced upon acute ex vivo stress or chronic in vivo stress, imaged at high-resolution via confocal microscopy and detected automatically, using a dedicated software.

Local Translation in Axons: When Membraneless RNP Granules Meet Membrane-Bound Organelles.

Eukaryotic cell compartmentalization relies on long-known membrane-delimited organelles, as well as on more recently discovered membraneless macromolecular condensates. How these two types of organelles interact to regulate cellular functions is still largely unclear. In this review, we highlight how membraneless ribonucleoprotein (RNP) organelles, enriched in RNAs and associated regulatory proteins, cooperate with membrane-bound organelles for tight spatio-temporal control of gene expression in the axons of neuronal cells. Specifically, we present recent evidence that motile membrane-bound organelles are used as vehicles by RNP cargoes, promoting the long-range transport of mRNA molecules to distal axons. As demonstrated by recent work, membrane-bound organelles also promote local protein synthesis, by serving as platforms for the local translation of mRNAs recruited to their outer surface. Furthermore, dynamic and specific association between RNP cargoes and membrane-bound organelles is mediated by bi-partite adapter molecules that interact with both types of organelles selectively, in a regulated-manner. Maintaining such a dynamic interplay is critical, as alterations in this process are linked to neurodegenerative diseases. Together, emerging studies thus point to the coordination of membrane-bound and membraneless organelles as an organizing principle underlying local cellular responses.