SummaryRibosome assembly occurs mainly in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding many ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Here, we report a physical and functional interaction between locally synthesized RPs and ribosomes in the axon. We show that axonal RP translation is regulated through a sequence motif, CUIC, that forms an RNA-loop structure in the region immediately upstream of the initiation codon. Using imaging and subcellular proteomics techniques, we show that RPs synthesized in axons join axonal ribosomes in a nucleolus-independent fashion. Inhibition of axonal CUIC-regulated RP translation decreases local translation activity and reduces axon branching in the developing brain, revealing the physiological relevance of axonal RP synthesis in vivo. These results suggest that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus in neurons.

The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains: sheets and interconnected tubules. These domains undergo dynamic reshaping in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status: The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven repositioning of lysosomes, which leads to both a redistribution of the ER tubules and a change of its global morphology. Therefore, lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.

2!SUMMARY Ribosomes are known to be assembled in the nucleolus, yet recent studies have revealed robust enrichment and translation of mRNAs encoding ribosomal proteins (RPs) in axons, far away from neuronal cell bodies. Using subcellular proteomics and live-imaging, we show that locally synthesized RPs incorporate into axonal ribosomes in a nucleolus-independent fashion. We revealed that axonal RP translation is regulated through a novel sequence motif, CUIC, that forms a RNA-loop structure in the region immediately upstream of the initiation codon. Inhibition of axonal CUICregulated RP translation leads to defects in local translation activity and axon branching, demonstrating the physiological relevance of the axonal ribosome remodeling. These results indicate that axonal translation supplies cytoplasmic RPs to maintain/modify local ribosomal function far from the nucleolus. INTRODUCTIONRNA localization and local translation play key roles in the assembly and maintenance of neuronal connections (Campbell and Holt, 2001;Holt and Schuman, 2013;Wu et al., 2005).Recent genome-wide studies on the axonal transcriptome revealed that thousands of mRNAs are localized to the axon. A consistent but unexpected finding of these studies is the robust enrichment of mRNAs that encode ribosomal proteins (RPs), protein components of ribosomes. Axons are long neuronal processes that carry out many vital specific cellular functions far from their cell bodies, including translation, and must therefore maintain their protein synthetic machinery in good order. However, because most eukaryotic ribosome assembly is well known to occur in the nucleolus (Fromont-Racine et al., 2003;Lastick and McConkey, 1976), the physiological function of RP-coding mRNAs in a neuronal subcellular compartment far distant from the nucleus was enigmatic. RP-coding mRNAs have been abundantly detected in axons of a variety of neuron types, such as retinal ganglion cells (RGCs) (Zivraj et al., 2010), sympathetic neurons (Andreassi et al., 2010) and motor ! 5!In this study, we explore intra-ribosomal roles of axonally synthesized RPs using a range of technical approaches including live imaging, in vivo gene knockdown, bioinformatics, nascent protein labeling and mass spectrometry-based proteomics. We found that axonal translation of RPs coordinately peaks at the axon branching stage in RGCs in vivo, and their translation is regulated by a branch-promoting factor, Netrin-1, through a novel loop structure-forming sequence motif, CUIC, that is shared by ~70% of RP-coding mRNAs. Using nascent protein labeling and proteomic mass spectrometry analysis on ribosomes isolated from pure axons, together with live-imaging approaches, we show that axonally synthesized RPs are physically incorporated into axonal ribosomes in a nucleolus-independent fashion. Furthermore, we demonstrate the physiological importance of the axonal ribosome remodeling by showing that inhibition of axonal RP translation leads to a significant decrease in the level of axonal mRNA translation and s...

Inflammation may contribute to multiple brain pathologies. One cause of inflammation is lipopolysaccharide/endotoxin (LPS), the levels of which are elevated in blood and/or brain during bacterial infections, gut dysfunction and neurodegenerative diseases, such as Parkinson’s disease. How inflammation causes neuronal loss is unclear, but one potential mechanism is microglial phagocytosis of neurons, which is dependent on the microglial P2Y6 receptor. We investigated here whether the P2Y6 receptor was required for inflammatory neuronal loss. Intraperitoneal injection of LPS on 4 successive days resulted in specific loss of dopaminergic neurons (measured as cells staining with tyrosine hydroxylase or NeuN) in the substantia nigra of wild-type mice, but no neuronal loss in cortex or hippocampus. This supports the hypothesis that neuronal loss in Parkinson’s disease may be driven by peripheral LPS. By contrast, there was no LPS-induced neuronal loss in P2Y6 receptor knockout mice. In vitro, LPS-induced microglial phagocytosis of cells was prevented by inhibition of the P2Y6 receptor, and LPS-induced neuronal loss was reduced in mixed glial–neuronal cultures from P2Y6 receptor knockout mice. This supports the hypothesis that microglial phagocytosis contributes to inflammatory neuronal loss, and can be prevented by blocking the P2Y6 receptor, suggesting that P2Y6 receptor antagonists might be used to prevent inflammatory neuronal loss in Parkinson’s disease and other brain pathologies involving inflammatory neuronal loss.

28The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains, 29 sheets and interconnected tubules. These domains undergo dynamic reshaping, in response to 30 changes in the cellular environment. However, the mechanisms behind this rapid remodeling 31 within minutes are largely unknown. Here, we report that ER remodeling is actively driven by 32 lysosomes, following lysosome repositioning in response to changes in nutritional status. The 33 anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We 34 validate this causal link via the chemo-and optogenetically driven re-positioning of lysosomes, 35 which leads to both a redistribution of the ER tubules and its global morphology. Lysosomes sense 36 metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this 37 mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a 38 critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal 39 development. 40 41 Main text 42 43The structure of the ER is constantly adapted for the particular needs of the cell (1): the dynamic 44 transitions between ER sheets and tubules allow it to rapidly respond to the changing cellular 45 environment. A group of ER-shaping proteins have been identified as maintaining ER morphology 46 (1), mutations in which are linked to diseases such as hereditary spastic paraplegias (HSPs) (2). 47 (3, 4). Previous work has shown that ER tubule elongation can be driven by three mechanisms: 1/ 50 force generation by motors moving along microtubules (5), which can be classified as sliding, 2/ 51 coupling to microtubule growth using a tip assembly complex (TAC), and 3/ hitchhiking by 52 connecting to other organelles. Whether such reshaping in local domains of the ER tubules could 53 lead to the global reorganization and redistribution of ER remains an open question, and, if this is 54 the case, how is this process regulated? The ER is known to contact other motile organelles, 55 including endosomes, lysosomes, mitochondria, peroxisomes et cetera (6). Among these, 56 lysosomes are particularly interesting, as they make a great number of contacts with the ER (7) 57 and their positioning is regulated by different nutritional status (8). Although ER has been reported 58 to regulate lysosome motions (9), it is not clear whether lysosomes can modulate ER reshaping 59 and distribution, for example via coupled motion (4). We hypothesized that a causal link exists 60 between lysosome motion and ER redistributing and asked whether this provides a mechanism for 61 ER morphological response to nutritional status, given that lysosomes are known to act as signaling 62 hubs for metabolic sensing (10). 63 We first investigated the correlation of motions between lysosomes and the ER network by rapid 64 live-cell imaging. We visualized ER with GFP-tagged vesicle-associated membrane protein-65 associated protein A (VAP...

It is increasingly recognized that local protein synthesis (LPS) contributes to fundamental aspects of axon biology, in both developing and mature neurons. Mutations in RNA-binding proteins (RBPs), as central players in LPS, and other proteins affecting RNA localization and translation are associated with a range of neurological disorders, suggesting disruption of LPS may be of pathological significance. In this review, we substantiate this hypothesis by examining the link between LPS and key axonal processes, and the implicated pathophysiological consequences of dysregulated LPS. First, we describe how the length and autonomy of axons result in an exceptional reliance on LPS. We next discuss the roles of LPS in maintaining axonal structural and functional polarity and axonal trafficking. We then consider how LPS facilitates the establishment of neuronal connectivity through regulation of axonal branching and pruning, how it mediates axonal survival into adulthood and its involvement in neuronal stress responses.

These folds are determined by both protein sequence and solvent properties, and determine protein function. [2] Therefore, the diversity of protein folds in cells allows them to carry out a range of functions, such as catalysis of different reactions. Functional versatility within polypeptide chains is further increased through the presence of multiple independently folding sequences, defined as domains, each associated with distinct functions, such as dimerization or responsiveness to a regulator. However, it is now recognized that some proteins do not have defined conformations, or feature domains that are intrinsically disordered (intrinsically disordered regions, IDRs), and that these confer function by mediating context-dependent proteinprotein interactions. [3] The concept of self-organization of proteins is also applied to assemblies of multiple proteins, in which case it refers to formation of dynamic multi-component structures, [4] including certain oligomeric complexes, filaments, and phase-separated assemblies. In phase-separated assemblies, of which a range exist, IDR-containing proteins form a dense phase within the cytoplasm, commonly referred to as biomolecular condensates. [5] As for folds, the formation of these assemblies depends both on interactions between the phase-separating proteins, which can be mediated by their IDRs, and between these proteins and the surrounding solution. Within the cytoplasm, these phase-separated "droplets" commonly contain RNA, in which case they are referred to as ribonucleoprotein (RNP) granules. They can be considered to have "emergent properties:" certain characteristics can be ascribed to assemblies that are not properties of individual constituent proteins. [6] For instance, biomolecular condensates can display liquid-like properties such as fusion and wetting, which led to their identification as phase-separated droplets. [7] These properties allow them to function as dynamic membraneless organelles, or compartments. This compartmentalization is commonly referred to as occurring on the "mesoscale," which is defined as that range of lengths larger than the size of individual molecular machinery such as ribosomes, but smaller than that of the whole cell. [8] The sensitivity of protein folds and macromolecular interactions to local environmental conditions confers both regulatory potential and risk of loss of function in "extreme" conditions. This regulatory potential is exemplified by and has beenThe cytoplasm is an aqueous, highly crowded solution of active macromolecules. Its properties influence the behavior of proteins, including their folding, motion, and interactions. In particular, proteins in the cytoplasm can interact to form phase-separated assemblies, so-called biomolecular condensates. The interplay between cytoplasmic properties and protein condensation is critical in a number of functional contexts and is the subject of this review. The authors first describe how cytoplasmic properties can affect protein behavior, in particular condensate formation...

Large fields of view (FOVs) in total internal reflection fluorescence microscopy (TIRFM) via waveguides have been shown to be highly beneficial for single molecule localisation microscopy on fixed cells [1, 2]and have also been demonstrated for short-term live-imaging of robust cell types [3-5], but not yet for delicate primary neurons nor over extended periods of time. Here, we present a waveguide-based TIRFM set-up for live-cell imaging of demanding samples. Using the developed microscope, referred to as the ChipScope, we demonstrate successful culturing and imaging of fibroblasts, primary rat hippocampal neurons and axons of Xenopus retinal ganglion cells (RGC). The high contrast and gentle illumination mode provided by TIRFM coupled with the exceptionally large excitation areas and superior illumination homogeneity offered by photonic waveguides have potential for a wide application span in neuroscience applications.TIRFM provides an effective means for the spatially confined illumination of a sample close to the coverslip/substrate via evanescent fields [6][7][8]. It provides particular advantages for fluorescence imaging as out of focus signal is intrinsically avoided leading to high signal to noise ratios and image contrast. In addition to the molecular specificity afforded by fluorescence imaging and image contrast, TIRFM reduces the overall illumination dose on the sample. This minimises phototoxicity, making TIRF the method of choice for many live-cell imaging applications with delicate samples such as live neurons [9]. TIRFM is usually accomplished by using a large numerical aperture (NA) objective lens for both the excitation and detection paths. Unfortunately, the high magnification of lenses required for TIRFM limits the accomplishable FOVs and thus imaging throughput. This makes conventional TIRFM impractical for extended and often fast moving samples such as neurons and their organelles [10]. This FOV restriction is removed if waveguides are used for TIRF illumination and in principle, arbitrarily large areas could be achieved through appropriately designed waveguide geometries (width and length). Because the excitation and detection paths are completely decoupled from one another, full flexibility in choice of the imaging objective lens is retained, allowing for control over the FOV size, as illustrated on fibroblasts in Suppl. Figure S1. In the so called ChipScope microscopy system [1], multiple colours can be admitted simultaneously into the photonic chip, enabling the simultaneous TIRF excitation of multiple fluorophores (see Suppl. Figure S2).Waveguides have previously been shown to be a viable growth substrate for cell culture [3, 4], but to fully exploit the gentle TIRF illumination for live-cell image applications, especially in the neurosciences, additional considerations and adaptations must be made to maintain the cells alive under suitable conditions. The scope of this work was to adapt a waveguide TIRF microscopy set-up for the imaging of sensitive cell types like primary neurons, and...