RESUMEN
Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.
Asunto(s)
Esclerosis Amiotrófica Lateral , Axones , Demencia Frontotemporal , Mutación , Proteína FUS de Unión a ARN , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Axones/patología , Axones/metabolismo , Animales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Demencia Frontotemporal/metabolismo , Femenino , Masculino , Xenopus laevis , Conos de Crecimiento/metabolismo , Humanos , Modelos Animales de EnfermedadRESUMEN
From the first illustrations of neuronal morphology by Ramón y Cajal to the recent three-dimensional reconstruction of synaptic connections, the development of modern neuroscience has greatly benefited from breakthroughs in imaging technology. This also applies specifically to the study of neurodegenerative diseases. Much of the research into these diseases relies on the direct visualisation of intracellular structures and their dynamics in degenerating neural cells, which cannot be fully resolved by diffraction-limited microscopes. Progress in the field has therefore been closely linked to the development of super-resolution imaging methods. Their application has greatly advanced our understanding of disease mechanisms, ranging from the structural progression of protein aggregates to defects in organelle morphology. Recent super-resolution studies have specifically implicated the disruption of inter-organelle interactions in multiple neurodegenerative diseases. In this article, we describe some of the key super-resolution techniques that have contributed to this field. We then discuss work to visualise changes in the structure and dynamics of organelles and associated dysfunctions. Finally, we consider what future developments in imaging technology may further our knowledge of these processes.
Asunto(s)
Enfermedades Neurodegenerativas/patología , Neuronas/ultraestructura , Orgánulos/ultraestructura , Animales , Humanos , Imagenología Tridimensional , Microscopía Confocal , Microscopía Fluorescente , Imagen Individual de MoléculaRESUMEN
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.
Asunto(s)
Lipopolisacáridos/toxicidad , Neuronas/metabolismo , Neuronas/patología , Receptores Purinérgicos P2/deficiencia , Sustancia Negra/metabolismo , Sustancia Negra/patología , Animales , Línea Celular Transformada , Células Cultivadas , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Células PC12 , Ratas , Sustancia Negra/efectos de los fármacosRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/genética , Esclerosis Amiotrófica Lateral/genética , Axones/metabolismo , Discapacidad Intelectual/genética , Enfermedad de Parkinson/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Axones/patología , Regulación del Desarrollo de la Expresión Génica , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Mutación , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/metabolismo , Red Nerviosa/patología , Proteínas del Tejido Nervioso/clasificación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The 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, and then describe the functional implications of this interplay in three cellular contexts, which exemplify how protein self-organization can be adapted to support certain physiological phenotypes. The authors then describe the formation of RNA-protein condensates in highly polarized cells such as neurons, where condensates play a critical role in the regulation of local protein synthesis, and describe how different stressors trigger extensive reorganization of the cytoplasm, both through signaling pathways and through direct stress-induced changes in cytoplasmic properties. Finally, the authors describe changes in protein behavior and cytoplasmic properties that may occur in extremophiles, in particular organisms that have adapted to inhabit environments of extreme temperature, and discuss the implications and functional importance of these changes.
Asunto(s)
Proteínas , ARN , Proteínas/metabolismo , Citoplasma/metabolismo , Sustancias Macromoleculares/metabolismo , Citosol/metabolismo , ARN/metabolismoRESUMEN
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.
RESUMEN
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 (RGCs). 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.
Asunto(s)
Neuronas , Fotones , Animales , Microscopía Fluorescente , RatasRESUMEN
Ribosome 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.