Super-Resolution Microscopy Reveals Presynaptic Localization of the ALS/FTD Related Protein FUS in Hippocampal Neurons

Abstract

Fused in Sarcoma (FUS) is a multifunctional RNA-/DNA-binding protein, which is involved in the pathogenesis of the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A common hallmark of these disorders is the abnormal accumulation of mutated FUS protein in the cytoplasm. Under normal conditions FUS is confined to the nuclear compartment, in neurons, however, additional somatodendritic localization can be observed. In this study, we carefully analyzed the subcellular localization of endogenous FUS at synaptic sites of hippocampal neurons which are among the most affected cell types in FTD with FUS pathology. We could confirm a strong nuclear localization of FUS as well as its prominent and widespread neuronal expression throughout the adult and developing rat brain, particularly in the hippocampus, the cerebellum and the outer layers of the cortex. Intriguingly, FUS was also consistently observed at synaptic sites as detected by neuronal subcellular fractionation as well as by immunolabeling. To define a pre- and/or postsynaptic localization of FUS, we employed super-resolution fluorescence localization microscopy. FUS was found to be localized within the axon terminal in close proximity to the presynaptic vesicle protein Synaptophysin1 and adjacent to the active zone protein Bassoon, but well separated from the postsynaptic protein PSD-95. Having shown the presynaptic localization of FUS in the nervous system, a novel extranuclear role of FUS at neuronal contact sites has to be considered. Since there is growing evidence that local presynaptic translation might also be an important mechanism for plasticity, FUS – like the fragile X mental retardation protein FMRP – might act as one of the presynaptic RNA-binding proteins regulating this machinery. Our observation of presynaptic FUS should foster further investigations to determine its role in neurodegenerative diseases such as ALS and FTD.

FUS Mislocalization and Vulnerability to DNA Damage in ALS Patients Derived hiPSCs and Aging Motoneurons

“…In mature 42 days differentiated MN from control cell lines, FUS was not only localized in the nuclei (Japtok et al, 2015; Lenzi et al, 2015; Liu et al, 2015) but was also detectable as small punctae along the neurites. This observation was very similar to the FUS localization along neurites and within the presynaptic compartment in hippocampal neurons (Schoen et al, 2016). …”

“…In neurons, FUS is found at synaptic sites indicating that it might be important for local translation of transported mRNA (Fujii and Takumi, 2005; Fujii et al, 2005; Yasuda et al, 2013; Sephton et al, 2014) and involved in synaptic plasticity (Fujii et al, 2005; Aoki et al, 2012; Schoen et al, 2016). Therefore, we investigated the localization of FUS in control and patient-derived MNs and analyzed a FUS associated pathology in axons and/or dendrites.…”

“…FUS seems to be an important factor for the nuclear export of messenger RNA (mRNA) and the dendritic transport of mRNA for local translation in neurons (Fujii and Takumi, 2005; Fujii et al, 2005). Furthermore, FUS-positive granules co-localizing with synaptic markers are also present along dendrites of mouse neurons and also in the human brain, suggesting an additional role at synaptic sites (Belly et al, 2010; Aoki et al, 2012; Schoen et al, 2016). In this respect, it has been described that upon synaptic mGluR5 activation FUS is translocated to dendritic spines.…”

“…Young MNs harboring the most aggressive mutation presented FUS delocalization and spontaneous DNA-damage accumulation. Moreover, in control cells FUS was detectable in small punctae along neurites (Belly et al, 2010; Aoki et al, 2012; Schoen et al, 2016). In mutant MN, however, an increased number of large, densely packed FUS positive SG were formed spontaneously along the neurites.…”

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

“…Although fluorescence imaging of tissue sections has been completed using both confocal and superresolution microscopies1718193536, results with SMLM presented here are improved in many respects. The spatial resolution obtained here on FFPE sections with SMLM is ~30 nm laterally and ~70 nm axially, which is on par with previous work with STED19 and clearly better than conventional confocal microscopy (Figs 1 and 2).…”