Aberrant Phase Separation of FUS Leads to Lysosome Sequestering and Acidification

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology

“…2A ). Since proteins undergoing LLPS form condensates when fused to oligomerization domains that decrease their saturation concentration (i.e., the minimal concentration at which proteins phase separate), we fused N-WASP to the light-sensitive flavin adenine dinucleotide-binding protein cryptochrome-2 (Cry2) as in previous studies [ 33 , 34 ]. Optogenetic activation of Cry2-mCherry fused to N-WASP caused rapid cluster formation of the protein in HEK cells (Fig.…”

Emerging Roles for Phase Separation of RNA-Binding Proteins in Cellular Pathology of ALS

“…ALS-associated misfolded proteins are targeted to aggresomes ( Kawaguchi et al, 2003 ) subsequently degraded by autophagy ( Mateju et al, 2017 ). The aberrant phase separation of FUS leads to the formation of cytosolic aggregates in a concentration-dependent fashion and lysosomes are juxtaposed to these aggregates ( Trnka et al, 2021 ). In line with these findings, increased lysosomal activity and enhanced autophagy are also reported in astrocytes derived from ALS patients ( Rajpurohit et al, 2020 ).…”

“…Recent findings demonstrate the association of SGs with several membrane-linked compartments such as ER, lysosomes and mitochondria ( Liao et al, 2019 ; Lee et al, 2020 ; Amen and Kaganovich, 2021 ; Trnka et al, 2021 ) raising the interest in SG physiological properties and their implication in ER and oxidative stress in ALS. These are precisely the mechanisms targeted by the most recent drug AMX0035 (tauroursodeoxycholic acid and sodium phenylbutyrate), which is at the late stages of approval by FDA for clinical use.…”

“…For the roles of two well-studied RBPs, TDP-43 and FUS in frontotemporal dementia, we refer to the review by Carey and Guo within this Special Issue. The aberrant RBP condensates can even trap folded proteins which do not undergo LLPS such as SOD1 ( Mateju et al, 2017 ; Pakravan et al, 2021 ) and alter the intracellular organelle trafficking ( Ling et al, 2019 ; Trnka et al, 2021 ). Understanding the mechanisms how LLPS of several ALS-associated RBPs corroborates with the intracellular organelle trafficking ( Figure 1 ) opens novel directions for disease treatment.…”

Editorial: Protein Phase Separation and Aggregation in (Patho)Physiology of Neurons

“…In fact, ( Milicevic et al ) argue that LLPS-disrupting mutations are a common feature of many RNPs implicated in the pathology of ALS, such annexin 11, ataxin 2, hnRNPA1, hnRNPA2, and TIA-1. The cause-effect relationship between RNA granule assembly, their association with membrane-bound compartments such as ER, lysosomes or mitochondria, and their response to (oxidative) stress, are all important for the appropriate neuronal physiology ( Liao et al, 2019 ; Amen and Kaganovich, 2021 ; Trnka et al, 2021 ). The aberrant phase separation of RNPs induces the loss of their cellular function and also triggers the formation of inclusions that may trap folded proteins and membranes.…”