TDP-43 in nuclear condensates: where, how, and why.

TDP-43 is an abundant and ubiquitously expressed nuclear protein that becomes dysfunctional in a spectrum of neurodegenerative diseases. TDP-43's ability to phase separate and form/enter biomolecular condensates of varying size and composition is critical for its functionality. Despite the high density of phase-separated assemblies in the nucleus and the nuclear abundance of TDP-43, our understanding of the condensate-TDP-43 relationship in this cellular compartment is only emerging. Recent studies have also suggested that misregulation of nuclear TDP-43 condensation is an early event in the neurodegenerative disease amyotrophic lateral sclerosis. This review aims to draw attention to the nuclear facet of functional and aberrant TDP-43 condensation. We will summarise the current knowledge on how TDP-43 containing nuclear condensates form and function and how their homeostasis is affected in disease.

A toolkit for the identification of NEAT1_2/paraspeckle modulators.

Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.

ALS-linked cytoplasmic FUS assemblies are compositionally different from physiological stress granules and sequester hnRNPA3, a novel modifier of FUS toxicity.

Formation of cytoplasmic RNA-protein structures called stress granules (SGs) is a highly conserved cellular response to stress. Abnormal metabolism of SGs may contribute to the pathogenesis of (neuro)degenerative diseases such as amyotrophic lateral sclerosis (ALS). Many SG proteins are affected by mutations causative of these conditions, including fused in sarcoma (FUS). Mutant FUS variants have high affinity to SGs and also spontaneously form de novo cytoplasmic RNA granules. Mutant FUS-containing assemblies (mFAs), often called "pathological SGs", are proposed to play a role in ALS-FUS pathogenesis. However, structural differences between mFAs and physiological SGs remain largely unknown therefore it is unclear whether mFAs can functionally substitute for SGs and how they affect cellular stress responses. Here we used affinity purification to isolate mFAs and physiological SGs and compare their protein composition. We found that proteins within mFAs form significantly more physical interactions than those in SGs however mFAs fail to recruit many factors involved in signal transduction. Furthermore, we found that proteasome subunits and certain nucleocytoplasmic transport factors are depleted from mFAs, whereas translation elongation, mRNA surveillance and splicing factors as well as mitochondrial proteins are enriched in mFAs, as compared to SGs. Validation experiments for a mFA-specific protein, hnRNPA3, confirmed its RNA-dependent interaction with FUS and its sequestration into FUS inclusions in cultured cells and in a FUS transgenic mouse model. Silencing of the Drosophila hnRNPA3 ortholog was deleterious and potentiated human FUS toxicity in the retina of transgenic flies. In conclusion, we show that SG-like structures formed by mutant FUS are structurally distinct from SGs, prone to persistence, likely cannot functionally replace SGs, and affect a spectrum of cellular pathways in stressed cells. Results of our study support a pathogenic role for cytoplasmic FUS assemblies in ALS-FUS.

Calcium-responsive transactivator (CREST) toxicity is rescued by loss of PBP1/ATXN2 function in a novel yeast proteinopathy model and in transgenic flies.

Proteins associated with familial neurodegenerative disease often aggregate in patients' neurons. Several such proteins, e.g. TDP-43, aggregate and are toxic when expressed in yeast. Deletion of the ATXN2 ortholog, PBP1, reduces yeast TDP-43 toxicity, which led to identification of ATXN2 as an amyotrophic lateral sclerosis (ALS) risk factor and therapeutic target. Likewise, new yeast neurodegenerative disease models could facilitate identification of other risk factors and targets. Mutations in SS18L1, encoding the calcium-responsive transactivator (CREST) chromatin-remodeling protein, are associated with ALS. We show that CREST is toxic in yeast and forms nuclear and occasionally cytoplasmic foci that stain with Thioflavin-T, a dye indicative of amyloid-like protein. Like the yeast chromatin-remodeling factor SWI1, CREST inhibits silencing of FLO genes. Toxicity of CREST is enhanced by the [PIN+] prion and reduced by deletion of the HSP104 chaperone required for the propagation of many yeast prions. Likewise, deletion of PBP1 reduced CREST toxicity and aggregation. In accord with the yeast data, we show that the Drosophila ortholog of human ATXN2, dAtx2, is a potent enhancer of CREST toxicity. Downregulation of dAtx2 in flies overexpressing CREST in retinal ganglion cells was sufficient to largely rescue the severe degenerative phenotype induced by human CREST. Overexpression caused considerable co-localization of CREST and PBP1/ATXN2 in cytoplasmic foci in both yeast and mammalian cells. Thus, co-aggregation of CREST and PBP1/ATXN2 may serve as one of the mechanisms of PBP1/ATXN2-mediated toxicity. These results extend the spectrum of ALS associated proteins whose toxicity is regulated by PBP1/ATXN2, suggesting that therapies targeting ATXN2 may be effective for a wide range of neurodegenerative diseases.

Long non-coding RNA NEAT1_1 ameliorates TDP-43 toxicity in in vivo models of TDP-43 proteinopathy.

Pathological changes involving TDP-43 protein ('TDP-43 proteinopathy') are typical for several neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). FTLD-TDP cases are characterized by increased binding of TDP-43 to an abundant lncRNA, NEAT1, in the cortex. However it is unclear whether enhanced TDP-43-NEAT1 interaction represents a protective mechanism. We show that accumulation of human TDP-43 leads to upregulation of the constitutive NEAT1 isoform, NEAT1_1, in cultured cells and in the brains of transgenic mice. Further, we demonstrate that overexpression of NEAT1_1 ameliorates TDP-43 toxicity in Drosophila and yeast models of TDP-43 proteinopathy. Thus, NEAT1_1 upregulation may be protective in TDP-43 proteinopathies affecting the brain. Approaches to boost NEAT1_1 expression in the CNS may prove useful in the treatment of these conditions.

Multistep process of FUS aggregation in the cell cytoplasm involves RNA-dependent and RNA-independent mechanisms.

Fused in sarcoma (FUS) is an RNA-binding protein involved in pathogenesis of several neurodegenerative diseases. Aggregation of mislocalized FUS into non-amyloid inclusions is believed to be pivotal in the development of cell dysfunction, but the mechanism of their formation is unclear. Using transient expression of a panel of deletion and chimeric FUS variants in various cultured cells, we demonstrated that FUS accumulating in the cytoplasm nucleates a novel type of RNA granules, FUS granules (FGs), that are structurally similar but not identical to physiological RNA transport granules. Formation of FGs requires FUS N-terminal prion-like domain and the ability to bind specific RNAs. Clustering of FGs coupled with further recruitment of RNA and proteins produce larger structures, FUS aggregates (FAs), that resemble but are clearly distinct from stress granules. In conditions of attenuated transcription, FAs lose RNA and dissociate into RNA-free FUS complexes that become precursors of large aggresome-like structures. We propose a model of multistep FUS aggregation involving RNA-dependent and RNA-independent stages. This model can be extrapolated to formation of pathological inclusions in human FUSopathies.

Compromised paraspeckle formation as a pathogenic factor in FUSopathies.

Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of paraspeckles, in an RNA-dependent manner and responds in the same way as other paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and paraspeckle formation in cultured cells, and FUS deficiency leads to loss of paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.

Fused in sarcoma (FUS) protein lacking nuclear localization signal (NLS) and major RNA binding motifs triggers proteinopathy and severe motor phenotype in transgenic mice.

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1-359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1-359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5-4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1-359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.

RNP granules in ALS and neurodegeneration: From multifunctional membraneless organelles to therapeutic opportunities.

Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.

Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.

TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.

Modelling FUSopathies: focus on protein aggregation.

The discovery of a causative link between dysfunction of a number of RNA-binding proteins with prion-like domains and the development of certain (neuro)degenerative diseases has completely changed our perception of molecular mechanisms instigating pathological process in these disorders. Irreversible aggregation of these proteins is a crucial pathogenic event delineating a type of proteinopathy. FUS (fused in sarcoma) is a prototypical member of the class, and studies into the causes and consequences of FUSopathies have been instrumental in characterizing the processes leading to deregulation of RNA metabolism in neurodegeneration. In vivo models of FUSopathy have provided critical insights into the mechanisms of FUS toxicity and clues on the role of non-amyloid aggregates, which are hallmarks of these diseases. The present review summarizes the data on FUS aggregation signatures in available model organisms on the basis of overexpression of FUS variants.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function.

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

Selective pattern of motor system damage in gamma-synuclein transgenic mice mirrors the respective pathology in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.

Dimebon does not ameliorate pathological changes caused by expression of truncated (1-120) human alpha-synuclein in dopaminergic neurons of transgenic mice.

BACKGROUND: Recent clinical studies have demonstrated that dimebon, a drug originally designed and used as a non-selective antihistamine, ameliorates symptoms and delays progress of mild to moderate forms of Alzheimer's and Huntington's diseases. Although the mechanism of dimebon action on pathological processes in degenerating brain is elusive, results of studies carried out in cell cultures and animal models suggested that this drug might affect the process of pathological accumulation and aggregation of various proteins involved in the pathogenesis of proteinopathies. However, the effect of this drug on the pathology caused by overexpression and aggregation of alpha-synuclein, including Parkinson's disease (PD), has not been assessed. OBJECTIVE: To test if dimebon affected alpha-synuclein-induced pathology using a transgenic animal model. METHODS: We studied the effects of chronic dimebon treatment on transgenic mice expressing the C-terminally truncated (1-120) form of human alpha-synuclein in dopaminergic neurons, a mouse model that recapitulates several biochemical, histopathological and behavioral characteristics of the early stage of PD. RESULTS: Dimebon did not improve balance and coordination of aging transgenic animals or increase the level of striatal dopamine, nor did it prevent accumulation of alpha-synuclein in cell bodies of dopaminergic neurons. CONCLUSION: Our observations suggest that in the studied model of alpha-synucleinopathy dimebon has very limited effect on certain pathological alterations typical of PD and related diseases.

Connecting the "dots": RNP granule network in health and disease.

All cells contain ribonucleoprotein (RNP) granules - large membraneless structures composed of RNA and proteins. Recent breakthroughs in RNP granule research have brought a new appreciation of their crucial role in organising virtually all cellular processes. Cells widely exploit the flexible, dynamic nature of RNP granules to adapt to a variety of functional states and the ever-changing environment. Constant exchange of molecules between the different RNP granules connects them into a network. This network controls basal cellular activities and is remodelled to enable efficient stress response. Alterations in RNP granule structure and regulation have been found to lead to fatal human diseases. The interconnectedness of RNP granules suggests that the RNP granule network as a whole becomes affected in disease states such as a representative neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this review, we summarize available evidence on the communication between different RNP granules and on the RNP granule network disruption as a primary ALS pathomechanism.

Frameshift peptides alter the properties of truncated FUS proteins in ALS-FUS.

Mutations in the FUS gene cause a subset of ALS cases (ALS-FUS). The majority of FUS mutations are missense mutations affecting the nuclear localisation signal (NLS) of FUS. In addition, a number of frameshift mutations which result in complete NLS deletion have been described. Patients bearing frameshift mutations usually present with more aggressive disease, characterised by an early onset and rapid progression. Both missense mutations in the NLS coding sequence and complete loss of the NLS are known to result in cytoplasmic mislocalisation of FUS protein. However, in addition to the removal of FUS functional domains, frameshift mutations in most cases lead to the attachment of a "tail" of novel amino acids at the FUS C-terminus - a frameshift peptide. It is not clear whether these peptide tails would affect the properties of truncated FUS proteins. In the current study, we compared intracellular behaviour of disease-associated truncated FUS proteins with and without the corresponding frameshift peptides. We demonstrate that some of these peptides can affect subcellular distribution and/or increase aggregation capacity and stability of the truncated FUS protein. Our study suggests that frameshift peptides can alter the properties of truncated FUS variants which may modulate FUS pathogenicity and contribute to the variability of the disease course in ALS-FUS.

Long non-coding RNA Neat1 regulates adaptive behavioural response to stress in mice.

NEAT1 is a highly and ubiquitously expressed long non-coding RNA (lncRNA) which serves as an important regulator of cellular stress response. However, the physiological role of NEAT1 in the central nervous system (CNS) is still poorly understood. In the current study, we addressed this by characterising the CNS function of the Neat1 knockout mouse model (Neat1-/- mice), using a combination of behavioural phenotyping, electrophysiology and expression analysis. RNAscope® in situ hybridisation revealed that in wild-type mice, Neat1 is expressed across the CNS regions, with high expression in glial cells and low expression in neurons. Loss of Neat1 in mice results in an inadequate reaction to physiological stress manifested as hyperlocomotion and panic escape response. In addition, Neat1-/- mice display deficits in social interaction and rhythmic patterns of activity but retain normal motor function and memory. Neat1-/- mice do not present with neuronal loss, overt neuroinflammation or gross synaptic dysfunction in the brain. However, cultured Neat1-/- neurons are characterised by hyperexcitability and dysregulated calcium homoeostasis, and stress-induced neuronal activity is also augmented in Neat1-/- mice in vivo. Gene expression analysis showed that Neat1 may act as a weak positive regulator of multiple genes in the brain. Furthermore, loss of Neat1 affects alternative splicing of genes important for the CNS function and implicated in neurological diseases. Overall, our data suggest that Neat1 is involved in stress signalling in the brain and fine-tunes the CNS functions to enable adaptive behaviour in response to physiological stress.

Stress granules regulate paraspeckles: RNP granule continuum at work.

Eukaryotic cells contain several types of RNA-protein membraneless macro-complexes - ribonucleoprotein (RNP) granules that form by liquid-liquid phase separation. These structures represent biochemical microreactors for a variety of cellular processes and also act as highly accurate sensors of changes in the cellular environment. RNP granules share multiple protein components, however, the connection between spatially separated granules remains surprisingly understudied. Paraspeckles are constitutive nuclear RNP granules whose numbers significantly increase in stressed cells. Our recent work using affinity-purified paraspeckles revealed that another type of RNP granule, cytoplasmic stress granule (SG), acts as an important regulator of stress-induced paraspeckle assembly. Our study demonstrates that despite their residency in different cellular compartments, the two RNP granules are closely connected. This study suggests that nuclear and cytoplasmic RNP granules are integral parts of the intracellular "RNP granule continuum" and that rapid exchange of protein components within this continuum is important for the temporal control of cellular stress responses. It also suggests that cells can tolerate and efficiently handle a certain level of phase separation, which is reflected in the existence of "bursts", or "waves", of RNP granule formation. Our study triggers a number of important questions related to the mechanisms controlling the flow of RNP granule components within the continuum and to the possibility of targeting these mechanisms in human disease.

Stress granules regulate stress-induced paraspeckle assembly.

Eukaryotic cells contain a variety of RNA-protein macrocomplexes termed RNP granules. Different types of granules share multiple protein components; however, the crosstalk between spatially separated granules remains unaddressed. Paraspeckles and stress granules (SGs) are prototypical RNP granules localized exclusively in the nucleus and cytoplasm, respectively. Both granules are implicated in human diseases, such as amyotrophic lateral sclerosis. We characterized the composition of affinity-purified paraspeckle-like structures and found a significant overlap between the proteomes of paraspeckles and SGs. We further show that paraspeckle hyperassembly is typical for cells subjected to SG-inducing stresses. Using chemical and genetic disruption of SGs, we demonstrate that formation of microscopically visible SGs is required to trigger and maintain stress-induced paraspeckle assembly. Mechanistically, SGs may sequester negative regulators of paraspeckle formation, such as UBAP2L, alleviating their inhibitory effect on paraspeckles. Our study reveals a novel function for SGs as positive regulators of nuclear RNP granule assembly and suggests a role for disturbed SG-paraspeckle crosstalk in human disease.

Antiviral Immune Response as a Trigger of FUS Proteinopathy in Amyotrophic Lateral Sclerosis.

Mutations in the FUS gene cause familial amyotrophic lateral sclerosis (ALS-FUS). In ALS-FUS, FUS-positive inclusions are detected in the cytoplasm of neurons and glia, a condition known as FUS proteinopathy. Mutant FUS incorporates into stress granules (SGs) and can spontaneously form cytoplasmic RNA granules in cultured cells. However, it is unclear what can trigger the persistence of mutant FUS assemblies and lead to inclusion formation. Using CRISPR/Cas9 cell lines and patient fibroblasts, we find that the viral mimic dsRNA poly(I:C) or a SG-inducing virus causes the sustained presence of mutant FUS assemblies. These assemblies sequester the autophagy receptor optineurin and nucleocytoplasmic transport factors. Furthermore, an integral component of the antiviral immune response, type I interferon, promotes FUS protein accumulation by increasing FUS mRNA stability. Finally, mutant FUS-expressing cells are hypersensitive to dsRNA toxicity. Our data suggest that the antiviral immune response is a plausible second hit for FUS proteinopathy.