Academic literature on the topic 'MicroRNA, RNA metabolism, ALS, FUS'

Beyond traditional approaches in understanding amyotrophic lateral sclerosis (ALS), multiple recent studies in RNA-binding proteins (RBPs)—including transactive response DNA-binding protein (TDP-43) and fused in sarcoma (FUS)—have instigated an interest in their function and prion-like properties. Given their prominence as hallmarks of a highly heterogeneous disease, this prompts a re-examination of the specific functional interrelationships between these proteins, especially as pathological SOD1—a non-RBP commonly associated with familial ALS (fALS)—exhibits similar properties to these RBPs including potential RNA-regulatory capabilities. Moreover, the cytoplasmic mislocalization, aggregation, and co-aggregation of TDP-43, FUS, and SOD1 can be identified as proteinopathies akin to other neurodegenerative diseases (NDs), eliciting strong ties to disrupted RNA splicing, transport, and stability. In recent years, microRNAs (miRNAs) have also been increasingly implicated in the disease, and are of greater significance as they are the master regulators of RNA metabolism in disease pathology. However, little is known about the role of these proteins and how they are regulated by miRNA, which would provide mechanistic insights into ALS pathogenesis. This review seeks to discuss current developments across TDP-43, FUS, and SOD1 to build a detailed snapshot of the network pathophysiology underlying ALS while aiming to highlight possible novel therapeutic targets to guide future research.

Abstract Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

Amyotrophic lateral sclerosis (ALS) is a fatal human neurodegenerative disease affecting primarily motor neurons. Two RNA-binding proteins, TDP-43 and FUS, aggregate in the degenerating motor neurons of ALS patients, and mutations in the genes encoding these proteins cause some forms of ALS. TDP-43 and FUS and several related RNA-binding proteins harbor aggregation-promoting prion-like domains that allow them to rapidly self-associate. This property is critical for the formation and dynamics of cellular ribonucleoprotein granules, the crucibles of RNA metabolism and homeostasis. Recent work connecting TDP-43 and FUS to stress granules has suggested how this cellular pathway, which involves protein aggregation as part of its normal function, might be coopted during disease pathogenesis.

Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease, which is universally fatal. While the causes of this devastating disease are poorly understood, recent advances have implicated RNA-binding proteins (RBPs) that contain predicted prion domains as a major culprit. Specifically, mutations in the RBPs TDP-43 and FUS can cause ALS. Cytoplasmic mislocalization and inclusion formation are common pathological features of TDP-43 and FUS proteinopathies. Though these RBPs share striking pathological and structural similarities, considerable evidence suggests that the ALS-linked mutations in TDP-43 and FUS can cause disease by disparate mechanisms. In a recent study, Couthouis et al. screened for protein candidates that were also involved in RNA processing, contained a predicted prion domain, shared other phenotypic similarities with TDP-43 and FUS, and identified TAF15 as a putative ALS gene. Subsequent sequencing of ALS patients successfully identified ALS-linked mutations in TAF15 that were largely absent in control populations. This study underscores the important role that perturbations in RNA metabolism might play in neurodegeneration, and it raises the possibility that future studies will identify other RBPs with critical roles in neurodegenerative disease.

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.

TAR DNA-binding protein-43 KDa (TDP-43) and fused in sarcoma (FUS) as the defining pathological hallmarks for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), coupled with ALS-FTD-causing mutations in both genes, indicate that their dysfunctions damage the motor system and cognition. On the molecular level, TDP-43 and FUS participate in the biogenesis and metabolism of coding and noncoding RNAs as well as in the transport and translation of mRNAs as part of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules. Intriguingly, many of the RNA targets of TDP-43 and FUS are involved in synaptic transmission and plasticity, indicating that synaptic dysfunction could be an early event contributing to motor and cognitive deficits in ALS and FTD. Furthermore, the ability of the low-complexity prion-like domains of TDP-43 and FUS to form liquid droplets suggests a potential mechanism for mRNP assembly and conversion. This review will discuss the role of TDP-43 and FUS in RNA metabolism, with an emphasis on the involvement of this process in synaptic function and neuroprotection. This will be followed by a discussion of the potential phase separation mechanism for forming RNP granules and pathological inclusions.

Fused in sarcoma (FUS) is a DNA/RNA binding protein that is involved in RNA metabolism and DNA repair. Numerous reports have demonstrated by pathological and genetic analysis that FUS is associated with a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and polyglutamine diseases. Traditionally, the fibrillar aggregation of FUS was considered to be the cause of those diseases, especially via its prion-like domains (PrLDs), which are rich in glutamine and asparagine residues. Lately, a nonfibrillar self-assembling phenomenon, liquid–liquid phase separation (LLPS), was observed in FUS, and studies of its functions, mechanism, and mutual transformation with pathogenic amyloid have been emerging. This review summarizes recent studies on FUS self-assembling, including both aggregation and LLPS as well as their relationship with the pathology of ALS, FTLD, and other neurodegenerative diseases.

Abstract Alterations of RNA metabolism caused by mutations in RNA-binding protein genes, such as transactivating DNA-binding protein-43 (TDP-43) and fused in sarcoma (FUS), have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). Unlike the accumulation of TDP43, which is accepted as a pathological hall mark of sporadic ALS (sALS), FUS pathology in sALS is still under debate. Although immunoreactive inclusions of FUS have been detected in sALS patients previously, the technical limitation of signal detection, including the necessity of specific antigen retrieval, restricts our understanding of FUS-associated ALS pathology. In this study, we applied a novel detection method using a conventional antigen retrieval technique with Sudan Black B treatment to identify FUS-positive inclusions in sALS patients. We classified pathological motor neurons into 5 different categories according to the different aggregation characteristics of FUS and TDP-43. Although the granular type was more dominant for inclusions with TDP-43, the skein-like type was more often observed in FUS-positive inclusions, suggesting that these 2 proteins undergo independent aggregation processes. Moreover, neurons harboring FUS-positive inclusions demonstrated substantially reduced expression levels of dynactin-1, a retrograde motor protein, indicating that perturbation of nucleocytoplasmic transport is associated with the formation of cytoplasmic inclusions of FUS in sALS.

Over 30% of patients with amyotrophic lateral sclerosis (ALS) exhibit cognitive deficits indicative of frontotemporal dementia (FTD), suggesting a common pathogenesis for both diseases. Consistent with this hypothesis, neuronal and glial inclusions rich in TDP43, an essential RNA-binding protein, are found in the majority of those with ALS and FTD, and mutations in TDP43 and a related RNA-binding protein, FUS, cause familial ALS and FTD. TDP43 and FUS affect the splicing of thousands of transcripts, in some cases triggering nonsense-mediated mRNA decay (NMD), a highly conserved RNA degradation pathway. Here, we take advantage of a faithful primary neuronal model of ALS and FTD to investigate and characterize the role of human up-frameshift protein 1 (hUPF1), an RNA helicase and master regulator of NMD, in these disorders. We show that hUPF1 significantly protects mammalian neurons from both TDP43- and FUS-related toxicity. Expression of hUPF2, another essential component of NMD, also improves survival, whereas inhibiting NMD prevents rescue by hUPF1, suggesting that hUPF1 acts through NMD to enhance survival. These studies emphasize the importance of RNA metabolism in ALS and FTD, and identify a uniquely effective therapeutic strategy for these disorders.

MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND. In the present paper we review recent developments showing a functional link between SMN (survival of motor neuron), the causative factor of SMA (spinal muscular atrophy), and FUS (fused in sarcoma), a genetic factor in ALS (amyotrophic lateral sclerosis). SMN is long known to have a crucial role in the biogenesis and localization of the spliceosomal snRNPs (small nuclear ribonucleoproteins), which are essential assembly modules of the splicing machinery. Now we know that FUS interacts with SMN and pathogenic FUS mutations have a significant effect on snRNP localization. Together with other recently published evidence, this finding potentially links ALS pathogenesis to disturbances in the splicing machinery, and implies that pre-mRNA splicing may be the common weak point in MND, although other steps in mRNA metabolism could also play a role. Certainly, further comparison of the RNA metabolism in different MND will greatly help our understanding of the molecular causes of these devastating diseases.

Project1: Unraveling the impact of microRNA on Amyotrophic Lateral Sclerosis pathogenesis. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that specifically affects upper and lower motor neurons leading to progressive paralysis and death. There is currently no effective treatment. Thus, identification of the signaling pathways and cellular mediators of ALS remains a major challenge in the search for novel therapeutics. Recent studies have shown that microRNA have a significant impact on normal CNS development and onset and progression of neurological disorders. Based on this evidence, in this study we test the hypothesis that misregulation of miRNA expression play a role in the pathogenesis of ALS. Hence, we exploited human neuroblastoma cell lines expressing SOD(G93A) mutation as tools to investigate the role of miRNAs in familiar ALS. To this end, we initially checked the key molecules involved in miRNAs biogenesis and processing on these cells and we found a different protein expression pattern. Subsequently, we performed a genome-wide scale miRNA expression, using whole-genome small RNA deep-sequencing followed by quantitative real time validation (qPCR). This strategy allowed us to find a small group of up and down regulated miRNA, which are predicted to play a role in the motorneurons physiology and pathology. We measured this group of misregulated miRNA by qPCR on cDNA derived from (G93A) mice at different stage of disease and furthermore on cDNA derived from lymphocytes from a group of sporadic ALS patients. We found that mir-129-5p was up-regulated in cells, mice and in patients and we validated that HuD as mir129-5p target. It has been reported that ELAVL4/HuD plays a role in neuronal plasticity, in recovery from axonal injury and multiple neurological diseases. Furthermore, we generated stable cell line overespressing mir129-5p and we found a reduction in neurite outgrowth and in the expression of differentiation markers in compare to control cells. Taken together these data strongly suggest that microRNAs play a role in ALS pathogenesis and in particular that mir129-5p can affect neuronal plasticity by modulating ELAVL4/HuD level. Project 2: FUS/TLS depletion leads an impairment of cell proliferation and DNA Damage Response. FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function hypothesis (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line we performed growth proliferation and survival assays. From these experiments emerged that FUS-depleted cells display alterations in cell proliferation. In order to explain this observation, we tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than control cells. Based on the notion that FUS interacts with the miRNA processing proteins (Morlando et al. 2012), to explain this phenotype, we looked at miRNAs expression and we found an up-regulation of mir-7. Interestingly, this up-regulation is also observed in cells that express the ALS-linked FUS R521C mutation. Finally, since an increasing number of work correlated FUS with DNA damage and repair we explored the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). We found that FUS depletion had an effect on the initial level of DNA damage by inducing the phosphorylation of H2AX in basal condition and that it delayed DSB repair when acute DNA damage occurs. Interestingly, genotoxic treatment resulted in changes in the subcellular localization of FUS in normal cells. We are currently exploring on one hand the mechanism by which FUS depletion leads to DNA damage, and on the other the functional significance of FUS relocalization after genotoxic stress. Taken together, these studies may contribute to the knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.