The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization,Stress Granules,and Detergent-Insoluble Aggregates of FUS/TLS

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.     

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.     

R521C and R521H mutations in FUS result in weak binding with Karyopherinβ2 leading to Amyotrophic lateral sclerosis: a molecular docking and dynamics study

Fused in sarcoma (FUS) gene encodes the RNA binding protein FUS. This gene is mapped to chromosome 16p11.2. The FUS protein binds with karyopherineβ2 (Kapβ2) through its proline/tyrosine nuclear localization signal (PY-NLS) that helps in the localization of FUS protein within the nucleus. Arginine residue in 521 position (R521) of PY-NLS plays a vital role in the binding of FUS protein with Kapβ2. Mutations in this position (R521C and R521H) are the most predominant mutations associated with amyotrophic lateral sclerosis (ALS). However, the mechanism by which these mutations lead to ALS is poorly understood. We examined the binding behaviour of the mutants FUS (R521C) and FUS (R521H) with Kapβ2 through protein–protein docking and molecular dynamics simulation. The binding patterns of mutants were compared with the binding behaviour of wild FUS–Kapβ2. Our results suggest that these mutants have relatively weak binding affinity with Kapβ2 when compared with wild FUS–Kapβ2 as indicated by the lesser number of interactions found between the mutant FUS and Kapβ2. Hence, these mutations weakens the binding and this results in the cytoplasmic mislocalization of mutant FUS; and thereby it increases the severity of ALS.     

Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro

FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.

     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

ULK2 Ser 1027 Phosphorylation by PKA Regulates Its Nuclear Localization Occurring through Karyopherin Beta 2 Recognition of a PY-NLS Motif

Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif (⁷⁷⁴gpgfgssppGaeaapslRyvPY⁷⁹⁵) in the S/P space domain of ULK2, which is similar to the consensus PY-NLS motif (R/K/H)X _2–5PY. Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant (the cytoplasmic localization form) was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.     

Stress signaling from the endoplasmic reticulum: A central player in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the misfolding and aggregation of distinct proteins in affected tissues, however, the pathogenic cause of disease remains unknown. Recent evidence indicates that endoplasmic reticulum (ER) stress plays a central role in ALS pathogenesis. ER stress activates the unfolded protein response (UPR), a homeostatic response to misfolded proteins. The UPR is initially protective by up-regulation of specific ER stress-regulated genes and inhibition of general protein translation. However, long-term ER stress leads to cell death via apoptotic signaling, thus providing a link to neurodegeneration. Activation of the UPR is one of the earliest events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant superoxide dismutase 1 (SOD1). Recently, genetic manipulation of ER stress in several different SOD1 mouse models was shown to alter disease onset and progression, implicating an active role for the UPR in disease mechanisms. Furthermore, mutations to vesicle-associated membrane protein-associated protein B (VAPB), an ER transmembrane protein involved in ER stress regulation, also cause some cases of familial ALS. ER stress also occurs in spinal cord tissues of human sporadic ALS patients, and recent evidence suggests that perturbation of the ER could occur in ALS cases associated with TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS) and valosin containing protein (VCP). Together these findings implicate ER stress as a potential upstream mechanism involved in both familial and sporadic forms of ALS.     

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid–liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.     

Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid droplets in vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.     

The FUS about arginine methylation in ALS and FTLD

EMBO J (2012) 31 22, 4258–4275 doi:10.1038/emboj.2012.261; published online September112012In a time where links between amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) neurodegeneration are becoming increasingly clear, it is important to establish the convergent and divergent mechanisms responsible for this. Accordingly, Dormann et al (2012) have identified that methylation of the Fused in sarcoma (FUS) RGG3 domain is involved in the cytoplasmic mislocalisation of ALS-FUS mutants, through a transportin-dependent mechanism. By contrast, hypomethylation in this domain may play a role in the aberrant accumulation of FUS in FTLD-FUS. This work showcases arginine methylation as a phenomenon to watch out for in neurodegenerative pathology.The nuclear RNA/DNA-binding protein, FUS, first burst onto the ALS scene in 2009 when it was discovered that mutations in this protein are causative for familial ALS (Kwiatkowski et al, 2009; Vance et al, 2009). It is now thought to be responsible for 4% of familial (and rare sporadic) ALS cases. In ALS patients with FUS mutations (ALS-FUS), the FUS protein is deposited in abnormal protein inclusions in neurons and glia and nuclei often show a reduced FUS staining (Lagier-Tourenne et al, 2010). Fascinatingly, this abnormal FUS deposition is also observed in several subtypes of FTLD, subsequently termed as FTLD-FUS. However, unlike in ALS-FUS, there are no known FUS mutations in this disease (Da Cruz and Cleveland, 2011). Dormann et al (2012) now broaden our knowledge of how ALS-causing mutations in FUS lead to its abnormal cytoplasmic deposition in neurons and glia, and for the first time suggest a mechanism through which this could also occur in FTLD-FUS, in the absence of FUS mutations.The majority of pathogenic mutations identified in ALS-FUS are located at the C-terminus of the protein within a region identified to be a proline-tyrosine nuclear localization signal (PY-NLS) (Figure 1). The PY-NLS binds to the nuclear import receptor transportin (TRN), which facilitates FUS transport into the nucleus. Pathogenic FUS mutations affect key residues of the PY-NLS or completely delete the signal sequence and thus impair nuclear import of FUS (Dormann et al, 2010). This nuclear transport defect is likely directly involved in pathogenesis, as mutations that cause a very severe nuclear import deficit (e.g., FUS-P525L), are associated with earlier disease onset and a rapid disease course (Dormann et al, 2010).Open in a separate windowFigure 1(A) Schematic diagram showing the domain structure of FUS. SYGQ-rich, serine, tyrosine, glycine, glutamine-rich domain; RRM, RNA recognition motif; ZnF, zinc finger; PY, proline-tyrosine nuclear localization signal (PY-NLS). (B) Schematic diagram summarizing the divergent mechanisms by which arginine methylation (or absence thereof) may bring about FUS mislocalization and accumulation in ALS-FUS and FTLD-FUS. In ALS-FUS caused by FUS mutations, neuronal cytoplasmic inclusions contain methylated FUS, but are negative for EWS, TAF15 and TRN. Dormann et al (2012) propose that these FUS-specific inclusions result from the combination of a genetic defect (point mutations in the PY-NLS that impair transportin binding) and post-translational modification (arginine methylation in RGG3 domain that also impairs transportin binding). By contrast in FTLD-FUS, neuronal cytoplasmic inclusions contain all three FET proteins and transportin, but are not immunoreactive with meFUS-specific antibodies. It is therefore possible that hypomethylation of the FET proteins and thus increased transportin binding may be involved in the co-deposition of these proteins in FTLD-FUS.FUS and other related PY-NLS-containing FET proteins such as Ewing sarcoma (EWS) protein and TATA-binding protein-associated factor 15 (TAF15) have been described to undergo extensive asymmetric dimethylation in their arginine-glycine-glycine (RGG) domains (Araya et al, 2005; Hung et al, 2009; Jobert et al, 2009). In recent times, it has been established that this arginine methylation can affect their nuclear localization (Araya et al, 2005; Jobert et al, 2009; Tradewell et al, 2012). In this issue, Dormann et al (2012) sought to delve further into the mechanism by which this occurs. They began their studies in a similar fashion to Tradewell and colleagues, by confirming that inhibiting global arginine methylation using the general methylation inhibitor, adenosine-2,3-dialdehyde (AdOx) could restore the lost nuclear localization of both HA-tagged cytoplasmic ALS-causing mutants and cytoplasmic EWS and TAF15 point mutants in HeLa cells. As AdOx is capable of inhibiting protein, DNA and lipid methylation, they also investigated the effect of specifically preventing protein methylation on the localization of the severe FUS-P525L ALS mutant. In these experiments, siRNA-mediated silencing of PRMT1, the protein arginine methyltransferase responsible for the majority of cellular protein arginine dimethylation, successfully restored the nuclear localization of FUS-P525L, thus confirming the importance of arginine methylation in the cytoplasmic localization of ALS-FUS mutants. After performing this groundwork, Dormann and colleagues then began an elegant set of experiments to unravel the mechanism behind their results. They first wanted to identify whether TRN was involved. Co-expression of GFP-tagged TRN inhibitor peptide with HA-tagged FUS-P525L in HeLa cells lead to a complete prevention of the normal nuclear accumulation of FUS-P525L upon AdOx treatment. Thus, identifying a critical role for TRN in the observed nuclear import of ALS-FUS mutants upon demethylation.Dormann et al (2012) next sought to determine how arginine methylation may impact the nuclear import of FUS by TRN. Their first step was to determine which arginine residues in FUS were involved. With this came the ground-breaking finding that it is actually arginine residues in the RGG3 motif N-terminal to the PY-NLS (Figure 1), rather than in the PY-NLS itself that can modulate TRN-dependent nuclear import of mutant FUS. Through NMR spectroscopy, Dormann and colleagues were then able to show that arginine residues in the FUS RGG3 motif can bind directly to TRN. These observations were taken further via studies of the interaction of recombinant FUS-RGG3 domain or synthetic FUS peptides with TRN by isothermal titration calorimetry. Here, they showed that the FUS-P525L mutant bound weakly or not at all to TRN, but in the presence of the unmethylated RGG3 domain the binding affinity of the mutant was rescued to WT-like levels. These observations were validated by the fact that the unmethylated RGG3 domain alone was able to bind TRN in the absence of a C-terminal PY-NLS with an affinity similar to that of the WT PY-NLS. Methylation of the RGG3 peptide completely prevented this binding. Through these experiments Dormann et al (2012) were able to show for the first time that residues outside of the PY-NLS can be involved in FUS nuclear import. Furthermore, they were able to establish a working model by which this occurs: they propose that in normal situations, the PY-NLS anchors the FUS C-terminus to TRN and the adjacent RGG repeats stabilize the interaction. Methylation of the RGG repeats interferes with TRN binding, but in WT FUS the affinity of the PY-NLS for TRN is sufficient to allow nuclear import to continue. By contrast, in the methylated P525L mutant, weak binding of both the methylated RGG domain and the c-terminus of the PY-NLS to TRN abrogates FUS nuclear import, thus causing its cytoplasmic accumulation.On the basis of their model, Dormann et al (2010) predicted that cytoplasmically mislocalized ALS FUS mutants would be methylated in their RGG3 domains. To test this hypothesis, they generated two monoclonal antibodies specific to the methylated RGG3 domain (meFUS antibodies). Cytosolic FUS mutants, such as FUS-P525L, were recognized by the meFUS-specific antibodies in HeLa cells, thus strongly suggesting that methylation of FUS mutants is a contributing factor to their cytoplasmic retention.Having generated the valuable tools of meFUS-specific antibodies, Dormann et al (2010) next turned their attention to the analysis of whether the intriguing shared property of cytoplasmic FUS accumulation in ALS-FUS and FTLD-FUS could be related to arginine methylation. In direct support of their cell-culture experiments, they discovered that there was a strong and consistent co-labelling of all FUS-positive cytoplasmic neuronal and glial inclusions with the meFUS antibody in post-mortem tissue from ALS-FUS patients. However, the surprise came when they began analysing post-mortem tissues from various subtypes of FTLD-FUS patients. Here, they could not see any labelling of FUS-positive neuronal and glial cytoplasmic and intranuclear inclusions with the meFUS-specific antibody. This strongly suggests that a different FUS cytoplasmic retention mechanism is in play in FTLD-FUS patients compared to ALS-FUS patients. In support of this, inclusions in ALS-FUS patients have recently been identified to contain only the FUS protein, while inclusions in FTLD-FUS show a clear co-deposition of all FET proteins (FUS, EWS, and TAF15) along with TRN itself (Neumann et al, 2012). From this, Dormann et al (2012) conclude that mislocalization of FUS in ALS is likely a phenomenon resulting directly from mutations in the nuclear localization signal that is exacerbated by arginine methylation in the RGG3 domain, whereas FUS mislocalization in FTLD-FUS may result from a more general defect in TRN-mediated nuclear import of FET proteins (Figure 1). Indeed, they speculate that in FTLD, hypomethylation of the FET proteins by a yet to be determined mechanism may lead to excessively tight binding of these proteins to TRN. This in turn may lead to impaired dissociation of FET-TRN complexes, which could over time lead to the co-deposition of FET proteins and TRN in cytoplasmic and nuclear inclusions in FTLD patients. The results of future experiments performed to test this hypothesis will doubtless be of huge significance for the FTLD field. Furthermore, this study raises many questions about the normal cellular role of FUS arginine methylation. Is it for example involved in the fine tuning of the shuttling of FUS into and out of the nucleus and if this is the case could arginine methylation in the RGG1 and RGG2 domains, one of which (RGG1) is close to the FUS nuclear export signal, also be involved in regulating FUS localization? All this remains to be seen…     

Novel mutations in TARDBP (TDP-43) in patients with familial amyotrophic lateral sclerosis

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43-positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the approximately 25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.     

Understanding the role of TDP-43 and FUS/TLS in ALS and beyond

Dominant mutations in two DNA/RNA binding proteins, TDP-43 and FUS/TLS, are causes of inherited Amyotrophic Lateral Sclerosis (ALS). TDP-43 and FUS/TLS have striking structural and functional similarities, implicating alterations in RNA processing as central in ALS. TDP-43 has binding sites within a third of all mouse and human mRNAs in brain and this binding influences the levels and splicing patterns of at least 20% of those mRNAs. Disease modeling in rodents of the first known cause of inherited ALS-mutation in the ubiquitously expressed superoxide dismutase (SOD1)-has yielded non-cell autonomous fatal motor neuron disease caused by one or more toxic properties acquired by the mutant proteins. In contrast, initial disease modeling for TDP-43 and FUS/TLS has produced highly varied phenotypes. It remains unsettled whether TDP-43 and FUS/TLS mutants provoke disease from a loss of function or gain of toxicity or both. TDP-43 or FUS/TLS misaccumulation seems central not just to ALS (where it is found in almost all instances of disease), but more broadly in neurodegenerative disease, including frontal temporal lobular dementia (FTLD-U) and many examples of Alzheimer's or Huntington's disease.     

A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro

TAR DNA-binding protein-43 (TDP-43) is a highly conserved, ubiquitously expressed nuclear protein that was recently identified as the disease protein in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS). Pathogenic TDP-43 gene (TARDBP) mutations have been identified in familial ALS kindreds, and here we report a TARDBP variant (A90V) in a FTLD/ALS patient with a family history of dementia. Significantly, A90V is located between the bipartite nuclear localization signal sequence of TDP-43 and the in vitro expression of TDP-43-A90V led to its sequestration with endogenous TDP-43 as insoluble cytoplasmic aggregates. Thus, A90V may be a genetic risk factor for FTLD/ALS because it predisposes nuclear TDP-43 to redistribute to the cytoplasm and form pathological aggregates.     

Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro

Mutations in the RNA binding protein FUS (fused in sarcoma) have been linked to a subset of familial amyotrophic lateral sclerosis (ALS) cases. The mutations are clustered in the C-terminal nuclear localization sequence (NLS). Various FUS mutants accumulate in the cytoplasm whereas wild-type (WT) FUS is mainly nuclear. Here we investigate the effect of one ALS causing mutant (FUS-ΔNLS, also known as R495X) on pre-mRNA splicing and RNA expression using genome wide exon-junction arrays. Using a non-neuronal stable cell line with inducible FUS expression, we detected early changes in RNA composition. In particular, mutant FUS-ΔNLS increased calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) at both mRNA and protein levels, whereas WT-FUS had no effect. Chromatin immunoprecipitation experiments showed that FUS-ΔNLS accumulated at the CAMK2N2 promoter region, whereas promoter occupation by WT-FUS remained constant. Given the loss of FUS-ΔNLS in the nucleus through the mutation-induced translocation, this increase of promoter occupancy is surprising. It indicates that, despite the obvious cytoplasmic accumulation, FUS-ΔNLS can act through a nuclear gain of function mechanism.     

Profilin 1 mutants form aggregates that induce accumulation of prion-like TDP-43

Mutations in the profilin 1 (PFN1) gene have been identified as a cause of familial amyotrophic lateral sclerosis (ALS), and neuropathological studies indicate that TDP-43 is accumulated in brains of patients with PFN1 mutation. Here, we investigated the role of PFN1 mutations in the formation of prion-like abnormal TDP-43. Expression of PFN1 with pathogenic mutations resulted in the formation of cytoplasmic aggregates positive for p62 and ubiquitin, and these aggregates sequestered endogenous TDP-43. TDP-43 accumulation was facilitated in the presence of proteasome or lysosome inhibitor. Co-expression of mutant PFN1 and TDP-43 increased the levels of detergent-insoluble and phosphorylated TDP-43, and this increase required the C-terminal region of TDP-43. Moreover, detergent-insoluble fractions prepared from cells expressing ALS-linked mutant PFN1 induced seed-dependent accumulation of TDP-43. These findings indicate that expression of PFN1 mutants induces accumulation of TDP-43, and promotes conversion of normal TDP-43 into an abnormal form. These results provide new insight into the mechanisms of TDP-43 proteinopathies and other diseases associated with amyloid-like protein deposition.