TDP-43 induces mitochondrial damage and activates the mitochondrial unfolded protein response.

Mutations in or dys-regulation of the TDP-43 gene have been associated with TDP-43 proteinopathy, a spectrum of neurodegenerative diseases including Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS). The underlying molecular and cellular defects, however, remain unclear. Here, we report a systematic study combining analyses of patient brain samples with cellular and animal models for TDP-43 proteinopathy. Electron microscopy (EM) analyses of patient samples revealed prominent mitochondrial impairment, including abnormal cristae and a loss of cristae; these ultrastructural changes were consistently observed in both cellular and animal models of TDP-43 proteinopathy. In these models, increased TDP-43 expression induced mitochondrial dysfunction, including decreased mitochondrial membrane potential and elevated production of reactive oxygen species (ROS). TDP-43 expression suppressed mitochondrial complex I activity and reduced mitochondrial ATP synthesis. Importantly, TDP-43 activated the mitochondrial unfolded protein response (UPRmt) in both cellular and animal models. Down-regulating mitochondrial protease LonP1 increased mitochondrial TDP-43 levels and exacerbated TDP-43-induced mitochondrial damage as well as neurodegeneration. Together, our results demonstrate that TDP-43 induced mitochondrial impairment is a critical aspect in TDP-43 proteinopathy. Our work has not only uncovered a previously unknown role of LonP1 in regulating mitochondrial TDP-43 levels, but also advanced our understanding of the pathogenic mechanisms for TDP-43 proteinopathy. Our study suggests that blocking or reversing mitochondrial damage may provide a potential therapeutic approach to these devastating diseases.

FUS interacts with ATP synthase beta subunit and induces mitochondrial unfolded protein response in cellular and animal models.

FUS (fused in sarcoma) proteinopathy is a group of neurodegenerative diseases characterized by the formation of inclusion bodies containing the FUS protein, including frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Previous studies show that mitochondrial damage is an important aspect of FUS proteinopathy. However, the molecular mechanisms by which FUS induces mitochondrial damage remain to be elucidated. Our biochemical and genetic experiments demonstrate that FUS interacts with the catalytic subunit of mitochondrial ATP synthase (ATP5B), disrupts the formation of ATP synthase complexes, and inhibits mitochondrial ATP synthesis. FUS expression activates the mitochondrial unfolded protein response (UPRmt). Importantly, down-regulating expression of ATP5B or UPRmt genes in FUS transgenic flies ameliorates neurodegenerative phenotypes. Our data show that mitochondrial impairment is a critical early event in FUS proteinopathy, and provide insights into the pathogenic mechanism of FUS-induced neurodegeneration.

PINK1 and Parkin are genetic modifiers for FUS-induced neurodegeneration.

Dysregulation of Fused in Sarcoma (FUS) gene expression is associated with fronto-temporal lobar degeneration (FTLD), and missense mutations in the FUS gene have been identified in patients affected by amyotrophic lateral sclerosis (ALS). However, molecular and cellular defects underlying FUS proteinopathy remain to be elucidated. Here, we examined whether genes important for mitochondrial quality control play a role in FUS proteinopathy. In our genetic screening, Pink1 and Park genes were identified as modifiers of neurodegeneration phenotypes induced by wild type (Wt) or ALS-associated P525L-mutant human FUS. Down-regulating expression of either Pink1 or Parkin genes ameliorated FUS-induced neurodegeneration phenotypes. The protein levels of PINK1 and Parkin were elevated in cells overexpressing FUS. Remarkably, ubiquitinylation of Miro1 protein, a downstream target of the E3 ligase activity of Parkin, was also increased in cells overexpressing FUS protein. In fly motor neurons expressing FUS, both motility and processivity of mitochondrial axonal transport were reduced by expression of either Wt- or P525L-mutant FUS. Finally, down-regulating PINK1 or Parkin partially rescued the locomotive defects and enhanced the survival rate in transgenic flies expressing FUS. Our data indicate that PINK1 and Parkin play an important role in FUS-induced neurodegeneration. This study has uncovered a previously unknown link between FUS proteinopathy and PINK1/Parkin genes, providing new insights into the pathogenesis of FUS proteinopathy.

FUS Interacts with HSP60 to Promote Mitochondrial Damage.

FUS-proteinopathies, a group of heterogeneous disorders including ALS-FUS and FTLD-FUS, are characterized by the formation of inclusion bodies containing the nuclear protein FUS in the affected patients. However, the underlying molecular and cellular defects remain unclear. Here we provide evidence for mitochondrial localization of FUS and its induction of mitochondrial damage. Remarkably, FTLD-FUS brain samples show increased FUS expression and mitochondrial defects. Biochemical and genetic data demonstrate that FUS interacts with a mitochondrial chaperonin, HSP60, and that FUS translocation to mitochondria is, at least in part, mediated by HSP60. Down-regulating HSP60 reduces mitochondrially localized FUS and partially rescues mitochondrial defects and neurodegenerative phenotypes caused by FUS expression in transgenic flies. This is the first report of direct mitochondrial targeting by a nuclear protein associated with neurodegeneration, suggesting that mitochondrial impairment may represent a critical event in different forms of FUS-proteinopathies and a common pathological feature for both ALS-FUS and FTLD-FUS. Our study offers a potential explanation for the highly heterogeneous nature and complex genetic presentation of different forms of FUS-proteinopathies. Our data also suggest that mitochondrial damage may be a target in future development of diagnostic and therapeutic tools for FUS-proteinopathies, a group of devastating neurodegenerative diseases.

USP33 mediates Slit-Robo signaling in inhibiting colorectal cancer cell migration.

Originally discovered in neuronal guidance, the Slit-Robo pathway is emerging as an important player in human cancers. However, its involvement and mechanism in colorectal cancer (CRC) remains to be elucidated. Here, we report that Slit2 expression is reduced in CRC tissues compared with adjacent noncancerous tissues. Extensive promoter hypermethylation of the Slit2 gene has been observed in CRC cells, which provides a mechanistic explanation for the Slit2 downregulation in CRC. Functional studies showed that Slit2 inhibits CRC cell migration in a Robo-dependent manner. Robo-interacting ubiquitin-specific protease 33 (USP33) is required for the inhibitory function of Slit2 on CRC cell migration by deubiquitinating and stabilizing Robo1. USP33 expression is downregulated in CRC samples, and reduced USP33 mRNA levels are correlated with increased tumor grade, lymph node metastasis and poor patient survival. Taken together, our data reveal USP33 as a previously unknown tumor-suppressing gene for CRC by mediating the inhibitory function of Slit-Robo signaling on CRC cell migration. Our work suggests the potential value of USP33 as an independent prognostic marker of CRC.

An ALS-mutant TDP-43 neurotoxic peptide adopts an anti-parallel ß-structure and induces TDP-43 redistribution.

TDP-43 proteinopathies are clinically and genetically heterogeneous diseases that had been considered distinct from classical amyloid diseases. Here, we provide evidence for the structural similarity between TDP-43 peptides and other amyloid proteins. Atomic force microscopy and electron microscopy examination of peptides spanning a previously defined amyloidogenic fragment revealed a minimal core region that forms amyloid fibrils similar to the TDP-43 fibrils detected in FTLD-TDP brain tissues. An ALS-mutant A315E amyloidogenic TDP-43 peptide is capable of cross-seeding other TDP-43 peptides and an amyloid-ß peptide. Sequential Nuclear Overhauser Effects and double-quantum-filtered correlation spectroscopy in nuclear magnetic resonance (NMR) analyses of the A315E-mutant TDP-43 peptide indicate that it adopts an anti-parallel ß conformation. When added to cell cultures, the amyloidogenic TDP-43 peptides induce TDP-43 redistribution from the nucleus to the cytoplasm. Neuronal cultures in compartmentalized microfluidic-chambers demonstrate that the TDP-43 peptides can be taken up by axons and induce axonotoxicity and neuronal death, thus recapitulating key neuropathological features of TDP-43 proteinopathies. Importantly, a single amino acid change in the amyloidogenic TDP-43 peptide that disrupts fibril formation also eliminates neurotoxicity, supporting that amyloidogenesis is critical for TDP-43 neurotoxicity.

Alternative Pre-mRNA Splicing, Cell Death, and Cancer.

Alternative splicing is one of the most powerful mechanisms for generating functionally distinct products from a single genetic loci and for fine-tuning gene activities at the post-transcriptional level. Alternative splicing plays important roles in regulating genes critical for cell death. These cell death genes encode death ligands, cell surface death receptors, intracellular death regulators, signal transduction molecules, and death executor enzymes such as caspases and nucleases. Alternative splicing of these genes often leads to the formation of functionally different products, some of which have antagonistic effects that are either cell death-promoting or cell death-preventing. Differential alternative splicing can affect expression, subcellular distribution, and functional activities of the gene products. Molecular defects in splicing regulation of cell death genes have been associated with cancer development and resistance to treatment. Studies using molecular, biochemical, and systems-based approaches have begun to reveal mechanisms underlying the regulation of alternative splicing of cell death genes. Systematic studies have begun to uncover the multi-level interconnected networks that regulate alternative splicing. A global picture of the complex mechanisms that regulate cell death genes at the pre-mRNA splicing level has thus begun to emerge.

PSF suppresses tau exon 10 inclusion by interacting with a stem-loop structure downstream of exon 10.

Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA-protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5' splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.

Expression of human FUS protein in Drosophila leads to progressive neurodegeneration.

Mutations in the Fused in sarcoma/Translated in liposarcoma gene (FUS/TLS, FUS) have been identified among patients with amyotrophic lateral sclerosis (ALS). FUS protein aggregation is a major pathological hallmark of FUS proteinopathy, a group of neurodegenerative diseases characterized by FUS-immunoreactive inclusion bodies. We prepared transgenic Drosophila expressing either the wild type (Wt) or ALS-mutant human FUS protein (hFUS) using the UAS-Gal4 system. When expressing Wt, R524S or P525L mutant FUS in photoreceptors, mushroom bodies (MBs) or motor neurons (MNs), transgenic flies show age-dependent progressive neural damages, including axonal loss in MB neurons, morphological changes and functional impairment in MNs. The transgenic flies expressing the hFUS gene recapitulate key features of FUS proteinopathy, representing the first stable animal model for this group of devastating diseases.

An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity.

Mutations in TARDBP, encoding TAR DNA-binding protein-43 (TDP-43), are associated with TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We compared wild-type TDP-43 and an ALS-associated mutant TDP-43 in vitro and in vivo. The A315T mutant enhances neurotoxicity and the formation of aberrant TDP-43 species, including protease-resistant fragments. The C terminus of TDP-43 shows sequence similarity to prion proteins. Synthetic peptides flanking residue 315 form amyloid fibrils in vitro and cause neuronal death in primary cultures. These data provide evidence for biochemical similarities between TDP-43 and prion proteins, raising the possibility that TDP-43 derivatives may cause spreading of the disease phenotype among neighboring neurons. Our work also suggests that decreasing the abundance of neurotoxic TDP-43 species, enhancing degradation or clearance of such TDP-43 derivatives and blocking the spread of the disease phenotype may have therapeutic potential for TDP-43 proteinopathies.

Expression of human FUS/TLS in yeast leads to protein aggregation and cytotoxicity, recapitulating key features of FUS proteinopathy.

Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have been associated with amyotrophic lateral sclerosis (ALS). FUS-positive neuropathology is reported in a range of neurodegenerative diseases, including ALS and fronto-temporal lobar degeneration with ubiquitin-positive pathology (FTLDU). To examine protein aggregation and cytotoxicity, we expressed human FUS protein in yeast. Expression of either wild type or ALS-associated R524S or P525L mutant FUS in yeast cells led to formation of aggregates and cytotoxicity, with the two ALS mutants showing increased cytotoxicity. Therefore, yeast cells expressing human FUS protein recapitulate key features of FUS-positive neurodegenerative diseases. Interestingly, a significant fraction of FUS expressing yeast cells stained by propidium iodide were without detectable protein aggregates, suggesting that membrane impairment and cellular damage caused by FUS expression may occur before protein aggregates become microscopically detectable and that aggregate formation might protect cells from FUS-mediated cytotoxicity. The N-terminus of FUS, containing the QGSY and G rich regions, is sufficient for the formation of aggregates but not cytotoxicity. The C-terminal domain, which contains a cluster of mutations, did not show aggregation or cytotoxicity. Similar to TDP-43 when expressed in yeast, FUS protein has the intrinsic property of forming aggregates in the absence of other human proteins. On the other hand, the aggregates formed by FUS are thioflavin T-positive and resistant to 0.5% sarkosyl, unlike TDP-43 when expressed in yeast cells. Furthermore, TDP-43 and FUS display distinct domain requirements in aggregate formation and cytotoxicity.

RNA helicase p68 (DDX5) regulates tau exon 10 splicing by modulating a stem-loop structure at the 5' splice site.

Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5' splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5' splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.

A Drosophila model for TDP-43 proteinopathy.

Neuropathology involving TAR DNA binding protein-43 (TDP-43) has been identified in a wide spectrum of neurodegenerative diseases collectively named as TDP-43 proteinopathy, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). To test whether increased expression of wide-type human TDP-43 (hTDP-43) may cause neurotoxicity in vivo, we generated transgenic flies expressing hTDP-43 in various neuronal subpopulations. Expression in the fly eyes of the full-length hTDP-43, but not a mutant lacking its amino-terminal domain, led to progressive loss of ommatidia with remarkable signs of neurodegeneration. Expressing hTDP-43 in mushroom bodies (MBs) resulted in dramatic axon losses and neuronal death. Furthermore, hTDP-43 expression in motor neurons led to axon swelling, reduction in axon branches and bouton numbers, and motor neuron loss together with functional deficits. Thus, our transgenic flies expressing hTDP-43 recapitulate important neuropathological and clinical features of human TDP-43 proteinopathy, providing a powerful animal model for this group of devastating diseases. Our study indicates that simply increasing hTDP-43 expression is sufficient to cause neurotoxicity in vivo, suggesting that aberrant regulation of TDP-43 expression or decreased clearance of hTDP-43 may contribute to the pathogenesis of TDP-43 proteinopathy.

Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.

Similar to many genes involved in programmed cell death (PCD), the caspase 2 (casp-2) gene generates both proapoptotic and antiapoptotic isoforms by alternative splicing. Using a yeast RNA-protein interaction assay, we identified RBM5 (also known as LUCA-15) as a protein that binds to casp-2 pre-mRNA. In both transfected cells and in vitro splicing assay, RBM5 enhances the formation of proapoptotic Casp-2L. RBM5 binds to a U/C-rich sequence immediately upstream of the previously identified In100 splicing repressor element. Our mutagenesis experiments demonstrate that RBM5 binding to this intronic sequence regulates the ratio of proapoptotic/antiapoptotic casp-2 splicing isoforms, suggesting that casp-2 splicing regulation by RBM5 may contribute to its tumor suppressor activity. Our work has uncovered a player in casp-2 alternative splicing regulation and revealed a link between the alternative splicing regulator and the candidate tumor suppressor gene. Together with previous studies, our work suggests that splicing control of cell death genes may be an important aspect in tumorigenesis. Enhancing the expression or activities of splicing regulators that promote the production of proapoptotic splicing isoforms might provide a therapeutic approach to cancer.

Polypyrimidine tract-binding protein (PTB) differentially affects malignancy in a cell line-dependent manner.

RNA processing is altered during malignant transformation, and expression of the polypyrimidine tract-binding protein (PTB) is often increased in cancer cells. Although some data support that PTB promotes cancer, the functional contribution of PTB to the malignant phenotype remains to be clarified. Here we report that although PTB levels are generally increased in cancer cell lines from multiple origins and in endometrial adenocarcinoma tumors, there appears to be no correlation between PTB levels and disease severity or metastatic capacity. The three isoforms of PTB increase heterogeneously among different tumor cells. PTB knockdown in transformed cells by small interfering RNA decreases cellular growth in monolayer culture and to a greater extent in semi-solid media without inducing apoptosis. Down-regulation of PTB expression in a normal cell line reduces proliferation even more significantly. Reduction of PTB inhibits the invasive behavior of two cancer cell lines in Matrigel invasion assays but enhances the invasive behavior of another. At the molecular level, PTB in various cell lines differentially affects the alternative splicing pattern of the same substrates, such as caspase 2. Furthermore, overexpression of PTB does not enhance proliferation, anchorage-independent growth, or invasion in immortalized or normal cells. These data demonstrate that PTB is not oncogenic and can either promote or antagonize a malignant trait dependent upon the specific intra-cellular environment.

An intronic signal for alternative splicing in the human genome.

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.

Pre-mRNA splicing and retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous retinal diseases and a common cause of blindness. Among the 12 autosomal dominant RP (adRP) genes identified, four encode ubiquitously expressed proteins involved in pre-mRNA splicing, demonstrating the important role that pre-mRNA splicing plays in the pathogenesis of retinal degeneration. This review focuses on recent progress in identifying adRP mutations in genes encoding pre-mRNA splicing factors and the potential underlying molecular mechanisms.

NSSRs/TASRs/SRp38s function as splicing modulators via binding to pre-mRNAs.

The genes for neural-salient serine/arginine-rich (NSSR) proteins 1 and 2 have been cloned from the neuronal differentiated embryocarcinoma cell line, P19. NSSRs contain an RNA recognition motif (RRM) at the N-terminal and several SR rich regions at the C-terminal resembling RS domains. We found that NSSRs associated with U1-70k, and determined the exon inclusion activity of NSSRs' C-terminals. First, the RRM was changed to the MS2 coat protein (MS2CP) and then, MS2 RNA stem-loops were inserted in the middle of the exon N of the clathrin light chain B minigene as an artificial exonic splicing enhancer to be recognized by the MS2CP. The modified exon N of the pre-mRNA was included by the MS2CP switched NSSR 1, but it was excluded by the MS2CP switched NSSR 2. The deletion analysis of the MS2CP switched NSSR 1 showed that the middle SR rich region was responsible for the activity of the modified exon N inclusion. Furthermore, the RRM domain of NSSRs recognized mRNAs. NSSRs were expressed in the nervous system, especially in cerebellar and hippocampal primordia, ventricular zone of the neocortex and olfactory bulb primordia, retina, and olfactory epithelium at E15.5, all containing undifferentiated neural stem cells. Taken together, our results showed that NSSRs modulate alternative splicing via binding to premRNAs during neural differentiation.

Regulation of the alpha-fetoprotein gene by the isoforms of ATBF1 transcription factor in human hepatoma.

We investigated mechanisms regulating expression of alpha-fetoprotein (AFP) in 3 human hepatoma cell lines, HuH-7, HepG2, and huH-1, producing high, medium, and low levels of AFP, respectively. The silencer, a negative cis-acting element of the AFP gene, was highly activated in huH-1 and HepG2 to repress AFP enhancer activity by 91%, whereas only 26% repression was observed in HuH-7. To account for the difference in AFP production between HepG2 and huH-1, we investigated the roles of two isoforms of the AT motif-binding factor 1 (ATBF1) transcription factor, ATBF1-A and -B. Cotransfection assays showed that the ATBF1 isoforms regulated the AFP gene differently in HepG2 and huH-1. In huH-1 and HuH-7, both ATBF1 isoforms suppressed strongly enhancer activity and slightly promoter activity. In HepG2, on the other hand, ATBF1-A suppressed the enhancer and promoter activities, but surprisingly, ATBF1-B was found to stimulate enhancer activity while showing no effect on the promoter. Levels of ATBF1-A mRNA were similar in all 3 cell lines, whereas the expression ATBF1-B mRNA varied greatly, with the highest level seen in HepG2 followed by huH-1 and HuH-7. These results suggest that, in HepG2, ATBF1-B may have a dominant negative effect to relieve the transcriptional repression caused by its isoform. In support of this view, we found that the N-terminal region specific to the ATBF1-A molecule possessed transcriptional repressor activity. Thus, the use of the ATBF1 variants as well as the silencer may provide a unique mechanism that contributes to the determination of AFP levels in human hepatoma cell lines.