Background-Tumor necrosis factor-␣ (TNF-␣) and angiotensin II (Ang II) modulate heart failure in part by provoking the hypertrophic response. Signal transduction pathways of those factors are implicated in reactive oxygen intermediates (ROIs). Therefore, we hypothesized that TNF-␣ and Ang II might cause myocyte hypertrophy via the generation of ROIs. Methods and Results-To test the hypothesis, we tested whether TNF-␣ and Ang II could induce the generation of ROIs and whether antioxidants such as butylated hydroxyanisole (BHA), vitamin E, and catalase might inhibit the hypertrophy in cultured neonatal rat cardiac myocytes. ROIs were measured by the ROI-specific probe 2Ј,7Ј-dichlorofluorescin diacetate in cultured cardiac myocytes. We demonstrated that TNF-␣ and Ang II induced the generation of ROIs in a dose-dependent manner. TNF-␣ (10 ng/mL) and Ang II (100 nmol/L) enlarged cardiac myocytes and increased [ 3 H]leucine uptake, and BHA (10 mol/L) significantly inhibited both effects. Other antioxidants, such as vitamin E (1 g/mL) and catalase (100 U/mL), also inhibited the enlargement of cardiac myocytes induced by TNF-␣. Conclusions-These

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.

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. Recent studies show that TDP-43 is a major protein component of neuronal inclusion bodies in the affected tissues in a range of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD) (6, 7), Alzheimer's disease (AD) (8-10), and other types of dementia (10-13). Decreased protein solubility, hyperphosphorylation, abnormal cleavage, and cytoplasmic mislocalization of TDP-43 have been associated with TDP-43 proteinopathy (14-16). It is not clear whether TDP-43 proteinopathy is caused by loss-of-function of TDP-43 or gain-of-function neurotoxicity. Here, we report the generation and characterization of transgenic flies expressing human TDP-43. In different types of neurons, including photoreceptors, mushroom bodies, or motor neurons, simply overexpressing hTDP-43 by itself is sufficient to cause protein aggregate formation and neuronal loss in an agedependent manner, suggesting that increased hTDP-43 expression or aberrant accumulation of hTDP-43 may lead to TDP-43 proteinopathy. Our transgenic flies recapitulate important pathological and clinical features of ALS, representing a powerful animal model for TDP-43 proteinopathy. ResultsGeneration of Transgenic Flies Expressing Human TDP-43. To study human TDP-43 (hTDP-43) in vivo, we used Drosophila, a powerful genetic model widely used to study neurodegeneration (17, 18). We generated transgenic flies expressing monomeric red fluorescent protein (RFP) as a control or hTDP-43 fused to RFP in different populations of neurons using UAS/Gal4 system (19) (Fig. S1C).We also generated transgenic flies expressing a mutant hTDP-43, T202, containing the carboxy...

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 (UPR mt ) 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-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.

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.alternative splicing regulation ͉ cancer ͉ cell death ͉ RNA binding protein

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.

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.