Idiopathic Pulmonary Fibrosis is a lethal fibrotic disease characterized by the unrelenting proliferation and persistence of fibroblasts in a type I collagen-rich matrix that result in an expanding reticular network of fibrotic tissue. However, the underlying mechanism responsible for the persistence of myofibroblasts in IPF remains unclear. During normal tissue repair, unwanted fibroblasts are eliminated during collagen-matrix contraction by a mechanism whereby high PTEN activity suppresses Akt. We have previously found that FoxO3a, a transcriptional activator of apoptosis-inducing proteins, is inactivated in IPF fibroblasts resulting from aberrantly high PI3K/Akt activity due to inappropriately low PTEN activity. Here we demonstrate that this low FoxO3a activity confers IPF fibroblasts with resistance to collagen-mediated apoptosis. We show that the mechanism by which low FoxO3a activity confers IPF fibroblasts with an apoptotic resistant phenotype involves suppression of Fas expression as a result of down regulation of cav-1 expression via a PTEN/Akt-dependent pathway. We demonstrate that PTEN over-expression or Akt inhibition increases FoxO3a expression in IPF fibroblasts, resulting in up-regulation of caveolin-1. We show that FoxO3a binds to the cav-1 promoter region and ectopic expression of FoxO3a transcriptionally increases cav-1 mRNA and protein expression. In turn, we show that overexpression of caveolin-1 increases Fas levels and caspase-3/7 activity and promotes IPF fibroblast apoptosis on polymerized type I collagen. We have found that the expression of caveolin-1, Fas and cleaved caspase-3 proteins in fibroblasts within the fibroblastic foci of IPF patient specimens is low. Our data indicate that the pathologically altered PTEN/Akt axis inactivates FoxO3a down-regulating cav-1 and Fas expression. This confers IPF fibroblasts with an apoptosis-resistant phenotype and may be responsible for IPF progression. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disorder of unknown etiology The FoxO3a transcription factor controls the expression of proteins regulating both the cell cycle and cell viability. Active FoxO3a functions as a powerful inhibitor of the cell cycle and also promotes apoptosis During normal tissue repair, excess fibroblasts are eliminated by apoptosis. The mechanism involves collagen contraction-mediated activation of PTEN thereby suppressing phosphorylated Akt levels This study involves the analysis of human IPF patient specimens. Primary fibroblast lines were obtained from unused, existing pathological human tissue samples, and therefore is exempt (exemption 4). Tissue samples were stripped of all identifiers and designated as waste. All patients underwent procedures for diagnostic or therapeutic procedures. Written informed consent was obtained on all patients prior to the procedure being performed. Use of human tissues was approved by the Institutional Review Board (IRB) at the University of Minnesota.Cell lines were derived from lungs removed at the time of transplantation or death. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high resolution chest computed tomography findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological findings of usual interstitial pneumonia. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society (ATS) and the European Respiratory Society (ERS). For this study, new primary IPF fibroblast lines were generated as tissue became available. To reduce technical variability, we routinely utilize cells between passages 5 and 7 because of concern that the phenotype of the cells is altered at higher passage. To address concerns of biological variability, we studied 8 IPF cell lines and 6 control cells lines.8 IPF primary human lung fibroblast lines were established and analyzed for this study. Six control primary adult human lung fibroblast lines were established from histologically normal lung tissue distant from carcinoid tumor or radiation-induced fibrotic lung tissue or anatomically normal lung tissue not used at the time of transplantation. Primary lung fibroblast lines were generated by explant culture and cultured in high glucose DMEM containing 10% FCS. Fibroblasts were used between passages five and seven. Cells were characterized as fibroblasts as previously described 4 of control or IPF fibroblasts were cultured on type I polymerized collagen coated 96 well plates in 100 µl of serum free medium as a function of time. 20 µl of Cell Titer Blue reagent was then added to each well and further incubated for 1 to 2 h. Cell viability was measured by a 96 well plate reader with a fluorescence filter set 560(Ex)/590(Em). For caspase-3/7 activity assay, Apo-ONE Homogeneous Caspase-3/7 assay kit was used . 3×104 of control or IPF fibroblasts in 100 µl of serum free DMEM buffer cultured on polymerized collagen on 96 well plates were incubated with 100 µl of the mixture of caspase substrate and Apo-one caspase-3/7 buffer for 12 h. The fluorescence of each well with a filter set 485(Ex)/527(Em) was used to measure caspase-3/7 activity in samples.To precisely measure viable cells on collagen matrix, Cell-Titer-Blue Cell Viability Assay  was used. Briefly, 3×10CAG CCA CCC GAG ATTT GAG CA 3′), human reverse 18S rRNA (5′-TAG TAG CGA CGG GCG GTG TG 3′), mouse forward 18S rRNA (5′-AGG GGA GAG CGG GTA AGA GA-3′), mouse reverse 18S rRNA (GGA CAG GAC TAG GCG GAA CA-3′), human forward cav-1 (5′- TGA CTG AGA AGC AAG TGT ATG ACG-3′), human reverse cav-1 (5′- GCA GAA GGT ATG GAC GTA GAT-3′), mouse forward cav-1 (5′-TGA CTG AGA AGC AAG TGT ATG ACG-3′), mouse reverse cav-1 (5′-GCA GAA GGT ATG GAC GTA GAT-3′) were prepared from the Biomedical Genomic Center at the University of Minnesota. Quantitative PCR was performed using a Light Cycler FastStart DNA Masterplus SYBR Green I Kit. Samples were quantified at the log-linear portion of the curve using LightCycler analysis software and compared to an external calibration standard carve. Cav-1 levels were normalized to 18S rRNA. Quantitative PCR parameters were as follows. Mouse 18SrRNA, 60°C annealing, 10 second extension for 40 cycles; mouse cav-1, 56°C annealing, 15 second extension for 40 cycles; human 18SrRNA, 62°C annealing, 11 second extension for 40 cycles; human cav-1, 56°C annealing, 15 second extension for 40 cycles. For the analysis of putative FoxO3a binding site on the cav-1 promoter region, MATCH program (version 1.0) was used. This software is designed to search potential binding sites for transcription factor nucleotide sequences using the library of mononucleotide weight matrices from TRANSFACT 6.0. The search is performed using 0.75 and 0.70 as cutoffs for core and matrix similarity, respectively using MATCH program. For the confirmation of FoxO3a binding to the cav-1 promoter, 2×105 IPF fibroblasts were infected with 1×106 PFU of adenovirus expressing wild type FoxO3a and agarose ChiP kit from Pierce was used according to manufacturer’s protocol. After crosslinking and micrococcal nuclease digestion steps, FoxO3a bound complex was immunoprecipitated with or without FoxO3a antibody (2 µg). Rabbit IgG was also used as a negative control. After DNA was recovered, C1 forward primer containing putative FoxO3a binding sites , 5′-CTC CAC CCC TGC TGA GAT GAT-3′ and C1 reverse primer 5′-AGT GAG AAC GTT GTC CCG CGC TGG-3′ were used for ChIP-PCR. PCR was carried out with 2 µl of recovered DNA using 0.25 µM of C1 primers as follows. 94°C for 7 min for 1 cycle, then 94°C for 30s, 55°C for 40 s and 72°C for 40 s for 35 cycles and 72°C for 1 cycle for 7 min. Amplified products were analyzed using 2% agarose gel. For the analysis of FoxO3a dependent transcriptional regulation, a luciferase reporter construct that contains Forkhead consensus binding sites (FHRE-Luc) were obtained from Addgene, MA. 2×105 IPF and control fibroblasts expressing wild type FoxO3a, dominant negative FoxO3a or empty vector were transfected with 2 µg of pGL3-FHRE-Luc plasmid using FuGene HD transfection reagent . Cells were then cultured on polymerized collagen in serum free medium for 24 h and lysed in 250 µl of lysis buffer. The luciferase activity of one fifth of the samples were assayed using Bright-Glo™ Luciferase Assay system  according to manufacturer’s protocol using Lumat LB 9507 luminometer.For Western analysis, PTEN, p-Akt (s473), cav-1, Fas antibodies were obtained from Cell Signaling Technologies, MA. GAPDH and actin antibodies and FoxO3a antibody for immunoprecipitation for ChIP assay were purchased from Santa Cruz biotechnologies. FoxO3a and inactive FoxO3a (S253 phosphorylated FoxO3a) antibodies for Western analysis were obtained from Millipore, MA and Cell Signaling Technologies, MA. Anti-human Fas activating clone CH11 was also obtained from Millipore. Immunohistochemical analysis of myofibroblasts within the fibroblastic foci of IPF patient specimens was performed using an α smooth muscle actin antibody . Cav-1, Fas siRNA and non-coding scrambled control siRNA were purchased from Life Technologies and Santa Cruz Biotechnologies, respectively using X-treme GENE siRNA transfection reagent (Roche Applied Science) according to manufacturer’s protocol. At 24 h post transfection, fibroblasts were cultured on polymerized collagen in serum free medium and assay was performed as described. For quantitative RT-PCR assay, total RNA was extracted from cells using Trizol  according to manufacturer’s protocol. cDNA was prepared using the Taqman Reverse Trascription kit (Life Technologies) with random hexamer primers and 1 µg of total RNA. Human forward 18S rRNA  and adenovirus titer was measured by plaque method in 0.5% soft agar. The cells were infected with adenoviral vectors at a multiplicity of infection of 1∶20. For the transient transfection assay, plasmids expressing wild type (WT), mutant (S253A) or triple mutant FoxO3a (TM) which three phosphorylation residues recognized by Akt were mutated to alanine were obtained from Addgene. Adenovirus expressing HA-tagged Akt with c-src myristolyation sequence fused in frame to the N terminus (Hyper active Akt), HA-tagged Akt dominant mutant  and wild type FoxO3a were purchased from Vector Biolabs . Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs. Control or IPF fibroblasts were infected with adenovirus expressing PTEN, Akt or empty vector in 2 ml of tissue culture dishes. 1 to 2×105 control or IPF fibroblasts per ml of DMEM medium were infected with 1×106 PFU of each adenovirus for assay.For PTEN adenovirus construction, wild type (WT) PTEN cDNA was generated from normal human lung fibroblasts (HLF-210) by RT-PCR. The primers used for wild type PTEN, which span the entire coding region of PTEN are Patient specimens were fixed overnight with paraformaldehyde (4%) at RT. The specimens were rinsed in 6% sucrose/0.1 M Dulbeccos PBS (DPBS) at RT, infiltrated with increasing concentrations of Sucrose/DPBS with rotation at 4°C over a 72 h period, and cryoprotected first with 20% sucrose/DPBS overnight, then in a 1∶1 solution of 20% sucrose in DPBS/O.C.T , and rotated overnight at 4°C. After cryoembedding in O.C.T., 4 µ sections were cut and placed on silane-coated slides using a Leica 1950 Cryostat. Cryosections were fixed in 100% methanol at −20°C for 10 minutes, air dried and then quenched with 0.3% Hydrogen peroxide in methanol for 15 minutes at RT, and again air dried. They were then rinsed in PBS, placed in a steamer for 10 min with Citrate Buffer for antigen retrieval, and blocked for 30 min with normal serum determined by the host species of the antibody, to block nonspecific binding of secondary antibodies. Endogenous Avidin and Biotin binding sites were blocked by sequential incubation with Avidin/Biotin Blocking Kit . The sections were then incubated overnight at 4°C in their respected concentrations for each antibody, α- smooth muscle actin (1∶100) (Vector Labs), p-FoxO3A (1∶150) , cleaved caspase-3 (1∶100) and cav-1(1∶1000)  and Fas (1∶100) . For detection of the primary antibody, the LSAB Method was used. The sections were first rinsed with PBS, then placed in a species specific biotinylated secondary antibody for 30 min (1∶500), and transferred to the HRP-Streptavidin  conjugate solution for 30 min. The enzyme was then visualized with the application of the chromagen substrate 3, 3′ Diaminobenzene and counterstained with Hematoxylin. Control specimens were processed with the identical protocol minus the primary antibody to produce the negative controls.Data are expressed as the mean ± S.D. Experiments were performed three times. Paired evaluations were made for experimental and control conditions within each experiment, and significance was determined by Student's t test. Significance level was set at p<0.05.We have previously found that normal fibroblasts undergo apoptosis during their interaction with and contraction of polymerized collagen matrices Active FoxO3a can promote apoptosis. Since FoxO3a function is abnormally low in IPF fibroblasts, we next examined whether up-regulation of FoxO3a expression in IPF fibroblasts alters their morphology and viability when they interact with polymerized collagen. When FoxO3a was overexpressed in IPF fibroblasts on collagen matrix, they adopted a round appearance and apoptotic cells were readily apparent whereas IPF fibroblasts treated with GFP empty vector maintained their fibroblast morphology . The perRecent studies have demonstrated that caveolin-1 (cav-1) regulates the Fas-mediated apoptotic pathway Prior studies indicate that the cav-1 promoter contains putative FoxO consensus binding sites (5′-GTAAA(C/T)A-3′) To further verify whether FoxO3a is responsible for low cav-1 protein expression in FoxO3a −/− cells, we re-constituted wild type FoxO3a in FoxO3a−/− cells, and cav-1 expression was measured. Cav-1 protein level was high when wild type FoxO3a was expressed compared to that of cells expressing GFP , dominant negative FoxO3a (DF) or GFP in IPF fibroblasts cultured on polymerized collagen and measured cav-1 levels. Cav-1 protein expression increased in IPF fibroblasts in which wild type FoxO3a was over-expressed fibrolasts in the fibroblastic foci in vivo cell environment. Pepsin extracted process removes nonhelical telopeptides situated at the N- and C-terminal ends of native collagen molecules Although we found that IPF fibroblasts display an apoptotic resistant phenotype on type I polymerized collagen, it is possible that the pepsin extracted collagen that we used for our studies may not fully simulate the polymerized cross-linked collagen that exists in the Although we focused on the role of FoxO3 in regulating IPF fibroblast function, FoxO3a is also thought to be involved in aging. Since IPF is a disease of older individuals with a mean age of ∼65 years In summary, we demonstrate that IPF fibroblasts display an apoptotic resistant phenotype on polymerized collagen. The mechanism for this resistance involves inactivation of FoxO3a by Akt which down-regulates cav-1 expression . Decreas


The deduced electrical noise equivalent power is 1.2 A bolometer is a device that makes an electrical resistive response to the electromagnetic radiation resulted from a raise of temperature due to heating. The combination of the extremely weak electron-phonon interactions along with its small electron heat capacity makes graphene an ideal material for applications in ultra-fast and sensitive hot electron bolometer. However, a major issue is that the resistance of pristine graphene weakly depends on the electronic temperature. We propose using disordered graphene to obtain a strongly temperature dependent resistance. The measured electrical responsivity of the disordered graphene bolometer reaches 6 × 10 One of many astonishing properties of graphene is its weak electron-phonon (e-p) coupling. In normal conductor, e-p scattering quickly dominates as the temperature increases, resulting in a diminishing carrier mobility. In contrast, e-p scattering in graphene is negligible even at room temperature1346791011121314151819This present work demonstrates a new approach for implementing a graphene HEB. The main key is to drive the electronic system into the strong localization regime by adding disorder. Note that disorder here in general denotes the scattering potentials of various forms, such as lattice defects, charge impurities, etc. The divergence of the resistance at low temperature will allow for sensitive temperature measurement. The high resistance, together with an as-grown Boron Nitride (BN) tunneling barrier between graphene and electrical contacts, also reduces thermal dissipation out of the electron gas via diffusion. The bolometer fabricated with such method is found highly sensitive and with a very low background noise. Moreover, this approach does not require graphene with high mobility. The device structure with merely a graphene bar in contact with two electrodes can be easily fabricated with standard photo-lithography technique and extended into a 2D detector array.D peak in the Raman spectrum in Fig. 1(a)23The samples used in this study were Graphene/BN bilayer grown by CVD methodμm wide BN/Graphene ribbon on the metal electrodes, see R against T−1/3 in a semilog scale, where a linear dependence spans from the lowest temperature of 1.5 K to 300 K. The data suggest that electrons are strongly localized and the transport is dominated by the so-called variable range hopping (VRH)ρ(T) ∝ exp[(T0/T)1/3]. Here, T0 is a characteristic temperature, T0 = 12/πkBg(EF)ξ2, where kB is the Boltzmann constant, g(EF) the density of states at the Fermi level EF, ξ the localization length. We have performed a linear fit for the ln R versus T−1/3 plot. The slope of the fit yields a localization length ξ of 50 nm. Both the Raman results and the transport data indicate that the graphene film is disordered. The sharp increase of the resistance at low temperature due to localization is the key ingredient of our bolometer, as the responsivity of our bolometer is proportional to the slope of the R – T curve, dR/dT. R/dT as a function of temperature. At 2 K, dR/dT reaches 22 kΩ/K. Moreover, a high electrical resistance means a high thermal resistance, which prevents heat from dissipation via diffusion and also helps the responsivity.Our bolometer devices consist of a 5 Idc to generate heat in the electron gas. As shown in V/I = R0 exp(−eEl/BTk) when eEl/BTk < 1R0 is the zero bias resistance, E the electric field, e the electron charge and l a fraction of the hopping distance mr, i.e., l = 0.18mr. Since mr = ξ(T0/T)1/3, we have l = 29 nm at 1.57 K. A fit of the data to an exponential field dependence is very poor, see To see how the bolometer responses to heating, we apply a d.c. bias current Te will be determined by the strength of the e-p scattering and nearly uniform along the sample. We now show that this is indeed true in our devices. For a uniform temperature, Te at different biases can be obtained from the resistance R based on the R – T curve. We have measured R – Idc in magnetic fields of 0, 1, 5 T . It linearly follows ρ. The highest value in our experiment reaches 6 MΩ/nW. The corresponding responsivity of the device, which is 2.5 μm long and 5 μm wide, is 6 × 106 V/W at an excitation current of 2 nA.The responsivity of a graphene HEB depends mainly on two factors. One is the e-p coupling strength, which determines the amplitude of the temperature increase in response to a certain power input. The other is the voltage response to the temperature change, which is the sensitivity of graphene as a thermometer. In i.e., graphene/BN/SiO2/Si, the transfer matrix of the structure is deducedV at different temperatures are plotted in To test the actual bolometric response of the device, we employed a red LED to illuminate the device. The LED emits light with a peak light wavelength of 650 nm. Since the whole device is under uniform and constant illumination, photovoltaic and photothermoelectric signals will not be picked up by the lock-in (See Method). Considering a multilayer structure, R – Vdc plot into a Te – P plot and then numerically compute the derivative dTe/dP, as plotted in Te, which is expected by theoretical work on e-p scattering in graphene. By fitting the linearity, we have Tph is the phonon(lattice) temperature, while TBG is Bloch-Grüneisen temperature, defined as kBTBG = 2kFvph, where kF is the Fermi wave vector, vph the speed of soundvph = 2 × 104 m/s, TBG ≈ 102 K, much higher than Te and Tph. Considering a deformation potential D = 18 eVvF = 106 m/s, we find that for a graphene film with an area of 12.5 μm2, We have shown that the thermal dissipation pathway is mainly dominated by e-p scattering. Therefore, the thermal resistance due to e-p scattering can be computed by τ of the bolometer can now be estimated by ρ ≈ θCeR, where eC is the electronic heat capacity. −20 J/K, resulting in τ ≈ 1.4 nS. Apparently, the intrinsic speed of graphene bolometer is extremely fast. However, in practical application, the actual response time is also limited by the RC time constant of the whole circuit, which is τ = RC. Here, R and C are the circuit resistance and capacitance, respectively. Take the sample above as an example. Considering the total area of four metal electrode pads and the graphene sample, the capacitance to the back gate is estimated to be 7 pF. The total capacitance of our measurement cables is roughly about 250 pF, while the total resistance, including the sample resistance and two contact resistances, is 94 kΩ. We then have τ ≈ 24 μs. Therefore, it is important to minimize the parasitic capacitance of the bolometer circuit and the resistance of graphene in order to approach the intrinsic speed of graphene HEBs.Having known the value of the thermal resistance, the response time θR ≈ 58 K/nW at 1.5 K. Thus, NEP by the thermal fluctuations is R = 54 kΩ plus the total contact resistance of 40 kΩ. The corresponding NEP is the noise divided by the responsivity, Besides the responsivity, noise equivalent power (NEP) is a key factor that determines the performance of a bolometer. There are three noise sources, the measurement circuit noise, the thermal fluctuations and the Johnson noise6 × 0.027 ≈ 1.6 × 105 V/W and a NEP of When estimating the responsivity and NEP of our devices, we have used the absorbed power. Although this gives the ultimate performance of the device, it is useful to discuss the optical responsivity and NEP, estimated by taking the incident radiant power as the input. Taking into account the 2.7% absorptance of the device, we get an optical responsivity, 6 × 10e.g., the ratio of the device area to the contact area. In fact, for disordered graphene, the resistivity is relatively high so that the heat dissipation through diffusion can be substantially reduced. As seen in the We now discuss the role of the BN layer. As we have explained, it increases the contact resistance, therefore reduces thermal dissipation by diffusion. As a result, the temperature is uniform along the device, which allows for straightforward derivation of the electron temperature and the thermal conductance and analysis of e-p scattering. However, the temperature uniformity is not a requirement for our bolometer, although it is helpful to gain the responsivity by suppression of heat dissipation into contacts. In addition to increasing the contact resistance, it can be achieved by enhancing the graphene resistivity or by modifying the device geometry, 2/Si substrate. Similar 2D array is the basic structure for 2D bolometric imaging, where each element works independently to provide a pixel. The graphene ribbon is 5 μm long and 2 μm wide. The fabrication procedure is similar to the one for making an individual device (see the Method). Although the e-beam lithography was used for this demonstration, photo-lithography is well suited for fabrication of the array. Note that the structure of the detector array has not been optimized, i.e., the optical fill-factor is very low. We want to point out that the simplicity of the device structure and the aspect ratio dependent responsivity and resistance, offer significant flexibility in device design to meet applications.The basic structure of a disordered graphene HEB is comprised of only a graphene ribbon and two electrodes. It can be easily expanded to a detector array using conventional photo-lithography technique. In e.g., SdH oscillations, superconducting tunnel junctions and dual-gated bilayer graphene, disordered graphene provides a rather simple, yet effective scheme to implement a graphene HEB. The bolometer has an electrical responsivity of 6 × 106 V/W, over an order of magnitude higher than other approaches135 V/W. This value is much higher than 1 × 103 V/W for the dual-gated bilayer graphene deviceCompared with previous approaches, The responsivity of disordered graphene HEB rivals Si bolometers and composite bolometersh-BN samples in this work were synthesized via low pressure chemical vapor deposition on 25 um Cu foils (purchased from Alfa Aesar)h-BN was initially grown on Cu foil at 1000°C, using ammonia borane  as the precursorh-BN was achieved by introducing carbon sources into the system. Depending on the graphene growth parameters, i.e., growth temperature and carbon source inlet flow rate, the quality of graphene can be controlled. In order to obtain defective graphene, low temperature (800°C) and high carbon source flux were chosen to increase densities of defects and nucleation in graphene film. The bolometer devices were fabricated by e-beam lithography. 20 nm Ti/50 nm Au electrodes were first deposited on Si/SiO2 (300 nm) substrates. The graphene/BN films were then transferred onto the substrateBilayer structure of defective graphene/X.S.W. conceived the experiment. X.S.W. and D.P.Y. supervised the experiment. Q.H. fabricated devices and performed low temperature measurements. T.G. synthesized BN/graphene samples. Y.F.Z. and Z.F.L. supervised sample synthesis. X.S.W., Q.H., Y.C. and G.R.L. wrote the manuscript. X.S.W., Y.C. and G.R.L. performed data analysis. R.Z. and J.H.C. helped with the experiment. All authors participated in data discussion and reviewed the manuscript.Supplementary Info


Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays an important role in feeding behavior. It activates two G-protein-coupled receptors, MCHR1 and MCHR2, of which MCHR1 is the primary regulator of food intake and energy homeostasis in rodents. In mammalian cells transfected with MCHR1, MCH is able to activate multiple signaling pathways including calcium mobilization, extracellular signal-regulated kinase activation, and inhibition of cyclic AMP generation through Gi/o- and Gq-coupled pathways. Further evidence suggests that MCHR1 is regulated through posttranslational modifications, which control its intracellular localization and provide appropriate cellular responses involving G-protein signaling. This review summarizes the current data on the control of MCHR1 function through glycosylation and phosphorylation, as related to cell function. Especially, a series of mutagenesis study highlights the importance of complete glycosylation of MCHR1 for efficient trafficking to the plasma membrane. The G-protein-coupled receptor (GPCR) gene family is one of the largest families in the mammalian genome. GPCRs are typical heptahelical receptors composed of an extracellular N-terminus, an intracellular C-terminus, and seven transmembrane bundles connected by three intracellular loops and three extracellular loops. Activation of GPCRs induces second messenger responses that change the biochemical properties of the recipient cell and can modulate not only its electrophysiological responsiveness, but also its transcriptional activity. Thus, the diverse signaling and wide array of functions have allowed GPCRs to be employed in the physiological regulation of nearly all biological functions. These features coupled with ligands that are chemically highly specific for the receptors have resulted in the extensive utilization of GPCR-targeted drug design. Furthermore, the dynamic posttranslational modifications may provide tissue-specific functions, since distinct cellular environments or agonists can mediate different effects on receptor signaling or regulation of a number of GPCRs –5. ConseMCH gene is overexpressed upon fasting and in ob/ob leptin-deficient mice  was SLC-1/GPR24, an orphan GPCR, by applying orphan receptor strategies and reverse pharmacology –19. MCHRXenopus tropicalis, information for four MCH receptors has been obtained (A second MCH receptor (MCHR2) was subsequently identified by six groups using human genomic sequence searches . It sharobtained . Phylogeobtained . Therefoin vivo potency in the rodent. Comprehensive review of desired selectivity and efficacy of MCHR1 antagonist in vivo should consult some recent reviews  localized in the C-terminal cytoplasmic tail. The insertion of palmitate into the cytoplasmic leaflet of the plasma membrane can create a fourth loop named helix 8, thereby profoundly affecting the GPCR structure and consequently the interactions with intracellular partner proteins. Although a putative palmitoylation site has not been identified in mammalian MCHR1 , the exiXenopus, mouse, and human orthologs , six for protein kinase C , and one for protein kinase casein kinase 2 (Ser/Thr–X–X–Asp/Glu: T342) Figure . Howevers Figure , while ts Figure .The MCH-MCHR1 system has integral roles in many cellular events, and represents an important therapeutic target. For example, it may be targeted in strategies for developing treatments against obesity and mood disorders, and possibly also for inflammatory diseases. Therefore, the regulation of the MCH system by posttranslational modifications remains an emerging area at the nexus of endocrinology with important implications for drug development. However, our understanding of the regulation of MCHR1 signaling by glycosylation and phosphorylation is limited Table , 57, 58.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TAR-DNA binding protein 43 (TDP-43) is a heterogeneous ribonucleoprotein (hnRNP) identified as a major protein constituent of cytosolic inclusions in spinal cord of patients with motor neuron disease. The inclusions are formed by movement of TDP-43 from its predominantly nuclear localization to the cytosol, followed by accumulation of ubiquitinated and phosphorylated C-terminal TDP-43 fragments. The mechanisms controlling TDP-43 trafficking and accumulation are not well known, however, it has been demonstrated previously that kinases control movement and accumulation of hnRNPs. Therefore, we investigated whether kinases also controlled TDP-43 accumulation during cell stress.Neuronal cultures were treated with the mitochondrial inhibitors paraquat or sodium arsenite, or transfected with C-terminal TDP-43 or full length TDP-43 constructs and examined for kinase control of TDP-43 localization.We found that stress induction induced robust cytosolic accumulation of C-terminal TDP-43 into RNA stress granules, some of which progress to ubiquitinated inclusions. Inhibitors of c-Jun N-terminal kinase (JNK) and cyclin-dependent kinase 2 (CDK2) specifically inhibit and/or reverse the cytosolic accumulation of TDP-43 with little effect on ubiquitous stress granule formation. Our studies have shown active CDK2 co-localizes with accumulated TDP-43 and heterogeneous ribonucleoprotein K (hnRNP K). CDK2 inhibition blocks phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to Interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevents accumulation of TDP-43. We have also determined that specific C-terminal mutations to TDP-43 alter expression of hnRNP K during cell stress. Preliminary studies indicate that this may involve altered hnRNP K phosphorylation.Due to the recently identified role for hnRNPs in motor neuron disease and frontotemporal dementia, further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.


Tardbp leads to a dramatic increase in the levels of splicing variants of mouse sortilin 1 and translin. However, the levels of these two abnormal splicing variants are not increased in hTDP-43 transgenic mice despite significant downregulation of mTdp-43 in these mice. Moreover, further downregulation of mTdp-43 in hTDP-43 hemizygous mice, which are asymptomatic, to the levels equivalent to that of mTdp-43 in hTDP-43 homozygous mice does not induce the pathological phenotypes observed in the homozygous mice. Lastly, the number of dendritic spines and the RNA levels of TDP-43 RNA targets critical for synapse formation and function are significantly decreased in symptomatic homozygous mice. Together, our findings indicate that mTdp-43 downregulation does not lead to a loss of function mechanism or account for the pathological phenotypes observed in hTDP-43 homozygous mice because hTDP-43 compensates for the reduction, and associated functions of mTdp-43. Rather, expression of hTDP-43 beyond a certain threshold leads to abnormal metabolism of TDP-43 RNA targets critical for neuronal structure and function, which might be responsible for the ALS or FTLD-like pathologies observed in homozygous hTDP-43 transgenic mice.Tar DNA binding protein 43 (TDP-43) is the major component of pathological deposits in frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in amyotrophic lateral sclerosis (ALS). It has been reported that TDP-43 transgenic mouse models expressing human TDP-43 wild-type or ALS-associated mutations recapitulate certain ALS and FTLD pathological phenotypes. Of note, expression of human TDP-43 (hTDP-43) reduces the levels of mouse Tdp-43 (mTdp-43). However, it remained unclear whether the mechanisms through which TDP-43 induces ALS or FTLD-like pathologies resulted from a reduction in mTdp-43, an increase in hTDP-43, or a combination of both. In elucidating the role of mTdp-43 and hTDP-43 in hTDP-43 transgenic mice, we observed that reduction of mTdp-43 in non-transgenic mice by intraventricular brain injection of AAV1-sh TARDBP, the gene encoding TDP-43, are associated with sporadic and familial ALS, indicating that TDP-43 can directly cause neurodegeneration Tar DNA-binding protein 43 (TDP-43) is the principal component of ubiquitinated inclusions in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) in vitro and in vivoin vivo and observed that mice developed early embryonic lethality, suggesting an important role for TDP-43 in development WT) or mutant hTDP-43, leads to pathological phenotypes consistent with certain TDP-43 proteinopathies. These phenotypes may include some of the following: increased ubiquitination, truncation, aggregation and phosphorylation of TDP-43, cytoplasmic TDP-43 inclusions, neuronal degeneration, motor dysfunction, learning and memory deficits, and mitochondrial abnormalities Tardbp. Additionally, recent studies demonstrate that TDP-43 autoregulates itself to decrease its expression Tardbp levels through this mechanism. However, it remains unclear whether the reduction in mTdp-43 (loss of function) or the overexpression of hTDP-43 (gain of function), or combined events are responsible for the ALS or FTLD-like pathologies observed in hTDP-43 transgenic mice.Previous studies have explored the TDP-43 loss of function hypothesis Tardbp mRNA observed in our hTDP-43 transgenic mice results in significant reduction of mTdp-43 protein. We found that this reduction in mTdp-43 does not increase the levels of splicing variants known to be inhibited by TDP-43. In particular, the levels of mouse Ex17b-containing sortilin 1 (mSort1+Ex17b) and Ex5-deleted translin (mTsn_ΔEx5) mRNAs are significantly upregulated when mTdp-43 protein is reduced in non-transgenic (NT) mice but not in hTDP-43 mice, suggesting that hTDP-43 has similar biological functions to that of mTdp-43 in vivo. Moreover, a further reduction of mTdp-43 in asymptomatic hemizygous hTDP-43 (hTDP-43hemi) mice to the levels equivalent to that of symptomatic homozygous hTDP-43 (hTDP-43homo) mice did not lead to the development of pathological phenotypes indicating that downregulation of mTdp-43 is not sufficient to induce the pathological phenotypes observed in hTDP-43homo mice. Finally, we provide evidence that high expression levels of hTDP-43 beyond a certain threshold lead to alterations in TDP-43 RNA substrates critical for maintaining neuronal structure and function and this, in turn, might cause neuronal dysfunction and the development of pathological phenotypes.To address the aforementioned question, we first generated an antibody that specifically detects mTdp-43 protein, and confirmed that downregulation of mouse hemi and hTDP-43homo transgenic mice. The levels of mTdp-43 protein in hTDP-43hemi and hTDP-43homo mice respectively decreased to ∼55% and 45% of NT mice levels (Tardbp mRNA and mTdp-43 protein levels in both hemizyogous and homozygous hTDP-43M337V mice (data not shown). Of importance, TDP-43M337V and TDP-43WT display similar biological activity in autoregulating the levels of TDP-43 in vivo, suggesting the ALS-associated M337V mutation does not lead to loss of function.Recently, we and others have reported that the mRNA levels of mTdp-43 are significantly decreased in hTDP-43 transgenic mice e levels , suggestin vivosortilin 1 (Sort1) translin (Tsn) mSort1+Ex17b (sortilin 1 including exon 17b) and mTsn_ΔEx5 (translin with exon 5 deletion) , the levels of mSort1+Ex17b and mTsn_ΔEx5 increased 3.7- and 5.2-fold, respectively, when mTdp-43 levels are reduced -shRNA targeting mTdp-43 in neonatal mice expressing only mTdp-43 (NT mice) or mTdp-43 and hTDP-43 (hTDP-43hemi mice). Note that efficient downregulation of mTdp-43 protein and RNA was achieved in both NT and hTDP-43hemi transgenic mice (mSort1+Ex17b and mTsn_ΔEx5 (hemi mice transduced with a control shRNA (shCtrl), the levels of mSort1+Ex17b and mTsn_ΔEx5 were not increased after further reduction of mTdp-43 in shTardbp-treated hTDP-43hemi mice. Similar results were observed in hTDP-43M337V mice . Overall our data suggest that a decrease in mTdp-43 protein in hTDP-43 transgenic mice is not responsible for the observed phenotype in these mice. Rather, it does seem that the human TDP-43 may be driving these events.We have previously reported that hTDP-43n levels . To invemice see . Since homo mice . At 1 mogregates , phosphogregates , and abngregates . These pemi mice  and NT mhomo mice to a certain point, beyond which the overexpression of hTDP-43 induces toxicity, we sought to investigate whether the highly abundant hTDP-43 protein present in these mice would alter the levels of certain TDP-43 mRNA targets and consequently account for the TDP-43 associated pathologies in hTDP-43homo mice. To this end, we measured the levels of several neuronal TDP-43 RNA targets that are critical for neuronal survival and function homo mice compared to NT mice and hTDP-43hemi mice , presenilins (PSEN1 and PSEN2) or microtubule associated protein tau (MAPT) have been overexpressed in animals to make Alzheimer’s disease (AD) mouse models, which are characterized by the production and accumulation of amyloid-β into plaques or of hyperphosphorylated tau into neurofibrillary tangles Generation of transgenic mice is a widely used research technique to model human diseases, given transgenic mice recapitulate key pathological features of disease and also serve as valuable tools to explore the role of disease-causing molecules (e.g. TDP-43). More importantly, the models provide opportunities to evaluate potential therapeutic strategies for translational research. For instance, mutations in genes encoding amyloid precursor protein  and mTsn lacking exon 5 (mTsn_ΔEx5) increase dramatically. These two opposite splicing patterns upon mTdp-43 downregulation (exon inclusion in mSort1 and exon exclusion in mTsn) make our assay more comprehensive. Of note, the levels of mSort1+Ex17b and mTsn_ΔEx5 are not increased in hTDP-43 transgenic mice, despite the fact that these mice present lower levels of mTdp-43 protein than in AAV1-shTardbp injected NT mice, which do show a significant increase in these aberrant splicing isoforms. Moreover, further reduction of mTdp-43 in hTDP-43hemi mice to the levels equivalent to that of hTDP-43homo mice do not lead to an increase in mSort1+Ex17b nor mTsn_ΔEx5 isoforms. As a result, our data indicate that loss of mTdp-43 function in hTDP-43 mice is compensated by the expression of hTDP-43. These results are not surprising since mouse and human TDP-43 proteins are 96% homologous WT and hTDP-43M337V develop phenotypes characteristic of FTLD and ALS diseases. However, hTDP-43hemi transgenic mice are virtually indistinguishable from NT controls homo mice hTDP-43hemi mice have ∼75% hTDP-43 protein or ∼25% less hTDP-43 protein. As such, we hypothesize that expression of hTDP-43 over a certain threshold will disrupt the processing of TDP-43 RNA targets that will consequently cause the neuropathologies observed in hTDP-43homo mice. While we were unable to detect differences in TDP-43 RNA targets known to be altered by mTdp-43 downregulation, we found significant changes in TDP-43 RNA targets that are critical for neuronal function and survival in hTDP-43 mice overexpressing high levels of hTDP-43. In particular, the mRNA levels of synaptic markers Psd-95, Bsn, Gap-43, and Syt7 are significantly decreased in hTDP-43homo mice, but not in hTDP-43hemi mice. Moreover, the number of dendritic spines in the hippocampus of hTDP-43homo mice is also significantly decreased compared to NT. More important, the mRNA levels of the above mentioned targets and the number of dendritic spines are not altered in AAV1-shTardbp treated hTDP-43hemi mice, excluding the possibility that low levels of mTdp-43 are responsible for the synaptic dysfunction in hTDP-43homo mice. Together, our results suggest that expression of total levels of TDP-43 protein (human and mouse) above 2.5 fold of endogenous mouse Tdp-43 in NT mice might disrupt RNA metabolism of several synaptic targets To further determine whether the lower levels of mTdp-43 protein in our hTDP-43 transgenic mice result in loss of mTdp-43 function, we examined the levels of abnormal splicing variants of two of its RNA targets: homo mice. In addition, there is no difference between wild-type and M337V hTDP-43 proteins regarding their ability to regulate RNA splicing. Our findings suggest that the current hTDP-43 transgenic mouse models might not be the most suitable tools to investigate loss of mTdp-43 function. Knock-in transgenic mouse models expressing either wild-type hTDP-43 or ALS-associated TDP-43 mutations at similar levels to mTdp-43 should be generated to understand the roles of TDP-43 in disease pathogenesis.In conclusion, although expression of hTDP-43 reduces the levels of mTdp-43 in hTDP-43 transgenic mice, this reduction does not lead to loss of mTdp-43 function because hTDP-43 compensates for functional losses in mTdp-43. Moreover, expression of hTDP-43 above a certain threshold disrupts RNA metabolism, especially of genes critical for neuronal survival and function, which consequently causes the pathological phenotypes observed in hTDP-43The hTDP-43 transgenic mice expressing wild-type or M337V mutant hTDP-43 have been previously reported The polyclonal mTdp-43 antibody is produced by using synthetic peptide corresponding to residues 203–221 of mTDP-43 (TAEELQQFFCQYGEVVDVF) to immunize rabbits. The resulting sera were affinity purified and the specificity of the antibody was characterized using Western blot.Tardbp, mSort1+Ex17b and mTsn_ΔEx5 mRNA values were normalized to Gapdh values, an endogenous transcript control. The data was analyzed by using Software RQ Manager 1.2 (Applied Biosystems). The primers used were: Gapdh: 5′-CATGGCCTTCCGTGTTCCTA-3′ and 5′-CCTGCTTCACCACCTTCTTGAT-3′; Tardbp: 5′-GGGGCAATCTGGTATATGTTG-3′ and 5′-TGGACTGCTCTTTTCACTTTCA-3′; mSort1+Ex17b: 5′-AAATCCCAGGAGACAAATGC-3′ and 5′-GAGCTGGATTCTGGGACAAG-3′; mTsn_ΔEx5∶5′GGTCTTCCTGGCAGCATTTG-3′ and 5′-TTGACAGACAGCCTCGATGC-3′. To quantify the RNA levels of TDP-43 synaptic targets in human TDP-43 transgenic mice, we also performed q-PCR. There primers used were: Psd-95∶5′-CGCTACCAAGATGAAGACACG-3′ and 5′-CAATCACAGGGGGAGAATTG-3′; Bsn: 5′-TGTGGCTTTAACCCAACACC-3′ and 5′-TTTGGCAGTTCAGACAGAGC-3′; Dlg1∶5′-TCATTCTCATATCTCACCAATAAAGC-3′ and 5′-CAGGGGTCACAGGGACAA-3′; Dlg2∶5′-TCAACTCCCTACCCCCACTA-3′ and 5′-GCAGTACTGTGCTGGGAATG-3′; Dvl1∶5′-CCATGGACCAGGACTTCG-3′ and 5′-GGCAACTTGGCATTGTCAT-3′; Gap-43∶5′-AGAGGATGCTGCCACCAA-3′ and 5′-GGCTTCGTCTACAGCGTCTTT-3′; Syt7∶5′-GAGGCTTGGACATGAAATCC-3′ and 5′-TCCGAAAGCCCTAATACCAG-3′; Snap25∶5′-GCTCCTCCACTCTTGCTACC-3′ and 5′-CAGCAAGTCAGTGGTGCTTC-3′; Syt1∶5′-AAGGAGATTCCAAAAGGAACAA-3′ and 5′-TTTTGGTTCAAGCGGAATG-3′.TRIzol  and Pure Link™ RNA Mini Kit  were used for RNA extraction. A total of 3 µg of RNA were used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The qRT- PCR was performed using an HT7900 analyzer (Applied Biosystems). Each 5 µl reaction contained: 2 µl of cDNA diluted 1∶20, 0.5 µl primer mix (1 µM for each primer), and 2.5 µl SYBR green (Invitrogen). The qRT-PCR program was as follows: 50°C 2min, 95°C 2min, 40 cycles of 95°C 15s and 60°C 1min. For dissociation curves, a dissociation stage of 95°C 15s, 60°C 15s was added at the end of the program. Sort1∶5′-CAAATGCCAAGGTGGGATGAA-3′ and 5′-TTGAATCCAAAGCCTCTACGCC-3′; Tsn: 5′-CCCGAGAGGCTGTTACAGAG-3′ and 5′-CCTCGGATGGAAAGGTCATA-3′.For semi-quantitative PCR, 10 µl of 1∶40 diluted cDNA was used for PCR amplification in a final volume of 20 µl using Taq polymerase (Qiagen). PCR cycling conditions were as follows: 94°C, 3min; 94°C, 30s; 60°C, 30s; 72°C, 30s (10 cycles); 94°C, 30s; 50°C, 30s; 72°C, 30s (20 cycles); 72°C, 10min. PCR products were electrophoresed on 2% agarose gels. The primers used were: Tissues were homogenized at 10 ml/g (volume/weight) in lysis buffer . Following centrifugation, protein concentration of the supernatant was assessed by BCA assay . Following Western blotting, membranes were incubated with rabbit polyclonal mouse specific Tdp-43 antibody that we have generated(1∶1000), mouse monoclonal human specific TDP-43 antibody , rabbit polyclonal GFP antibody , and mouse monoclonal Gapdh antibody  overnight at 4°C. Following incubation with an appropriate secondary antibody, immunoreactivity was visualized by ECL and exposure to film.2O. Antigen retrieval was performed in a dH2O steam bath for 30 min. Tissues were immunostained with cytochrome oxidase subunit IV  and pS403/S404-phosphorylated TDP-43  antibodies, using the DAKO Autostainer (Dako Auto Machine Corporation) and the DAKO EnVision+ HRP system. DAKO Liquid DAB Substrate–Chromogen system was the chromogen. After immunostaining, sections were briefly counterstained with hematoxylin to visualize cell nuclei and coverslipped. Paraffin-embedded sections were also stained with hematoxylin and eosin.Tissues were embedded in paraffin, sectioned (5 µm thick) and mounted on glass slides. Sections were deparaffinized in xylene and rehydrated in a graded series of alcohol, followed by dHMice were deeply anesthetized with a mixture of ketamine  and xylazine  and perfused with 4% paraformaldehyde (PFA). Brains were removed and put into 4% PFA for additional 20 minutes and then washed overnight in PBS at 4°C. Vibratome sections of 200 µM were prepared and immersed in PBS at 4°C. The Helios Gene Gun System  was used to deliver gold (1.0 µm) and tungsten particles coated with lipophilic dyes into perfusion-fixed brain slices. Preparations of the DiOlistic bullets were performed according the methods described previously Tardbp and AAV1-shControl was prepared by standard methods. Briefly, AAV vectors expressing shTardbp and shControl under the control of the cytomegalovirus enhancer/chicken β-actin promoter, a woodchuck post-transcriptional regulatory element, and the bovine growth hormone, poly (A), were generated by plasmid transfection with helper plasmids in HEK293T cells. All plasmids were sequence verified as described above. Forty-eight hours after transfection, the cells were harvested and lysed in the presence of 0.5% sodium deoxycholate and 50 U/ml Benzonase  by freeze thawing, and the virus was isolated using a discontinuous iodixanol gradient. The genomic titer of each virus was determined by qRT-PCR.AAV1-shOne-way ANOVA with Tukey’s posthoc analysis were used to compare measures among 3 groups. Two-tailed student’s t test was used to compare measures among 2 groups. For data presentation, normalized values were averaged and presented as mean ± standard error of mean (SEM). Values of p<0.05 were considered statistically significant.Figure S1Intraventricular injection of the neonatal mouse brain with AAV1-shTardbpM337V transgenic mice, but cannot cause loss of mTdp-43 function. can further decrease mTdp-43 levels in the brain of hTDP-43 (A–B) Intraventricular injection of AAV1-shTardbp decreases mTdp-43 protein levels (A–B) and RNA levels (C) in the brain of both non-transgenic (NT) and hTDP-43M337V hemizygous mice. There are no significant difference of the protein RNA levels of mTdp-43 between AAV1-shTDP-43 injected hemizygous mice and homozygous mice. (D–E) Injection of AAV1-shTardbp significantly increases the mRNA levels of mSort1+Ex17b (D) and mTsn_ ΔEx5 in the brain of NT mice, but not in hTDP-43M337V hemizygous mice. mSort1+Ex17b and mTsn_ΔEx5 RNA levels between AAV1-shTardbp injected hemizygous mice and homozygous mice are not significantly different. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, N.S. no significance, as assessed by one-way ANOVA with Tukey’s posthoc analysis.(TIFF)Click here for additional data file.Figure S2WT hemizygous mice injected with AAV1-shNo alternations of RNA of synaptic proteins and dendritic spines in hTDP-43Tardbp. (A) Intraventricular injection of AAV1-shTardbp in the brain of hTDP-43WT hemizygous mice does not alter the RNA levels of PSD-95, Bsn, GAP-43 and Syt7. (B–C) Intraventricular injection of AAV1-shTardbp in hTDP-43WT hemizygous mice does not decrease dendritic spine number in the brain. Data shown are the means ± SEM of 3–5 mice per group; **p<0.001, as assessed by one-way ANOVA with Tukey’s posthoc analysis.(TIFF)Click here for additional data file.


The estimated median survival of patients with IPF is 2–5 years following diagnosis, and the mortality rate is greater than that associated with numerous malignancies ,2.This has presented significant challenges in patient management and until recently there were no pharmacological treatments approved for patients with IPF in Europe.The timing of this meeting was therefore particularly relevant as there were a number of important advances and new findings during 2011, which included the publication of the updated guidelines from the ATS/ERS/JRS/ALAT Committee and the approval of pirfenidone for the treatment of mild-to-moderate IPF in Europe ,4.In addition, a press release from the National Institutes of Health reported that one arm of an ongoing trial, PANTHER-IPF, had been stopped because patients with IPF receiving a currently used triple-drug therapy consisting of prednisone, azathioprine, and N-acetylcysteine (NAC) had worse outcomes than those who received matching placebo. The interim results from PANTHER-IPF have since been published .Respiratory Research reported the proceedings from this first European IPF meeting, with key articles selected by the AIR Scientific Committee, which were considered essential reading for all clinicians managing patients with IPF in Europe.This supplement to Articles in this supplement included a review of the recently updated ATS/ERS/JRS ALAT diagnostic criteria by Professor Athol Wells, and discussion on how these can facilitate early diagnosis of IPF. It also considered issues that may remain unclear despite the updated guidance. Linked to the diagnosis of IPF, Nicola Sverzellati gave a master class on HRCT imaging and practical requirements for optimising CT scanning techniques.Presentations then focused on the management of IPF. Professor Luca Richeldi discussed the Cochrane Collaboration approach to analysing clinical trial data and the findings of Cochrane meta-analyses that have been performed to determine the effect of treatments used in the management of IPF. This was followed by Professor Vincent Cottin, who summarised the outcomes from prior clinical trials in IPF and the recently published Phase III studies of pirfenidone that led to its approval in Europe. Following these updates, Professor Jürgen Behr and Luca Richeldi discussed how these data can be interpreted in light of the updated recommendations of the ATS/ERS/JRS/ALAT Committee regarding treatment of IPF. Professor Carlo Albera outlined the implications of the press release from the NIH regarding the PANTHER-IPF study.Hopefully the selected proceedings from this meeting will provide IPF clinicians with new insights and support in the management of their patients. The intention is for the AIR meeting to become a fixed date in the calendar for respiratory physicians and researchers dedicated to improving knowledge and bringing new hope for IPF patients.U. Costabel has received consultancy fees from Actelion, Boehringer Ingelheim, Centocor, Gilead and InterMune, and has received lecture fees from InterMune.J. Behr has received fees for speaking from Actelion, Bayer-Schering, Boehringer-Ingelheim, Encysive, GSK, Pfizer, Lilly, Nycomed, InterMune, Novartis, MSD and has served as consultant/advisor for Actelion, Bayer-Schering-Pharma, Lilly, Pari-Pharma, GSK, Pfizer, Optima, Gilead. J. Behr has also received research grants from Actelion, Bayer-Schering, InterMune, Pari-Pharma and attending international and national congresses sponsored by Actelion, Boehringer-Ingelheim, AstraZeneca, Bayer.V. Cottin has received fees for speaking from InterMune, Boehringer Ingelheim, and Actelion, and has participated as a member of steering committees, a member of data safety monitoring boards or as an investigator to clinical trials sponsored by Actelion, Boehringer Ingelheim, Gilead, and InterMune Inc.L. Richeldi has received consultancy fees from Boehringer Ingelheim, InterMune, Celgene and Gilead, and lecture fees from InterMune.The authors thank C. Trenam, I. Mandic and M. Smith of IntraMed Communications for editorial assistance in the preparation of the manuscript. Development of this article was supported by InterMune AG.


In this study we examined the effect of diacetylbis(-methylthiosemicarbazonato)copperII (CuII(atsm)) and glyoxalbis(-methylthiosemicarbazonato)copperII (CuII(gtsm)) on TDP-43-positive SGs induced in SH-SY5Y cells in culture. We found that the CuII(btsc)s blocked formation of TDP-43-and human antigen R (HuR)-positive SGs induced by paraquat. The CuII(btsc)s protected neurons from paraquat-mediated cell death. These effects were associated with inhibition of ERK phosphorylation. Co-treatment of cultures with either CuII(atsm) or an ERK inhibitor, PD98059 both prevented ERK activation and blocked formation of TDP-43-and HuR-positive SGs. CuII(atsm) treatment or ERK inhibition also prevented abnormal ubiquitin accumulation in paraquat-treated cells suggesting a link between prolonged ERK activation and abnormal ubiquitin metabolism in paraquat stress and inhibition by Cu. Moreover, CuII(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These results demonstrate that CuII(btsc) complexes could potentially be developed as a neuroprotective agent to modulate neuronal kinase function and inhibit TDP-43 aggregation. Further studies in TDP-43 animal models are warranted.Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, motor neuron disease with no effective long-term treatment options. Recently, TDP-43 has been identified as a key protein in the pathogenesis of some cases of ALS. Although the role of TDP-43 in motor neuron degeneration is not yet known, TDP-43 has been shown to accumulate in RNA stress granules (SGs) in cell models and in spinal cord tissue from ALS patients. The SG association may be an early pathological change to TDP-43 metabolism and as such a potential target for therapeutic intervention. Accumulation of TDP-43 in SGs induced by inhibition of mitochondrial activity can be inhibited by modulation of cellular kinase activity. We have also found that treatment of cells and animal models of neurodegeneration, including an ALS model, with bioavailable  Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease in which death of upper and lower motor neurons leads to progressive brain and spinal cord deterioration. Patients with ALS rarely survive more than a few years after diagnosis with respiratory failure the most common cause of death In 2006, TAR DNA binding protein 43 (TDP-43) was identified as the major protein constituent of ubiquitinated neuronal inclusions in non-SOD1 ALS cases TDP-43 is a 414 amino acid protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and consists of two RNA recognition motifs and a C-terminal glycine rich region The hallmark neuropathological features of primary TDP-43 proteinopathies include clearing of TDP-43 from the nucleus and accumulation of TDP-43 in cytoplasmic inclusions. These inclusions are enriched in ubiquitinated and hyperphosphorylated (phospho-Ser409/410) CTF-TDP-43 The early disease processes associated with abnormal TDP-43 metabolism are not well understood. Recent studies have shown that an early change to TDP-43 processing and metabolism in ALS may involve its localization to RNA stress granules (SGs) in vivo hallmarks of disease While these studies have begun to delineate some of the initial processes in TDP-43 accumulation, it is not known whether this early stage of TDP-43 aggregation can be developed into a therapeutic target. Recently, we developed a cell model involving paraquat-induced mitochondrial inhibition in neurons and demonstrated that this stress induced changes to TDP-43 that re-capitulated II complexes (CuII(btsc)s) on TDP-43 SG formation. These complexes have traditionally been investigated for their potential as imaging agents for hypoxic tumors II(btsc)s have a potent neuroprotective effect in several different mouse models of neurodegenerative disorders, including models of Alzheimer’s disease, Parkinson’s disease and ALS II(btsc)s is likely related to their ability to modulate kinase signaling mechanisms including Akt, ERK and JNK in both cell culture and animal models bis(methylthiosemicarbazonato)-copperII (CuII(atsm)) has a potent protective effect in an animal model of ALS (SOD1G93A) II(atsm) also modulated TDP-43 processing in the SOD1G93A murine model. Treatment of SOD1G93A mice with CuII(atsm) inhibited accumulation of phosphorylated TDP-43 in the spinal cord of these mice II(btsc)s and other Cu-complexes in animal and cell models II(btsc)s could modify the processing and localization of TDP-43 to SGs. We found that CuII(atsm) and glyoxalbis(methylthiosemicarbazonato)-copperII (CuII(gtsm)) were potent inhibitors of SG formation, blocking accumulation of TDP-43 and HuR into SGs caused by treatment with paraquat. Further studies showed that the protective action was associated with inhibition of stress-induced ERK phosphorylation by CuII(atsm). The protective effect of CuII(atsm) on TDP-43 aggregation was also associated with an inhibitory effect on accumulation of ubiquitinated proteins and decreased paraquat neurotoxicity. Moreover, CuII(atsm) reduced accumulation of C-terminal (219–414) TDP-43 in transfected SH-SY5Y cells. These studies show for the first time that treatment of neuronal-like cells with a copper-complex can inhibit stress-induced cell toxicity and prevent early aggregation of TDP-43 into SGs. This may offer a potential insight into novel targets and therapeutic treatments for TDP-43 proteinopathies.In the present study, we have used this cell model to examine the effect of bis(thiosemicarbazonato)-copper4′,6′ Diamino-2-phenylindole dihydrochloride (DAPI) was obtained from Invitrogen . N,N′-Dimethyl-4,4′-bipyridinium dichloride (paraquat), rotenone, 1-methyl-4-phenylpyridinium (MPP+) and sodium arsenite, were from Sigma Aldrich  and LDH assay kit was purchased from Roche Diagnostics . SP600125 and PD98095 were purchased from Merck Biosciences .Polyclonal TDP-43 antisera were purchased from Proteintech Group . Antisera to ubiquitin were from Santa Cruz Biotechnology . Monoclonal antisera to HuR were obtained from Invitrogen . Antisera to total and phosphorylated forms of ERK and JNK, as well as antibodies to actin and GAPDH were purchased from Cell Signalling Technologies .2 at 37°C.The cell lines used in this study were the human neuroblastoma SH-SY5Y cell line and human epithelial HeLa cell line. Cells were passaged and maintained in DMEM plus 5% FBS (HeLa cells) or DMEM/F12 plus 10% FBS (SH-SY5Y cells) as previously reported An assay for cell death (LDH) was performed as previously described II(btsc)s were synthesized as previously described II(btsc)s were prepared as 10 mM stock solutions in DMSO and added at indicated concentrations. CuCl2 and FeCl2 10 mM stock solutions were prepared in dH2O and added at indicated concentrations.Cu2O and added at indicated concentrations and the medium was briefly mixed by aspiration. Incubations were performed for periods stated in individual experiments. Where indicated, cells were co-treated with kinase inhibitors (SP600125 (JNK) or PD98095 (ERK)) from stock solutions prepared at 10 mM in DMSO. Control cultures were treated with vehicle alone. For immunoblotting, cells were harvested into Phosphosafe Extraction Buffer  containing protease inhibitor cocktail (Roche Diagnostics) and stored at −80°C until use. For immunofluorescence studies, cells were grown on glass coverslips and fixed after treatment in 4% w/v paraformaldehyde for 30 min.Cells were grown in 24 or 6-well plates or on 15 mm coverslips (for immunofluorescence) for 2–3 days before experiments (∼80% confluent). Paraquat, rotenone, MPP+ or sodium arsenite were prepared in dHCell lysates were analyzed for ATP content using an ATP Assay kit (Sigma-Aldrich) as previously reported 2) multiwell plates . After treatment with paraquat and or CuII(btsc)s or CuCl2, cell samples were collected and centrifuged at 720 g for 10 min. Supernatant was removed and the cell suspension was re-suspended in 60 µl Phosphosafe Extraction Buffer with 5% (v/v) DNAse Sigma Aldrich  and 1% phenylmethylsulfonyl fluoride (v/v). The suspension was then centrifuged at 15,000 g and the supernatant was collected and normalized to 8 µg/µl using the BCA assay. The cell suspension was then diluted 1∶2 in sample buffer  bromophenol blue in dH2O). 10 µl was loaded per well of non-denaturing polyacrylamide gels without SDS. Gels were run in Tris-glycine running buffer at 150 V for 1 hr. Gels were incubated in the dark in nitrotetrazolium blue chloride (Sigma Aldrich) solution (2 mg per ml dH2O) for 20 min on a rocking platform in the dark, at room temperature. Nitrotetrazolium blue chloride solution was removed from gels and replaced with developer solution  for 1 hr. Gels were subsequently imaged using a Fujifilm LAS3000 Imager . Quantification of SOD activity was done using Image J analysis, comparing levels of SOD activity based on nitrotetrazolium blue reduction.Cells were grown and treated in 6-well  against TDP-43. For detection of total and phospho-forms of JNK and ERK, membranes were probed with anti-JNK or anti-ERK  and antisera to phospho-forms of each protein . Secondary antiserum was rabbit-HRP at 1∶5,000 dilution. Blots were developed using GE Healthcare ECL Advance Chemiluminescence and imaged on a Fujifilm LAS3000 imager. Expression of GAPDH or actin was determined using antisera at 1∶5,000 and 1∶3,000 respectively for protein loading controls where necessary.SH-SY5Y cells were grown on 15 mm diameter coverslips and treated as indicated. Cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After blocking for 1 hr with 10% normal goat serum, cells were incubated with primary antibody , ubiquitin (1∶150) or HuR (1∶50), for 2 hr at room temperature or overnight at 4°C. This was followed by labeling with secondary AlexaFluor or FITC goat anti-mouse or anti-rabbit antisera at 1∶500 for 2 hr at room temperature or overnight at 4°C. After washing, the coverslips were incubated with DAPI at 0.5 ng/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Images shown are representative of multiple fields and triplicate coverslips per experiment. TDP-43 and HuR-positive stress granules (SGs) were counted in cultures where indicated. A minimum of 500 cells was counted across multiple fields of view (and multiple coverslips) for each treatment. The number of TDP-43 and HuR-positive SGs were counted in these cells. The total number of cells was divided by the total number of SGs to provide a measure of mean SGs per cell. SGs were not observed in untreated cells.Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer’s instructions. DNA was quantified and TDP-43 inserts were identified positively by digestion with BamHI and XhoI.Plasmid DNA corresponding to GFP-tagged full-length wild-type (WT) TDP-43 (pEGFP-TDP WT) and the C-terminal fragment of TDP-43, (pEGFP-TDP 219–414) or empty expression vector pEGFP-C1 were prepared as described by Nonaka et. al. 51 cells per well in 24 well–plates on coverslips. Cells were transfected 24 hr after seeding with the pEGFP-C1 empty vector, pEGFP-TDP WT and pEGFP-TDP 219–414 using Dreamfect (Qiagen) according to manufacturer’s instructions. After 48 hr incubation, cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After washing, the coverslips were incubated with DAPI at 0.5 ng/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Expression of TDP-43 was determined by the EGFP-tagged construct. Efficiency of transfection with pEGFP-C1 vector was approximately 20–25% SH-SY5Y cells were seeded at 2×10t-test and ANOVA where appropriate.All data described in graphical representations are mean ± standard error of the mean (SEM) from a minimum of three experiments. Densitometric analysis of immunoblots was performed using NIH ImageJ 1.43 software on two-three separate blots and adjusted for loading control (GAPDH or actin). Results were analysed using a two-tailed Student’s II(atsm) (II(gtsm) (II(gtsm) has a higher potential to induce toxicity than CuII(atsm) due to the fact that CuI is readily released in the intracellular reducing environment from CuII(gtsm) II(atsm) is more resistant to reduction of CuII to CuI under normoxic conditions and therefore is thought to release less CuIII(gtsm) was used at a concentration 20-fold less than CuII(atsm) due to its higher potential to release bioavailable Cu.To induce TDP-43-positive SGs, SH-SY5Y cells were exposed overnight to 1 mM paraquat . As reported previously, this exposure to paraquat induces only mild loss of cell viability (∼15% as determined by MTT assay) and no cell death (as determined by LDH assay) II(atsm)  or 50 nMII(gtsm) . These cII(atsm) or CuII(gtsm) substantially inhibited formation of TDP-43 and HuR-positive SGs induced by paraquat (II(atsm) and CuII(gtsm) with CuCl2 at 1 µM and 100 µM and FeCl2 at 100 µM. ZnCl2 was not examined as we have reported previously that ZnCl2 induces nuclear aggregates of TDP-43 2 had no effect on TDP-43 or HuR SGs (2 induced an effect indistinguishable to 1 µM CuII(atsm) and 50 nM CuII(gtsm) (II(btsc)s are highly efficient at delivering intracellular Cu when compared to CuCl2 and therefore a higher level of CuCl2 is required to obtain the same increase in cellular bioavailable Cu 2 at 100 µM (2 ligand had no effect on SG formation (data not shown). These findings demonstrated that co-treatment of SH-SY5Y neurons with low concentrations of CuII(btsc)s inhibited paraquat-mediated SG formation, including TDP-43-positive SGs and this was likely to be related to the ability of the CuII(btsc)s to efficiently deliver bioavailable Cu into the cell. Subsequent studies were performed using CuII(atsm) due to the significant potential of this compound as a therapeutic agent for ALS based on efficacy in our preliminary in vivo studies Co-treatment with either Cuparaquat . To dete HuR SGs , howeverII(gtsm) . These ft 100 µM . SimilarII(atsm) and 1 mM paraquat overnight blocked formation of SGs as observed in SH-SY5Y cells (data not shown). To determine if CuII(atsm) could inhibit against TDP-43-positive SGs generated by another common inhibitor of mitochondrial function, we treated HeLa cells with 500 µM sodium arsenite for 1 hr. This is the most common method used for SG induction and we have shown previously that sodium arsenite induces robust TDP-43 and HuR SGs in HeLa cells. However, co-treatment with 1 µM CuII(atsm) for 1 hr, 4 hr, or overnight prior to the 1 hr sodium arsenite treatment had no effect on the formation of TDP-43 and HuR-positive SGs (Next we examined if paraquat-mediated TDP-43-positive SGs could be inhibited in HeLa cells. Co-exposure of HeLa cells with 1 µM Cutive SGs . The reaII(atsm) inhibited TDP-43 and HuR-positive SGs, we first determined if there was any effect on TDP-43 or HuR expression. Cells treated with 1 µM CuII(atsm) alone overnight revealed no substantial alterations in full length TDP-43 or HuR expression (data not shown). Interestingly whilst paraquat induced a slight increase in 35 kDa TDP-43 (II(atsm) further increased this expression (II(atsm) did not affect 35 kDa CTF-TDP-43 levels in the absence of paraquat treatment (data not shown). The effect on 35 kDa CTF-TDP-43 was not observed with 1 µM CuCl2 (2 did slightly increase the expression of the 35 kDa band (II(atsm) lead to elevated 35 kDa CTF-TDP-43 levels. HuR expression was not substantially altered in any condition tested (II(atsm).To examine how Cua TDP-43 , co-expopression . Using NµM CuCl2 , althougkDa band . We repon tested . These rII(atsm) on kinases. As reported previously II(atsm) inhibited ERK activation (II(atsm) did not inhibit levels of phosphorylated ERK when examined after 2 hr of incubation (II(atsm) having no effect on JNK phosphorylation after 2 hr incubation with paraquat (II(atsm), both ERK and JNK phosphorylation were significantly inhibited compared to paraquat alone (II(atsm) alone had no effect on basal ERK or JNK phosphorylation over the same time period  on sodium arsenite-treated HeLa cells. Exposure of HeLa cells to 500 µM sodium arsenite for 1 hr induced significant activation of JNK and ERK (II(atsm) did not substantially affect activation of either kinase (II(atsm) in sodium arsenite-treated cells  was found to inhibit ERK phosphorylation in SH-SY5Y cells treated with paraquat, we compared the effect of CuII(atsm) and an ERK inhibitor on SG formation. We found that both CuII(atsm) and PD98059 (ERK inhibitor) induced the same inhibition of TDP-43 and HuR-positive SGs when co-treated with paraquat (compare II(atsm) with II(atsm) has an analogous effect to inhibition of ERK, the results contrast with the more specific action of JNK inhibition on TDP-43 alone (II(atsm) could inhibit TDP-43 and HuR-positive SG formation through inhibition of prolonged ERK activation. This is consistent with our observations that paraquat does not induce TDP-43/HuR-positive SGs until after 8 hr of exposure II(atsm) did not block SGs induced by 1 hr treatment with sodium arsenite, as prolonged exposure to CuII(atsm) e.g. overnight in the presence of the mitochondrial inhibitor, is needed to obtain a protective response from the copper complex.As Cu43 alone . These fII(atsm) only inhibited ERK (and JNK) activation after a prolonged exposure with paraquat, we further investigated the potential neuroprotective mechanism of CuII(atsm) that might account for this. We examined the effect of CuII(atsm) on paraquat-induced changes to ubiquitin. Previous reports have demonstrated that paraquat treatment leads to altered proteasomal function and can result in accumulation of ubiquitinated proteins II(atsm) resulted in complete abrogation of the ubiquitin accumulation in paraquat-treated cultures  may have a broad neuroprotective effect leading to decreased cell stress and therefore lack of SG formation.Due to the observation that Cucultures . Moreovecultures . As expebiquitin . This waII(atsm) to protect against paraquat-induced cell death. As 1 mM paraquat did not induce substantial cell death, we exposed cultures to 10 mM paraquat overnight (II(atsm) significantly reduced this neurotoxic action to 35% (II(gtsm) (50 nM) also significantly decreased paraquat-mediated toxicity -like activity, we examined the SOD activity using an in-gel assay. Exposure of SH-SY5Y cells to 1 µM CuII(atsm), 50 nM CuII(gtsm) or 1 µM CuCl2 in the presence or absence of 1 mM paraquat resulted in no significant increase in SOD-like activity in treated cells as compared to untreated controls (II(btsc)s mediated through inhibition of protein accumulation and kinase activation rather than through enhanced SOD or SOD-like activity.To confirm this protective action, we measured the ability of Cuvernight . This trvernight . Co-trean to 35% . In additoxicity  (28%). Acontrols . These fII(atsm) could inhibit endogenous TDP-43-containing SGs induced by mitochondrial inhibition, we were interested to know if this action could be extended to prevention of pathological aggregation of TDP-43 associated with ALS. To investigate this, we transfected SH-SY5Y cells with wild-type (WT) full length TDP-43-GFP and a C-terminal fragment known to aggregate in cultured neurons (CTF-TDP-43 219–414-GFP) II(atsm) either at the time of CTF-TDP-43 219–414 transfection or 24 hr later. At 48 hr after transfection, cells were assessed for the level of CTF-TDP-43 219–414 aggregation. CuII(atsm) significantly reduced the number of TDP-43 aggregates in transfected cells when added at time of transfection (0 hr) or at 24 hr post-transfection  had a greater inhibitory effect when added after 24 hr, reducing the number of cells with aggregates by ∼50% (II(atsm) from the cell before accumulation of CTF-TDP-43 219–414 when added at time of transfection. Most aggregates form in the second 24 hr after transfection. As we have shown previously that JNK inhibition, but not ERK inhibition, significantly inhibits accumulation of CTF-TDP-43 219–414 II(atsm) inhibition of CTF-TDP-43 aggregation here is mediated through its inhibitory effect on JNK (associated with cell stress induced by CTF-TDP-43 aggregation). However, these studies show that CuII(atsm) can significantly reduce cellular accumulation of both endogenous and transfected TDP-43.Finally, although we had shown that Cusfection . CuCl2 aormation . Interes by ∼50% . This maII(btsc) Cu-complexes to inhibit stress-mediated TDP-43-positive SGs in neuronal cell culture. We found that CuII(atsm) and CuII(gtsm) inhibited paraquat-mediated sustained ERK phosphorylation resulting in abrogation of SG formation and improved survival of cells treated with paraquat. In addition, both CuII(atsm) and the ERK inhibitor, PD98059, inhibited accumulation of ubiquitin in paraquat-treated cells indicating that this may be a protective action of CuII(atsm) mediated through inhibition of prolonged ERK activation. CuII(atsm) also reduced aggregation of CTF-TDP-43 in transfected cells.In this study, we investigated the ability of CuII(btsc)s have been investigated extensively as hypoxia imaging agents and as potential anti-cancer compounds for delivery of radionuclides to tumors II(atsm) has a relatively negative CuII/I reduction potential (0’E = −0.59 V vs SCE) and this limits reduction of the metal ion to CuI and the subsequent release of bioavailable CuI into cells under normal cell conditions but CuI release is enhanced under hypoxia, cellular energy impairment and probably within select sub-cellular compartments within the cell II(gtsm), it is easier to reduce the CuII to CuI (0’E = −0.43 V vs SCE) so the complex is able to release considerable CuI under basal cellular reducing conditions. In previous studies, we have harnessed these properties to develop CuII(btsc)s as potential therapeutic agents to deliver small levels of bioavailable Cu to the brain. This is achieved through the ability of these compounds to cross the blood brain barrier II(btsc)s can modulate cell signaling pathways including phosphorylation and activity of phosphoinositol-3-kinase (PI3K), ERK, glycogen synthase kinase 3 (GSK3) and JNK II(btsc)s II(atsm) significantly delayed disease onset and extended life span in a low transgene copy SOD1G93A murine model of ALS. We observed that CuII(atsm) delayed disease onset, extended survival and inhibited oxidative damage and glial activation in treated mice II(atsm), the therapeutic action of this complex in the SOD1G93A mice remains unknown II(atsm) did inhibit accumulation of phosphorylated full length and C-terminal TDP-43 in the treated SOD1G93A mice. Our data here and previously II(atsm) has also protected against neurodegeneration and improved motor performance in models of Parkinson’s disease CuII(btsc)s could inhibit formation of TDP-43-positive SGs induced by the mitochondrial inhibitor, paraquat.We previously reported that treatment of neuronal and non-neuronal cell cultures with paraquat overnight induced a robust induction of TDP-43-positive SGs and that this was mediated through ERK and JNK activation II(atsm) to inhibit TDP-43-and HuR-positive SG formation during paraquat-mediated stress was likely due to its ability to inhibit ERK phosphorylation. Although CuII(atsm) also inhibited prolonged JNK phosphorylation, we have shown that inhibition of JNK in this model stops TDP-43 association with SGs but not HuR SG formation. In this study, CuII(atsm) and the ERK inhibitor, PD98059, both prevented formation of HuR-and TDP-43-positive SGs making the inhibition of ERK potentially more important to these protective effects than JNK inhibition in this cell model. However, the JNK inhibitory action of CuII(atsm) was perhaps more likely to be involved in the reduced aggregation of CTF-TDP-43 219–414 in transfected cells. This is because we reported previously that JNK, but not ERK inhibition, reduced accumulation of CTF-TDP-43 219–414 and 162–414 in the same cell type II(atsm) could mediate inhibition of CTF-219–414 TDP-43 aggregation through an unrelated mechanism.The ability of CuII(atsm) did not inhibit ERK or JNK phosphorylation after 2–4 hr exposure to the Cu-complex but did inhibit both kinases after an overnight treatment. These findings suggested that CuII(atsm) had a delayed effect and prevented prolonged activation. This may be due to inhibition of complex upstream pathways and could be associated with the prevention of ubiquitin accumulation observed in cultures treated with paraquat. The fact that both ERK inhibition and CuII(atsm) blocked the ubiquitin aggregation suggested that ERK inhibition is upstream of changes to ubiquitin in paraquat-treated cells. Interestingly, other mitochondrial inhibitors did not induce effects on ubiquitin or induce SGs supporting a close connection between ubiquitin changes and SG formation, possibly controlled by ERK. It has been reported elsewhere that ERK (and JNK) have important roles in the toxic effects of paraquat Interestingly, CuII(atsm) is inhibiting ERK is not known but is likely to involve release of Cu as we observed the same effect with CuII(gtsm), which is known to release intracellular Cu. We have also found CuII(gtsm) to potently inhibit ERK at low concentrations in other cell lines including PC12 neurons and primary cortical neurons (data not shown). Interestingly, we have shown that under certain conditions, CuII(atsm) can also activate ERK rather than inhibit its activity II(atsm)-mediated inhibition of ERK may initially appear contradictory, it is likely that the different outcomes of CuII(atsm) treatment are related to basal ERK activity in each model system. Our unpublished studies have shown that if ERK phosphorylation is low, CuII(atsm) (under conditions of energy inhibition) can increase phosphorylation, whereas if ERK phosphorylation is already high (as in paraquat-treated cultures in this study) CuII(atsm) can inhibit ERK. The reason for this dynamic effect is not known but may be related to changes in subcellular Cu pools.How CuII(atsm) and CuII(gtsm) in this system were supported by our finding that both compounds inhibited cell death induced by paraquat.It has been reported elsewhere that Cu can enhance function of ubiquitin-associated activities, although excess Cu can also induce abnormal ubiquitination II(atsm) inhibited paraquat-induced TDP-43 and HuR SGs, there was no effect on SGs formed by sodium arsenite treatment. The reason for this may be the time frame required for the Cu-complex to act. We tested CuII(atsm) against sodium arsenite by using pre-treatment regimes of overnight or 4 hr but these had no effect, indicating that the CuII(atsm) should be present at the same time as the stress and that the effect is then mediated through a relatively slow action. Whether this is related to Cu release from the complex under the paraquat-induced stress or the need for Cu to induce protein synthesis is unknown. However, as ALS is a slowly progressing disease, these complexes may have therapeutic potential. We have shown that CuII(atsm) inhibits disease progression in a SOD1 model of ALS II(atsm) inhibited formation of phosphorylated cleavage fragments of TDP-43 in the treated SOD1 mice II(atsm) protects mice from disease induced by overexpression or mutation of TDP-43 While CuII(bstc) complexes can inhibit formation of TDP-43-positive SGs and prevent paraquat-mediated cell death. This neuroprotection appeared to be associated with inhibition of ERK phosphorylation and may be mediated through modulation of cellular ubiquitination by Cu. Further studies are warranted to determine if CuII(atsm) or related CuII(btsc)s are potential therapeutics for ALS and other neurodegenerative diseases through kinase modulation and maintenance of ubiquitin homeostasis.In summary, we have found that co-treatment of cells with CuFigure S1ATP levels in paraquat-treated cells. SH-SY5Y cells were treated overnight with 1 mM paraquat and total cellular ATP levels were determined in cell lysates. *p<0.05 compared to control cells.(TIF)Click here for additional data file.Figure S22 on TDP-43 expression.Effect of CuClA: Cells were treated with 1 mM paraquat overnight in the presence or absence of 1 µM CuII(atsm) or 100 µM CuCl2. Cells were examined for expression of full length TDP-43 and 35 kDa CTF-TDP-43 by western blot. B: Effect of CuII(atsm) on ERK and JNK phosphorylation. Cells were treated overnight with 1 µM CuII(atsm) and examined for expression of phosphorylated ERK and JNK.(TIF)Click here for additional data file.


Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated  Group II introns have not yet been identified in the nuclear genomes of eukaryotes, but their predicted descendants , are highly abundant in eukaryotes, comprising together more than half of the genome in mammals.Interestingly, many plant organellar group II introns lack an ORF capable of specifying a maturase, or they encode a degenerate maturase protein which is probably unable to promote intron mobility  and a site-specific endonuclease (D/En) motif at the C-terminus. Genetic and biochemical data show that the processing of group II introns in bacteria is facilitated by a single maturase-ORF encoded within DIV of the intron itself  for nMAT4  , and five new nuclear maturase loci in Physcomitrella . Building upon this new information a comprehensive phylogeny was constructed to determine the evolutionary relationships among the different maturase sequences; a simplified version of this phylogeny, displaying only the relationships among the seed plant (six maturase genes), Marchantia (eight related genes) and Selaginella maturases, is shown in Figure A recent study by Guo and Mower  has provmat genes fall into two main categories . It is likely, therefore, that the angiosperm type I nMat1 and nMat2 genes resulted from a nuclear gene duplication, following the translocation to the nucleus of a single mitochondrial maturase related to Marchantia maturase mat-Mpo1   Figure . It is f) Figure ; this ev) Figure , this po) Figure .Different RNA-binding proteins were shown to function in the splicing and processing of introns in plant organellar genomes (see below). Some, such as a DEAD RNA-helicase (PMH2) and a CRM-domain protein (mCSF1) are required for optimal splicing of a large set of introns, whereas others  rather appear to be specific for a single, or few, individual introns. Although this review focuses mainly on maturases, this section summarizes the information available about other types of mitochondrial group II intron splicing factors.E.coli, YhbY is mainly associated with the maturation of the pre-50S ribosomal subunit  proteins are characterized by a conserved RNA-binding domain of ~10 kDa (Pfam-PF01985), which is related to a conserved bacterial ORF (YhbY)   domain which is represented in a small family of proteins in angiosperms (15 in Arabidopsis and 17 in rice)   which binds to chromatin and interacts with the nuclear Ran GTPase Dasso, . SimilarNicotiana sylvestris (nms1) was shown to disrupt the splicing of the first intron in the nad4 transcript  often results in severe developmental defects.Plants possess some of the most complex organelle compositions of all known eukaryotic cells. As sessile organisms, land-plants must cope with multiple environmental stresses. During their evolution, plants have acquired complex regulatory mechanisms to coordinate cellular functions with environmental stresses. The participation of nuclear-encoded factors in organelle RNA metabolism may therefore provide means to control the biogenesis of the respiratory (and photosynthetic) machineries, and thus to link cellular metabolism and environmental and developmental signals. Following this idea, the splicing of both plastidial Barkan,  and mitoccmFc, cox2 and the ribosomal rpl2 and rps3 genes, the splicing mutants are strongly affected in respiratory associated functions and cellular metabolism  (reviewed in Millar et al., The importance of these stages is further reflected in plant mutants affected in genes required for the splicing of mitochondrial introns. Such mutants exhibit reduced germination and seedling establishment, and are often affected in growth and development (see Keren et al., Chlamydomonas reinhardtii demonstrated that the trans-splicing of the two group II introns in its chloroplast genome, is also facilitated by a large set of nuclear-encoded RNA binding cofactors (Goldschmidt-Clermont et al., Clostridium tetani, was shown to undergo alternative splicing reactions resulting with five alternative mRNAs products, each encoding a distinct protein isoform (McNeil et al., trans” in a primitive spliceosome, and the group II structural features of the target primordial introns degenerated. Interestingly, features of some plant mitochondrial introns reflect possible stages that may have occurred in such a process. Several undergo trans-splicing: different portions of the intron are assembled from two and in one case, three different transcripts. At least one trans-splicing intron undergoes a significant degree of mis-splicing, and this is lost in plants in which the intron is further fragmented, a process that may have played a role in the formation of the primordial spliceosome. Also, several introns lack key structural features, such as the bulged adenosine in the DVI stem, which serves as the nucleophile in the first splicing step (Figure Based on the structural features, the similarity of exon-intron boundaries and a common splicing-mechanism, group II introns are proposed to be ancestors of the eukaryotic spliceosomal introns Sharp, . While bp Figure  and may trans.” The homology of group II maturases to the core spliceosomal splicing factor (Dlakic and Mushegian, Maturases and their accessory splicing factors in plant mitochondria, thus represent a highly versatile set of proteins which differ from their presumptive monospecific ancestors. These features may correspond to a further evolutionary link between mitochondrial group II introns and the spliceosomal machinery in nuclear genomes of most eukaryotic cells. Likewise, the recruitment of different protein cofactors to assist with the splicing of mitochondrial introns in plants (see Figure The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.