Glioma, especially glioblastoma, is pathologically characterized by high aggressiveness, which largely contributed to the ineffectiveness of current therapies. It has been recently reported that intrinsic PD-L1 can regulate tumor malignancy, whereas underlying mechanisms remain mostly unclear. Here, we report a novel mechanism by which PD-L1 promotes glioma cell infiltration. In orthotopic glioma models, PD-L1 expression was up-regulated predominantly in glioma cells in the infiltrating front. For PD-L1-overexpressed glioma cells, PI3K/Akt and actin regulations were among the top six most altered signaling pathways as detected by RNA-sequencing. PD-L1 significantly activated Akt/F-actin signaling while suppressed autophagic signaling upon cell starvation. Mechanistically, PD-L1 preferentially bound to Akt among various PI3K/Akt signaling proteins. Serial truncation identified the interaction between the 128-237aa fragment of PD-L1 and the 112-480aa fragment of Akt, which facilitates the membrane translocation/activation of Akt, and was unaffected by Perifosin (specific p-Akt inhibitor targeting Akt PH-domain). Taken together, our data indicate that in glioma cells, PD-L1 is induced to prevent autophagic cytoskeleton collapse via Akt binding/activation, facilitating glioma cell invasion upon starvation stress. Glioma, the most common primary intracranial tumor, originates from various types of cells. The most malignant glioma is glioblastoma, or glioblastoma multiforme (GBM), which accounts for around 21% of glioma and can be primary or secondary from grade II-III glioma. Recently, glioma is also classified into three types based on its molecular markers: 1p/19q deletion, IDH mutation and TERT promoter mutation. Among them, grade IV glioma mainly has the TERT promoter mutation accompanied by EGFR/PTEN mutation. Current treatments for glioma remain conventional surgery, radiotherapy and chemotherapy. However, glioma is extremely easy to relapse after treatment, with an average survival time of only 14 months for GBM patients after treatment  is a key negative regulator for immune inhibitory axis signaling controlling T-lymphocyte infiltration inside solid tumors. Previous studies reported that PD-L1 is widely expressed in glioma cell lines , 13 and PD-L1 is recently considered to be oncogenic. PD-L1 knockdown significantly decreases tumor volume in murine ovarian cancer, melanoma , murine in vivo. Our data revealed that PD-L1 promoted glioma cell infiltration via starvation-induced Akt/autophagy/F-actin signaling. Particularly, we dissected PD-L1-Akt binding fragments and elucidated how PD-L1/Akt interactions activated its downstream cascades.In the present study, we first reported that PD-L1 was mostly prominent in those highly aggressive infiltrating glioma cells +/K+ ATPase α1 , β-tubulin  were commercially purchased.Cloroquine  was used at a final concentration of 25 μM. LY294002  was used at a final concentration of 0–50 μM. Perifosine  was used at a final concentration of 0–25 μM. Primary antibodies against phospho-Akt , total Akt (#9272), phospho-mTOR, total mTOR,pP62, phospho-P70S6K, LC3B were purchased from Cell Signaling Technology . Antibodies against PD-L1/CD274 , PD-L1 , GST , Beclin1 , N-terminal GFP (Sigma), β-actin , NaGFP-LC3 plasmid was a gift from Dr. He Li (Huazhong University of Science and Technology). GST-kRas/PTEN/Akt1/Akt2/Akt3/EGFR VIII/PKA plasmids were gifts from Dr. Haian Fu (Emory University). PD-L1-EGFP expressing-plasmid was purchased from GeneChem . Full-length and truncated human PD-L1 coding cDNA was PCR amplified from PD-L1-EGFP plasmids and cloned into NV  vector including NV-PD-L1 FL (full length 1-290 aa), NV-PD-L1-T1-259 (truncate 1-259aa), T128-259, T128-239, T1-178. PD-L1 cloned into pDEST-26 or p-FU-Venus vector was used to express GST-PD-L1 and Venus-PD-L1 FL1-290 or T19-290. Full-length and truncated human Akt1 cDNA was PCR amplified from GST-Akt1 plasmid and cloned into p-FU-Venus vector for Venus-Akt1, Akt1-T1-111, and Akt1-T122-480. Akt1-Y176A and Akt1-K14R mutants were constructed by using overlapping-PCR from p-FU-Venus-Akt1 plasmid. Transfection of plasmids was performed by using Lipofectamine 2000  according to the manufacturer's instructions.P-values were adjusted using the Benjamini and Hochberg's approach for controlling the false discovery rate. Differently expressed genes (DEGs) with P < 0.005, adjusted P < 0.05 (Padj) and absolute changing fold ≥1.2 were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis. KEGG pathways and GO terms with Padj < 0.05 were considered significantly enriched by DEGs. We have submitted our data to NCBI (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE107581).Three 100-mm dishes of U251/PD-L1 and U251/Vec stable cell lines were subjected to total mRNA isolation, cDNA libraries construction and sequencing at Illumina HiSeq sequence platform (PE150) with 6G clean data by Novogene Bioinformatics Institute . Differential gene expression analysis between U251/PD-L1 and U251/Vec groups was performed using the DESeq2 R package (1.10.1). The resulting ad libitum access to food and water. Briefly, adult Kunming male mice (18–20 g) were anesthetized with chloral hydrate (350 mg/kg) and a burr hole was drilled in the skull 0.5 mm posterior to the bregma and 2.0 mm lateral to the midline. A 10-μl Hamilton syringe  containing 20,000 G422 cells (mouse GBM cell line) in 1 μl of PBS was advanced to a depth of 3.5 mm from the skull surface and then withdrawal 0.3 mm. Cell suspension was delivered at the rate of 1 μl/min. After cell implantation, the needle was left in place for 6 min before withdrawal. After 6–14 d of cell inoculation, the mice were perfused with 4% paraformaldehyde (PFA) and the brains were paraffin-embedded.All animal handling and experiments were performed in accordance with NIH guidelines and approved by the Ethics Committees of Huazhong University of Science and Technology. The mice were group housed in the Animal Core Facility of Tongji Medical College under a 12 h light/dark cycle with IHC was performed as previously reported . ParaffiHuman glioblastoma cell lines U251, LN229, and human embryonic kidney 293T cell line were purchased from American Tissue Culture Collection  or China Center for Type Culture Collection . U251, LN228, U87MG with stable PD-L1 overexpression  or knockout (U251/sgGFP or sgPD-L1) were generated as previously described  were considered autophagy-positive. An average percentage of puncta-positive cells from nine fields/culture  was calculated and used for statistical analysis.GST-pull down assay was performed as previously described . 293 cel+/K+ ATPase α1 and β-tubulin as internal control of membrane and cytosol proteins respectively.U251 cells were transfected with p-FU-Venus or p-FU-Venus-PD-L1 plasmid for 24–36 h. Membrane and cytosol proteins were extracted using Membrane and Cytosol Protein Extraction Kit  according to manufature's instructions. Membrane and cytosol proteins were then subjected to Western blotting with Nain vitro experiments. Paired Student's test was used to compare between two groups of animal experiments. Comparisons among multiple groups were performed through one-way ANOVA with Student–Newman–Keuls post-test. P < 0.05 was considered statistically significant.All experiments were repeated independently for at least three times. The values were expressed as means ± SEM. Unpaired Student's test was used to compare between two groups of To ascertain the relationships between intrinsic PD-L1 and glioma cell invasive behavior during GBM development, we examined PD-L1 expression in the whole brain at various stages of GBM development in a highly aggressive orthotopic mouse G422 model . PD-L1 a+-GBM cells in the infiltration frontier, mRNA sequencing was conducted in PD-L1-overexpressed U251 glioma cells. Differential expressed genes (DEGs) between U251/PD-L1 and U251/Vec cells  were selected by three criteria  and the selected DEGs were subjected to various systemic analysis. DO (Disease Ontology) enrichment showed that PD-L1-altered gene expression was significantly associated with malignant glioma (http://gepia.cancer-pku.cn) revealed that PD-L1 (CD274) was negatively associated with the prognosis of glioma patients  of Akt based on its structure  , it is iFinally, we examined the effects of PD-L1 on F-actin structure in starved glioma cells. Fluorescent images of Palloidin staining clearly showed that F-actin formation was evidently increased in PD-L1-overexpressed U251 cells under normal culture conditions as well as EBBS-12 h incubation . Taken tIn our present study, we discovered that PD-L1 elevation occurred predominantly in highly aggressive glioma cells. RNA sequencing revealed that high levels of PD-L1 in glioma cells mainly mediated cell migration and PI3K/Akt/actin signaling. PD-L1 reduced starvation-induced Akt inhibition, autophagic influx and F-actin collapse in glioma cells. Mechanistically, PD-L1 directly interacted with Akt, and this process involved PD-L1 128-237aa and Akt 112-480aa. The binding of PD-L1 to Akt facilitated membrane-translocation of Akt and thus elicited downstream biological effects in frontier glioma cells. These findings reveal a novel invasive mechanism in GBM.+-glioma cells were highly aggressive .The datasets generated for this study can be found in the NCBI (The animal study was reviewed and approved by Experimental Animal Ethics Committee, Tongji Medical College, Huazhong University of Science and Technology.RC, XX, XQ, FL, CL, YL, XL, and GJ performed the experiment. RC, FP, XQ, and XC data analysis, wrote the manuscript, and contributed to discussion. FH and DL contributed to discussion. RC, FP, XQ, and XC designed and wrote the manuscript.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.


The stretchability and the volumetric capacitance are increased to 150% and 86 mF/cm3 (0.181 mF/cm2), respectively. This additive nanofabrication method is highly desirable for the development of self-sustainable stretchable energy storage integrated with wearable technologies. The flexible and stretchable energy storage with a high energy density opens the new opportunity for on-chip sensing, imaging, and monitoring.Direct laser writing with an ultrashort laser beam pulses has emerged as a cost-effective single step technology for realizing high spatial resolution features of three-dimensional structures in confined footprints with potential for large area fabrication. Here we present the two-photon direct laser writing technology to develop high-performance stretchable biomimetic three-dimensional micro-supercapacitors with the fractal electrode distance down to 1 µm. With multilayered graphene oxide films, we show the charge transfer capability enhanced by order of 10 The development of on-chip in-plane micro-supercapacitors (MSCs) using laser scribed graphene (LSG) has attracted tremendous attention due to the integration possibilities with the small-scale devices3. However, the deliverable aerial capacitance from that MSCs4 is minimal for powering micro-devices which limit the potential of autonomous systems.Energy storage demands for the technological advancements in micro-stretchable, wearable, and portable applications5. However, the current research in this area has been mainly focused on the use of pseudocapacitive materials6 and gold nanoparticles for LSG7, which makes those MSCs lacking the stretchability8 essential for wearable applications9 due to the fragile nature of gold. Although direct laser writing (DLW) with ultrashot femtosecond (fs) laser beams has been adopted to generate 3D MSCs8, two-photon excitation induced in the focus of a high-numerical objective has not been explored. Due to the 3D confinement in the tight focus10, this technology has opened the enormous potential for the industrial scale production11. As a result, this single-step fabrication technique has generated various complex 3D and free-standing structures at micron and sub-micron scales in telecommunications12, biomedical applications13, free-form optics14 and displays15.To address this challenge, three-dimensional (3D) energy storages have emerged as a new concept which can potentially enable the miniaturization and integrability in a single platform without losing the performance of energy storages by the effective utilization of the volumetric capacitance performance4. On the other hand, the MSCs fabricated using electron beam lithography (EBL) limits the possibilities of extension to three-dimensional which inturn affects the capacitance16 in limited aerial footprints.In this paper, we demonstrate the fabrication of a bioinspired 3D two-photon-induced (2PI)-graphene MSC directly on an elastomeric polydimethylsiloxane (PDMS) substrate, using the two-photon DLW approach to generate the electrode inter-distance down to 1 mm Fig.  in 40 mi2 and the energy storage density exceeding the current lithium-ion batteries. More importantly, the stretchability and the volumetric capacitance reach 150% and 86 mF/cm3 (0.181 mF/cm2), respectively, which are enhanced by more than three times and 100 times compared with the state of the art results17.The adoption of PDMS substrate, in addition, allows us to achieve the stretchable feature. Our result shows the charge transfer capability enhanced by order of 104 19 which is the measure of the local size of a set of numbers and can be calculated by using the Box-counting method20. The relationship between HD, the linear scaling (L) and the resulting increase in size (S) which is the number of self-similar units generated after scaling for a layer by layer plane construction of 3D object can be written as below1 for a total 2PI-graphene film thickness of 23 µm, as shown in Fig. The 2PI multilayered electrode structure is achieved with the 3D extension of the two-dimensional structure based on the fractal pattern derived from fern tree leaves4 Fig.  with int2. Beyond the miniaturization aspect, the high spatial resolution of the focused fs-laser beams can lead to the theoretically predicted ideal inter-electrode distance below 25 µm in the MSC for the optimum electrolyte ionic path21.We use a fs-laser beam to achieve the multilayer graphene oxide (MLGO) reduction for the fabrication of a 3D 2PI-graphene MSC in a footprint of 1 mm22, which involve the multiphoton absorption within the time scales of less than picosecond resulting in the suppression of heat diffusion and the elimination of the heat affected zone. As a result, this fabrication method can lead to structures with high-resolution. However, the graphene oxides photoreduction at a wavelength of 800 nm may be contributed by a combination of photothermal and photochemical23 assisted with shock waves as the pulse repetition rate becomes higher. In order to understand the phenomenon in detail, we conducted a detailed analysis of the photoreduction of graphene oxide film under different repetition rates of 80 MHz, and 10 kHz which can be found in Supplementary Notes and the best conditions obtained under 80 MHz is considered for the fabrication of 2PI-graphene MSCs.It has been demonstrated that the microstructures fabricated by fs pulses are mainly a non-thermal fabrication process24. The PL measurements in single-layer graphene oxide film as a function of the laser fluence under a repetition rate of 80 MHz confirms the two-photon absorption  measurementsion Fig.  during tion Fig. . The temThe 2PI phenomena lead to polycrystalline nature for the obtained 2PI-graphene film as observed from the transmission electron microscopy (TEM) image Fig. . In addiD/IG ratios obtained were 1.39 and 1.08 for positive and negative graphene oxides respectively which indicates the increase of defects in the positive graphene oxide layers. The Raman spectra of MLGO were given in Fig. D/IG ratio was calculated to be 1.26.For the improvement of the MSC energy storage density, the thickness of the graphene oxide film is a significant factor. In our studies, we observe that the focused laser beam with an objective of NA 1.4 generates the stronger thickness reduction in the 2PI-graphene film compared with an objective of NA 0.6 Fig. . The optNanoscribe with a pulse width of 100 fs. The temperature involved in the reduction process of graphene oxides using 80 MHz is calculated to be 800 °C  spectroscopies  shows an almost quasi-rectangular behavior upto a 150% stretchability as given in Fig. −1 with a step size of 0.1 mV and the obtained volumetric capacitance is around 86 mF cm−3  substrate by using a commercial direct writing setup Nanoscribe which has a wavelength of 800 nm, pulse width 120 fs and repetition rate of 80 MHz The PDMS substrate  was prepared in a mixing ratio of 10:1. The air was evacuated from the PDMS mixture using the vacuum. The polymer was crosslinked at 80 °C for one hour in a convection oven. The laser fluence used for writing the graphene oxide film was 0.18 J/cm2 with a 20x objective of 0.6 NA. The fabrication of 2PI-graphene MSCs was followed by filling the MSCs with electrolyte ionic gel and allowed to dry overnight. 41. 1.3 mg/ml of each graphene oxides were dispersed in water and drop cast on a glass substrate. Each layer of graphene oxides was dried at 60 °C, and the process was repeated until the desired number of alternate layers of graphene oxides (MLGO) was achieved.Graphene oxide films were prepared through the layer by layer assembly using the electrostatic attraction between the positive and negative graphene oxide layers. Hummer’s method was used to synthesize the negatively charged graphene oxides (Sigma-Aldrich)2] (Sigma-Aldrich) in a ratio of 0.03 g/1.0 g and the mixture was stirred under a nitrogen atmosphere for about 5 hours. The electrochemical window of the electrolyte is 2.5 V.We used an ionic gel electrolyte. The ionogel was prepared by mixing fumed silica nanopowder  with the ionic liquid (1-butyl-3-methylimidazolium bis (trifluoromethyl sulfonyl) imide [BMIM] [NTfRaman spectra were obtained for different layers of graphene oxides using a Renishaw Raman spectrometer equipped with an Nd-YAG laser beam of wavelength 514 nm. Scans were performed between 1,000 and 2,700 wavenumbers with a laser spot size of 1 µm. The background corrected spectrum was normalized by dividing the data by the maximum intensity. The XPS measurements were conducted using the Thermo K-alpha X-ray Photoelectron Spectrometer (XPS) using monochromatic, Micro-focused Al K-α source.−9 Torr and TEM measurements were conducted using the JEOL 2010 with samples fixed on a copper grid. Porosity measurements of the obtained film is determined using three methods: the diameter analysis of the SEM images using Image J software, BJH method using Surface Area and Porosity Analyser  and Small-angle-X-ray scattering (SAXS) measurements. SAXS was conducted on a microcalix SAXS system (Bruker) using Cu Ka radiation . The sample was placed into a 1.5 mm outer diameter quartz capillary. Background measurement was on an empty capillary. Measurements were carried out under vacuum at 20 °C with an exposure time of 2 hours. Primary masking, data reduction and background subtraction and were carried out in BioXTAS Raw42. The pores were modeled as a collection of polydisperse spheres of air within the 2PI graphene matrix28, and fitting was carried out in SasView43 using a standard model44 with a Gaussian distribution. The best fit yielded an average radius of 18.7 nm with a polydispersity of 14.6%.SEM images were conducted using Philips XL30 SEM with a vacuum of 10Two-point conductivity measurements on 2PI-graphene films reduced under different repetition rates were conducted using the Keithley 2400 series source meter wired through two micro-positioners using tungsten probes of tip diameter 5 µm. Measurements were taken by varying the applied voltage between 0.1 V to 4.5 V. The rectangle patterns of area 300 µm × 300 µm were connected to gold electrical pads on each side. The electrical conductivity study was performed by keeping the probes on each side of gold electrical pads. With the thickness information, the bulk conductivity was evaluated using the following equation:2 as the current collectors. Open-circuit measurements were performed initially for about one hour before the electrochemical tests to ensure the stable conditions. We used an ionic gel electrolyte to study the performance of obtained 3D 2PI-MSCs under stretched and non-stretched conditions. The CV measurements with a step size of 0.1 mV from 0 to 2.5 V at a scan rate of 1–3,000 mV s−1 with an average of 10 cycles.The electrochemical measurements were undertaken using the Keithley ECE 2400 source meter with a tungsten probe of diameter 5 µm and an exposed area 2.25 mm21,i is the applied current (A), and dV/dt is the slope of the discharge curve .The capacitance was calculated from the CC curves at different current densities using the formula25. The volume specific capacitance was calculated using the formula,total is the total capacitance obtained from the 3D 2PI-graphene electrode connected with the gold probe electrodes, C (Gold+MLGO) is the capacitance contributed by the gold and MLGO film separator, V3D 2PI is the volume of the 3D 2PI-graphene MSCs.The energy and power densities of the 3D 2PI-graphene MSCs were calculated by taking away the effect of the capacitance from the gold pad, and MLGO filmden, and the power density, Pden (rate of charge transfer) were calculated as the following:The energy density, Etotal, and Ptotal of the device can be calculated by the formula:total is the energy density in Wh/cm3, Cv is the volumetric capacitance, ΔE is the operating voltage window in Volts, RESR is internal voltage drop at the beginning of the discharge, Vdrop, at a constant current density (i) calculated from the CC measurements and given asTotal energy, ESupplementary Information


To enhance the wettability between Ag atoms and graphene of graphene-reinforced silver-based composite filler, the adsorption behavior of Ag atoms on graphene was studied by first-principles calculation. This was based on band structure analysis, both p-type doping and n-type doping form, of the vacancy-defected and Ce-doped graphene. It was verified by the subsequent investigation on the density of states. According to the charge transfer calculation, p-type doping can promote the electron transport ability between Ag atoms and graphene. The adsorption energy and population analysis show that both defect and Ce doping can improve the wettability and stability of the Ag-graphene system. Seen from these theoretical calculations, this study provides useful guidance for the preparation of Ag-graphene composite fillers. Silver-based filler and graphene are the two main materials that will be discussed in this study. Silver-based filler is a very important kind of brazing material, which has the advantages of a high strength, good mechanical properties, a high electrical conductivity, corrosion resistance, and so on ,2,3. It In this study, the adsorption behavior of Ag atoms on vacancy-deficient graphene (VG) and Ce-doped graphene was calculated by first-principles theory. By analyzing the adsorption energy, band structure, and population, the effects of vacancy defects and Ce doping on the adsorption behavior of Ag on graphene were investigated. The interaction between Ag and graphene and electron transfer were discussed. This research will help to improve the wettability between graphene and silver-based composite fillers, thus providing theoretical guidance and technical support for the application of graphene-reinforced composite fillers.−5 eV/atom.The calculations were performed using the CASTEP code , which iC) site directly above a C atom, and the top (TCe) site above the Ce atom. The models of VG and Ce-doped graphene are shown in A 4 × 4 supercell with a periodic boundary condition along the x-y plane was employed for the infinite graphene sheet model. The vacuum was set with 20 Å in the z direction, which could avoid the interaction between periodic graphene layers . By remoadE) can be computed byAg-grapheneE, AgE, and grapheneE represent the total energy of the graphene-Ag system, the energy of the Ag atom, and the energy of graphene, respectively.The adsorption energy of an Ag atom on graphene  is energetically favored for Ag atom adsorption in comparison with the adsorption on either hollow (H) or bridge (B) sites in intrinsic graphene and Ce-doped graphene . This re−3 in −3, which proves that the wettability of the system has been improved.The system change of VG adsorption of Ag atoms is the largest, as shown in To further investigate the mechanism and effect of doping, different band structures were computed, including the intrinsic graphene, VG, and Ce-doped graphene before and after Ag adsorption. The Dirac point ,45 formsThe partial density of states (DOS) in −1, indicating that the defect causes chemical adsorption between Ag atoms and graphene, and there is a stable chemical bond, which is consistent with the previous adsorption energy, adsorption height, and DOS analysis. Compared with intrinsic graphene, the population of the Ce-doped graphene-Ag system between the Ce and Ag atoms is 0.28, and its number of population per unit bond length is 0.933 charge·nm−1, implying the formation of a stable chemical bond between them.To further study the interaction between graphene and the adsorbed Ag atom, population analysis was completed for intrinsic graphene, VG, and Ce-doped graphene after adsorption of the Ag atom. The results are summarized in Vacancy defects in graphene trigger electron deficiency and p-type doping, which could significantly improve the adsorption energy, charge transfer, and wettability between graphene and the adsorbed Ag atom. Conversely, for Ce-doped graphene, n-type doping formed and thus increased the Fermi level of graphene, which might hinder the electron transfer between Ag and graphene. However, the adsorption energy and population analysis shows that Ce doping could enhance the stability of the Ag–graphene system and wettability.The adopted research methods, mechanism cognitions, and obtained conclusion in this study might contribute to the future development of efficient and stable brazing materials, and provide useful guidance for the preparation of graphene-reinforced Ag filler with fine properties.


Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving loss of motor neurons and having no known cure and uncertain etiology. Several studies have drawn connections between altered retrotransposon expression and ALS. Certain features of the LINE-1 (L1) retrotransposon-encoded ORF1 protein (ORF1p) are analogous to those of neurodegeneration-associated RNA-binding proteins, including formation of cytoplasmic aggregates. In this study we explore these features and consider possible links between L1 expression and ALS.C9orf72 gene-mutated ALS patients.We first considered factors that modulate aggregation and subcellular distribution of LINE-1 ORF1p, including nuclear localization. Changes to some ORF1p amino acid residues alter both retrotransposition efficiency and protein aggregation dynamics, and we found that one such polymorphism is present in endogenous L1s abundant in the human genome. We failed, however, to identify CRM1-mediated nuclear export signals in ORF1p nor strict involvement of cell cycle in endogenous ORF1p nuclear localization in human 2102Ep germline teratocarcinoma cells. Some proteins linked with ALS bind and colocalize with L1 ORF1p ribonucleoprotein particles in cytoplasmic RNA granules. Increased expression of several ALS-associated proteins, including TAR DNA Binding Protein (TDP-43), strongly limits cell culture retrotransposition, while some disease-related mutations modify these effects. Using quantitative reverse transcription PCR (RT-qPCR) of ALS tissues and reanalysis of publicly available RNA-Seq datasets, we asked if changes in expression of retrotransposons are associated with ALS. We found minimal altered expression in sporadic ALS tissues but confirmed a previous report of differential expression of many repeat subfamilies in Here we extended understanding of the subcellular localization dynamics of the aggregation-prone LINE-1 ORF1p RNA-binding protein. However, we failed to find compelling evidence for misregulation of LINE-1 retrotransposons in sporadic ALS nor a clear effect of ALS-associated TDP-43 protein on L1 expression. In sum, our study reveals that the interplay of active retrotransposons and the molecular features of ALS are more complex than anticipated. Thus, the potential consequences of altered retrotransposon activity for ALS and other neurodegenerative disorders are worthy of continued investigation.The online version of this article (10.1186/s13100-018-0138-z) contains supplementary material, which is available to authorized users. With the discovery in 1950 of transposable elements (TEs) genomes began to seem far more dynamic than hitherto conceived  and manyin trans of thousands of processed pseudogenes and a million non-autonomous Short Interspersed Elements (SINEs), including Alu and SVA (composite SINE/VNTR/Alu) elements [Retrotransposon TEs include long terminal repeat (LTR) and non-LTR class elements. Both retrotranspose by a “copy and paste” mechanism involving reverse transcription of an RNA intermediate and insertion of its cDNA copy at a new site in the genome. LTR-retrotransposons, including human endogenous retroviruses (HERVs), are remnants of past germ line infections by retroviruses that subsequently lost their ability to reinfect cells. While the HERV-K(HML-2) group includes some polymorphic proviral loci , 4, humaelements . The 6.0elements –9. The gThere are 145 fully intact L1s in the human genome of which cell culture retrotransposition assays suggest about 100 remain potentially mobile in any individual diploid genome . There abona fide genomic L1 insertions and contaminating extrachromosomal L1-derived nucleic acids. Some studies may therefore warrant additional verification . ALS is a fatal neurodegenerative disease involving loss of upper and lower motor neurons and afflicts 2 in 100,000 people each year. Death typically follows 2 to 3 years after onset and, while about 90% of cases are sporadic, the rest have a family history of the disease. There are no current means to reverse the course of ALS, and treatment involves efforts to slow progression of symptoms . ALS haspol gene transcripts and RT protein in cortical neurons of some ALS patients. Hadlock et al. [C9orf72) ALS gene but not in sporadic ALS cases or controls [Increased reverse transcriptase activity from an unknown source is detectable in sera and cerebrospinal fluids of non-HIV-infected ALS patients –48. Douvk et al.  noted elk et al. . Howevercontrols .The accumulation of neuronal RNA and protein aggregates, including cytoplasmic stress granules (SGs), is a pathogenic hallmark of a number of neurodegenerative diseases. Pathological aggregation of RNA-binding proteins has been implicated in ALS, FTLD, Alzheimer's disease, spinocerebellar ataxia, Huntington’s disease, and inclusion body myositis. In the case of ALS, there is increasing evidence that abnormal RNA processing and abnormal self-aggregation of proteins, leading to altered RNA granule formation and malfunction of protein pathways, contribute to motor neuron death . A key pTARDBP gene) is especially interesting as a unifying pathological marker of both FTLD and ALS. Mutations in TARDBP are involved in about 4% of familial (fALS) and 1% of sporadic ALS (sALS) cases. However, even lacking a mutation, TDP-43 protein, while typically nuclear in healthy cells, is cleaved and hyperphosphorylated and accumulates in ubiquitinated cytoplasmic inclusions in almost all ALS and almost half of FTLD patients . TDP-43iewed in , Alzheimiewed in  and Huntiewed in  diseasesiewed in .C9orf72 gene-related ALS samples. While so far the evidence is not compelling for ALS, we believe the potential for altered non-LTR retrotransposon expression playing a role in neurodegenerative disorders is worthy of continued investigation.Several features of LINE-1-encoded ORF1p are reminiscent of neurodegeneration-associated proteins. ORF1p is an ubiquitinated and phosphorylated RNA-binding protein prone to forming cytoplasmic aggregates, including SGs –63. TherEarly studies showed that endogenous ORF1p concentrates in cytoplasmic granules in cultured cells or fixed tissues , 64. OveConfirming previous results , constitPreviously, it was shown that overexpressed ORF2p and L1 RNA also colocalize with ORF1p in cytoplasmic aggregates , 63, 71.Many RNA-binding proteins that form SGs have intrinsically disordered prion-like domains rich in glutamine and asparagine (Q-N) residues. Aggregation of prion-like domain proteins is characteristic of various neurodegenerative disorders including ALS (reviewed in ). No priPreviously, we and others , 61 showWe introduced the R159H change into ORF1-EGFP-L1-RP, a construct with CMV promoter and ORF1 C-terminally tagged with EGFP followed by intact downstream L1 sequence, and as expected observed a 30% decline in the number of HEK 293T cells with ORF1p cytoplasmic granules Fig. . We nextFinally, we considered the possibility that the abundance of R159 polymorphisms might be due to a CpG dinucleotide methylation hotspot. Following genome bisulfite conversion, PCR amplification, cloning of the amplicons and Sanger sequencing, we queried the methylation status of nine CpGs within a 436-nt stretch (1169-1604) of ORF1 surrounding the R159 codon. Although CpGs were methylated (16 to 64%), we observed no preference for methylation at the ORF1 R159 codon 4 probe [31 RNA granules in nuclei and cytoplasm of some cells 4 probe . As withlls Fig. .Thus, being a promiscuous RNA-binding protein, it is possible that L1 ORF1p is able to bind and sequester many cellular RNAs in granules present in both the cytoplasm and nucleus.0 phase but disagreeing on the extent of retrotransposition loss in G1/S-arrested cells . Mitdecline;  recentlydecline; , and simdecline; . Since Odecline; , we chos2102Ep cells are nullipotent, manifesting a stable phenotype; indeed, they are used as a reference to characterize newly derived hESC lines . However1/S phase transition using aphidicolin, an inhibitor of DNA polymerase α, or hydroxyurea, which inhibits ribonucleotide reductase causing a loss of deoxyribonucleotides [1/S had no significant effect on the average percentage of cells with endogenous ORF1p nucleolar localization , a G S phase , or Gemi S phase , and the S phase  that oveNext, we considered if LINE-1 ORF1p shuttles between the nucleus and cytoplasm, as is the case with TDP-43 and some other prion-domain RNA-binding proteins associated with neurodegenerative diseases . ShuttliPreviously, we fused a suspected ORF1p leucine-rich NES  and linker to the C-terminus of EGFP. While a functional NES should cause EGFP, which is normally both cytoplasmic and nuclear, to become more cytoplasmic , we fail1/S phase arrest, our results suggest this is not due to failure of ORF1p RNPs to enter nuclei, at least in 2102Ep cells, which are known to accommodate cell culture retrotransposition [In contrast to our results, Mita et al.  reportedposition . Moreoveposition  this doeSOD1) [C9orf72 is by far the most frequent gene accounting for about 35% of fALS, 25% of fFTD, and 6% of sALS cases [FUS) and TARDBP mutations each account for about 4% of fALS cases. Other ALS-associated genes, including alsin (ALS2), angiogenin (ANG), heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), optineurin (OPTN), sequestosome-1 (SQSTM1/P62), ubiquilin 2 (UBQLN2), TANK binding kinase 1 (TBK1), valosin-containing protein (VCP) and VAMP-associated protein B and C (VAPB) among others, account for only a small percentage of cases so confounding treatment strategies. ALS animal models of neurodegeneration have mostly examined the toxic effects of overexpressing disease-related aggregation-prone proteins. Mutants of several ALS-associated RNA-binding proteins are known to shift localization from the nucleus to the cytoplasm and form RNA foci in the disease state [To date at least 25 genes have been linked to ALS , 53. TheSOD1) , is mutaLS cases , 93, 116se state , 118.Previously, in a yeast two-hybrid screen we identified FUS protein as an ORF1p interaction partner, and we confirmed that the two wild-type proteins colocalized in cytoplasmic granules of a minor percentage of stressed human nTERA-2 embryonal carcinoma cells . Here weHNRNPA1 and HNRNPA2B1 are prion domain proteins that bind TDP-43 and have been linked with some ALS cases , 123. BoC9orf72 ALS gene can undergo non-conventional repeat-associated non-ATG (RAN) translation and generate dipeptide repeats that form inclusions in cerebellum, neocortex, and hippocampal neurons of C9 patients and toxic cytoplasmic aggregates in cultured neuronal cells or Drosophila models  values for a subset of mostly evolutionarily young primate-specific non-LTR retrotransposons, including L1, Alu and SVA, and LTR5_Hs (HERV-K(HML-2)) and LTR7Y (HERV-H) subfamilies. Study SRP057819 showed an increase (approximately 15%) in RPKM values for the TDP-43 knockdown (KD) versus control HeLa cell lines for L1s only (including L1PA2 and human-specific L1-Ta and L1-pre-Ta subfamilies) Fig. , left. HWe then analyzed the GSE77702 dataset with TEtranscripts , a softwTherefore, and in contrast to some reports (see discussion), our analyses do not indicate a clear TDP-43-specific link with elevated activity of TEs, particularly LINE-1 retrotransposons. In fact, overexpression of wild-type TDP-43 strongly inhibits cell culture retrotransposition of both human L1 and mouse IAP elements.TDP-43 contains a NLS and NES, two RNA-recognition motifs (RRM1 and RRM2) that bind nucleic acids, and a C-terminal glycine-rich region that mediates protein interactions Fig. , top 13. A revieANG mutations have been implicated in ALS and Parkinson's disease [We also considered the effect of angiogenin mutations on cell culture L1 retrotransposition. ANG is a member of the pancreatic RNase A superfamily and a potent mediator of neovascularization, as well as being a host defense factor against some microorganisms  and an e disease . Overexp disease –161. NotSimilarly, we examined the effect of mutations in FUS protein. Exogenous expression of wild-type FUS had no effect on cell culture retrotransposition nor obvious cytotoxicity, but ALS-related mutations in its C-terminal NLS (R514G and H517Q) inhibited retrotransposition over 20% Fig.  S4D, E. In sum, increased expression of some neurodegeneration-related proteins may decrease retrotransposon activity, while some disease-related mutations can modify these effects.To further determine if changes in expression of non-LTR class retrotransposons are associated with ALS, we performed RT-qPCR analyses of 108 bulk spinal cord and brain tissue samples of 38 ALS patients and 27 non-affected controls , and the cellular heterogeneity of the tissues analyzed. TE activation may occur in only a subset of cells within bulk tissue samples, so limiting sensitivity of detection, and in the case of motor neurons these cells may be progressively eliminated in the disease state. In sum, however, no major differences in TE expression where detected in ALS patients when compared with controls.C9orf72-associated ALS (C9ALS), and 10 sALS individuals and analyzed these for differentially expressed genes. A subsequent reanalysis of the same datasets using the HOMER analyzeRepeats program revealed significantly increased global expression of repetitive element types in frontal cortex but not cerebellum of C9ALS compared with sALS patients and healthy controls [Prudencio et al  generatecontrols . SettingWe sought to assess further the degree to which TEs are differentially expressed in sALS-associated tissues by using TEtranscripts  to analyThe GSE76220 dataset consists of single-end 50-bp sequence of total RNA isolated from laser-capture microdissected lumbar spinal cord sections of 13 sALS patients and 8 control individuals . There wWe then reanalyzed the GSE67196 RNA-Seq dataset of Prudencio et al. , 171 usiWe also analyzed for the first time TE expression for the NeuroLINCS dbGaP Study phs001231 (SRP098831). This dataset consists of poly(A)+ non-stranded mRNA of iPSC-derived motor neurons from 4 C9ALS and 3 SMA patients (3 sequencing replicates each) and 3 unaffected controls (2 or 3 replicates each). Transcripts of some TE types are not polyadenylated and so are likely underrepresented in this dataset following poly(A)+ selection. However, although Alu elements are transcribed by RNA polymerase III and not polyadenylated, they contain both internal and 3'-end poly(A) stretches guaranteeing capture of their transcripts. An MDS plot showed C9ALS samples clustered away from SMA and control samples, while clustering of SMA from control samples was less evident Fig. , left. T5 TE loci mapped  the particular loci that contribute to repeat family transcription differences between diseased and healthy states, (ii) the coding capacity of transcribed repeat loci of possible relevance for a specific disease, and (iii) potentially variant retrotransposon proteins and RNAs that should be considered when studying disease relevance. We therefore applied a recently developed locus-specific TE mapping pipeline , as described in the Methods section, to reanalyze the GSE67196 data set . The numCaveats of this type of analysis should be noted. For example, most of the significant DE L1 loci were likely not transcribed from their own promoters, since 1) almost 95% of those mapped were less than 5600 bp in length and so lacked much of their 5' UTRs, and 2) 70% of DE L1s were within genes, and so may be transcribed as part of a longer gene transcript. Moreover, only 2 younger primate-specific L1P1 and 4 L1PA2 L1 loci were differentially expressed . As for DE Alu loci, 81% were within genes . Furthermore, most mapped Alu loci were older elements, with only 5% of them AluY subfamily members, a bias likely due to the inability of currently available algorithms to confidently map short sequence reads to young highly similar TEs. In general, designing RNA-Seq analysis pipelines that efficiently map short sequence reads of young highly similar TEs to their source loci has to date been difficult for reasons discussed below.C9orf72 ALS patients, as previously reported for the GSE67196 dataset [C9orf72-associated ALS disease.In summary, RNA-Seq analysis of the SRP098831 NeuroLINCs dataset suggests widespread upregulation of TE sequences from numerous subfamilies in  dataset . HoweverSelf-aggregation of RNA-binding proteins is a leitmotif of neurodegenerative diseases, including amyotrophic lateral sclerosis. The ORF1 protein of LINE-1 retrotransposons is also an aggregation-prone RNA-binding protein. Of the approximately 500,000 L1s in the human genome, about 5000 are full-length, or about one percent of DNA , 174. MaAnalogous to gene products associated with certain neurodegenerative diseases, L1 ORF1p RNPs are prone to forming cytoplasmic RNA granules. In unstressed cells of some lines, ORF1p constitutively forms cytoplasmic granules that are only faintly and partially marked by canonical stress granule proteins. As shown in Fig. A functional role for L1-associated cytoplasmic RNA granules in retrotransposition remains unknown. This begs the question, what are these constitutively expressed ORF1p aggregates? ORF1p fails to associate with Golgi, lysosome, or endoplasmic reticulum marker proteins , 177. EnEndogenous ORF1p may concentrate as perinuclear, nuclear, or nucleolar. We showed in Fig. 1/S phase transition did not obviously alter ORF1p nuclear localization. Non-blocked cells showed significant concentration of ORF1p in nucleoli, whether the cells were in G1 phase or not. Our results in part contradict a recent study proposing a strong cell cycle bias for ORF1p accumulation in the nucleus during mitosis where it remains during G1 phase [We therefore examined the cell cycle as one possible mechanism controlling ORF1p subcellular localization. Blocking 2102Ep cell cycling at GG1 phase . This diG1 phase  tested aG1 phase , 168, whPreviously, we showed that ORF1p point mutations and C-terminal and N-terminal deletions increased nuclear accumulation . Thus, Might there be cellular consequences for misregulated expression or mislocalization of aggregation-prone LINE-1 proteins? L1 ORF1p is a promiscuous RNA-binding protein able to capture many cellular RNAs. Co-IP experiments with tagged L1 RNPs have identified numerous bound RNAs, including SVA and Alu SINEs and small non-coding RNAs of importance for the cell , 189. Dipol and TARDBP genes to be strongly and positively correlated, and their encoded proteins colocalized in ALS neurons. Douville and Nath [Previous studies have considered links between TDP-43 and retrotransposon expression. TDP-43 was first identified as a transcriptional repressor that binds the RNA regulatory element TAR of HIV-1 proviruses to inhibit their expression . Howeverand Nath  also linand Nath  found thand Nath  and endoand Nath  showed aand Nath . While iand Nath  that TDPand Nath  found wiand Nath .We found that, while TDP-43 binds and colocalizes with the L1 ORF1p RNP, its increased expression strongly represses rather than derepresses human L1 and mouse IAP cell culture retrotransposition (without attendant cytotoxicity). On the other hand, inhibition of endogenous TDP-43 had no effect on L1 retrotransposition in HEK 293T cells. Although altered levels of TDP-43 were associated with modestly increased methylation of endogenous L1 promoters, this was not accompanied by a change in exogenous ORF1p expression or promoter effects in a luciferase assay. Moreover, a recent study by Prudencio et al.  found noC9orf72 and sporadic ALS tissue samples (GSE67196). In all three datasets, we failed to find significant misregulation of TE subfamilies in sALS vs controls, consistent with the previous analysis of GSE67196 [We also reanalyzed for TE subfamily expression two RNA-Seq datasets of sALS tissue samples not previously examined for TE expression (GSE76220 and SRP064478) and one previously tested dataset of both GSE67196  and withGSE67196  that TE bona fide L1 transcripts originating from L1 5' UTR promoters were limited to only a small number of highly expressed full-length L1 loci.Several pitfalls exist for RNA-Seq analyses of differential TE expression: conclusions should be drawn with care. High copy number, close sequence similarity, and especially the frequent embedment of TE sequences in longer gene transcripts  can lead to misinterpretation. While expression of a TE subfamily may appear misregulated, a change in expression observed may in fact be due to altered expression of a gene in which a member of that TE subfamily resides. In their analysis of RNA-Seq data from HEK 293T cells, for example, Deininger et al.  mapped gFurthermore, we have speculated that cell conditions that induce elevated expression of L1s or HERVs, and therefore their encoded reverse transcriptases, could induce promiscuous reverse transcription of cytoplasmic RNAs  and HERV-W LTR retrotransposons by direct Sanger sequencing of cloned cDNAs and RT-qPCR and Western blot analyses, but failed to find significant differences when comparing ALS and controls . It is cn genome .31 ; pcDNA5ergata"; ); GFP-; IAP-ne Roussy; ); pmRFP- Roussy; ); pcDNA3 Roussy; ), mCherr Roussy; ); EGFP-T Roussy; ); pRK5-M Roussy; ); SOD1 W Roussy; ), pCS2 . The fo Roussy; , ORF1-T7 Roussy; , ORF1-EG1-WT-GFP , and pc-1-WT-GFP .Ultimate ORF cDNA clones (Invitrogen) were cloned with V5-epitope tags and tobacco etch virus (TEV) protease cleavage sites on their N-termini by shuttling them from pENTR221 vector into pcDNA3.1/nV5-DEST vector using Gateway Technology (Invitrogen). Ultimate ORF Clone ID numbers were V5-ALS2 (IOH62502), V5-ANG (IOH29453), V5-OPTN (IOH57143), V5-SMN2 (IOH10903), V5-SOD1 (IOH4089), V5-SQSTM1 (IOH5103), V5-TAF15 (IOH40855), V5-TBK1 (IOH21006), V5-VAPB (IOH4934), and V5-VCP (IOH52832). FL-TDP-43 was generated using Ultimate ORF Clone IOH45677 and Gateway vector pEZYflag . TDP-43-V5-WT and TDP-43-FL were generated by PCR-amplification of TDP-43 from pRK5-Myc-TDP-43 with AAG linker and C-terminal V5- and FLAG-tags, respectively, and cloned in pcDNA6/myc-His B . C9ORF72-FL with AAG linker and C-terminal FLAG tag was amplified from Ultimate ORF Clone IOH45695 and cloned in pcDNA6/myc-His B.The helper-dependent adenovirus for construct A/RT-pgk-L1RP-EGFP (Ad-L1) , 217 wassiRNAs , human cervical cancer HeLa-JVM cells , human ePost-mortem brain and spinal cord frozen tissues were obtained from the University of Maryland Brain and Tissue Bank of the NIH NeuroBioBank, the Target ALS Multicenter Postmortem Tissue Core at Johns Hopkins University, and the Department of Neurosciences of the University of California San Diego School of Medicine, as indicated in Additional file Commercial antibodies included rabbit (rb) α-ATG12 (D88H11), rb α-ATG16L1 (D605), rb α-CDT1 (D10F11), rb α-cyclin B1, mouse (ms) α-DYKDDDDK (FLAG)-tag (9A3) , goat (gt) α-eIF3η , rb α-Geminin , ms α-p70 S6 kinase  .For protein extracts, tissues or cells were lysed in RIPA buffer (Sigma) with Mammalian Protease Inhibitor Cocktail and phenylmethanesulfonyl fluoride (Sigma) and homogenized with a Diagenode Bioruptor. In the case of tissues, 2 mm zicronium silicate beads (Next Advance) were added to the tubes. Samples were centrifuged at 11K at 4For RNA extracts, all brain tissue and some spinal cord tissues were disrupted and homogenized in 500 ml of Trizol (Invitrogen) using the TissueLyser LT (Qiagen). Briefly, 30 mg of sample were transferred to a 2 ml tube containing 250 μl of Trizol and one 5 mm stainless steel bead. The TissueLyser LT program used was 50Hz for 1 min. After a spin, the supernatant was collected and another 250 μl were added to the sample to repeat the same procedure. Finally, both fractions were combined and RNA purification with Trizol followed the manufacturer`s instructions. Some spinal cord samples were homogenized in 500 μl of Trizol and zicronium silicate beads using a Benchmark BeadBlaster24. Following centrifugation, the supernatant was further purified using an RNeasy Mini Kit with On-column DNase digestion with RNase-Free DNase Set (Qiagen).Next, the RNA was treated with RQ1 RNase-free DNAse (Promega) for 30 min, purified with ultrapure phenol:chloroform:isoamyl alcohol mixed at 25:24:1 (v/v/v) (Ambion) and precipitated with 3 volumes of ice cold 100% ethanol and 0.1 volume 3M sodium acetate. To assure absence of cross-contaminating genomic DNA, 1 μg of total RNA was treated again with another round of RNase-free DNase I (Invitrogen) for 15 min.GAPDH and could detect no significant effect of RIN when the various tissue types were considered separately. However, a mild effect (R2=0.38) of RNA quality on Ct-levels is acknowledged when combining RIN and Ct values from all samples. Importantly, omission of samples with lower RINs did not affect our conclusions.RNA Integrity numbers (RINs) are shown in Additional file TNF element reporter plasmid was cotransfected with 0.5 μg of empty vector or test plasmid in HeLa-JVM cells. At eighteen hours post-transfection, the cells were expanded from six-well plates to T75 flasks, and two days later selection for retrotransposition events with 500 μg/ml of G418 was begun. After 15 days of selection, cells were fixed, stained with Giemsa, and colonies were counted.The EGFP L1 cell culture retrotransposition assay was conducted as previously described , 222. Th12 viral particles/mL [SH-SY5Y and NSC-34 cells were infected with Ad-L1 at ∼8 × 10icles/mL , 217.Bisulfite analysis was performed as described , 146 usi(GACCTTTCTCTCTAACTACCCTTAAC). PCR amplicons analyzed for Additional file 75 flasks and selection with 5 μg/ml blasticidin was begun. After 12 days, cells were fixed, stained with Giemsa and colonies were counted. Similarly, we co-transfected in HeLa cells pcDNA3, a neomycin (neo)-resistant vector, together with either empty vector (pcDNA6/myc-His B) or test expression constructs, followed by selection of cells with 500 μg/ml Geneticin .To test potential protein toxicity . Use of the MultiTox-Fluor Multiplex Cytotoxicity Assay kit (Promega) followed manufacturer's instructions. This assay simultaneously measures cell viability and cytotoxicity in a single-reagent reaction, permitting ratios of live to dead cell readings to be calculated.GAPDH was used as an internal normalization control .RT-qPCRs were conducted as previously described , 224. A qPCR cycling parameters were as follows: 10 min at 95°C, 40 cycles of 15 sec at 95°C, followed by 60 sec at 60°C. Melting curve analysis was performed to confirm the identity of the amplified product. We employed the ΔΔCt method  to deterRT-PCR reactions used GoTaq Green Master Mix (Promega) and primer pairs 1) L1 5'UTR forward (ACGGAATCTCGCTGATTGCTA) and L1 5'UTR reverse (AAGCAAGCCTGGGCAATG) , which ahttps://www.geneious.com; [L1Base 2  was usedous.com; ). OccurrPublicly available RNA-Seq datasets were analyzed by TEtranscripts software package . TDP-43-The Library of Integrated Network-Based Cellular Signatures (LINCS)-NeuroLINCS dGAP dataset  includes RNA-Seq of iPSC-derived motor neurons from 4 C9ALS and 3 SMA patients (3 sequencing replicates each), and 3 unaffected healthy controls (2 or 3 replicates each). It has been reported that L1 activity in iPSCs can vary with cell passage, increasing during reprogramming but subsequently subsiding , 230. HoTo generate RPKM plots, raw data was aligned to the consensus sequences for a selected group of younger retrotransposons present in RepBase , includiMDS plots were generated in R script using the edgeR package .http://labshare.cshl.edu/shares/mhammelllab/www-data/TEToolkit/ (including 1181 TE types). Following the generation of a count table for gene and TE transcripts, the differential expression analysis closely followed the DESeq2 package [TEtranscripts is a software package that estimates both gene and TE transcript abundances in RNA-Seq data and conducts differential expression analysis on the resultant count tables . Sequenc package  for modeThe pipeline to map TEs to individual genomic loci used the alignment algorithm HISAT2  to map sAdditional file 1:Figure S1. Patterns of L1 ORF1p expression in various cell lines using multiple antibodies for detection. Figure S2. Frequency of polymorphisms and DNA methylation at the L1 ORF1p R159 residue. Figure S3. ORF1p does not contain a CRM1-dependent nuclear export signal. Figure S4. Cell culture toxicity assays. Figure S5. Overexpression or knockdown of TDP-43 protein does not alter L1 expression. Figure S6. Methylation analyses of the CpG island of the 5' UTR promoter of endogenous L1 elements show effects of altered levels of TDP-43. Figure S7. Expression levels of L1 Additional file 3:Table S2. Tissue samples used in this study. (PDF 65 kb)Additional file 4:Table S3. Summary of significant individual DE TE loci in the GSE67196 RNA-Seq dataset. (XLSX 774 kb)


Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pulmonary disease with a median survival of 3–5 years after diagnosis. Recent evidence identifies mucins as key effectors in cell growth and tissue remodeling processes compatible with the processes observed in IPF. Mucins are classified in two groups depending on whether they are secreted (secreted mucins) or tethered to cell membranes (transmembrane mucins). Secreted mucins  are released to the extracellular medium and recent evidence has shown that a promoter polymorphism in the secreted mucin MUC5B is associated with IPF risk. Otherwise, transmembrane mucins  have a receptor-like structure, sensing the external environment and activating intracellular signal transduction pathways essential for mucosal maintenance and damage repair. In this context, the extracellular domain can be released to the external environment by metalloproteinase action, increased in IPF, thus activating fibrotic processes. For example, several studies have reported increased serum extracellular secreted KL6/MUC1 during IPF acute exacerbation. Moreover, MUC1 and MUC4 overexpression in the main IPF cells has been observed. In this review we summarize the current knowledge of mucins as promising druggable targets for IPF. Interstitial lung diseases (ILDs) are a group of disorders characterized by cellular proliferation, interstitial inflammation, fibrosis, or a combination of these conditions within the alveolar wall . If no cThe course of IPF is variable and somewhat unpredictable. Despite the recent approval of two treatments (pirfenidone and nintedanib), the median survival time from diagnosis is 3–5 years . IPF biors35705950) associated with IPF development. This polymorphism has been reported in 30%–35% of IPF patients [KL-6 is a high-molecular-weight glycoprotein classified as a human transmembrane MUC1. Several studies have reported increased serum KL-6 levels during acute IPF exacerbation and a recent study demonstrated that serial increases in serum KL-6 levels are associated with a rapid decline in predicted forced vital capacity (FVC), and further demonstrated that higher KL-6 levels are correlated with lower survival rates . The usepatients . These fAirway mucus is the layer that covers, protects and lubricates the respiratory tract. This mucus is composed of water, ions, lung secretions, serum protein transudates, anti-microbial proteins and mucus glycoproteins (mucins) ,36,37. MMucins are classified into two groups depending whether they are secreted (secreted mucins) or tethered to epithelial cell membranes (transmembrane mucins (TM)). Secreted mucins are further subdivided into gel-forming and non-gel-forming mucins .MUC1, MUC4, MUC12, MUC13, MUC14, MUC15, MUC16, MUC20, MUC21, MUC22); 4 secreted gel-forming mucins ; and 2 secreted, non-gel-forming mucins ) [Mucins are encoded by MUC genes. To date, 21 human MUC genes have been identified , of whic, MUC8)) ,40,41. T, MUC8)) .Secreted mucins are the most abundant glycoprotein component of mucus . They arSecreted gel-forming mucins are characterized by their high molecular weight (5–40 MDa), large size (600–900 nm), high proportion of glycosylation (50%–80%) and the capacity to oligomerise and build up viscoelastic gel, which covers the respiratory epithelium by forming a matrix where bacteria are trapped, thus providing a barrier function ,37,41. TAs noted above, MUC5AC and MUC5B are the major, and best-described, secreted gel-forming mucins. Elevated levels of MUC5AC and MUC5B have been reported in asthma patients , and eleTM mucins are large glycoproteins that localize to the apical surfaces of epithelial cells exposed to relatively harsh environments . Cell suSeveral lines of evidence link mucins to IPF disease, suggesting that they are key effectors of the disease. However, the exact role of mucins in IPF development processes remains unknown, as do their activation mechanisms and ability to mediate intracellular signalling. Thus, knowledge of these activation processes, and of intracellular fibrotic processes mediated by mucins, would be helpful for establishing IPF diagnostic tools, to elucidate IPF development and to identify promising IPF drug targets .MUC5B is mainly produced in mucous cells of the submucosal glands . Thus, iMUC5B gene has a particularly large central exon of 10,713 base pairs (bp), encoding a 3571-amino acid peptide [The  peptide . This si peptide , most of peptide . This ce peptide  and acts peptide . Desseyn peptide  suggeste peptide , includi peptide )  and activating protein 1 (AP-1) , which bMUC5B expression has been found to be 14.1 times higher in subjects with versus without IPF , and it rs35705950) located on the p-terminus of chromosome 11, 3 kb upstream of the MUC5B transcription start site. In fact, a Genome-wide association study (GWAS) found that 38% of subjects with IPF had a minor (T) allele of the single-nucleotide polymorphism (SNP) rs35705950. Moreover, this polymorphism was also associated with 34% subjects with familial interstitial pneumonia and it is linked to 37.4-fold MUC5B upregulation in the lung in unaffected subjects and 5.7-fold MUC5B upregulation in patients with IPF [rs35705950 variant through the ERN2-XBP1S pathway. Moreover, MUC5B overexpression in the bronchiolo-alveolar epithelia is enhanced by the MUC5B promoter variant rs35705950 [rs35705950 show improved prognosis, lower bacterial burden and improved survival compared to those without this variant [The most important genetic risk factor for IPF is a common variant  residues interrupted by poorly glycosylated cysteine-rich domains. The 5′ flanking region consists of the cysteine-rich domains D1, D2, D and D3, which show sequence similarity with the Von Willebrand factor (vWF), as well as a putative leucine zipper motif. The central region encodes a single large exon containing nine poorly glycosylated cysteine-rich domains, in which cysteines 1–5 are interspersed with heavily glycosylated mucin-like domains rich in PTS residues without any repetitive sequences. Cysteine domains 5–9 are interspersed with four TR domains of eight amino acid residues . The cenMUC5AC is the main mucin observed in healthy airway secretions , and thers34474233 and rs34815853) have been cited as risk factors for IPF. These variants result in the same missense amino acid change in MUC5AC, which was observed in 4.4% of controls and up to 13.8% of IPF patients. Thus, MUC5AC can be considered another biologically plausible IPF susceptibility gene [Recently, two tightly linked low-frequency MUC5AC single-nucleotide variants (SNVs) on the p-terminus of chromosome 11  in ovarian and breast cancer cell lines . This fiMUC1 is activated by extracellular ligands such as intracellular adhesion molecule-1 (ICAM-1), which interacts with the protein component of the MUC1 VNTR or MUC1 N-terminal domain , and whiMUC1-CT is phosphorylated by several cell surface growth factor receptors involved in proliferation, apoptosis, transformation and transcription of various genes. For instance, platelet-derived growth factor receptor beta (PDGFRβ) , fibroblMUC1-CT can also directly interact with intracellular kinases such as c-Src, Lyn and Lck, which are activated by growth factors that are elevated in IPF , as wellIn terms of the detailed MUC1-CT phosphorylation mechanism, it has been observed that Thr41 (1224) phosphorylation at the TDR amino acid site by protein kinase Cδ (PKCδ), as well as Tyr46 (1229) phosphorylation at the YEKV amino acid site by c-Src, Lyn and Lck tyrosine kinases, FGF3, or EGFR , induce MUC1-CT also includes a CQC sequence necessary for the formation of MUC1-C oligomers . In carcAs noted above, MUC1 is one of the best-studied tumour-associated antigens, and is abnormally expressed in several malignant conditions. MUC1-targeting vaccines are currently in various stages of clinical trials, including: TG4010  , TecemotMUC4 is localized mainly in the cilia . In the MUC4 gene have been identified, which encode an assortment of secreted and membrane-bound forms of the protein. For example, some splice events in the region downstream of the central TR domain have been identified based on deletions and insertions in this region. Alternative splicing lacking the TR domain or a high degree of polymorphism was observed in the central TR region, with allele sizes ranging from 23.5 to 10.0 kb [MUC4 shows greater allelic diversity than other mucin genes, with a very large uninterrupted TR region containing 48-bp repeat units . Nearly  10.0 kb ,150. Int 10.0 kb .MUC4 is derived from a single gene that is post-translationally processed into an O-glycosylated extracellular subunit of approximately 600–800 kDa, and a largely N-glycosylated fragment of approximately 120 kDa. This heterodimer is a homolog of ascites sialoglycoprotein (ASGP)-1 and ASGP-2, which form the rat sialomucin complex. The ASGP-1-like extracellular domain, or MUC4α, contains an N-terminal signal sequence followed by three imperfect repeats of 126–130 residues, a unique sequence, three putative functional domains including a TR domain of 16 amino acid residues repeated up to nearly 400 times, a cysteine-rich domain, nidogen-like domain (NIDO), adhesion-associated domain in MUC4 and other proteins (AMOP) and a von Willebrand factor type D (vWD) domain. The GDPH cleavage site is located in this vWD domain. Furthermore, the ASGP-2-like or MUC4β subunit is rich in N-glycosylation sites and contains up to three EGF-like domains, a hydrophobic transmembrane region, and a 22-amino-acid CT containing a single tyrosine residue and two serine residues susceptible to phosphorylation ,42 Figu.Overexpression of MUC4 has been observed in the pulmonary arteries of patients with IPF and pulmonary hypertension (PH) . This suMUC4 has been suggested as another pathway by which growth factors increase MUC4 expression in cancer cells [MUC4 expression through the NF-κB and JAK/STAT pathways, respectively [Multiple pathways that regulate MUC4 expression through the binding of various transcription factors or cytokines to the MUC4 promoter have been reported over the years ,157. Forer cells . Combineectively . Recentlectively   are currently approved, and both have limited efficacy. As lung mucins are broadly associated with the airway defence, cell growth and tissue remodelling processes compatible with the processes observed in IPF disease, it is reasonable to analyse the expression, distribution, activation and function of mucins in pulmonary fibrosis, which might provide a useful tool for IPF diagnosis, follow-up and treatment. However, mucin biology is complex; thus, in-depth study is required before mucins can be considered as a new therapeutic approach for IPF.


Mechanical exfoliation of graphite via sonication produced graphene sheets, which were stabilized by the surface adsorption of a cationic surfactant (CTAB). In a subsequent step, varying amount of a cobalt complex [sodium hexanitrocobaltate(III)] was added which selectively bound with the positively charged head of surfactant. In the last step, cobalt complex was reduced with sodium borohydride to obtain Co/G NSs catalyst. The catalyst showed lower overpotential (280 mV) as compared to benchmark catalysts and decent stability and turnover frequency (TOF: 0.089 s One of the renewable and alternative energy sources is water, which could undergo photochemical and electrochemical splitting to produce oxygen and hydrogen green fuel5. In comparison to two-electron hydrogen evolution reaction (HER)7, four-electron oxygen evolution reaction (OER) possesses sluggish kinetics requiring higher overpotential (η) for the achievement of benchmark 10 mA/cm2 current density8. In the past decade various molecular9 and heterogeneous catalysts12 have been developed to achieve oxidation of water at lower overpotential. Focus has been on the earth-abundant metal catalysts15 that could possibly replace the benchmark OER catalysts based on scarcely available Pt, Ir and Ru16. Among other abundantly available transition metals, much attention has been gained by Co catalyst since the pioneer work of Nocera17 and several catalysts based on oxides, chalcogenides, phosphides and phosphates of cobalt have been developed for water splitting22 and energy storage27. However these catalysts based on earth-abundant metals need to be improved to achieve low overpotentials, low Tafel slopes, high Faraday efficiency, high turnover frequency (TOF) and stability28. Among others, carbon29 and graphene-supported cobalt heterostructures have shown high current density, low overpotential and significant stability for OER35. Graphene, a hexagonally arranged two dimensional material made up of sp2-carbon atoms possesses extraordinary properties like room temperature electron mobility, high thermal conductivity and ability to withstand high current densities36. Very high mechanical strength, surface area (>2600 m2/g) and density of surface active sites make graphene particularly suitable for catalysis39. Electrochemical properties of graphene greatly depend upon the synthetic method and can potentially be enhanced by the incorporation of heteroatoms in the graphene lattice which modulates the spin density and charge distribution of carbon atoms thereby creating the catalytically active sites for OER42. However conventional synthesis of graphene by Hummer’s method43 and its modifications45 involves the use of hazardous chemicals or their products whose removal, reuse and disposal is an issue. Chemical vapor deposition (CVD)50 and liquid-phase exfoliation (LE)53 are the only alternative (green) methods55 for the fabrication of highest-quality graphene known for its superior properties. The former method however, is still relatively expensive as it demands large energy and the removal of the substrate36. Recently materials based on cobalt(II) hydroxide has shown potential in electrochemical water splitting58. However poor electrical conductivity of the material needs to be improved in order to obtain a significant OER activity63. Inspired by the pioneer work of Coleman et al.68 we report a one-pot procedure for the solution-phase synthesis69 of graphene-Co(OH)2 nanosheets (Co/G NSs) which catalyze water oxidation at remarkably low overpotential of 280 mV providing high TOF of 0.089 s−1.The present energy resources are insufficient to fulfill global energy in near future due to their rapid depletion, therefore new technological advances to quest clean, sustainable and viable energy resource are highly demanded65. The surfactant gets adsorbed on the surface of so produced graphene via Van der Wall’s interactions and stabilizes the 2-dimensional material 6]: CoL) was added whose anion ([Co(NO2)6]3-) electrostatically binds with the the positive head of the surfactant , to produce graphene flakes in water by mechanical exfoliation of graphite via ultrasonication Fig. 65. The sl)71. For the calculation of extinction coefficient (α), a graphene dispersion (500 ml) was prepared using graphite (3 mg/mL) and CTAB (0.1 mg/mL). After centrifugation and decantation, absorption spectrum was taken at 660 nm. 60 mL of this dispersion was filtered through pre-weighted filter units, dried and weighed to find the graphene concentration. The value of absorptivity constant was found to be 1256 mL/mg/l which is close to the reported value of 1390 mL/mg/l65.UV-vis absorption spectrum of graphene dispersion was found featureless and flat Fig.  at and aG) and CTAB (CCTAB) were also optimized and optimum yield of graphene was found with CG = 3 mg/mL and CCTAB = 0.1 mg/mL  was purchased from Sigma Aldrich. All the solutions were prepared in deionized water (DI) and methanol.Graphite obtained from used zinc-carbon batteries was soaked and washed with hydrochloric acid to leach metal impurities. Cetyl trimethylammonium bromide (CTAB), Sodium borohydride  and surface morphology were examined by using FEI NOVA nano SEM 450 equipped with Oxford EDX detector. Raman spectra were taken using Raman Microscope Renishaw, UK. Surface analysis of the catalysts was conducted using X-ray photoelectron spectrometer . Charging of the spectra was corrected against the graphitic-like C 1 s peak (from the instrument itself) taken at 284 eV and used as a reference. CASAXPS software (version 2.3.16) was used for peak fitting.Transmission electron microscopy (TEM) was carried out by using a Varian LEO 9220 (200 kV) and a JOEL JEM-2200FS instrument. The sample was suspended in chloroform and sonicated for 5 min. Subsequently a drop of the suspended sample was placed on a grid (Plano S 166-3) and allowed to dry.Electrochemical analysis was executed by using Gamray Reference 600 Potentiostat/galavanostat/ZRA with three-electrode system.4 was added and dispersion was stirred at room temperature for 2 hours.Graphene was prepared by a simple mechanical exfoliation method. Typically, graphite and CTAB were mixed in 3.3:1 mass ratio in DI water and the mixture was sonicated for 10 hr in ultrasonic bath (Elma X-tra 70 H) whose temperature was kept below 50 °C. CTAB-stabilized graphene suspension was centrifuged (Centrifuge -5810R) for one hour to remove undissolved graphite and unstable graphene. In the next step, Cobalt complex was added to graphene dispersion (Co to graphene mass ratio = 1:100) and sonicated for 30 min. Afterwards, freshly prepared solution of NaBHFor characterization, graphene dispersion was filtered through syringe filter (0.22 μm) and vacuum dried. For TEM and Raman analysis, dispersions were precipitated at 14000 rpm for 30 minutes.2) electrode (GCE) was scrubbed with alumina powder (0.05 µm) before using as the working electrode. A silver/ silver chloride electrode (Ag/AgCl) was used as reference electrode and platinum wire as counter electrode. All the potentials were converted into RHE by using following Nernst equation.All the electrochemical experiments were performed at room temperature (25 °C) using standard three-electrode system. A glassy carbon  and current density (j) by using Tafel equation.2) and ‘m’ is the loading mass of electrocatalyst (0.017 mg/cm2).Theoretically, mass activity was calculated by using the following equation.−1) and n is the number moles of electrocatalyst.The turnover frequency (TOF) can also be theoretically calculated by considering that every metal atom is involved in catalysis.96.Electrochemically surface area (ECSA) for each system can be measured from double layer charging capacitance of electrocatalytic surface determined from non-faradic region of cyclic voltammetry at multiple scan rates from 10 mV/sec to 200 mV/sec of cyclic voltamogramdl) which yield a straight line equation with a slope which is equal to Cdl according to following equation97 (Fig. c is the double layer charging current, v is the scan rate and Cdl is the double layer charging capacitance. The value of Cdl for electrodeposited Co/G NSs catalyst is 1.17 mF obtained from CV of different scan rates (Fig It is assumed that non- faradic current is due to double layer charging capacitance Figs  and S5. n97 Fig. .\documenates Fig . Then, Es is the specific capacitance due to double layer of smooth surface of deposited sample on electrode. Values of Cs for different metal electrodes have been reported in literature for acidic and alkaline solutions lies in the range of 0.022−0.130 mF cm−2 for KOH and NaOH. The reported value of specific capacitance for cobalt surface is 0.043 mF cm−2 in 0.1 M KOH and consider it as a ‘typical’ value for such materials. The calculated electrochemically active surface area of Co/G NSs is 16 cm2 according to our calculations by using Cdl value.Here CSupplementary Information


GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin–sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin–sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients.Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin ( The ubiquitous TDP-43 RNA-binding protein (RBP) is the major component of the pathological aggregates described in the autoptic brain tissues of the majority of amyotrophic lateral sclerosis (ALS) patients and of a subset of frontotemporal lobar dementia (FTLD) cases ,2. PathoGrn) mRNA as a cytoplasmic target in murine experimental models with TDP-43 binding to its 3′UTR sequence [Grn mRNA stability is increased, leading to an upregulation of progranulin (Pgrn) protein content [TDP-43 regulates different steps of RNA metabolism both in the nucleus, where it is mainly localized and controls splicing, and in the cytoplasm, where it is involved in mRNA transport, stability and local translation . We and sequence ,7. We alGRN gene are causative of familial (5–20%) and sporadic (1–5%) FTLD cases [GRN haploinsufficiency and TDP-43 pathology. Both in animal and cell models, Pgrn depletion was shown to induce cytoplasmic TDP-43 mislocalization or accumulation of its C-terminal fragments and to severely compromise neuronal survival and neurite formation [Progranulin is a secreted glycoprotein expressed in the nervous system both in neuronal and glial cells, where it mainly modulates neuroinflammation and acts as a neurotrophic factor, promoting cell survival and neurite/axon growth . AutosomLD cases , which aLD cases , suggestormation ,12,13,14Grn knock-out mice [GRN LOF mutations [SORT1 gene expression [The maintenance of progranulin levels is also important for lysosome activity, which is severely affected in out mice  and in nutations ,17. In nutations , recentlutations . Interespression  and altepression ,21,22. IGRN pathology aim to restore PGRN levels by the inhibition of the SORT1–PGRN interaction. Indeed, the pharmacological or gene inhibition of SORT1 protein levels has been associated with an increase of extracellular PGRN levels [Therapeutic approaches for FTLD-N levels ,23. MoreN levels  and is aN levels ,26,27.GRN and SORT1 RNA, in this study we further investigated the progranulin–sortilin axis in TDP-43 LOF cell models, evaluating if the secreted progranulin levels, important for both its neurotrophic and lysosomal functions, are affected. By comparing human and murine TDP-43 LOF neuronal cell models, we provide evidence that TDP-43-associated regulatory mechanisms differ between mice and humans having a different impact on progranulin bioavailability.Given the TDP-43 regulatory activity on both Grn gene expression  model obtained by the ectopic expression of the pathological TDP-43 C-terminal fragments (25 kDa and 35 kDa). Unlike the full-length TDP-43 protein, which localized only in the nucleus as expected, we confirmed that the truncated 35 kDa and 25 kDa TDP-43 proteins formed aggregates with a prevalent cytoplasmic localization a, mimickPOLDIP3 ,30. WhenPOLDIP3 . In contPOLDIP3 . When weGRN mRNA as already proven in mice [GRN 3′UTR sequences showed an overall 70% identity 6 motif 6 motif .GRN 3′UTR region (GRN 3′UTR similarly to the murine one (GRN mRNA.We performed a UV-crosslinking immunoprecipitation (UV-CLIP) assay using a riboprobe encompassing the human full-length R region  and cellrine one a, suggesGRN mRNA levels . By immunofluorescence analysis, we observed that the ectopic full-length TDP-43 protein localized in the nucleus and the 35 kDa and 25 kDa TDP-43 C-terminal fragments formed mainly cytoplasmic aggregates a as in tSort1ex17b isoform with unchanged overall expression of the Sort1 gene , whose alternative splicing is described to be differently regulated by TDP-43 in mice and humans ,31. We art1 gene a, similart1 gene , and conescribed , mainly  isoform b as the an cells . By anal17 cells .When we analyzed Sort1 at the protein level in murine LOF cells, we observed that the total protein amount did not change in comparison with cells transfected with a control siRNA . InsteadAs previous literature data showed that both the human and murine sortilin protein may be proteolytically cleaved and extracellularly released by the shedding mechanism ,32, we aSortilin gene and by the subsequent different distribution of the sortilin receptor both intracellularly and extracellularly  [NEFL, HDAC6 and Add2) [We previously described that TDP-43 post-transcriptionally regulates the 34 cells , confirmGrn mRNA . Given tl models . In rodeand Tau) ,34 or stnd Add2) ,36,37 efGRN mRNA at the protein level, we observed that an increase in mRNA content was associated with higher levels of intracellular progranulin in both human and murine TDP-43-depleted cells, thus supporting similarity between humans and mice in progranulin regulation. As progranulin is a secreted glycoprotein, whose main functions depend on its extracellular bioavailability [Sortilin gene expression and splicing mediated by TDP-43 might account for the different effects on extracellular progranulin availability [When we also investigated the effects of TDP-43 LOF on lability , we alsolability ,21 .Human neuroblastoma M17 and SK-N-BE cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 2 g/l glucose, 100 U/mL penicillin and 100 μg/mL streptomycin. Trypsin-EDTA (Sigma-Aldrich) was used to split NSC-34, M17 and SK-N-BE cells.All neuronal-like cell lines were used in experiments without inducing further neuronal differentiation.Human embryonic kidney (HEK) 293T cell line was maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. All media and reagents were from Thermo Scientific if not differently specified.5 cells/well were plated in 6-well plates and silenced in a double round using Lipofectamine 2000 (Thermo Fisher Scientific). NSC-34 and Neuro-2a cells were harvested after 96 h of gene silencing with the siRNA duplex (40 nM), while M17 and SK-N-BE cells were silenced with siRNA duplex (80 nM) for 72 h. For quantification of secreted progranulin levels, the culture medium was replaced by Optimem (Thermo Fisher Scientific) 16 h before harvesting cells. For loss-of-function experiments, gene silencing was performed using siRNA duplexes for mouse TDP-43 (Sigma) and for human TDP-43 , as reported in ,20. Stea5 NSC-34 or M17 cells/well were seeded in 6-well plates for Western blotting and on glass coverslips for the immunostaining assay and transiently transfected with 3 μg of pGFPC2 empty vector , pGFPC2-hTDP-43, pGFPC2-hTDP-35 or pGFPC2-hTDP-25 . After a 48 h transfection, cells for immunostaining were fixed in 4% paraformaldehyde in phosphate buffered saline  for 20 min at room temperature (RT). Differently, for Western blot analyses, cells were harvested, used to generate lysate and processed as described below.For the gain-of-function experiments, 2 × 106 HEK293T cells were seeded in a 100 mm dish, transiently transfected with 6 μg of pFlag-CMV2-hTDP-43 , harvested 24 h after transfection and used to generate lysate for protein extraction, as described below.For UV-CLIP assays, 1.5 × 10Protein lysates were obtained by homogenizing cells in lysis buffer . Protein lysates were incubated for 15 min on ice and then quantified by BCA protein assay (Thermo Fisher Scientific). Protein samples (25 μg) were resolved by SDS-PAGE on 10% NuPAGE Bis-Tris pre-cast polyacrylamide gels (Thermo Fisher Scientific) and transferred to nitrocellulose membranes .g for 5 min was performed to remove debris and dead cells and the supernatants were collected. One-fifth of the total medium was precipitated overnight at −20 °C in four volumes of cold acetone. The day after, media were centrifuged at 15,000× g at 4 °C for 30 min and pellets were resuspended in SDS-loading buffer and resolved by SDS-PAGE as described above.For the quantification of secreted progranulin, centrifugation of the conditioned media at 4000× Specific primary antibodies  was used to quantify the Pgrn protein content in the conditioned media of NSC-34 cells upon TDP-43 knockdown and a 1/25 of the total conditioned medium was used for quantification according to the manufacturer’s instruction. Total RNA was obtained using TriZol reagent (Thermo Fisher Scientific) following the manufacturer’s instructions. Total RNA from NSC-34/N2a (1 μg) and M17/SK-N-BE (3 μg) cells was treated with 1 U DNaseI (Roche) for 20 min at 37 °C and retro-transcribed using oligodT and 1 U SuperScript II-RT (Thermo Fisher Scientific).Grn, total Sort1, Sort1∆ex17b and Sort1ex17b isoforms, and RPL10a genes are listed in Ct) of the housekeeping RPL10a gene were used to normalize the Ct value (ΔCt) for each tested gene and every experimental sample was referred to the mean ΔCt of the controls (ΔΔCt). Fold change values were expressed as 2Ct−ΔΔ.Real time PCR was performed with SYBR Green PCR Master mix  on QuantStudio 12 K Flex (Applied Biosystems) and the primer sequences used for human and mouse Cells were fixed as described above were then washed twice in 1× PBS for 10 min and permeabilized in 0.3% Triton X-100 (Sigma) in 1× PBS  for 5 min at RT. After blocking buffer  in PBS,) for 30 min at RT, incubation with anti-GFP antibody  was performed for 90 min at 37 °C. Anti-mouse IgG Alexa Fluor 488  was used as secondary antibody and coverslips were mounted onto glass slides using Prolong Gold antifade reagent with DAPI (Thermo Fisher Scientific). A confocal inverted microscope (Nikon Eclipse C1) was used to acquire Z-stacks images (0.2 μm) with a 60× magnification. GRN 3′UTR was performed by the online pairwise sequence alignment EMBOSS Needle software. The online RBPmap software (http://rbpmap.technion.ac.il [GRN 3′UTR. A stringent cut-off (≥5) was set for the Z-score value. The nucleotide sequence alignment of human (NM_002087.3) and murine (NM_008175.5) on.ac.il ) was useGRN (322 pb) was amplified by RT-PCR from SK-N-BE total RNA using the primers listed in Grn 3′UTR probe [32P-radiolabeled riboprobes, as previously described [The 3′UTR region sequence of human TR probe  was usedescribed . Immunopt test or one-way ANOVA, followed by appropriate post-hoc tests to compare two or multiple groups, using the GraphPad PRISM 5 software package. Data were presented as mean ± standard error of mean (s.e.m.) of at least three independent experiments and significance value was defined as * p < 0.05, ** p < 0.01, *** p < 0.001.Statistical analyses were performed using two-tailed unpaired student’s Progranulin mRNA is conserved, the different control of sortilin gene expression, splicing, protein cleavage and extracellular release strongly influences the bioavailability of extracellular progranulin in the two species. As the progranulin–sortilin axis has been proposed as a novel therapeutic target in ALS/FTLD, inter-species differences in regulatory mechanisms associated to the TDP-43 protein should be carefully considered when translating preclinical data obtained in mouse disease models to patients.Our results show that the progranulin–sortilin axis is differently affected by TDP-43 loss-of-function in mice and humans because although the TDP-43-dependent regulation of


Escape from immune control must be important in the natural course of B-cell lymphomas, especially for those with activation of NF-κB. The pre-clinical LMP1/CD40-expressing transgenic mouse model is characterized by B-cell specific CD40 signaling responsible for NF-κB continuous activation with a spleen monoclonal B-cell tumor after 1 year in 60% of cases. LMP1/CD40 tumors B-cells expressed high levels of PD-L1. This expression was dependent on activation of either NF-κB, JAK1/JAK2 or BTK pathways since these pathways were activated in tumor B-cells and ex vivo treatment with the inhibitory molecules PHA-408, ruxolitinib and ibrutinib led to decrease of its expression. Treatment of LMP1/CD40-expressing lymphomatous mice with an anti-PD-L1 monoclonal antibody induced tumor regression with decreased spleen content, activation and proliferation rate of B-cells as well as a marked increase in T-cell activation, as assessed by CD62L and CD44 expression. These results highlight the interest of therapies targeting the PD-1/PD-L1 axis in activated lymphomas with PD-L1 expression, with possible synergies with tyrosine kinase inhibitors.The online version of this article (10.1186/s12964-019-0391-x) contains supplementary material, which is available to authorized users. Aberrant expression of the programmed death-ligand 1  checkpoint molecule has been reported in many cancers such as breast, lung and colon tumors as well as during chronic viral infections like those with Epstein-Barr virus (EBV) for example . NF-κB, LMP1/CD40-expressing mice have been already described . AnimalsSpleen from mice were collected and immune cells were filtered through a sterile nylon membrane. Cell suspensions were stained at 4 °C in FACS Buffer  with the following fluorescent-labelled antibodies: anti B220-BV421 , anti CD4-FITC , anti CD8a-APC , anti CD62L-BV421 , anti CD44-PE , anti-PD-L1-PE , anti PD-1-PECy7 , anti CD80-APC  and anti CD86-FITC . Stained cells were analyzed using a BD-Fortessa SORP flow cytometer . Results were analyzed using Kaluza Flow Cytometry software 1.2 .For in vivo proliferation, mice were injected intraperitoneally with 2 mg BrdU , 18 h before isolating cells. Splenocytes were stained for B220 and phases of cell cycle were analyzed by measuring BrdU and Propidium Iodide (PI)-incorporation, using the FITC-BrdU Flow Kit .Total RNA was extracted from CD19_Cre or LMP1/CD40 splenocytes and complementary DNAs were reverse transcribed from 1 μg of total RNA samples using the High Capacity cDNA Archive Kit . PCR products were amplified from each cDNA using the TaqMan® Universal PCR Master Mix and TaqMan® Gene Expression Assays: IL-10/Mm01288386_m1, PD-L1/Mm03048248_m1, GAPDH/Mm99999915_g1. The GAPDH gene was used as a reference gene for the control of amplification. The calculated relative gene expression level was equal to 2 − DDCT, where DDCT is the delta delta cycle threshold. Gene expression fold changes were calculated as the ratio of the test condition to its control.6 cells were lysed and sonicated in Laemmli sample buffer. Lysates were separated on 12% SDS-PAGE gel and transferred to PVDF membrane. Revelation were done using anti-TRAF1 antibody (N-19) purchased from Santa Cruz Biotechnology; anti-STAT3 antibody (124H6) and anti-phosphoSTAT3 antibody (Tyr 705) (D3A7) from Cell Signaling Technology; and anti-ERK (p-44/42) antibody and anti-phosphoERK antibody (Thr 202/Tyr 204) from Cell Signaling Technology. Signals were visualized with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). The amount of loaded protein was standardized against GAPDH using an anti-GAPDH antibody from R&D Systems. Membranes were numerized with ChemiDoc™ Touch Imaging System (Bio-Rad) and images were analysed with the Image Lab Software (Bio-Rad).10 × 10Our previous transcriptome studies from LMP1/CD40-expressing mice suggested that those tumors might express high levels of CD274/PD-L1 . We thusTo address in vivo the role of PD-L1 in these B-cell lymphoma, LMP1/CD40-expressing mice were treated with an antibody blocking PD-1/PD-L1 signaling for 3 weeks according to a methodology already described , 9. We oIn addition to be able to activate the classical pathway, CD40 is a strong inducer of the alternative NF-κΒ activating pathway as already shown in LMP/CD40 mice . CD40 coAltogether, these results show that CD40, IL-10 and BCR signaling pathways have complex interplays. As summarized in Fig. Expression of PD-1 and PD-L1 in B-cell lymphomas and effect of immune therapies against the immune checkpoint axe has been recently reviewed . ExpressHere, our results show that NF-κB activated B-cell lymphoma of the LMP1/CD40-expressing mouse model exhibited an immune escape gene signature involving expression of PD-L1 and PD-L2, which expression was co-clusterized with IL-10. Over-expression of PD-L1 not only involved NF-κB activation by CD40 but also BTK and JAK/STAT signaling, the latter probably being indirectly regulated via an autocrine loop with participation of IL-10 for example. Indeed, PD-L1 expression could be down-regulated after treated with NF-κB, JAK1/JAK2 and BTK inhibitors. Expression of PD-L1 was very likely to be associated to tumor immune escape as demonstrated for numerous solid cancers such as melanoma of lung cancers . PD-1 exOur results indicate that therapies against the PD-L1/PD-1 axis may work in lymphomas as long as the tumor cells express PD-L1. Our results also suggest that combination of immune therapy targeting the PD-1/PD-L1 axis and TKI specific for the JAK/STAT or the BCR/BTK pathway could be of interest. Even if these chemical inhibitors such as ruxolitinib or ibrutinib could have their own inhibitory effect on actors of the anti-tumor response such as cytotoxic T-cells or macrophages, our results also raise the question of the effect of these molecules on the local reactivation of the immune system.Additional file 1:Table S1. Immune Escape Gene signature adapted to the Affymetrix HG-U133_Plus_2 array. Table S2. Immune Escape Gene signature adapted to the HT MG-430 PM Array. (DOCX 32 kb)Additional file 2:Table S3. Normalized expression levels of genes belonging to the immune escape gene signature of splenocytes from LMP1/CD40 and CD19_Cre mice. (DOCX 25 kb)Additional file 3:Figure S1. Absolute number of spleen granulocytes (A) and T-cells (B) in LMP1/CD40-expressing mice after injection of isotype control (Ctrl) or anti-PD-L1 (αPD-L1) antibody. For the PD-L1 treatment, LMP1/CD40-expressing mice were injected avery 4 days for three weeks with 200 µg anti-PD-L1 antibody in On VivoPure Dilution Buffer . ns, non-significant. Figure S2. IL-10 mRNA expression in splenocytes from LMP1/CD40-expressing mice treated with the PHA-408, ruxolitinib and ibrutinib inhibitors. Results are expressed in logarithm of fold change by comparison with the control. Figure S3. Analysis of NF-κB/TRAF1, JAK/STAT3, and ERK pathways by western blot of protein extracts from splenocytes of control CD19_Cre mice after 48 h in vitro treatment with the PHA-408, ruxolitinib and ibrutinib inhibitors. GAPDH was used as loading control. Figure S4. Analysis of PD-L1 expression by western blot of protein extracts from splenocytes of CD19_Cre mice i) after 48 h in vitro CD40 (R&D Systems), CD40 plus IL-4 (Peprotech), IL-10 (R&D Systems), IgM (Jackson ImmunoResearch) stimulations (lane 2 to 5), and ii) after 24 h in vitro CD40, CD40 plus IL-4, IL-10, IgM stimulations followed by 24 h treatment with the PHA-408, ruxolitinib and ibrutinib inhibitors (lanes 6 to 9). GAPDH was used as loading control. (PPTX 6393 kb)