IK1). Additionally, our computer simulation results shed new light on the novel cellular mechanism underlying AF by indicating that the imbalance between suppressed outward current IK1 and increased inward sodium-calcium exchanger current (INCX) resulted from SR calcium leak leads to spontaneous depolarizations. Furthermore, our simulation results suggest that these arrhythmogenic triggers can be potentially suppressed by inhibiting sarcoplasmic reticulum (SR) calcium leak and reversing remodeled IK1. More importantly, this study has clinically significant implications on the drugs used for maintaining SR calcium homeostasis, whereby drugs such as dantrolene may confer significant improvement for the treatment of AF patients with TBX5/PITX2 insufficiency.Transcription factors TBX5 and PITX2 involve in the regulation of gene expression of ion channels and are closely associated with atrial fibrillation (AF), the most common cardiac arrhythmia in developed countries. The exact cellular and molecular mechanisms underlying the increased susceptibility to AF in patients with TBX5/PITX2 insufficiency remain unclear. In this study, we have developed and validated a novel human left atrial cellular model (TPA) based on the ten Tusscher-Panfilov ventricular cell model to systematically investigate how electrical remodeling induced by TBX5/PITX2 insufficiency leads to AF. Using our TPA model, we have demonstrated that spontaneous diastolic depolarization observed in atrial myocytes with TBX5-deletion can be explained by altered intracellular calcium handling and suppression of inward-rectifier potassium current ( Current clinical treatment of AF is suboptimal, as a single one-size-fits-all treatment formula is used regardless of the different preconditions that lead to AF2. AF is a complex disease and known to be associated with multiple risk factors including hypertension, obesity2, diabetes mellitus3, ischemic heart disease, heart failure and stroke3. Recent human genome-wide association studies (GWAS) suggest that AF is also a heritable disease4. Over the past decade, some common genetic variants underlying AF in the general population, e.g., a T-box transcription factor TBX5 and a paired-like homeodomain transcription factor 2 PITX2, have been discovered14.Atrial fibrillation (AF), the most common cardiac arrhythmia, causes substantial mortality, morbidity and impaired quality of life in an aging population15. GWAS have discovered that the lack of TBX5 is associated with abnormalities in action potential, which leads to an increased risk of developing AF16. Various research laboratories have demonstrated that TBX5 potentially regulates AF through the modulation of multiple ion-channel genes, suggesting that electrical remodeling is a major contributor to cellular arrhythmogenic triggers20. In addition, the loss of function of PITX2 was observed in the TBX5-deletion atria, signifying that PITX2 takes part in TBX5 insufficiency induced-AF21. Multiple studies have revealed that PITX2 on human 4q25 locus is associated with AF where reduced PITX2 promotes an arrhythmogenic substrate26. Pro-arrhythmogenic effects of PITX2 are twofold: 1) it regulates the expression of genes involved in ion channels27, leading to increased electrical-remodeling-related risk of AF28, and 2) since PITX2 plays a crucial role in left-right atrial asymmetry during cardiac development29, PITX2-deletion can cause malformation of the pulmonary veins30, which play a substantial role in initiating and maintaining spontaneous AF31. Therefore, the reduced function of PITX2 in TBX5-deleted atrial myocytes may also contribute to AF, and collectively, electrical remodeling induced by the insufficiency of PITX2 and TBX5 may be a major contributor of the increased susceptibility to AF35.TBX5 plays important roles in heart development and cardiac rhythm control36 provide a powerful tool for assessing the impact of individual remodeled ion currents on the action potential (AP) and calcium dynamics which is not possible in experimental and clinical studies. To achieve this, in this study a robust computer model of human atrial cellular kinetics (TPA) based on the ten Tusscher-Panfilov (TP) ventricular cell model37 was developed, validated, and utilized to systematically illustrate the cellular and molecular mechanisms underlying spontaneous AF in patients with TBX5/PITX2 insufficiency.How electrical remodeling, regulated by the transcription regulation network governed by TBX5 and modulated by PITX2, contributes to AF has not been thoroughly investigated. Computational models21 and simulated AP profiles using the TPA model are shown in Fig. et al.21 by carefully studying atrial myocytes isolated from Hom-Tbx5 versus control mice, where their reported RMP and the number of triggered beats were −75.47 ± 0.94 mV versus −77.88 ± 2.4 mV and 24/30 versus 0, respectively  human atrial myocytes, computer simulations were conducted using our human atrial TPA model under both control and Hom-Tbx5 models at a pacing frequency of 1 Hz. Experimental AP recordings of murine atrial myocytesCa2+]i) is involved in TBX5 insufficiency-induced triggered activity, [Ca2+]i in the Hom-Tbx5 model was set to a steady value (0.07 µM) based on data collected in an experimental study21. Figure Ca2+]i observed in an experimental21 (black) and our modeling (red) study. Our computer simulation results are consistent with experimental observations that RMP increased from −83.51 mV (Control-Clamp) to −82.30 mV (Hom-Tbx5-Clamp) and action potential duration at 90% repolarization (APD90) was prolonged by 12.7% (from 232.9 ms to 262.5 ms). The comparison showed that the Hom-Tbx5 model has obtained AP characteristics similar to those from TBX5-mutant atrial myocytes observed in experimental studies21.To confirm the hypothesis that the cytosolic calcium concentration  Jup, calcium flow through the ryanodine receptor (RyR) Jrel, and inward rectifier potassium current IK1, caused AP prolongation, abnormal autonomous depolarization and elevated diastolic [Ca2+]i. To evaluate the contribution of each individual remodeled ion current to these APs and Ca2+ handling abnormalities, we have conducted a series of computer simulations by incorporating each individual ionic remodeling into the control model , RMP and APD90 under different conditions , homozygous PITX2-knockout (Hom-Pitx2), heterozygous PITX2-knockout (Het-Pitx2) and heterozygous knockout of both PITX2 and TBX5 (Het-Pitx2-Tbx5) conditions (for details see Methods) and the effects of TBX5/PITX2 interplay on APs and [Ca2+]i were examined , in comparison to the control condition. For heterozygous knockout of both PITX2 and TBX5 atrial cells, i.e., Het-Pitx2-Tbx5, APD90 and Cadiast were comparable to those of the control condition  in atrial myocytes from patients with AF38. To evaluate the role of Jleak in TBX5 insufficiency-induced arrhythmias, we have conducted computer simulations in which Jleak was blocked in Hom-Tbx5 atrial cells. As shown in Fig. Jleak was completely inhibited, spontaneous depolarizations, disrupted [Ca2+]i and diastolic Jrel frequency in Hom-Tbx5 atrial cells were decreased. However, APD90 and RMP of the Hom-Tbx5-block model were not well restored compared with the control model. These data suggest that SR calcium leak partly contributes to TBX5 insufficiency-induced arrhythmias.Dantrolene is a drug used to stabilize RyR for the treatment of malignant hyperthermia. Previous studies have shown that dantrolene beneficially influences disrupted sarcoplasmic reticulum (SR) calcium homeostasis by inhibiting SR calcium leak (Jleak was blocked by 80% (Hom-Tbx5-dantrolene) to mimic the treatment with dantrolene (10 µM) in left human atrial cardiomyocytes from patients with AF  significantly reduced total SR calcium leak of human AF cardiomyocytes by 56% ± 28%. To examine whether dantrolene has antiarrhythmic effects on spontaneous depolarizations induced by TBX5 insufficiency, we conducted simulations in which  AF Fig. . Simulat13. However, the exact mechanisms, particularly how electrical remodeling induced by these transcription factors contribute to the increased susceptibility to AF, remain unclear due to its nature of complexity and limitation of clinical/experimental studies39. To our knowledge, this is the first study in silico to show that TBX5 insufficiency-induced electrical remodeling predicts the incidence of triggered activity in human atrial cells and lends support to the concept of a crucial pathophysiological role of TBX5 insufficiency in the development of AF.Many studies over the past several decades have demonstrated that transcription factors, e.g., TBX5 and PITX2, can directly or indirectly influence atrial rhythm by regulating the expression of membrane effector genes37, we developed a human atrial cellular model, i.e., TPA, to use in our study by taking into account ionic differences between atrial and ventricular cells41. The calcium transient and AP profile of the TPA model are comparable to the Grandi et al. model40 and Courtemanche et al. model (CRN)42, respectively. The APD restitution curve of the TPA model is more accurate compared with experimentally measured data by Bosch et al.43. Most importantly, modifications to calcium-induced calcium release for reproducing early afterdepolarization (EAD)44 and delayed afterdepolarization (DAD) , which was developed by combining calcium handling of the TPA model with the transmembrane currents of the CRN model  required for calcium handling21, suggesting that SR calcium release is impaired in atrial cells of homozygous TBX5-knockout atria  is significantly down-regulated and its precise role in AF is unclear21. In our study, the role of suppressed IK1 in the development of TBX5 insufficiency-induced spontaneous depolarizations was assessed. Our simulation results showed that spontaneous depolarizations can be induced by TBX5 insufficiency-remodeled IK1 and that reversing remodeled IK1 can rescue TBX5 insufficiency-induced spontaneous depolarizations 21, KCNQ1 (encoding for IKs)55, RyR (encoding for Jrel)56 and SERCA (encoding for Jup)56, and downregulation of KCNJ2 (encoding for IK1)57 and CACNA1C (encoding for ICaL)56. On the other hand, TBX5 insufficiency-induced electrical remodelling includes reductions of INa, Ito, IKur, IK1, RyR and SERCA21. Further studies demonstrated that TBX5 and PITX2 antagonistically regulate INa, Jrel and Jup21. Our results demonstrate that electrical remodeling induced by PITX2 insufficiency causes AP abbreviation, in line with the experimental observation in heterozygous PITX2 mice, linking PITX2 insufficiency to AF28. Simulated results from this study also showed that electrical remodeling caused by TBX5 insufficiency leads to APD prolongation and triggered activity, indicating AF susceptibility in the presence of TBX5 insufficiency. PITX2 insufficiency or TBX5 insufficiency causes opposite effects on the human atrial APD, yet both cause the increased susceptibility to AF  and afterdepolarizations are a common observation in cellular pathophysiology48. Here, we show that normal calcium dynamics is necessary for normal APs and suppression of diastolic SR calcium leak can rescue TBX5 insufficiency-induced spontaneous depolarizations. This may suggest that calcium extrusion caused by SR calcium leak via INCX favors phase-4 depolarization and triggered activity. Experimental data showed that dantrolene, a drug known to stabilize RyR, can decrease the frequency of calcium waves, diastolic SR calcium leak and spontaneous calcium release, and significantly suppress DADs in human AF cardiomyocytes38. Our simulation results suggest that dantrolene may beneficially influence disrupted calcium handling, specifically diastolic SR calcium leak, and prevent the occurrence of TBX5 insufficiency-induced spontaneous depolarizations.In patients with AF, impaired calcium handling  critical to atrial conduction are significantly down-regulated, and atrial conduction and reentrant arrhythmias at the organ level were observed in TBX5 insufficiency mouse atria21. Although this study focuses on the cellular basis of atrial arrhythmogenesis, the initiation and maintenance of AF at the tissue level should be further investigated in future. Nevertheless, whilst it is important to make explicit the potential limitations of the approaches adopted in the present study, these potential limitations are not expected to influence fundamental conclusions that can be drawn from our data on the mechanisms by which suppressed IK1 and inward INCX due to increased SR calcium leak contribute to the generation of spontaneous depolarizations in human atrial cells of homozygous TBX5-knockout atria.Several limitations specific to this study are addressed here. Firstly, in the absence of the required human experimental data, when simulating effects of TBX5 insufficiency on ion channels, electrical remodeling was assumed to be identical in both human and animal atria, and characteristics of the simulated human APs were compared with experimental data of mouse cells for validation. These assumptions warrant further investigations and special attention must be paid to explain these simulated data. Secondly, although the IK1 and inward INCX due to increased SR calcium leak causes spontaneous depolarizations in human atrial cells. More importantly, our study suggests that these arrhythmogenic triggers can be potently suppressed by inhibiting SR calcium leak or reversing remodeled IK1. Therefore, the outcomes of this study may lead to more targeted patient-specific treatment for AF in the near future.In conclusion, we have demonstrated that electrical remodeling induced by TBX5 insufficiency causes disrupted SR calcium handling, AP prolongation and spontaneous depolarizations in human atrial cells. For the first time, we propose a novel cellular mechanism underlying TBX5 insufficiency induced-AF: that the imbalance between suppressed alias TPA was developed based on the TP model of ventricular epicardial cells. The TP model was chosen as the base model for modification as it incorporates human cell data and is also able to reproduce human AP morphology, APD rate dependence and detailed calcium handling37. The RyR channel model of the TP model was further modified with the combination of calcium-induced-calcium release flow Jcicr and SR calcium leak flow Jleak45 to reproduce triggered activity, i.e., EAD, DAD and spontaneous depolarizations. In our study, the SR calcium flow (Jrel) of RyR is given byVrel (0.102 mM/ms) is the maximal Jcicr conductance, Vsp (0.00036 mM/ms) is the maximal Jleak conductance, O is the proportion of open RyR channels, R is the proportion of closed RyR channels, [Ca2+]SR is the free SR calcium concentration and [Ca2+]SS is the free dyadic subspace calcium concentration59.For this study, a new human atrial cellular model 41. These modifications were based on most recent experimental data from human (wherever data was available) and from animals (where human data was unavailable), and our approach is consistent with those used in previous modeling studies41. Briefly, the maximal conductance of ICaL41, Ito60, IK160, IKs, IKr, INCX61, sodium-potassium pump current (INaK)61 and Jup62 were modified  potential for potassium ion. Activation gate XKur and inactivation gate YKur are given byt is the time variable. XKur,∞ and YKur,∞ denote for the steady-state for activation and inactivation, respectively, and τX/τY is the time constant for XKur,∞/YKur,∞. The mathematical expressions of XKur,∞ and YKur,∞ are given byThe TP model was then adjusted to generate our human atrial cell model TPA by incorporating experimentally documented ionic differences between human atrial and ventricular cellsIK1, the introduction of IKur, and the modifications to ICaL, Ito, IKr, IKs, IKur, INaK and INCX, respectively. This model represents the left atrial cells. All supporting data and computer simulation source code (http://models.cellml.org/workspace/520) from this study are available in the Supplementary Materials.Intracellular structure . During phase 0 of AP, sodium channels recover more slowly from inactivation, resulting in a smaller INa and a reduced maximal depolarization velocity (122.11 mV/ms) that is comparable to that measured in experiments of ~140 mV/ms63. Changes in Ito and IKur contribute to a more negative notch in the TPA (versus TP) model during the phase 1 of AP. During phases 2 and 3 of AP, the combination of ICaL and potassium currents  in the TPA model generates a smaller net current, leading to a less pronounced dome. In addition to the higher RMP, the more negative notch, and the less pronounced dome, the TPA model also exhibits a shorter APD of 233.6 ms compared with that of the TP model.The higher RMP in the TPA model occurs during phase 4 of AP where the reduction of Ca2+]i trace of the TPA model was comparable to that of Grandi et al. model40  and Cadiast in the TPA model were similar to those (marked with stars) reported in the Grandi et al. model and the Koivumaki et al. model64 which are both known for their more accurate calcium dynamics. In details, Cadiast (0.138 µM) and Catran (0.498 µM) obtained from the TPA model were similar to the measured data from Neef et al.35 (Cadiast = ~0.1566 µM) and Grandi et al.40 (Catran = ~0.468 µM). In terms of APD rate dependence  by Bosch et al.43 where the APD90 restitution curve of the TPA model was compared to that of the Koivumaki et al. model. All these comparison results have demonstrated that the TPA model can reliably reproduce the key properties of human atrial myocytes.The TPA model was fully assessed by comparing with existing human atrial models, particularly in terms of APs and calcium transient kinetics, since they are essential to this study Fig. . The [Cal40 Fig. , whereasl42 Fig. . The advnts Fig. , the calnce Fig. , the sim65, five different scenarios were considered: Hom-Tbx5, Het-Tbx5, Hom-Pitx2, Het-Pitx2 and Het-Pitx2-Tbx5 conditions. For Hom-Pitx2 and Het-Pitx2 models, the relative transcript expression of TBX5 was not changed significantly (100% versus 111 ± 17%)54, and these models were mainly used to investigate the pro-arrhythmic effects of PITX2 insufficiency in a dose-dependent manner (~20% versus ~45%). For Hom-Tbx5 and Het-Tbx5 models, the relative gene expression in TBX5 was ~15% and ~55%21 respectively, so effects of TBX5 insufficiency at a different scale on human atrial electrophysiology were examined. In addition, Nadadur et al. have demonstrated that TBX5 directly activates PITX2 and reduced TBX5 leads to decreased PITX2 expression21. The relative gene expression in PITX2 for Hom-Tbx5 was set at ~53%, whereas that for Het-Tbx5 was ~91%21. The Het-Pitx2-Tbx5 model was designed to investigate the role of TBX5/PITX2 interplay in human atrial electrophysiology. In the Het-Pitx2-Tbx5 model, the relative gene expression of PITX2 was set at ~54%, whereas the relative gene expression of TBX5 was ~64%21. Based on experimental data on TBX5/PITX2 insufficiency-induced changes in gene expression of ion channels (see Supplementary Table INa, Ito, IKur, IK1, Jup and Jrel were identified in TBX5-knockout atrial cells21, whereas remodeling of INa, ICaL, IKs, IK1, Jup and Jrel were identified in PITX2-knockout atrial cells65. For each model, relative transcript expression of TBX5 and PITX2 and modifications to ionic currents were described (see Fig. According to changes in gene expression of TBX5/PITX2 in experimentsSupplementary Video_S1Supplementary Video_S2Supplementary Video_S3Supplementary Information


The MMAS-8 is one of the most commonly used self-report adherence questionnaires. The Chinese version of MMAS-8 has been validated among a convenience sample of 176 patients in China [31]. The scale showed acceptable internal consistency (Cronbach’s α = 0.77) and test-retest reliability (r = 0.88), and good construct validity.’In the Methods section, along with the references mentioned in the statement:‘The analysis was adjusted for the effect of medication adherence via the Morisky 8-item medication adherence scale’.In the respective captions of Tables 3 and 4:The original article [1] contains a number of statements that the authors would like to be disregarded, noted ahead:These statements as well as the references contained within them are to be disregarded as it was discovered that the patients included in the analyses did not receive the MMAS and thus, the analyses did not adjust for the MMAS scores .


L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to  Growing tumors stimulate the formation of new blood vessels to supply the oxygen and nutrients the cancerous cells need to stay alive. Stopping tumors from forming the blood vessels could therefore help us to treat cancer. To do so, we need to understand how different proteins control when and how blood vessels develop.Cells make proteins by first ‘transcribing’ genes to form RNA molecules. In many cases, the RNA then goes through a process called alternative splicing. Proteins known as splicing factors cut out different segments of the RNA molecule and stick together the remaining segments to form templates for protein production. This enables a single gene to produce many different variants of a protein.Angiolini, Belloni, Giordano et al. have now studied mouse and human versions of the cells that line the blood vessels grown by tumors. This revealed that a splicing factor called NOVA2 targets a protein called L1CAM, which is normally responsible for gluing adjacent cells together. Angiolini et al. found that NOVA2 splices L1CAM into a form not seen before. Instead of remaining anchored to cell surfaces, the newly identified form of L1CAM is released into the blood circulation, where it stimulates new blood vessels to grow.Samples taken from the blood vessels of human ovarian tumors showed high levels of both NOVA2 and the modified form of L1CAM, while blood vessels in healthy tissue contain no, or very low levels of both proteins. Therefore, if the new form of L1CAM can be detected in the blood, it could be used to help cancer diagnosis, and to indicate which patients would benefit from treatments that restrict the growth of blood vessels in tumors. Further work is now needed to explore these possibilities. L1CAM. It is a cell surface glycoprotein with an extracellular portion that comprises six Ig-like domains, five fibronectin type III repeats, a short transmembrane (TM) domain, and a conserved cytoplasmic tail. A disintegrin and metalloproteinase (ADAM)10-mediated cleavage at membrane proximal site induces shedding of the L1CAM ectodomain  that function in a coordinate manner to promote or inhibit the inclusion of specific exons into the mRNA (Alternative splicing (AS) produces different mature transcripts (mRNAs) from a single primary pre-mRNA. AS decisions are modulated by a number of the mRNA . More ththe mRNA  giving rthe mRNA . Notablythe mRNA . In partthe mRNA . These ‘the mRNA , while tthe mRNA . Howeverthe mRNA  and propthe mRNA . HoweverRecently, we described the SRF Neuro-Oncological Ventral Antigen 2 (NOVA2) as a prominent regulator of AS during vascular development . NOVA2 wHere, we report a novel isoform of L1CAM expressed in ECs as the result of a NOVA2-induced AS event that removes the exon encoding the transmembrane domain of the protein. This gives rise to a soluble L1CAM variant, referred to as L1-ΔTM, that is released by ECs and is able to stimulate angiogenesis via autocrine/paracrine mechanisms. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer and correlate with poor outcome and tumor vascularization, respectively. Our findings, therefore, implicate the novel NOVA2/L1-ΔTM axis in EC pathophysiology and in ovarian cancer aggressiveness.L1CAM accounts for, or at least contributes to, its peculiar role in ECs. A bioinformatics analysis with the ExonMine program (http://www.imm.fm.ul.pt/exonmine/) (L1CAM exon 25 (a 135-nucleotide cassette exon) is excluded from the mature mRNA (L1CAM exon 25 by RT–PCR (L1CAM exon 25 (89% identity), we refer to it as exon 25 also in mouse L1cam. The AS of this exon was examined in normal mouse tissues, mouse EC lines and freshly purified murine ECs. As shown in L1CAM devoid of exon 25.We have recently reported the novel function of L1CAM in vascular endothelium . Since Aonmine/)  identifiure mRNA . We theny RT–PCR . In addiL1CAM mRNA, which lacks exons 2 and 27  or with L1-ΔTM. Immunoblotting of total extracts from L1-FL-expressing cells showed the expected doublet pattern (-expressing moEC showed a single band that migrated slightly faster than the lower band of L1-FL (Skipping of exon 25 results in an in-frame deletion of a 44-amino acid sequence (45 in mouse) that encompasses the entire transmembrane (TM) domain of L1CAM . This su2 and 27 . Thus, e pattern , with th pattern . In contof L1-FL , consistof L1-FL . Based oof L1-FL . Of noteof L1-FL . This coof L1-FL . Both thof L1-FL .Interestingly, L1-ΔTM was detected in the culture medium as a single band that migrated slower than the protein found in the cell lysate . We hypoL1cam  (Previous studies implicated fibroblast growth factor receptor (FGFR) signaling as an effector of L1CAM in different cellular contexts . Howevert shown) , as prevt shown) . To detet shown) , L1-ΔTM-t shown) . Thus, oL1cam in endothelium, we analyzed the sequence of mouse L1cam exon 25 and its flanking intronic regions, using SFmap (http://sfmap.technion.ac.il/) (To gain further insights into the molecular mechanisms regulating the AS of .ac.il/)  to searc.ac.il/) , three f.ac.il/) .L1cam, we first performed a splicing assay in HeLa cells co-transfected with a minigene (p-L1) encompassing exons 24, 25, and 26 of L1cam along with the flanking intron sequences  motifs, which represent putative binding sites for NOVA proteins  the higher expression of NOVA2 in freshly purified ECs from mouse lung and in lu2EC as compared with total mouse lung or melanoma cell line B16, respectively, was accompanied by the skipping of L1cam exon 25 down ECs   in the pre-mRNA targets . In part exon 25 , consistsequence . Accordiuman ECs .L1cam, we carried out UV crosslinking and immunoprecipitation (CLIP), which allows to identify direct protein-RNA interactions in live cells  as a suitable model system. In fact, we found a markedly higher number of NOVA2-positive vessels in OC  than in healthy ovaries (https://www.proteinatlas.org/) (L1CAM in ECs isolated from OC (HOC-EC). As shown in L1-ΔTM isoform was readily detected in HOC-EC from seven independent OC samples. To test whether vascular L1-ΔTM in OC is associated with tumor angiogenesis, we measured the vessel density in a small cohort of OC samples pre-classified as L1-ΔTM-positive or negative by RT-qPCR , measured by CD31 immunostaining  program. As shown in NOVA2 correlated with shorter overall survival of the patients . Taken together, these results suggest that NOVA2 promotes AS of the L1CAM pre-mRNA in OC vessels, thus accounting for the vascular expression of L1-ΔTM, and highlight the proangiogenic role and the prognostic value of the NOVA2/L1-ΔTM axis in OC.We have recently described the expression of NOVA2 in vascular endothelium . Further ovaries . The abuas.org/)   . The peras.org/) . In addias.org/) . Thus, w RT-qPCR . A signie tumors . Furtherstaining . These fOur data implicated for the first time the splicing factor NOVA2 in the generation of a novel, EC-specific isoform of the cell adhesion molecule L1CAM, referred to as L1-ΔTM. Due to NOVA2-induced skipping of exon 25 that encodes the TM domain, L1-ΔTM is no longer associated to the cell surface and, hence, is released in the extracellular space. Consistent with the expression of NOVA2 in vascular ECs , the latbona fide angiogenic factor which, however, belongs to a class of molecules highly divergent from the classic polypeptide growth factors that exert this function . To our knowledge, L1-ΔTM provides the first example of an immunoglobulin-like cell adhesion molecule converted into a soluble angiogenic factor through AS removal of the exonic sequence that encodes the TM domain. AS of the TM domain occurs in a broad spectrum of human genes . This supports the notion that extracellular L1-ΔTM can promote angiogenesis via both autocrine and paracrine stimulation. The latter mode of action, in particular, might represent a way to amplify the angiogenic signal provided by vascular L1CAM. Indeed, in most cases only a fraction of vessels within tumor and inflammatory tissues (i.e. the most prominent conditions where vascular L1CAM is detected), exhibits L1CAM expression . Thus, tIn different cellular contexts, such as neurons, L1CAM frequently engages in homophilic L1CAM-L1CAM interactions between adjacent cells, which underlie its function in cell-cell adhesion . HoweverL1CAM exons 2 and 27  and colorectal carcinoma . This imThe translational implications of our findings extend beyond the therapeutic area. Based on its specific up-regulation in OC vessels (and likely in other cancer types as well), we speculate that L1-ΔTM could serve as a new circulating biomarker of cancer-associated vasculature. In this case, it may become necessary to discriminate between vessel-derived L1-ΔTM and the ectodomain of transmembrane L1CAM which is released by various cell types, including tumor cells, upon proteolytic cleavage . Assays In summary, we provide evidence that L1-ΔTM is a novel, soluble isoform of L1CAM generated in ECs through NOVA2-mediated AS. L1-ΔTM exhibits proangiogenic activity and is up-regulated in tumor vessels, which may have high translational and clinical relevance.Human cervix carcinoma HeLa cells  were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 2 mM L-glutamine (Lonza). Human embryonic kidney (HEK) 293 cells  were grown in FreeStyle 293 Expression Medium (Gibco). N-acetylglucosaminyltransferase I-deficient HEK293 cells [GnTI(-)]  were groVascular endothelial (VE) cadherin-positive ECs, here referred to as moEC, have been described in Mouse EC lines derived from whole embryo (EmbEC), lu2EC and moEC were cultured in DMEM-High Glucose (Lonza) with 10% FBS, 2 mM L-glutamine, 100 U/L penicillin/streptomycin (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 100 μg/ml heparin  and 50 μg/ml EC growth supplement . LuEC were cultured in MCDB131 medium (Sigma-Aldrich) supplemented with 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate (Gibco), 100 μg/ml heparin and 50 μg/ml ECGS as previously described . To enhaHuman ovarian cancer-derived EC (HOC-EC) have been described previously. HOC-EC cultures were found to contain at least 99% endothelial marker-positive  cells, and <1% alpha-smooth muscle actin-positive cells .Human immortalized cerebral microvascular ECs (hCMEC/D3) were provided by PO Couraud and cultured as described previously . hCMEC/DMouse melanoma B16F10 cells (hereafter referred to as B16) were purchased from ATCC and cultured in DMEM, 10% FBS.All cells were routinely tested for mycoplasma with a PCR-based method. Whenever applicable, cell line authentication was performed with the GenePrint STR System typing kit (Promega).L1cam  and its TM—deleted L1cam isoform  (p-L1 WT minigene (L1cam cassette was amplified with primers p-L1_F/R and then cloned into the EcoRI and XbaI restriction sites of pcDNA3.1(+) (Invitrogen). In p-L1 Mut minigenes (Mut3 and Mut5), the NOVA2-binding sites TCAT were replaced with ACAT by PCR-mediated mutagenesis of p-L1 WT minigene. HA-tagged human NOVA2 cDNA was PCR amplified with primers pcDNA-NOVA2_F/R and cloned into the BamH1 and XhoI restriction sites of the pcDNA3.1(+), whereas T7-tagged hnRNP A1 expression vector was generated as described previously   were gen and 27)  and thenminigene , the geneviously . The expL1-ΔTM cDNA was amplified with pUPE-L1_F/R primers from pcDNA Hygro-L1-ΔTM. The resulting amplicon, bearing in-frame 5'-BamHI and 3'-NotI restriction sites was then sub-cloned into pUPE.06.45 mammalian expression vector , bearing a cystatin signal peptide followed by N-terminal 6xHis, 3xStrepII tags, and a specific cleavage site for Tobacco Etch Virus protease (TEV) preceding the 5'-BamHI restriction site. All PCR products were verified by sequencing, whereas all primers are listed in To produce recombinant L1-ΔTM protein (see below), the HeLa cells were transiently transfected with Lipofectamine 3000 (Invitrogen), according to the manufacturer’s protocol. luEC were stably transfected with pcDNA-Hygro/L1-FL or pcDNA-Hygro/L1-ΔTM or the empty vector pcDNA-Hygro(-) (see below), followed by the selection of positive clones as previously described . HEK293 L1cam cDNAs were cloned in the pLenti-III-HA vector . Lentiviruses were produced using HEK293T as recipient cells . Cells were co-transfected with 10 μg of each packaging vector pMD2.G , pRSV-Rev , pMDLg/pRRE  and 10 μg of L1CAM-expressing lentiviral vectors by calcium phosphate precipitation method. After 24 hr, the medium containing the lentiviruses was filtered, supplemented with 8 μg/ml of polybrene (Sigma-Aldrich) and used to infect moEC.To obtain viruses expressing L1CAM isoforms, mouse NOVA2 cDNA  or shRNA for the mouse Nova2 gene (GIPZ shRNAs from Open Biosystems), respectively. After 48 hr of infection, the medium was refreshed and puromycin selection (3 μg/ml) was started. Since NOVA2 expression is regulated by EC density (L1CAM splicing NOVA2-knockdown moEC were used as confluent monolayers (500000 cells in 35 mm Petri dishes), whereas moEC overexpressing HA-tagged NOVA2 were tested at low density (500000 cells in 100 mm Petri dishes). hCMEC/D3 cells were transduced with lentiviral vectors carrying human HA-tagged NOVA2 cDNA  and after 48 hr, infected cells were selected with 3 μg/ml puromycin for 5 days.MoEC were transduced with HA-NOVA2 or with shRNA vectors as described in , using l density , for theNova2 in lu2EC, we used siRNA from Sigma-Aldrich (MISSION siRNA ID SASI_Mm01_00094763) and the corresponding negative control . Transfection was performed with Lipofectamine RNAiMax (Invitrogen) in accordance with the manufacturer’s instructions. Two subsequent transfections  were performed with 13 nM siRNA, and cells were collected 24 hr after the second transfection. To knock down NOVA2 in HUVEC/TERT, we used siRNA from Sigma-Aldrich (MISSION siRNA ID SASI_Hs01_00220812) and the corresponding negative control . Transfection was performed with Lipofectamine RNAiMax (Invitrogen) following the manufacturer’s instructions. Two subsequent transfections  were performed with 30 nM siRNA, and cells were collected 48 hr after the second transfection.To ablate CCTGTACATTTTCTAGGTTACCTGA-3’; GENE-TOOLS) at 15 μM plus 8 μM Endo-Porter PEG system (GENE-TOOLS) according to the manufacturer’s instructions. An irrelevant morpholino oligonucleotide  was used as control. After 16 hr, lu2EC were washed three times with PBS and starved for 24 hr in the following medium: DMEM High Glucose, 2 mM L-glutamine, 100 U/l penicillin streptomycin, 1 mM sodium pyruvate, 25 mM HEPES, 0.5% FBS. Conditioned media (CM) were collected and centrifuged for 10 min to remove cell debris.Subconfluent (80–85%) lu2EC cells were transfected with the MO-L1-SB oligonucleotide . Membrane permeabilization was obtained incubating coverslips in ice-cold PBS and 0.5% Triton X-100 for 3 min at 4°C. Cells were then incubated for 1 hr at RT in a humid chamber with blocking solution . Samples were then incubated for 2 hr with primary antibodies  diluted in blocking buffer, followed by the incubation with Cy3 donkey anti-Rabbit  secondary antibodies (45 min at RT). Finally, samples were washed in PBS and nuclei counterstained with DAPI solution . Confocal microscopy was performed with a Leica SP2 confocal microscope equipped with a motorized stage and violet (405 nm laser diode), blue (488 nm Argon), yellow (561 nm laser diode), and red (633 nm HeNe laser) excitation laser lines. For quantification purposes, at least five fields for each condition were counted and the average of the positive/negative cells was calculated. The statistical difference among groups was determined as described in the Statistical analysis section.2) using a proprietary tool of the Aperio ImageScope software (Leica Biosystems Imaging).Fresh tissue samples were obtained upon informed consent from patients undergoing surgery at the Gynecology Division of the European Institute of Oncology (Milan). Sample collection was performed under the protocol n. R789-IEO approved by the Ethics Committee of the European Institute of Oncology. The immunohistochemical analysis of L1CAM expression was carried out on a panel of high-grade serous ovarian carcinoma. Fresh samples were 4% PFA-fixed and paraffin embedded. After an overnight at 37°C, tumor sections (3 µm) were deparaffinized using Leica ST5020 Multistainer. Tissue sections were treated with the antigen unmasking solution EDTA pH 8 in pre-warmed water bath at 95°C for 50 min and then endogenous peroxidases were blocked using 3% Hydrogen peroxide solution (Carlo Erba). Tissue sections were incubated in blocking solution  for 1 hr. Samples were then incubated for 2 hr with primary antibodies  followed by secondary antibodies incubation (Dako EnVision + System HRP Labelled Polymer or Goat-on-Rodent HRP Polymer from Biocare) for 30 min at RT. Dako chromogen substrate (Liquid DAB +Substrate Chromogen System Ref. K3468) or Vina Green Chromogen Kit  were used for signal detection. Samples were counterstained using Hematoxylin solution (Leica). Double immunohistochemistry for NOVA2 and L1CAM (using the same primary antibodies as above) was performed as previously described . PictureL1cam transcripts with the exon included and the total intensity of the L1cam bands. Splicing of L1CAM in human normal tissues , normal human ovary and breast samples (Ambion) and human EC lines (HUVEC and hCMEC/D3) was analyzed with primers hL1E23_F and hL1E28_R , freshly purified ECs from mouse lung and EC lines was analyzed with primers mL1E23_F and mL1E28_R , while tissue RNA was extracted with AllPrep DNA/RNA FFPE (QIAGEN) according to manufacturer’s instructions. cDNA was obtained starting from 500 to 1000 ng of total RNA with Superscript IV RT cDNA synthesis kit (Invitrogen) according to the manufacturer’s instructions and an aliquot (1–2 μl) of cDNA was then PCR-amplified . The percentage of exon inclusion was calculated as the ratio between the intensity of the band of hL1E28_R . RT-PCR hL1E28_R  after semL1E28_R . Mouse t2 in a Stratlinker 2400 at 254 nm. Cell suspension was centrifuged at 4000 rpm for 3 min, and pellet was incubated for 10 min on ice in lysis buffer . Samples were briefly sonicated and incubated with 10 μl of 1:1000 RNase I  dilution and 2 μl of DNase  for 3 min at 37°C shaking at 1100 rpm, and then centrifuged at 15,000 g for 10 min at 4°C. One milligram of extract was immunoprecipitated using anti-NOVA2 antibody  or purified IgG (negative control) in the presence of protein A/G magnetic Dynabeads (Life Technologies). Immunoprecipitates were incubated overnight at 4°C under constant rotation. After stringent washes with high salt buffer , beads were equilibrated with PK buffer . An aliquot (10%) was kept as input lysate, while the rest was treated with 50 µg Proteinase K and incubated for 20 min at 37°C shaking at 1100 rpm. 7 M urea was added to the PK buffer and incubated for further 20 min at 37°C shaking at 1100 rpm. The solution was collected and phenol/CHCl3 (Ambion) was added. After incubation for 5 min at 30°C shaking at 1100 rpm, phases were separated by centrifuging for 5 min at 13000 rpm at RT. The aqueous layer was transferred into a new tube and precipitated by addition of 0.5 μl glycoblue (Ambion), 3 M sodium acetate pH 5.5 and 100% ethanol. After mixing, the solution containing retained RNA was precipitated overnight at −20°C. RNA extracted from both the input material and the immunoprecipitates was then analyzed by RT-qPCR as described in the above paragraph. The binding was expressed as percentage of the input material.CLIP assay was performed as previously described . moEC wep-L1 WT minigene and either 500 ng of protein expression vectors  or the empty vector. Five-hundred ng of each mutated minigene (Mut3 and Mut5) were transfected with 250 ng of HA-NOVA2 expression vector plus 250 ng of the empty vector; as a control, we used 500 ng of the empty vector. Total RNAs were extracted from HeLa cells after 24 hr and analyzed by RT-PCR with primers p-L1_F and BGH_R annealing to mouse L1cam exon 25 and to the Bovine Growth Hormone (BGH) polyadenylation site, respectively. Primers are listed in HeLa cells were transiently co-transfected by using Lipofectamine 3000 with 500 ng of For the production of secreted, recombinant L1-ΔTM , FreeStyConfluent mouse ECs were washed three times with PBS 1x and cultured for 48 hr in the following medium: DMEM High Glucose, 2 mM L-glutamine, 100 U/l penicillin streptomycin, 1 mM sodium pyruvate, 25 mM HEPES, 0.5% FBS. Conditioned media (CM) were collected and centrifuged for 10 min to remove cell debris.A Matrigel-based tubulogenesis assay was performed to assess the ability of ECs to form an organized capillary-like network. Before proceeding with the assay, 96-well plate was coated with 50 μl/well of Growth Factor-Reduced Matrigel (BD Biosciences) and left for 1 hr at 37°C for gelification. To assess the cell-autonomous effect of L1-ΔTM, transduced moEC were plated on Matrigel-coated plates in complete growth medium. Tube-like structures were manually counted under the microscope after 24 hr.To assess the paracrine effect of L1-ΔTM, confluent moEC were cultured overnight in the same medium used for CM production. The day after, moEC were seeded on polymerized Matrigel-coated wells in the appropriate CM or, where indicated, were treated with different concentrations of recombinant, purified L1-ΔTM. After 8 hr of incubation at 37°C, tubes-like structures were counted under the microscope.To block L1-ΔTM activity, transduced moEC were pre-incubated for 1 hr at 37 ˚C with 10 µg/ml of an anti-L1CAM blocking antibody [clone 324 ] or withAll animal studies were performed following a protocol approved by the fully authorized animal facility of European Institute of Oncology and by the Italian Ministry of Health  (IACUC n. 1256/2015) and in accordance to EU directive 2010/63. The sample size estimation was based on previous studies and pilot experiments. C57Bl/6 mice were injected into the right flank with 200 μl of CM derived from mouse ECs expressing either L1-ΔTM or L1-FL or from control ECs in a final volume of 600 μl of Growth Factor-Reduced Matrigel (BD Biosciences). Matrigel containing 0.5 μg of FGF2 was used as positive control. Groups were composed by three mice for each construct. Plugs were removed 7 days after injection, fixed in 4% PFA and paraffin embedded. Sections from fixed plugs were stained for CD31 as described above. The number of CD31-positive vessels that invaded Matrigel plugs were evaluated by manual counting of five different fields per section using Axioskop two microscope (Leica Biosystems).http://xena.ucsc.edu/). Survival plots were drawn using the Kaplan-Meier method and patients were stratified according to NOVA2 expression using median as threshold value. The log-rank Mantel-Cox test was employed to determine any statistical difference between the survival curves of the cohorts.To determine the prognostic relevance of NOVA2, overall survival curves of ovarian cancer patients were built analyzing the GDC TCGA dataset with the UCSC Xena web tool (vast-tools) (https://github.com/vastgroup/vast-tools). Then both replicates of each experimental point were pooled with vast-tools merge using the default parameters. This increased markedly the read coverage at each of the exon-exon junctions per experimental condition. Then, we applied vast-tools compare to perform a differential splicing analysis between the pooled control and the pooled knockdown samples, using a cutoff of delta percent spliced in (ΔPSI) of 15 with default settings.We previously performed RNA sequencing on NOVA2-knockdown moEC versus their parental control . To captt-tools)  . The correlation between the expression of L1-ΔTM and tumor vessel density was assessed using the Spearman rank correlation coefficient.Differences were considered significant at p<0.05. Asterisks correspond to p-value calculated by two-tailed, unpaired, t-test . The sample size estimation was based on previous studies and pilot experiments. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Patrick A Murphy (Reviewer #1).Thank you for submitting your work entitled "A Novel L1 Isoform with Angiogenic Activity Generated by NOVA2-mediated Alternative Splicing" for consideration by eLife.Based on discussions between the reviewers and editors and the individual reviews below, we regret that we are rejecting your paper for publication in All the reviewers and editors agreed that the basic results characterizing Nova2 regulation of L1 splicing and its possible role in tumor angiogenesis were quite interesting. However, there was general agreement that several key issues regarding angiogenic effects of the splice variant were not well supported. In particular, it is important to distinguish effects of the soluble splice variant from those of the extracellular domain that is cleaved from the typical isoform. Similarly, it is not clear whether the increased L1 and Nova2 expression observed in tumors was simply due to their increased vascularization and not causal as is proposed. To resolve this, the expression of the L1 variant and Nova2 need better quantification in normal and tumor endothelium. These issues are fundamental to the model that the L1 splice variant is driving tumor angiogenesis. The reviewers raise other valid points below.Reviewer #1:This is an interesting follow-up on previous work by this group, which had revealed an important function for the Nova2 splice factor in vascular development. Here, they reveal a connection between Nova2 and a target splice event in L1cam, resulting in a form of the protein with altered cellular localization and function. Furthermore, Nova2, which like L1cam is fairly endothelial specific in ovarian tumors, correlates with tumor progression. Overall, the work is well done and exciting, but I think there are a few points which could be addressed in greater detail.How much of the angiogenic properties of L1cam are due to altered splicing is a critical question. Rather than comparing angiogenic properties of supernatants of cells with exon25 deficient version of L1cam (L1-ΔTM) to vector control, I would have liked to see a comparison between cells expressing exon25 exclusive and inclusive versions of L1cam (L1- ΔTM and L1-FL). This would help address how much of the angiogenic effects observed are due to the change in splicing, rather than L1cam expression alone. It would be best to do these experiments in the matrigel plug assay, which more closely indicates angiogenic function than the tubulogenesis assay .A link between FGF and Exon25- negative L1cam is suggested but not confirmed by the experiments. Is there a change in FGF signaling that can be defined by immunofluorescence or Western blot? For example, could a difference in FGF receptor expression or phosphorylation be detected following treatment with soluble L1cam? One interpretation is that the FGF and L1cam signaling pathways are parallel inputs in the tube formation assay where both are required for tubulogenesis. Given these concerns and the use of the in vitro tube formation assay alone as an indication of their cross-talk, the claim that "L1-ΔTM promotes angiogenesis via FGFR1" seems overstated.Authors allude to data on increased secretion of L1cam in endothelial cells , but I do not see a data reference for this observation. Can they measure soluble L1cam with the C-terminus in the supernatant of their endothelial cell lines and some other L1 expressing cells which do not make the L1-ΔTM isoform? Or can they measure this in their endothelial cells with and without Nova2 suppression? This would help alleviate concerns about any artifacts due to overexpression of L1cam in moECs.If L1-ΔTM is specific to tumor endothelium, it is not surprising that tumor levels are correlated with vascular density. It would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization. There is some heterogeneity in the L1-ΔTM expression levels in isolated tumor endothelial cells , do those with lower L1-ΔTM come from less vascular tumors? Do they have lower NOVA2 expression? Perhaps that is what the authors looked at already, but this was not clear to me.Reviewer #2:L1 (L1CAM gene) is a cell-surface glycoprotein that undergoes ectodomain shedding via proteolytic cleavage at a membrane proximal site. This soluble ectodomain has previously been implicated in a wide array of cell signaling and adhesion processes involved in tumorigenesis.In this manuscript, Angiolini et al. identify a novel isoform of L1CAM with exon 25 skipped, termed L1-ΔTM. Because exon 25 contains the entire transmembrane domain of the protein, skipping of this exon results in a L1 protein isoform that no longer localizes on the cell surface and instead is found both in the cytoplasm and secreted into the extracellular space. The authors investigate whether this secreted full-length protein isoform of L1 (which does not undergo proteolytic cleavage and maintains the cytoplasmic tail) drives a pro-angiogenic program that supports aggressive tumorigenesis. They identify the well-characterized splicing factor, Nova2, as an important factor in promoting skipping of exon 25.This manuscript presents a novel alternative (non-proteolytic) mechanism for the release of the L1 ectodomain into the extracellular space and characterizes the role of Nova2 in producing this isoform. The observation that this novel splice variant occurs in endothelial cells and its regulation by Nova2, as well as the exogenous expression of the full-length and L1-ΔTM isoforms to produce glycosylated transmembrane and soluble proteins, respectively, are all compelling pieces of data. However, the major flaw in the manuscript is the lack of physiological evidence that the angiogenic effects observed upon overexpression of L1-ΔTM are specific to the isoform itself, or whether a similar pro-angiogenic affect would also result from overexpressing full length L1. Indeed, overexpression of membrane bound L1 (FL-L1) resulted in release of L1 into the extracellular space , presumably through proteolytic cleavage which would presumably have the same effect.The difficulty in separating the effect of the cleaved full-length protein compared to the exon 25 skipped splice variant is acknowledged. The authors could address this issue by generating a mutation in the proteolytic cleavage site that would render it inactive and determine whether the splicing variant can overcome this deficiency.A separate concern related to this criticism is that the manuscript relies almost entirely on a gain-of-function system. It is unclear how the level of overexpression of the L1-ΔTM isoform relates to endogenous expression either in cell lines or tumors, and without the L1-FL controls, one cannot assess the relative potency of this isoform being expressed.Reviewer #3:The manuscript by Angiolini et al. describes a novel alternatively spliced isoform of the cell-surface adhesion molecule L1CAM in endothelial cells (ECs). This novel isoform lacking exon 25, lacks the transmembrane domain, creating a soluble form of L1CAM. The authors show that this soluble L1-ΔTM isoform enhances the formation of tubes in mouse endothelial cells and this is inhibited by an inhibitor of FGFR1 signaling. They show direct binding of NOVA2 to exon 25 of the L1CAM transcript and in a splicing assay, show NOVA2 to be responsible for promoting skipping of L1CAM exon 25. Finally, they show a correlation between higher vessel density in L1-ΔTM-positive ovarian tumors, implying that alternative splicing of L1CAM correlates with tumor vascularization, supporting a proangiogenic function for L1-ΔTM.In general this manuscript is well written and easy to follow. The experimental data is clearly presented. The novelty of this report is the identification of a cell adhesion molecule converted into a soluble angiogenic factor through alternative splicing. This represents not only a novel target for future antiangiogenic strategies but also can be used for diagnostic and prognostic purposes. However, there are some things lacking in the paper that need to be addressed.1) Even though NOVA2 is an important splicing factor in EC cells, there is a bit of a logic "jump" caused by focusing only on NOVA2 as the regulator of L1CAM. The authors need to show other possible splicing factors from the SFmap/SpliceAid analysis, and possibly knockdown several of them  in their minigene splicing assay or RT-PCR on the endogenous transcripts.2) Figure 5 The authors need to normalize the amount of L1CAM and NOVA2 to the amount of blood vessels in the IHC. If any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor. The relevant question is if the blood vessels in the tumor express higher L1CAM and NOVA2 than normal? Also, it seems that additional cells are stained with NOVA2  which do not express L1CAM.3) In order to claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform, the authors need to show that in the presence of the cancer cells the EC cells express higher levels of NOVA2. Maybe this is through secretion of factors from the cancer cells or direct interaction, but the claim is not proven until some causative experiment is performed.4) Figure 4—figure supplement 3B and C should show endogenous levels of Nova2 in the transfected cells. The authors write in Materials and methods "Since NOVA2 expression is regulated by EC density , for the analysis of L1 splicing, NOVA2-knockdown moEC were used as confluent monolayers , whereas moEC overexpression HA-tagged NOVA2 were tested at low density ." If the expression is sensitive to cell density it should be shown for the specific experiments.5) Figure 4—figure supplement 3F. The effect of Nova2 overexpression on L1CAM splicing in human cells (82% inclusion) is not as strong as in mouse cells Figure 4B (27.2% inclusion). Can the authors provide an explanation for that?[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Douglas Black as Reviewing Editor and Jonathan Cooper as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Patrick A Murphy (Reviewer #1).Thank you for submitting your article "A Novel L1 Isoform with Angiogenic Activity Generated by NOVA2-mediated Alternative Splicing" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife.This manuscript is a resubmission of a previously rejected study. The reviewers agreed that the previous version was of significant interest but all had major criticisms and experiments that they felt were needed to support its conclusions. All of the reviewers now agree that the authors have made major changes that strengthen the paper. Assuming that the authors can adequately address the remaining points in these reviews, the editors are pleased to accept this paper for publication in Reviewer #1:The addition of new data has solidified key conclusions and addressed my original concerns. With these new pieces of data, the authors make a compelling argument that a reduction in the inclusion of exon 25, which is mediated by Nova2 in the endothelium, promotes the expression of a secreted L1 variant with angiogenic properties beyond the constitutive form of L1. I particularly like the addition of Supplementary Figure 2M and N, showing that a morpholino suppressing L1 exon 25 inclusion results in increased secretion of soluble L1. I think this should be considered for the main figures.Reviewer #2:This manuscript is a resubmission of a previously reviewed work that all reviewers agreed presents an important and novel biological mechanism in which alternative splicing essentially replaces proteolytic cleavage to release a polypeptide that is otherwise integrally associated with the plasma membrane. The authors have addressed most of the initial reviewers' comments and the paper is clearly much stronger now from the addition of new data and textual clarifications.I had originally suggested a making a mutation in the proteolytic site – Figure 2A showing the putative ADAM cleavage site as a box implies some knowledge of where cleavage occurs . If it is not clear where the cleavage occurs (and which enzymes are responsible), this figure is a bit misleading. The difficulties the authors cite regarding performing the suggested experiments are reasonable, but perhaps the figure could be clarified.The immunofluorescence data added to Figure 2C is much improved with regard to clearly showing the subcellular localization of each isoform. The data that has been added to Figure 3  are instrumental in addressing both my concerns and those of reviewer #1, this experiment adds a great deal of support to the central hypothesis of this paper.The authors have taken the criticism that the previous manuscript was overly dependent on gain-of-function experiments constructively in adding the new data presented in Figure 2—figure supplement 1. These experiments provide an elegant alternative approach to overexpression of individual isoforms by perturbing the splicing of the endogenous gene. This is a well-conceived and executed experiment.Figure 5B solidifies the assertion that NOVA2 and L1 are colocalized. This is a strong piece of evidence for the physiological relevance of the proposed mechanism within tumors.In summary, with this resubmission the authors have more than satisfactorily addressed the major points of concern raised in the original manuscript.Reviewer #3:The manuscript "A novel L1 isoform with angiogenic activity generated by Nova-2 mediated alternative splicing" has been revised. The authors have supplied additional data in several figures, new supplementary figures and table and two figures in their response to the reviewers. In addition, they have addressed the concerns of the reviewers in their letter.They have analyzed a cohort of 13 ovarian cancer samples for vessel density and expression levels of L1-ΔTM. They show that the levels of mRNA correlate with the degree of tumor vascularization . They have also redone the immunohistochemistry of NOVA2 in EC cells  showing that the immunoreactivity of NOVA2 is restricted to the nuclei of vascular ECs.The authors did not completely address point 3. Thus, the connection between L1-ΔTM and vascularization is left at the level of correlation rather than a causative effect. The authors should at least say that further experiments are required to determine if this a direct effect of L1-ΔTM. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:This is an interesting follow-up on previous work by this group, which had revealed an important function for the Nova2 splice factor in vascular development. Here, they reveal a connection between Nova2 and a target splice event in L1cam, resulting in a form of the protein with altered cellular localization and function. Furthermore, Nova2, which like L1cam is fairly endothelial specific in ovarian tumors, correlates with tumor progression. Overall, the work is well done and exciting, but I think there are a few points which could be addressed in greater detail.How much of the angiogenic properties of L1cam are due to altered splicing is a critical question. Rather than comparing angiogenic properties of supernatants of cells with exon25 deficient version of L1cam (L1-ΔTM) to vector control, I would have liked to see a comparison between cells expressing exon25 exclusive and inclusive versions of L1cam (L1-ΔTM and L1-FL). This would help address how much of the angiogenic effects observed are due to the change in splicing, rather than L1cam expression alone. It would be best to do these experiments in the matrigel plug assay, which more closely indicates angiogenic function than the tubulogenesis assay .We have performed the matrigel plug assay as recommended by the reviewer. As shown in Figure 3F, and described in the last paragraph of the subsection “L1-ΔTM regulates endothelial cell function”, the conditioned medium (CM) from L1-ΔTM- expressing ECs was significantly more angiogenic than the CM from L1-FL-expressing cells. Incidentally, we obtained similar results also in the tubulogenesis assay see . Based oThese additional experiments indicated that L1-ΔTM per seis endowed with angiogenic properties, while the mere expression of L1cam has little effect, further supporting the biological relevance of the splicing of exon 25 in endothelial cells.A link between FGF and Exon25- negative L1cam is suggested but not confirmed by the experiments. Is there a change in FGF signaling that can be defined by immunofluorescence or Western blot? For example, could a difference in FGF receptor expression or phosphorylation be detected following treatment with soluble L1cam? One interpretation is that the FGF and L1cam signaling pathways are parallel inputs in the tube formation assay where both are required for tubulogenesis. Given these concerns and the use of the in vitro tube formation assay alone as an indication of their cross-talk, the claim that "L1-ΔTM promotes angiogenesis via FGFR1" seems overstated.As suggested by the reviewer, we investigated whether soluble L1-ΔTM affects FGFR phosphorylation. Treating parental moEC with recombinant L1-ΔTM resulted in increased phospho-FGFR1, consistent with the L1-ΔTM-induced activation of FGFR1. These observations have been included in the Results .The reviewer is correct in pointing out that FGFR1 and L1CAM might activate parallel pathways that are both involved in tube formation. However, the observation that the FGFR inhibitor PD173074 reduces the tubulogenesis of L1-ΔTM-expressing moEC to the level of control cells (but has no effect on the L1-ΔTM-independent tube formation in control moEC), strongly suggests that in the context of tubulogenesis FGFR1 signaling acts as an effector of L1-ΔTM. Nevertheless, we agree with the reviewer’s concern about the overstatement and have modified the sentence accordingly .Authors allude to data on increased secretion of L1cam in endothelial cells , but I do not see a data reference for this observation. Can they measure soluble L1cam with the C-terminus in the supernatant of their endothelial cell lines and some other L1 expressing cells which do not make the L1-ΔTM isoform? Or can they measure this in their endothelial cells with and without Nova2 suppression? This would help alleviate concerns about any artifacts due to overexpression of L1cam in moECs.With regard to the concern about artifacts, we would argue that the data shown in Figure 2—figure supplement 1 reduce significantly the risk of artifacts due to the overexpression of L1CAM. The figure, indeed, shows in both moEC and luEC that the ectopic expression of L1-FL per se  does not result in the secretion of significant amounts of L1 with the C-terminus. The latter is detected occurs only in the CM of L1-ΔTM- expressing cells , supporting the specificity of our results.Nevertheless, the reviewer’s suggestion to compare endothelial cells with another L1- expressing cell type was very insightful. We compared mouse lung-derived EC, which express endogenous L1-ΔTM  and the mouse melanoma cell line B16, known to express L1 . We first verified by RT-PCR that B16 cells do not express the L1-ΔTM isoform , which is consistent with the lack of Nova2 expression , and then assayed for the secretion of C- terminus-containing L1 in the conditioned medium. Despite B16 cells express much higher levels of endogenous, cell-associated L1 than lu2EC , no protein was detectable in their conditioned medium with the antibody against the cytoplasmic tail, while lu2EC secreted high amounts of C-terminus-containing L1 . Together with the other data presented in the manuscript, these findings (described in the fourth paragraph of the subsection “Alternative splicing generates a novel soluble form of L1”) strongly support the increased secretion of L1- ΔTM in endothelial cells.If L1-ΔTM is specific to tumor endothelium, it is not surprising that tumor levels are correlated with vascular density. It would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization. There is some heterogeneity in the L1-ΔTM expression levels in isolated tumor endothelial cells , do those with lower L1-ΔTM come from less vascular tumors? Do they have lower NOVA2 expression? Perhaps that is what the authors looked at already, but this was not clear to me.r=0.84, p<0.001).We agree with the reviewer that “it would be more meaningful if the endothelial levels of L1-ΔTM expression were correlated with tumor vascularization”. Unfortunately, we could not assess the vascularization of the same tumors used to derive endothelial cells, as proposed by the reviewer, since the entire surgical specimens were processed to obtain the ECs themselves. Nevertheless, since our data strongly suggest that tissue expression of L1-ΔTM is largely confined to vascular endothelium , we performed RT-PCR for L1-ΔTM on a cohort of 17 archival samples of ovarian cancer. The same specimens were also stained for CD31 to measure vessel density. As shown in Figure 5D, the levels of L1-ΔTM mRNA correlated with the degree of tumor vascularization  mechanism for the release of the L1 ectodomain into the extracellular space and characterizes the role of Nova2 in producing this isoform. The observation that this novel splice variant occurs in endothelial cells and its regulation by Nova2, as well as the exogenous expression of the full-length and L1-ΔTM isoforms to produce glycosylated transmembrane and soluble proteins, respectively, are all compelling pieces of data. However, the major flaw in the manuscript is the lack of physiological evidence that the angiogenic effects observed upon overexpression of L1-ΔTM are specific to the isoform itself, or whether a similar pro-angiogenic affect would also result from overexpressing full length L1. Indeed, overexpression of membrane bound L1 (FL-L1) resulted in release of L1 into the extracellular space , presumably through proteolytic cleavage which would presumably have the same effect.We thank reviewer #2 for her/his positive comments and for the constructive criticisms that allowed us to improve our manuscript.With regard to the L1-ΔTM-specific effect, as specified also in our reply to reviewer #1, we have performed both in vivo matrigel plug and in vitro tube formation assays to compare cells expressing L1-ΔTM vs. full-length L1. As shown in Figure 3F and described in the last paragraph of the subsection “L1-ΔTM regulates endothelial cell function”, the conditioned medium (CM) from L1-ΔTM-expressing ECs was significantly more angiogenic than the CM from L1-FL-expressing cells. Similar results were obtained in the tubulogenesis assay see . As recoThe difficulty in separating the effect of the cleaved full-length protein compared to the exon 25 skipped splice variant is acknowledged. The authors could address this issue by generating a mutation in the proteolytic cleavage site that would render it inactive and determine whether the splicing variant can overcome this deficiency.We agree with the reviewer that this would be a very elegant and appropriate strategy. However, it would be quite a challenging task, given that the proteolytic cleavage of L1 can be carried out by a plethora of proteases . Moreover, L1 shedding has been investigated mostly in neurons, and the enzymes involved in the cleavage of L1 in endothelial cells remain elusive. Therefore, it would be quite difficult and time-consuming to identify such enzymes and perform the mutagenesis suggested by reviewer #2, and we feel that such an effort would fall out of the scope of our manuscript.A separate concern related to this criticism is that the manuscript relies almost entirely on a gain-of-function system. It is unclear how the level of overexpression of the L1-ΔTM isoform relates to endogenous expression either in cell lines or tumors, and without the L1-FL controls, one cannot assess the relative potency of this isoform being expressed.We thank the reviewer for asking these important controls, which lent further support to our conclusions. As described above in our response to reviewer #1, we have included the requested L1-FL controls. The results showed that L1-ΔTM-containing conditioned medium (CM) has a markedly higher angiogenic potential than L1-FL CM , were treated with a morpholino oligonucleotide that selectively interfered with the inclusion of exon 25 . As shown in Figure 3F (left), this resulted in increased skipping of exon 25 and release of endogenous L1-ΔTM in the extracellular compartment. Importantly, ECs exposed to the CM from morpholino-treated cells exhibited higher tube-forming activity than those exposed to control CM . These observations validated in an endogenous system our observations on the angiogenic activity of AS-generated L1-ΔTM.Reviewer #3:[…] 1) Even though NOVA2 is an important splicing factor in EC cells, there is a bit of a logic "jump" caused by focusing only on NOVA2 as the regulator of L1CAM. The authors need to show other possible splicing factors from the SFmap/SpliceAid analysis, and possibly knockdown several of them  in their minigene splicing assay or RT-PCR on the endogenous transcripts.L1cam exon 25 splicing . By using the minigene- based splicing assay as suggested by the reviewer, we found that the skipping of L1cam exon 25 occurred only upon overexpression of NOVA2, but not with hnRNP A1 or SRSF3 .As outlined in more details in the Results , the SFmap analysis identified NOVA2, hnRNP A1 and SRSF3 as possible candidate regulators of L1cam exon 25 , which revealed L1cam exon 25 as a novel target of NOVA2 in ECs .The rationale for investigating NOVA2-mediated AS of 2) Figure 5 The authors need to normalize the amount of L1CAM and NOVA2 to the amount of blood vessels in the IHC. If any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor. The relevant question is if the blood vessels in the tumor express higher L1CAM and NOVA2 than normal? Also, it seems that additional cells are stained with NOVA2  which do not express L1CAM.Figure 5A (left panel) shows indeed the percentage of NOVA2-positive vessels over the total number of vessels (determined by CD31 staining). We have now clarified this in the legend to Figure 5A as well as on the Y-axis of the graph. In addition, we now provide analogous measurements also for L1-positive vessels, confirming that they are markedly increased in cancer samples .r=0.84, p<0.001).Reviewer #3 argues that “if any blood vessel expresses L1CAM and NOVA2 then it is only a measure of higher vascularity in the tumor”. This concern was somehow similar to the last major comment of reviewer #1. To investigate in more detail the possible correlation between the expression of L1-ΔTM and tumor angiogenesis, we have analysed a cohort of 13 ovarian cancer samples both for vessel density and for the expression levels of L1-ΔTM . As shown in Figure 5D, the levels of L1-ΔTM mRNA correlated with the degree of tumor vascularization  and are able to show that the immunoreactivity of NOVA2 is restricted to the nuclei of vascular ECs . Finally, the vessel-specific immunoreactivity of NOVA2 is also shown in the images taken from the Human Protein Atlas website .3) In order to claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform, the authors need to show that in the presence of the cancer cells the EC cells express higher levels of NOVA2. Maybe this is through secretion of factors from the cancer cells or direct interaction, but the claim is not proven until some causative experiment is performed.We are afraid we did not understand completely the reviewer’s comment. In particular, we do not see the causal link between the effect of cancer cells on the endothelial expression of NOVA2 and our “claim that the blood vessels in the tumor express more NOVA2 and L1CAM isoform”. Along the same line, it is unclear to us why “the claim is not proven until some causative experiment [on the role of cancer cells] is performed”. The increased expression of NOVA2 and L1CAM in ovarian cancer vasculature is illustrated in Figure 5A, where we show that tumors contain 6 and 4-fold more vessels positive for NOVA2 and L1CAM, respectively, than their normal tissue counterpart. This phenomenon is independent on whether and how cancer cells regulate the expression of the two proteins in the endothelium.Having said that, we agree with the reviewer about the relevance of understanding the mechanisms that regulate the expression of NOVA2 in tumor-associated ECs. Nevertheless, we feel that investigating this issue would fall out of the scope of the present study. The experiments suggested by the reviewer, in addition, would be quite time-consuming in that they should not be limited to the cancer cells themselves, but should rather be extended to other components of the tumor microenvironment. It is conceivable, indeed, that different cell types and/or other microenvironmental factors account for (or at least contribute to) the induction of NOVA2 in the cancer vasculature. Along this line, a very recent paper reported that NOVA2 is upregulated in colorectal carcinoma vasculature and that endothelial NOVA2 expression is induced upon exposure to hypoxia , a condition shared by many solid tumors. This paper is now cited in our Discussion (sixth paragraph).4) Figure 4—figure supplement 3B and C should show endogenous levels of Nova2 in the transfected cells. The authors write in Materials and methods "Since NOVA2 expression is regulated by EC density , for the analysis of L1 splicing, NOVA2-knockdown moEC were used as confluent monolayers , whereas moEC overexpression HA-tagged NOVA2 were tested at low density ." If the expression is sensitive to cell density it should be shown for the specific experiments.Nova2 and L1-ΔTM in moEC cultured as sparse cells or at confluency. Confluent cells exhibit higher Nova2 expression as compared to sparse cells, at both mRNA and protein levels, thus demonstrating that Nova2 expression in moEC is sensitive to cell density, in agreement with previous data . The higher level of Nova2 in confluent moEC is mirrored by an increase in the exclusion of L1cam exon 25 .Figure 4—figure supplement 3A now shows the endogenous levels of 5) Figure 4—figure supplement 3F. The effect of Nova2 overexpression on L1CAM splicing in human cells (82% inclusion) is not as strong as in mouse cells Figure 4B (27.2% inclusion). Can the authors provide an explanation for that?L1CAM splicing. In this specific case, moEC derive from mouse lung, while hCMEC/D3 derive from human brain vessels. The heterogeneity of ECs from different districts, in fact, has been extensively documented and may reflect profoundly different phenotypic and functional properties .While it is possible that the discrepancy involves, at least in part, some species-specific features, also the origin of ECs may play a role in the relative efficiency of NOVA2 in NOVA2 knockdown resulted in a marked decrease in L1CAM splicing (with the inclusion of exon 25 going from 72% to 100%). This supports the hypothesis that the splicing of exon 25 in response to the genetic manipulation of NOVA2 is quantitatively different indifferent EC types. Of note, these data suggest that the activity of NOVA2 is cell context- dependent even within the same cell type .To further address the reviewer’s comment, we have reduced by shRNA the expression of endogenous NOVA2 in a different EC model, consisting of human umbilical vein ECs. As shown in Figure 4—figure supplement 3G, [Editors' note: the author responses to the re-review follow.]Reviewer #1:The addition of new data has solidified key conclusions and addressed my original concerns. With these new pieces of data, the authors make a compelling argument that a reduction in the inclusion of exon 25, which is mediated by Nova2 in the endothelium, promotes the expression of a secreted L1 variant with angiogenic properties beyond the constitutive form of L1. I particularly like the addition of Supplementary Figure 2M and N, showing that a morpholino suppressing L1 exon 25 inclusion results in increased secretion of soluble L1. I think this should be considered for the main figures.We thank reviewer #1 for his appreciation of the work we have done to improve our manuscript. We also thank him for his comments on the data presented in Supplementary Figure 2M and N. Following his suggestion, we have modified Figure 3 to include the morpholino data . In order to avoid overcrowding of the figure, the microscope pictures of the tubes previously shown in Figure 3A have been moved to Figure 3—figure supplement 1A.Reviewer #2:[…] I had originally suggested a making a mutation in the proteolytic site – Figure 2A showing the putative ADAM cleavage site as a box implies some knowledge of where cleavage occurs . If it is not clear where the cleavage occurs (and which enzymes are responsible), this figure is a bit misleading. The difficulties the authors cite in the letter regarding performing the suggested experiments are reasonable, but perhaps the figure could be clarified.We agree with the reviewer that Figure 2A was misleading. Therefore, following the reviewer’s suggestion to clarify the figure and considering that our manuscript does not deal with the proteolytic cleavage of L1CAM, we have removed the box indicating the putative ADAM cleavage site.Reviewer #3:[…] The authors did not completely address point 3. Thus, the connection between L1-ΔTM and vascularization is left at the level of correlation rather than a causative effect. The authors should at least say that further experiments are required to determine if this a direct effect of L1-ΔTM.We appreciate the reviewer’s thorough analysis of the new data that have been added in the revised manuscript. With regard to the last point, we have modified the Discussion to point out that further work should test the causal role of vascular L1-ΔTM in tumor vascularization .


In this paper, a dual-band terahertz absorber with two passbands is proposed. The absorber is composed of periodic patterned graphene arrays on the top of a SiO Metamaterials (MMs) are artificially designed sub-wavelength electromagnetic (EM) structures. MMs have attracted considerable attention over the past decade due to their exotic properties, such as negative refraction, double negative behavior, left-handed behavior and backward wave propagation ,2,3,4,5,Graphene is a one-atom-thick layer of carbon atoms arranged in a honeycomb lattice. One of the most significant properties of graphene is that its conductivity can be tuned by shifting the electronic Fermi level via electrostatic doping ,9. To obIn the microwave band, researchers have proposed periodic structures that can absorb and transmit waves in different frequency bands, which are called frequency selective rasorbers (FSRs). FSRs with lower or higher absorption band have been proposed in ,27,28,29In this paper, a graphene-based dual-band terahertz absorber with two passbands is proposed and investigated. The structure is composed of a SiOThe schematic of the structure is shown in y of 3.9 , a graphubstrate . The FSSubstrate . Four FSThe surface conductivity of graphene can be expressed as :(1)σ=σine is the electron charge, ℏ is the reduced Planck’s constant, T is the temperature. The surface conductivity is dominated by the intraband contribution in the terahertz band. In this paper, we assume the initial values of the relaxation time, chemical potential, and temperature to be The interband and intraband terms of the graphene surface conductivity can be defined as follows:In this study, the numerical simulation results are obtained by the frequency domain finite element method (FEM) solver of the CST Microwave Studio. Periodic boundary conditions are assigned along both the x-direction and y-direction while floquet ports are assigned along the z-direction. Local mesh refinement and adaptive mesh refinement are adopted to enhance the accuracy of the numerical simulation. In the simulation, graphene is modeled as an equivalent 2D surface impedance layer without thickness. To clarify the physical mechanism of our design, the surface current distribution of the FSS layer and the electric field distribution of our absorber are investigated in To better understand the underlying mechanism, the influence of the length of the major axis of the graphene ellipse and the thickness of the substrate are investigated. As shown in The transmission and absorption spectra with different chemical potentials are shown in Furthermore, the performance of our absorber under oblique incidence is investigated in In summary, we have demonstrated a dual-band terahertz absorber based on graphene with two passbands. The absorber is composed of a cross-elliptical graphene layer, a double-loop FSS layer and a SiO


TARDBP gene and the remaining (90–95%) are due to mutations in other genes such as: C9ORF72, SOD1, FUS, and NEK1 etc. Strikingly however, the majority of sporadic ALS patients (up to 97%) also contain the TDP-43 protein deposited in the neuronal inclusions, which suggests of its pivotal role in the ALS pathology. Thus, unraveling the molecular mechanisms of the TDP-43 pathology seems central to the ALS therapeutics, hence, we comprehensively review the current understanding of the TDP-43's pathology in ALS. We discuss the roles of TDP-43's mutations, its cytoplasmic mis-localization and aberrant post-translational modifications in ALS. Also, we evaluate TDP-43's amyloid-like in vitro aggregation, its physiological vs. pathological oligomerization in vivo, liquid-liquid phase separation (LLPS), and potential prion-like propagation propensity of the TDP-43 inclusions. Finally, we describe the various evolving TDP-43-induced toxicity mechanisms, such as the impairment of endocytosis and mitotoxicity etc. and also discuss the emerging strategies toward TDP-43 disaggregation and ALS therapeutics.TAR DNA binding protein 43 (TDP-43) is a versatile RNA/DNA binding protein involved in RNA-related metabolism. Hyper-phosphorylated and ubiquitinated TDP-43 deposits act as inclusion bodies in the brain and spinal cord of patients with the motor neuron diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While the majority of ALS cases (90–95%) are sporadic (sALS), among familial ALS cases 5–10% involve the inheritance of mutations in the  TAR DNA binding protein-43 (TDP-43) was identified in 1995 as a repressor protein associated with HIV-1 transcription, which binds to the trans-active response element DNA sequence of the viral genome and is critical for the regulation of the viral gene expression  and frontotemporal lobar degeneration (FTLD or FTLD-TDP) diseases                Rosen, , FUS  with a nuclear localization signal , two RNA recognition motifs: RRM1 (aa 104–176) and RRM2 (aa 192–262), a nuclear export signal , a C-terminal region (aa 274–414) which encompasses a prion-like glutamine/asparagine-rich (Q/N) domain (aa 345–366) and a glycine-rich region (aa 366–414)   (Cohen e366–414) . TDP-43 366–414) . In ALS,366–414) . Mitocho366–414) . Owing t domains  and its Accumulating evidence suggests that TDP-43 is natively dimeric or at least exists in a monomer-dimer equilibrium under normal physiological conditions  contain highly conserved RNA recognition motifs (RRMs), which are among the most abundant protein domains in the eukaryotes  is highly disordered and comprises of a glycine-rich region and also a segment enriched in uncharged polar amino acids, glutamine and asparagine (Q/N) . This unTDP-43 has versatile functions and it is involved in several steps of RNA metabolism such as: transcription, translation, mRNA transport, mRNA stabilization, microRNA (miRNA) and long non-coding RNA (lncRNA) processing etc. , TARDBP, FUS, SNCA (α-synuclein), HTT (Huntingtin), and APP (Amyloid precursor protein) etc.  granules which transport mRNA to distant locations. In the axonal cells, RNP granules are trafficked with assistance from microtubules  mRNA at the neuromuscular junctions , the membrane-less cytoplasmic foci of sizes ≤5 μm, ensues quickly upon exposure to stresses like: oxidative stress, heat shock, viral infection, and chemical exposure etc.  : AFM provides the surface contour by scanning using a molecular size cantilever and it provides surface topology of amyloid aggregates or fibers. AFM images can provide height features of amyloid fibrils/aggregates.CD (Circular Dichroism Spectroscopy): By measuring the differential absorbance of circularly polarized light, CD is widely used to characterize the protein's secondary structural elements. Amyloid-like aggregates tend to show higher β-sheet structure in comparison with the soluble, monomeric protein molecules and exhibits a negative peak around 215 nm.CR (Congo Red Birefringence): Upon binding with amyloid aggregates, absorbance maximum of CR shifts from 490 to 540 nm. Macroscopic amyloid aggregates bound to CR display apple-green birefringence when observed under cross-polarized light.Cryo-EM (Cryo-Electron Microscopy): An electron microscopic technique used for imaging frozen-hydrated specimens at cryogenic temperatures, where the specimens remain in their native state without the need for dyes or fixatives, allowing structure determination at high resolution. Cryo-EM generated micrographs have been used to distinguish various structural classes of amyloids.DLS (Dynamic Light Scattering): Fluctuation of intensity of scattered light with time due to Brownian motion of particles in solution are analyzed to detect diffusion of the molecules. DLS provides hydrodynamic radii of particles and can be used to evaluate the presence of amyloid aggregates and estimate their sizes.FCS (Fluorescence Correlation Spectroscopy): FCS records fluctuations in fluorescence intensity, providing information, such as diffusion coefficient and hydrodynamic radius which are used as a measure of size and concentration of monomers and aggregates in a solution.FRAP (Fluorescence Recovery After Photobleaching): A spectroscopic technique which is used to measure the diffusion of a population of fluorescently labeled molecules after photobleaching. It gives valuable insights into the mobility of intracellular aggregated species.FRET (Fluorescence Resonance Energy Transfer): FRET measures energy transfer from a donor fluorophore to acceptor fluorophore and can be used to detect the presence of small sub-population of oligomeric assemblies of misfolded proteins.FTIR (Fourier Transform Infrared Spectroscopy): Composition of secondary structural elements are determined by FTIR by measuring molecular bond vibrational frequencies. FTIR spectra can provide structural features of protein misfolding intermediates where the larger and rigid amyloids absorb near 1,620 cm−1 whereas the small and disordered fibers absorb at ~1,635 cm−1.MicroED (Micro- Electron Diffraction): A new method of cryo-EM where diffraction patterns are collected from submicron-thick 3D crystals using a focused low-dose electron beam under cryogenic temperatures and are deployed to visualize amyloid crystals with dimensions of few hundred nanometers.NMR spectroscopy (Nuclear Magnetic Resonance Spectroscopy): NMR is a spectroscopic technique to determine the molecular structure, dynamics and chemical environment of molecules by measuring magnetic fields of certain atomic nuclei. Since amyloids exhibit favorable nuclear spin relaxation, NMR is used in characterization of the overall symmetry of cross–β structures.SAXS : SAXS is used to determine the average particle size, shape, distribution, and surface-to-volume ratio by analyzing the elastic scattering of X-rays at small angles when passed through a specimen. This technique is widely used to characterize structural variations in amyloid fibrils.SLS (Static Light Scattering): SLS uses time-averaged intensity of scattered light to estimate molecular weight of particles in a solution and thereby helps in identifying the presence of higher molecular weight amyloid-like aggregates.Super-resolution Fluorescence Microscopy: In super-resolution microscopy, temporal or spatial modulation of the excitation or activation light helps to overcome the resolution limit to extract higher resolution information of the samples and provides detailed information on species morphology of oligomeric and fibrillary structures.TEM (Transmission Electron Microscopy): TEM provides morphological visualization of amyloid aggregates or fibers. First, the amyloid samples are negatively stained using metal compounds, such as uranyl acetate, before imaging.ThS (Thioflavin-S Fluorescence): Binding of ThS with amyloid aggregates displays a sharp fluorescence emission peak at ~520 nm when excited at 440 nm. It is also used to stain amyloid aggregates present in tissue sections and cell culture.ThT (Thioflavin-T Fluorescence): Binding of the planar dye Thioflavin-T to amyloid-like aggregates increases its fluorescence emission intensity at ~485 nm when excited at 445 nm.XRD (X-Ray Diffraction): Subjecting amyloid fibers to X-ray results in the display of a specific diffraction pattern known as the cross-β pattern in which β-strands run perpendicular to the fiber axis and β-sheets extend parallel to the fiber axis.TDP-43 protein is intricately associated with stress granule dynamics  in an isogenic cell line  and nuclear export signal (NES), regulate the nucleocytoplasmic shuttle of TDP-43  in TDP-43 are phosphorylation and ubiquitination  sequence. PAR binding was found to promote the TDP-43's phase separation in vitro and was also shown to be essential for the TDP-43's accumulation in the stress granules in the mammalian cells and neurons  is a post-translational modification that appears rapidly at the DNA damage sites, and has implications in cancer, cell cycle regulation, DNA repair pathways, and chromatin reorganization, etc. Bai, . Poly  present in the TDP-43 protein, four cysteine residues at the positions 173, 175, 198, and 244, are highly conserved  and can undergo oxidation and disulfide bond formation  and Congo red  of TDP-43, which could adopt the characteristic amyloid steric zipper structures  of TDP-43 in the presence of different Hofmeister series anions. We found that kosmotropic anions greatly accelerate whereas the chaotropic anions impede its amyloid-like aggregation rates -42 peptide's amyloid aggregation, a low net charge on the TDP-43 protein decreases its solubility and improves its aggregation, whereas, with high net charge the electrostatic repulsions dominate, which can impede the aggregation of TDP-43  and Kuru involve deposition of the infectious prion protein PrP in aggregated amyloid-like conformation in the affected brains   Shin an. SeveralPhase-separated droplets of the ALS-linked FUS mutants were found to display a propensity to mature into amyloid-like fibrillar aggregates  , autophagy and ER stress-activated unfolded protein response (UPR). Abnormal turnover of TDP-43 caused by mislocalization and aggregation appears as a key event for ALS and aberrations in the neuronal proteostasis have been identified in ALS , however, some of the ALS-linked TDP-43 mutations can abolish its ATG7 mRNA binding ability  or autophagy. Early studies suggested that the soluble as well as the aggregated TDP-43 are cleared by both the ubiquitin-proteasome system (UPS) and autophagy   and also as the major target of the ROS-induced damage. Protein oxidation/carbonylation, lipid peroxidation, depletion of anti-oxidants like glutathione, increase in the intracellular free iron content and damage to DNA, are widely used markers of oxidative stress  complex, even though SOD1 lacks a mitochondrial localization signal. Mutant SOD1 accumulates in the intermembrane space (IMS) and matrix of mitochondria and elicits toxicity , which is also implicated in ALS pathology, is transported to the mitochondria 2+-dependent ATP synthesis pathway and the transportation of mitochondria in the neuron  and the mitochondrial protein tyrosine phosphatase interacting protein (PTPIP51) and it also reduces the uptake of calcium by mitochondria, which has detrimental effects on the CaCytoplasmic accumulation of TDP-43, which is a pathological feature of ALS, results in unsolicited interaction with various cellular organelles, primarily the mitochondria  protein are linked to ALS, and the mutant CHCHD10 protein molecules are localized to the intermembrane space of mitochondria and are also found to interact with TDP-43  and CuII(gtsm), have shown potential to significantly improve the phenotypes of the TDP-43- and SOD1-associated toxicity in the transgenic mice and the neuronal cell models  and also into small oligomeric structures (20–30 nm)  is a master regulator of the expression of several heat-shock proteins during stress conditions  kinase is an important protein involved in the regulation of cell signaling, protein synthesis, and autophagy pathway. Several small molecules, like trehalose and rapamycin, can induce protective autophagy and improve the neuronal health. Rapamycin, a small molecule inhibitor of mTOR, stimulates autophagy through the formation of autophagosomes from the phagophore and enhances protein degradation  and CuII(atsm), which proposedly act by slowly releasing the CuII-ions within certain sub-cellular compartments like the stress granules , which are part of the nuclear pore machinery, have been shown to act as chaperones and disaggregases (Chook and Suel, Heat shock transcription factors have a significant role in maintaining the cellular proteostasis (Neef et al., The TDP-43 protein, by virtue of its versatile functions in RNA metabolism and homeostasis, has emerged as a vital protein for cellular health. Supporting its importance, aberrations in the TDP-43 homeostasis due to imbalance in its nucleocytoplasmic distribution, genetic mutations, aberrant post-translational modifications or aggregation, is increasingly being accepted as a causative of mis-regulation of RNA homeostasis and cytotoxicity. A few facets of the TDP-43-induced toxicity, such as mitotoxicity and proteosomal overload etc. have been unearthed, but how and in which sequence the toxicity mechanisms ensue leading to the neurodegeneration, remain poorly understood. The presence of TDP-43-positive inclusions in several other neurodegenerative diseases, in addition to ALS and FTLD, suggest of a more wide-spread and vital role of TDP-43 in the general process of neuro-degeneration. Thus, targeting the TDP-43 dyshomeostasis, may hold the key to finding common therapeutics, applicable to a multitude of neurodegenerative diseases.BP directed the manuscript preparation. AP, VB, VS, AG, and BP 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.