Following CNS demyelination, oligodendrocyte progenitor cells (OPCs) are able to differentiate into either remyelinating oligodendrocytes (OLs) or remyelinating Schwann cells (SCs). However, the signals that determine which type of remyelinating cell is generated and the underlying mechanisms involved have not been identified. Here, we show that distinctive microenvironments created in discrete niches within demyelinated white matter determine fate decisions of adult OPCs. By comparative transcriptome profiling we demonstrate that an ectopic, injury-induced perivascular niche is enriched with secreted ligands of the BMP and Wnt signalling pathways, produced by activated OPCs and endothelium, whereas reactive astrocyte within non-vascular area express the dual BMP/Wnt antagonist Sostdc1. The balance of BMP/Wnt signalling network is instructive for OPCs to undertake fate decision shortly after their activation: disruption of the OPCs homeostasis during demyelination results in BMP4 upregulation, which, in the absence of Socstdc1, favours SCs differentiation. This regenerative process can also involve the generation of remyelinating Schwann cells (SCs), the myelin-forming cells of the peripheral nervous system (PNS). The contribution of SCs to CNS remyelination have been reported in several clinical conditions including traumatic spinal cord injury, leukodystrophies , and mosThe underlying mechanism controlling this unusual CNS-to-PNS fate-switching of adult OPCs is unclear. SC-mediated remyelination of central axons is closely associated with localization of cells in the lesion and cellular composition of the surrounding tissue. We and others have reported that SCs can be predominantly found in the CNS areas from which astrocytes are absent . The cenFate decisions by adult multipotential cells are often regulated by a specialized microenvironment, termed the ‘niche’, associated with the vasculature . Injury-To get insight into molecular composition of post-injury niches we used a well-established model of demyelination induced by a stereotactic injection of ethidium bromide (EB) into the caudal cerebellar peduncle (CCP) of adult rats . Toxin iIn response to demyelination, OPCs become activated, proliferate, migrate and ultimately undergo terminal differentiation during several well-defined stages . First, We also found substantial regeneration of blood vessels and BBB within the areas undergoing remyelination. Blood vessels rebuilding their basement membrane and endothelial walls occurred in parallel with remyelination . Of notemyelin protein zero (MPZ) mRNA and immunostaining for periaxin. Strong signal from MPZ mRNA only occurred in cells closely associated with the vasculature, suggesting that an interaction between progenitors and blood vessels might be necessary for their alternative, SC differentiation (MPZ and PLP (encoding an oligodendrocyte specific protein) were mutually exclusive and spatially separated  for ntiation . Double eparated . Moreove lesions . Close e lesions .Our observations suggest that blood vessel itself or blood vessel-created microenvironment together with astrocyte absence are critical determinants for specific OPCs differentiation.The differences in cellular composition between distinct lesion areas prompted us to investigate their transcriptomes. We hypothesised that they would reflect microenvironmental cues regulating OPC differentiation. To address this hypothesis, we first examined differences between VN and non-VN transcriptomes at 6 and 10 dpl, assuming that key determinants of alternative OPC fate should be activated at the time when early differentiation decisions are made. We used laser capture microdissection (LCM) to precisely separate pre-defined discrete tissue compartments. Since LCM procedure requires good visualization of a structure of interest we developed fast RAC120 staining in fresh tissue sections  to definp-values<0.05; the source data are summarized in Using bioinformatic tools we identified genes differentially expressed in separated niches. We found 115 genes with significantly changed expression in the VN between 6 and 10 dpl, and 313 genes with significantly changed expression in non-VN  were significantly upregulated within the VN at 10 dpl. These data suggested that the distinctive VN transcriptome might be required for SC differentiation.Using differential gene expression (DEG) analysis, we identified a set of genes that were significantly regulated only in the VN and among them, several genes expressed exclusively in this microenvironment. A volcano plot, obtained by plotting the fold change of gene expression (log2 of 10 dpl/6 dpl ratio) against significance of the difference (-log10 adj.pv) revealed a prominent cluster of 18 such niche-specific genes at 6 dpl and 33 at 10 dpl . The definitions contained information not only about the presence of a given gene product, but also about interactions between the elements, co-regulation. Next, we placed the identified expression changes within pathways that contained significantly high number of differentially expressed genes. The most prominent group of genes clustered in the ‘hedgehog signalling network’, which includes interaction with elements of BMP and Wnt signalling pathways. Preserving the original pathway content and topology from KEGG database we generated a graphical representation of the expression data which visualizes relationship between genes expressed in VN and non-VN, respectively. It revealed the co-expression changes in the signalling network genes encoding for regulatory enzymes . Employing the same technique for niche separation, we examined differences in expression level of the genes of interest between VN and non-VN . We extended our qPCR analysis to samples from 14 dpl to examine broader kinetics. qPCR results showed upregulation of all transcripts in both niches compared to the intact tissue and confirmed differences in expression of genes encoding for e points . At 10 dBMP4 expression, the expression of Sostdc1 was significantly upregulated only in the non-VN  was the only BMP antagonist expressed within areas of demyelination; other BMP antagonists such as chordin, noggin, gremlin1, gremlin2, and follistatin were uniformly expressed at low levels in both compartments . At the time of highest e non-VN . BMP4 mRtagonist  and its tagonist .BMP4 mRNA expression was below the limit of detection, but was increased after demyelination. The number of BMP4-expressing cells reached a maximum at 10 dpl, then decreased around 14 dpl . BMP4 expression was confined to oligodendrocyte lineage as demonstrated by immunolabelling of nuclei of BMP4-positive cells with Olig2. Since the temporal profile of Olig2+ cells closely resembled that observed for Olig2+/Sox2+  (Vtn) and fibronectin (Fn1). A particularly highly expressed gene within the non-VN was connective tissue growth factor (CTGF). CTGF, a member of the CCN family of secreted matricellular proteins, can bind multiple ligands and interact with many other proteins in the remodeling of lesion environments. Its interactions with the ECM and integrins have been reported to affect oligodendrocyte differentiation  . In contntiation . Moreoventiation . As shown non-VN . The lowTo address whether ectopic SC differentiation contributed to CTGF production as occurs in the PNS, we specifically labelled peripheral glia in transgenic mice that allowed us to compare the level of CTGF production in CNS-derived and PNS-derived cells, separately.bona fide PNS glia  in which all neural crest progeny were labelled. We first demonstrated that Wnt1-Cre was active in SCs in peripheral nerves, but not in OPCs . We thenPNS glia .In this study, we demonstrate that CNS-derived SCs selectively occupied the perivascular area devoid of astrocytes and propose that differentiation of OPCs into SCs depends, at least partially, on vascular reorganization.We show that the tissue around the blood vessels promotes preferential accumulation of activated OPCs early after demyelinating insult, which correlates with remodelling of the capillary network. This is in agreement with data reporting that cerebral endothelium  promote Identification of a perivascular niche as a causative factor for alternative differentiation prompted us to employ transcriptome profiling approach in order to describe signals and processes that occur in the niche. Comparative analysis of global transcriptome in discrete areas of the same affected structure pointed towards BMP and Wnt signalling pathways as the crucial players with context-dependent functions. The implication of BMPs in healthy brain neurogenesis  and afteWe identified BMP4 as an OPCs-derived factor whose increased expression in the VN did not, however, correlate with astrogenesis. Moreover, as we showed previously, adult OPCs do not differentiate significantly into astrocytes in vivo, only occasionally we found OPC-derived astrocytes in the non-vascular areas . Our resBMP4 has been proposed to promote differentiation of engrafted adult OPCs primarily into cells with immunophenotypic and ultrastructural characteristics of myelinating SCs . SC diffsostdc1) binds directly to BMPs and antagonizes BMP signalling  mediates SCs remyelination by precursors of CNS origin after spinal cord injury . HoweverFinally, by using a transgenic approach we have provided novel evidence that CNS-derived SCs substantially differ from PNS myelinating cells, that is by different CTGF production. We show that CTGF participates in the pathological reaction of the astrocytic compartment which is in line with previous reports describing contribution of accumulating CTGF+ astrocytes to wound healing and glial scar formation in response traumatic brain injury .This study is the first to demonstrate the PNS-like OPC differentiation effect of BMP and Wnt signalling, both of which are signalling pathways associated with inhibition of OL differentiation and maturation. We propose that BMPs induce OPC priming and activation and, when combined with Wnt, instruct them to differentiate into peripheral myelin producing cells. This sets the stage for further investigations into the downstream effectors of the proposed signalling networks that govern alternative OPC differentiation during CNS remyelination.All research and animal care procedures were approved by the First Warsaw Local Ethics Committee for Animal Experimentation  and performed according to international guidelines on the use of laboratory animals.The animals were kept under standard laboratory conditions on a 12 hr light/dark cycle with constant access to food and water and were randomly assigned to experimental groups.Animals used:8–10 week-old female Wistar rats;ibidem);9–12 week-old female Wnt1-Cre transgenic mice expressing the Cre recombinase under control of the Wnt1 promoter elements described previously . Homozyg9–12 week-old female PDGFRα-CreERT2 transgenic mice expressing the Cre recombinase under control of the PDGFRα promoter described previously . HomozygP0-P2 pubs of transgenic eGFP-expressing rats Wistar-TgN(CAG-GFP)184Ys, National Bio Resource Project for the Rat in Japan, Kyoto University.2.Rat brain microvascular endothelial cells were purchased from Innoprot. Primary rat astrocytes and OPCs were isolated as described in the method details section, In vitro cell culture and treatment. Cells were maintained at 37°C in a humidified atmosphere of 5% CODemyelination in rats was induced bilaterally by stereotaxic injection of 4 μL of 0.01% ethidium bromide  into the caudal cerebellar peduncles  as previously described . EB was In mice, Cre recombination was induced by administering tamoxifen , dissolved in corn oil by sonication for 45 min at 30°C. Adult mice were given 300 mg per kg of body weight by oral gavage or by intraperitoneal injection, at 100 mg per kg of body weight, daily for four consecutive days. Tamoxifen treatment was stopped 5 days prior to inducing demyelination.Demyelination in mice was induced by injection of 1 μL of 0.1% ethidium bromide (EB) into the ventral or dorsal white matter funiculus at the level of T12 as previously described in detail . The posSostdc1 gene expression product) , and 3. OPCs pretreated with dorsomorphin . The sciatic nerve was re-exposed at the indicated time at the level of the previous injury and injected with 8 μL of cell suspension in density of 2 × 104 cells per microliter into four sites along the nerve over 4 mm proximal-distal to the lesion epicentre using a Hamilton syringe with a glass micropipette attached on the top, at a rate of 1.0 µL/min. Lentiviral-transduced cells were injected in the same manner but animals received cell suspension in density of 104 cells/μL to avoid cell clustering. The procedure was performed under a surgical microscope to visually confirm swelling of the nerve during intraneural injection. The animals in which the injected solution escaped from the needle track during injection or after withdrawal of the needle were omitted from the experiment. During all surgical procedures animals were anaesthetized with isoflurane supplemented with buprenorphine  for pain relief.For sciatic nerve crush in rats an incision was made over the length of the right hip and, after exposing the nerve, constant pressure was maintained for 30 s with using watchmaker’s forceps. For intraneural cell transplantation rats were randomly divided into groups receiving: 1. OPCs in control medium (0.1% BSA (wt/vol), 0.1% DMSO in DMEM), 2. OPCs pretreated with a recombinant human USAG1 protein , 100 U/mL penicillin, and 0.1 mg/mL streptomycin). After 8 days, microglial cells were removed by mild shaking over 1 hr and then cultures were shaken for additional 16 hr on a rotary shaker at 37°C at 250 rpm. OPCs were collected as the floating fraction. Adherent cells, astrocytes, were re-seeded on poly-L-lysine-coated culture plates at a concentration of 50 cells/mm2 and maintained for 48 hr before treatment in DMEM containing 10% fetal calf serum (with 2 mM glutamine and antibiotics).Primary mixed glial cultures were prepared from P0-P2 Wistar rat pups. Briefly, meninges were removed from brain hemispheres, forebrains were enzymatically (0.025% trypsin at 37°C for 20 min) and mechanically dissociated to a single cell suspension and cells were plated at a density of 3 × 10Astrocytes and endothelial cells were activated to proliferation and migration in wound healing conditions. Cells were plated on a 35 mm Petri dish, and the cell monolayer was scraped off the in five straight parallel lines using a 100 μl pipette tip to create ‘scratches’. After washing with phosphate-buffered saline (PBS) cells were maintained in standard medium for indicated time.4 cells/μL in the same medium containing respective compounds for implantation.For grafting into sciatic nerve GFP+ OPCs were isolated from primary glial cultures obtained from W-Tg(CAG-GFP)184Ys rats as described above and floating cells fraction were subsequently incubated with the tested compounds for 1 hr on rotary shaker at 100 rpm. Afterwards, the cells were carefully pelleted and re-suspended at the density of 2 × 106 per poly-l-lysine coated T-75 cm2 flask in OPC medium . Cells were infected with a set of three on-TARGET plus SMARTvector Lentiviral shRNAs for rat LRP6  and non-targeting shRNA (shRNA-control) synthesized by Dharmacon. All constructs were driven by constitutive cytomegalovirus (CMV) promoter and expressed TurboRFP fluorescent reporter. The sites targeted by the LRP6 siRNA lentivirus were: ATTATAACAAAGCGACTTG, ATGGGATCTAACACAATAG, and GGGTAATCTAAGCCGAACT, the titer was 108 TU/mL. For transduction, OPC were exposed to lentiviral particles at a multiplicity of infection (MOI) of 2. The cells were incubated in viral supernatants for 8 hr, and then the culture medium was replaced. At 72 hr the transduction efficiency was assessed as the ratio of the number of tRFP-positive cells to the total GFP-positive cells.For viral transduction GFP+ OPCs were seeded at a density of 2 × 104 per 24-wells plate and switched to differentiation in OPC medium without growth factors. Cells were fixed with 4% paraformaldehyde before being analysed for differentiation by immunostaining for myelin basic protein. Efficacy of gene knockdown was determined by western blot analysis.After 72 hr post infection OPCs were dissociated with papain solution (STEMCELL Technologies), blocked with 20% FCS in DMEM and subsequently suspended in ice-cold PBS. tRFP-positive cells were sorted out of GFP-positive OPC with FACS Aria II sorter using FACS Diva software (BD Biosciences). Cell aggregates were removed from analysis during each experiment by doublet exclusion using pulse processing-based FSC-H vs FSC-W method. Following sorting double positive cells were implanted into sciatic nerves at 24 hr after crush injury as described above. A portion of cells was plated at the density of 6 × 10Whole-cell protein extracts were separated on SDS-PAGE before electrophoretic transfer onto a nitrocellulose membrane (Amersham Biosciences). The membranes were incubated with primary antibodies diluted in a blocking buffer overnight and then with relevant secondary antibodies for one hour. Antibodies recognizing LRP6 (diluted 1:1000) was purchased from Abcam, tRFP (diluted 1:2000) from Evrogen, β-Actin antibody (diluted 1:2000) from Oncogene Research Products, horseradish peroxidase-conjugated anti-rabbit IgG (diluted 1:2000) from Vector Laboratories. Immunocomplexes were visualized by using ECL (Amersham). The molecular weight of proteins was estimated with pre-stained protein markers (ThermoScientific). Band intensities were determined by densitometric analysis of immunoblots with Molecular Imager FX and Quantity One software (BioRad).For fresh-frozen tissue isolation animals were terminally anaesthetized with pentobarbitone at appropriate time after demyelination (n = 5 for each time point), brains were immediately removed and frozen in isopentan alcohol. Brains were cryosectioned into 20 μm slices and mounted on PEN membrane  for further laser captured microdissection. For in situ hybridization and immunodetection animals were terminally anaesthetized with pentobarbitone and intracardially perfused with 4% (w/v) paraformaldehyde  in phosphate-buffered saline  at the indicated time after surgical procedure. Lesion containing tissue was dissected, post-fixed in 4% PFA overnight then immersed in 30% sucrose solution prepared with PBS for 48 hr before embedding with OCT (ThermoFisher Scientific). 12 μm coronal or longitudinal sections were thaw-mounted onto Poly-L-lysine-coated slides and stored at −80°C until further use.Ricinus Communis Agglutinin I  on ice for 5 mins and immediately dehydrated. All solutions were prepared using RNAse free water. Predefined areas within lesioned CCP were dissected form each section through the whole lesion volume with Laser Capture Microdissection System PixCell II . We defined a perivascular area  as an area around blood vessel with the diagonal length two times greater than the diagonal of a particular blood vessel.Brain vessels was visualized in tissue sections by incubation with rhodamine labelled Based on above, each predefined area around blood vessels as well as the remaining lesion area  were cut out using UV laser and capture with IR laser as quickly as less than 2 hr for one slide. Dissected fragments obtained from both CCP structures of each animal were pulled as VN and non-VN, separately and RNA was extracted using PicoPure RNA Isolation Kit (ThermoFisher Scientific). The quality and integrity of the RNA was determined using the Agilent 6000 RNA Nano kit and an Agilent Bioanalyzer 2100 (Agilent). RNA samples (100 ng) representing VN and non-VN from each animal was separately amplified and biotin-labelled employing the 3’IVT express kit (Illumina), and hybridized to RatRef-12 microarrays (Illumina) – a total of 20 hybridizations, with five biological replicates per condition. The microarray experiments were conducted at the microarray facility of the Cambridge Genomic Service, Cambridge, UK according to standard protocols. The microarray dataset has been deposited in the Gene Expression Omnibus repository, and is available with the accession number GSE93645.For verification of microarrays data, we used independent samples obtained using the same technique as described above. We enriched experimental conditions with the samples from intact controls  and animals 14 days after toxin injection (n = 3). Total RNA from sciatic nerve and cultured cells was extracted using Tri Reagent (Sigma) combined with column isolation RNeasy Mini Kit (Qiagen). mRNA was reverse transcribed using high capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) according to the manufacturer’s protocol. Quantitative real-time PCR (qPCR) reactions were performed using TaqMan Gene Expression Assays (ThermoFisher Scientific) with Applied Biosystems 7900HT Fast or QuantStudio 12K Flex Real-Time PCR System. We used the following primer/probe sets: Actb Rn00667869_m1; BMP4 Rn00432087_m1; CTGF Rn01537279_g1; Rn18s Rn03928990_g1; Sostdc1 Rn00596672_m1; Wnt2Rn01500736_m1; Wnt2b Rn00627297_m1.−(SΔCt−CΔCt) or as ratio of experimental sample/condition to intact tissue or control condition. For comparison of gene expression level in different cell types data were presented as ΔCT. Triplicate measurements were made on at least three biological replicates.For comparative quantification, the expression level of respective genes was normalized and expressed as a relative fold change. Delta CT values from qPCR analysis were calculated by subtracting the average value from housekeeping gene (18s RNA or Actb). The relative gene expression levels between the sample and control were determined using the formula 2BMP4 and Sostdc1 cDNA was amplified using total RNA isolated from injured rat spinal cord . The fragment of cDNA was subcloned into plasmid pSP73 (Promega) with flanking promoters for SP6 and T7 RNA polymerase. The sequences (5’→3’) of the PCR primers were: BMP4 forward CCTGGTAACCGAATGCTGAT, reverse CGATCGGCTAATCCTGACAT; Sostdc1 forward GAAAGAATTCACCGCCTCAG, reverse CCTGGCTTTCACTGCAAACT. Plasmids were sequenced and transcribed in vitro to obtain antisense and sense (control) probe used for hybridization. Briefly, to label cRNA probes, following linearization of plasmids with appropriate restriction enzyme DIG labelled antisense probes were synthesized using the DIG RNA labelling kit (Roche) with suitable RNA polymerases. The target mRNA-expressing cells were visualized as a dark blue-purple deposition with NBT/BCIP–alkaline phosphatase. Double labeling with two color in situ hybridization has been described previously (http://rsb.info.nih.gov/ij/).Riboprobes were generated based on rat genomic sequence from NCBI database. Specific sequences of rat mRNAs were chosen and later amplified by PCR reaction using Platinum Taq DNA Polymerase High Fidelity (ThermoFisher Scientific) to avoid false incorporation of nucleotides and cloned using TopoTA Cloning Kit (ThermoFisher Scientific). The rat eviously . BrieflyTissue was processed as described above. Sections or cells were incubated in sodium citrate  for antigen retrieval, blocked with PBS containing 0.3% (v/v) Triton X-100 and 10% (v/v) normal donkey serum , and incubated with primary antibody overnight, 4°C.Primary antibody used: Caspase3 R and D Systems AF835-SP; CTGF Abcam ab6992; GFAP DakoCytomation Z0334; GFAP BD Biosciences 556330; GFP Abcam ab6673; MPZ Abcam ab39375; Neurofilament NF DakoCytomation M0762; Olig2 Abcam ab33427; Olig2 Millipore MABN50; Periaxin Gift from Prof. Peter Brophy, University of Edinburgh, UK; SMI71 Calbiochem NE1026; Sostdc1 Abcam 99340; Sox10 Gift from Dr. Michael Wegner, Universität Erlangen-Nürnberg, Erlangen, Germany; Sox2 Santa Cruz Biotechnology sc-17320; vWF Abcam ab6994; Iba1 Wako 019–19741, MBP R@D Systems MAB42282, tRFP Evrogen AB233.Then, sections were incubated with appropriate AF488, AF555, or AT647- conjugated secondary antibodies . Nuclei were visualized with 4’, 6’-diamidino-2-phenylindole . Images were acquired with fluorescence microscope (Leica DM4000B) and confocal (Zeiss LSM700) microscopes.Microarray data were exported from Illumina BeadStudio software, processed and normalized using the R/Bioconductor beadarray, lumi and limma packages. Before normalization, probes that were not detected  were removed from further analysis. Remaining probes were mapped to gene identifiers from Ensembl database (gene_stable_id). For each gene, we computed a single average intensity profile from the profiles of all the probes mapped to it. The resulting average profile was then log2-transformed and used in statistical analysis and visualization. Two microarrays , which did not accomplish our highest standards of analysis have been excluded from comparison. Differential expression analyses were conducted using the limma Bioconductor package. False Discovery Rate (FDR) was used to adjust for multiple hypothesis testing . Genes wData analysis was performed using Prism 6 (GraphPad) software except microarray analysis. Data are represented as mean ± sd. To detect differences between experimental conditions Student’s t-test or Mann-Whithney U-test were performed. p<0.05 was considered to be statistically significant. Multiple group comparisons were conducted using one-way ANOVA followed by Newman-Keuls post hoc test. In vitro assays represent three independent experiments from individual culture preparations, unless otherwise indicated. 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 Marianne Bronner as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Injury-induced perivascular niche supports alternative differentiation of adult CNS progenitor cells" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this letter along with the Editor-in-Chief to help you consider what may be involved in satisfying the serious concerns raised in the reviews and the reviewer consultation session.Summary:In this manuscript, Justyna Ulanska-Poutanen and colleagues try to identify the molecular cues that cause adult OPCs to differentiate into Schwann cells or oligodendrocytes during CNS remyelination following ethidium bromide-induced demyelination in the caudal cerebellar peduncle. They argue that differentiation to Schwann cells (or better Schwann-like cells) is confined to injury-induced perivascular niches (VN) and induced by combined BMP/Wnt signals. These signals are derived from adult OPCs and endothelial cells and become dominant because of the low number of astrocytes and the low level of the astrocyte-derived dual BMP/Wnt inhibitor Sostdc1 in these areas.The topic of the study is very interesting and has the potential to shed light on remyelination processes. It is mostly written in an intelligible way. However, there are also several major drawbacks.Essential revisions:The manuscript falls short of providing a molecular mechanism of how combined BMP/Wnt signals cause the fate switch in OPCs.1) I am missing clear-cut evidence that the proposed signals really cause the fate switch in situ in the CNS. The presented data in favor of the model are first of all correlative CNS expression data. Additional support or stems from DRG co-culture experiments in which OPCs are treated with BMP4 and Wnt2b, and from experiments in which OPCs are grafted into the crushed sciatic nerve in the presence of signaling inhibitors. Neither of these experimental setups faithfully recreates CNS conditions. This is for instance evident from the fact that only Oct6 is induced in co-culture, not however any of the classical peripheral myelin genes.2) The argument for selective BMP inhibition in the non-vascular area appears soft. As shown in Figure 3C, there appears to be, at best, a delay in expression of Sostdc1 mRNA in the vascular niche, raising the question as to what happens to the phenotype of the Schwann-like cells or ongoing myelination within that zone subsequent to the 14 day time-point. In addition, is Sostdc1 protein diffusible within the microenvironment? The effects of DORSO and USAG1 upon the numbers of myelinated segments produced by the transplanted cells in the sciatic nerve should also have been quantified, rather than just by single photomicrographs.3) In addition, do the total numbers of Sox10 positive cells per unit area in the vascular niche at D10 and D14 post-lesion match  supporting a transformation argument. Otherwise, are there other potential explanations for the reduction in Olig2 positive cells such as migration into non-vascular areas or along newly generated blood vessels?4) It would be useful to have complementary myelin protein staining to MPZ in Figure 1F. Presumably MBP would be more widely expressed. What about PLP (an oligo specific protein); is this excluded from the perivascular niches?-if not it argues against an all or none phenomenon and/or suggests that the cells in this region are hybrids and as inconsistently conceded in the manuscript only 'Schwann cell like'.5) Whereas the expression profile of BMP4 is increased that of BMP2 is trending down. Is further, more complete interrogation of BMP profiling/signalling required before definitive comments about its relevance can be made?6) There will of course be some astrocytes in the perivascular area with end-feet contributing to the re-establishment of the blood-brain barrier. Is there suppression of their innate phenotype in this region or is it that these cells are fundamentally different to the reactive astrocytes in the non-vascular area? Essential revisions:The manuscript falls short of providing a molecular mechanism of how combined BMP/Wnt signals cause the fate switch in OPCs.1) I am missing clear-cut evidence that the proposed signals really cause the fate switch in situ in the CNS. The presented data in favor of the model are first of all correlative CNS expression data. Additional support or stems from DRG co-culture experiments in which OPCs are treated with BMP4 and Wnt2b, and from experiments in which OPCs are grafted into the crushed sciatic nerve in the presence of signaling inhibitors. Neither of these experimental setups faithfully recreates CNS conditions. This is for instance evident from the fact that only Oct6 is induced in co-culture, not however any of the classical peripheral myelin genes.We acknowledge that our experimental design did not focus on re-creating CNS conditions. Our specific aim was, however, to identify the causative cues that govern the alternate, PNS fate of OPC differentiation. To this end, we have primarily studied a CNS lesion model in which different OPCs differentiation fates occur in defined regions of the lesion. This led us to show that the alternate PNS fate was controlled by a perivascular microenvironment temporarily depleted of astrocyte-derived factors. Thus, a CNS environment containing reactive astrocyte supports canonical oligodendrocyte differentiation. Our logic in the current study was therefore to provide a PNS rather than a CNS environment to examine the ability of the candidate factors to induce the OPC alternative, PNS fate.Several lines of evidence have already suggested that BMPs are key players in lineage diversification during differentiation of neural stem cells and promoting a neural crest phenotype in CNS progenitors isolated from embryonic and adult spinal cord. Our microarray data proved the importance of BMP4 and shed light on Wnt signalling as a candidate for cooperative action. Therefore, our working hypothesis was that those two pathways, normally inhibited by astrocyte-derived factor/s in the extra vascular areas of demyelinated CNS white matter are responsible for PNS fate induction.We entirely agree that any of the classical peripheral myelin proteins were identified in our in vitro experiments . We observed an occurrence of myelin segments mainly in control conditions, which suggests that the other treatments caused OPC differentiation delayed.Therefore, in order to avoid any further confusion we propose to focus purely on in vivo studies in our revised manuscript. We now provide novel data to strengthen our conclusions and to confirm the causative role of Wnt signalling and BMP/Wnt cooperation in cell fate decision in vivo.Namely, we have inhibited Wnt signalling in transplanted OPCs with lentiviral shRNA against Lrp6  mimicking Sostdc1 action with regard specifically to Wnt. It has been shown that BMP4 does not interfere with Sostdc1-LRP6 binding, and functional assays showed that the ability of Sostdc1 to block Wnt activity through LRP6 is not impeded by BMP4 . Now, we are able to show that inhibition of Lrp6-dependent signaling prevents OPC-to-SC differentiation even in the high BMP4 background of injured sciatic nerve. Specifically, we have infected primary OPC with lentiviral shRNA against Lrp6 and transplanted them into nerves at 1 day after crush. Defective phenotype of cells transduced with LRP6 shRNA indicates that activation of WNT signalling is required and activation of both signalling is indispensable for initiation of PNS fate differentiation which has not been proposed yet. These new data are included in a new Figure 6.We do accept that the detailed mechanism of molecular control of the BMP/Wnt pathways cooperation in OPC alternative fate induction we describe remains unresolved. We recognize that solving this question will require for example, conditional genes tracing and OPC–specific overexpression of BMP4 and Wnt exclusively outside the vascular niche. Moreover, since we proposed Sostdc1 to be a central player to inhibit both signalling pathways the other experimental approach would be using inducible Sostdc1 knockout mice, which however, to our best knowledge, does not exist. On the other hand, rapid depletion of astrocyte, the main source of Sostdc1 protein, by which we could have reduced Sostdc1 production has been shown to cause central axonal damage.We therefore contend that since an important milestone has been now achieved, a detailed description of the molecular control of the signalling crosstalk and their downstream elements is beyond the scope of the current study. We very much hope that the reviewer will agree with us.2) The argument for selective BMP inhibition in the non-vascular area appears soft. As shown in Figure 3C, there appears to be, at best, a delay in expression of Sostdc1 mRNA in the vascular niche, raising the question as to what happens to the phenotype of the Schwann-like cells or ongoing myelination within that zone subsequent to the 14 day time-point. In addition, is Sostdc1 protein diffusible within the microenvironment? The effects of DORSO and USAG1 upon the numbers of myelinated segments produced by the transplanted cells in the sciatic nerve should also have been quantified, rather than just by single photomicrographs.We apologise for not having addressed these very valid points more clearly in our original submission.Our contention is that expression profiles of BMP4, Wnt2b and Sostdc1 are non-overlapping within defined lesion areas, and this disequilibrium, rather than BMP inhibition per se, is a key factor in determining OPC fate decision. In support of this, we have found that OPC-derived BMP4 is significantly increased in the vascular niche at 10 dpl, while expression of Sostdc1 is low at 10 dpl and limited to non-vascular niche areas. In other words, the delay of Sostdc1 expression in the vascular area allows the both signalling to be activated by their ligands present in the niche within the most critical time frame of OPC differentiation. Thus, the balance between activating and inhibitory signals and their fine-tuning affects the cell fate between 6 and 10 dpl (mostly around 10 dpl).Sostdc1 is definitely not restricted to the cell in which it is produced, since it is secreted molecule. However, at 14 dpl, when Sostdc1 is highly expressed, equally in both compartments of the lesion, most of OPCs are already specified to either an oligodendrocyte or Schwann cell fate .In our revised manuscript we have provided quantification of the effects of DORSO and USAG1 upon the numbers of myelinated segments produced by the transplanted cells in the host sciatic nerve, as the reviewer has requested. Moreover, we have performed experiments aimed to dissect BMP and Wnt impact on OPCs fate decision. Because Sostdc1 (or USAG1) serves as an inhibitory protein for both BMP and Wnt, our previous results might have not been fully conclusive in regard to Wnt involvement. Now we show clear evidence for Wnt involvement in induction of Schwann cell differentiation by genetic interference with Wnt signalling in differentiating OPCs .3) In addition, do the total numbers of Sox10 positive cells per unit area in the vascular niche at D10 and D14 post-lesion match  supporting a transformation argument. Otherwise, are there other potential explanations for the reduction in Olig2 positive cells such as migration into non-vascular areas or along newly generated blood vessels?The total number of Sox10 positive cells per area in the vascular niche does not differ significantly between 10 and 14 dpl: detailed quantification has now been provided in the Figure 1C.4) It would be useful to have complementary myelin protein staining to MPZ in Figure 1F. Presumably MBP would be more widely expressed. What about PLP (an oligo specific protein); is this excluded from the perivascular niches?-if not it argues against an all or none phenomenon and/or suggests that the cells in this region are hybrids and as inconsistently conceded in the manuscript only 'Schwann cell like'.PLP and MPZ mRNA identification have been now presented in Figure 1E.We and others already found MBP protein to be equally expressed in both CNS and PNS myelinating cells: however, we observed that MPZ and PLP are mutually exclusive within remyelinated CNS lesions. Appropriate images for Moreover, in the light of our results and those of others  we use “Schwann cells” instead of “Schwann-like cells” in our manuscript.5) Whereas the expression profile of BMP4 is increased that of BMP2 is trending down. Is further, more complete interrogation of BMP profiling/signalling required before definitive comments about its relevance can be made?We analysed the level of expression of different members of BMP family such as BMP4, BMP2, their receptors and antagonists  in local peri-vascular niche compared to non-vascular one. Microarray data have shown that only BMP4 was upregulated within the demyelinated lesion, regardless of the specific area examined. However, as we shown in Figure 3A, there was a significantly higher level of BMP4 in vascular areas versus non-vascular ones at 10 dpl (which we confirmed by qPCR).The data presented in Figure 3A show the ratio of expression between vascular niche and non-VN vascular niche, and suggest low expression of BMP2 in the vascular niche. We draw the reviewers’ attention to the fact that BMP2 transcripts are detected at a very low level and the difference in expression was not significant for either comparison (i.e.: vascular niche vs. non-vascular niche and at 6 dpl vs. 10 dpl). Figure 3 has now been supported by the data on BMP2 expression added as Figure 3—figure supplement 1. The raw data for BMPs expression are presented in We believe that any ambiguity can be removed by a more comprehensive description of the results and interpretation of our high-throughput data.The logic of the microarray was to give some initial insight into how the perivascular niche and how it differs from elsewhere in the lesion. In this regard, it proved very useful and helped provide the basis for subsequent hypothesis-led experiments about some  of the potentially relevant signalling pathways that emerged. We do agree that differential expression pattern between regions primarily reflects the difference in cellular composition. Indeed, we provided in vivo and in vitro evidences that the main sources of revealed differences in gene expression are OPCs in the VN and astrocytes in the non-VN. We acknowledge that we could do more to explain why some were chosen and others (Hedgehog for example) were not and have made the necessary amendments to our revised manuscript.6) There will of course be some astrocytes in the perivascular area with end-feet contributing to the re-establishment of the blood-brain barrier. Is there suppression of their innate phenotype in this region or is it that these cells are fundamentally different to the reactive astrocytes in the non-vascular area?As we showed in the Figure 1—figure supplement 1, astrocytes gradually repopulate the lesion areas . We found that at 6 dpl astrocytes were not present in the core of lesion, since they were likely killed by ethidium bromide. However, at 10 dpl some astrocytes, with processes juxtaposed to blood vessels and presumably contributing to the re-establishment of the blood-brain barrier, were occasionally observed. At 14 dpl, astrocytes are present in the vicinity of large blood vessels and by 28 dpl present throughout the entire remyelinated area (with the exception of the Schwann cell remyelinated areas) as we have now showed in the Figure 1—figure supplement 2. In such cases periaxin and GFAP were mutually exclusive, supporting our conclusion that astrocyte-derived factors inhibit the induction of OPC-to-Schwann cell differentiation within areas of the lesion in which astrocytes are present.


BDNF-AS) that has been shown to regulate BDNF expression. The role of BDNF-AS lncRNA in the molecular mechanisms of AUD is unknown. Here, we evaluated the expression and functional role of BDNF-AS in postmortem amygdala of either early onset or late onset alcoholics  and age-matched control subjects. BDNF-AS expression is increased in early onset but not in late onset AUD amygdala and appears to be regulated epitranscriptomically via decreased N6-methyladenosine on BDNF-AS. Upregulation of BDNF-AS is associated with a significant decrease in BDNF expression and increased recruitment of EZH2, which deposits repressive H3K27 trimethylation (H3K27me3) at regulatory regions in the BDNF gene in the early onset AUD group. Drinking during adolescence also contributed to significant decreases in activity-regulated cytoskeleton-associated protein (ARC) expression which also appeared to be mediated by increased EZH2 deposition of repressive H3K27me3 at the ARC synaptic activity response element. These results suggest an important role for BDNF-AS in the regulation of synaptic plasticity via epigenetic reprogramming in the amygdala of AUD subjects who began drinking during adolescence.Adolescent alcohol drinking is known to contribute to the development and severity of alcohol use disorders (AUDs) later in adulthood. Recent studies have shown that long non-coding RNAs (lncRNAs) are critical for brain development and synaptic plasticity. One such lncRNA is natural occurring brain-derived neurotrophic factor antisense ( Age of onset is one of the primary risk factors for the development of AUDs, as individuals who began drinking during adolescence are four times more likely to develop AUDs in adulthood2. Adolescence is a critical period in brain development and adolescent drinking decreases orbitofrontal cortex activity and increases amygdala activity3 leading to less executive control, more emotional impulsivity, alterations in decision-making, and have a higher risk to engage in risky behaviors and develop mental health problems later in life5. The amygdala acts as a hub for the processing of sensory signaling from cortical regions, and is involved in regulation of emotional reactivity, decision-making, and negative affective state of alcohol addiction7. During adolescence, the amygdala undergoes several key changes in specific inter- and intra-region connectivity8, and this process is thought to be disrupted by alcohol use9. Recent studies have begun to examine the molecular basis of these changes in the amygdala in preclinical models of adolescent alcohol exposure and have identified numerous potential molecular components, such as brain-derived neurotrophic factor (BDNF), that are involved in the development of alcohol dependence in adulthood12.Alcohol use disorders (AUDs) are chronic, debilitating psychiatric illnesses that contribute to considerable socioeconomic and health burden worldwide15. BDNF is associated with survival and differentiation of neurons, activity-dependent neuroplasticity, long-term potentiation, learning, memory, and anxiety16. BDNF is critical for normal brain development, as constitutive knockout mice die during second week after birth13 and is known to be involved in synaptogenesis and maintenance of dendrites during development and adulthood17. BDNF regulates synaptic plasticity through interactions with the tropomyosin-related kinase B (TrkB) receptor and induction of several signaling cascades that are involved in the regulation of synaptic plasticity and cAMP-responsive element-binding protein (CREB)-dependent gene expression13. Decreased expression of BDNF in the amygdala of rodent models of adolescent alcohol exposure has recently been implicated in anxiety-like and alcohol-drinking behaviors12. Furthermore, mutant versions of BDNF with decreased TrkB activity are associated with increase in alcohol consumption.18. A particularly important downstream target of BDNF signaling is activity-regulated cytoskeleton-associated protein (ARC) which is a member of the immediate-early gene family and is important in synaptogenesis and synaptic plasticity as well as anxiety and alcohol intake phenotypes21. BDNF activates ARC expression through CREB and other transcription factor binding to the synaptic activity response element (SARE) site located in the ARC promoter approximately 7 kb upstream from the transcription start site23.The neurotrophin BDNF has emerged as a critical molecular player in central nervous system (CNS) development, psychiatric disorders, and AUDsBDNF gene also has a complex regulatory and genetic structure, with 9 different promoters encoding at least 20 different splice variants sharing one common exon that encodes the BDNF protein (known as exon IX)24 and expression of these variants are temporal- and region-specific25. Studies in animal models have revealed that different BDNF transcript variants are downregulated in the amygdala by adolescent alcohol exposure and associated with alcohol intake in adulthood12.In addition to complex signaling, the 28. Most lncRNAs with described function are involved in regulating gene expression and chromatin structure through a number of different mechanisms including cis-tether, trans-regulation, allosteric modulators of epigenetic complexes  or transcription factors, or acting as a decoy either for transcription factors or repressors31. LncRNAs have recently been identified as being important in AUDs in adults32. Despite several advances in the understanding of lncRNA function, the function of the vast majority of lncRNAs are still unknown. A recent study characterized the role of the lncRNA BDNF-antisense (BDNF-AS) to be a negative regulator of BDNF exon IX expression and neurogenesis, and also described the overlap region between BDNF and BDNF-AS33. However, changes in BDNF and BDNF-AS expression and associated epigenetic regulatory mechanisms in the amygdala of human AUD subjects are currently unknown. We aimed to determine if there are epigenetic changes mediated by BDNF-AS that are operative in the regulation of BDNF expression in adolescence, and whether these changes persist into adulthood in the amygdala of human postmortem brains diagnosed with AUDs that began drinking before the age of 21. Here we report that the epitranscriptome and epigenetic mechanisms interact to regulate BDNF expression in the amygdala and are possibly involved in the pathophysiology of alcoholism that begins with drinking during adolescence.Long non-coding RNAs (lncRNAs) are broadly defined as RNAs that are greater than 200 bp and lack a functional open-reading frame. The majority of lncRNAs are found exclusively in the brain and are important in normal brain development, with temporal and regional specificity34. Detailed methods are provided in Supplementary Information.Human postmortem amygdala tissue was acquired from the New South Wales Brain Tissue Resource Center . Criteria for inclusion of AUD samples were: age >18 years, no history of major psychiatric disorders, and no history of other substance abuse disorders. Demographic characteristics are provided in Supplementary Table ACTB and GAPDH. Data are presented as average fold change of controls.Quantitative real-time PCR (qPCR) was run using standard methods with either Taqman probes or specific primers  following the manufacturer’s instructions. The optical density of each sample and standard was measured using the Spectra MR microplate reader (Dynex Technologies) and the amount of BDNF was calculated against the BDNF standard curve and expressed as pg/µg of total protein (Supplementary Methods).36. Purified DNA was analyzed by qPCR using primers (Supplementary Table Chromatin immunoprecipitation (ChIP) was run following previously published protocolsN6-methyladenosine Enrichment Kit (NEB) following the manufacturer’s instructions with some modifications. Purified RNA was sheared to <350 bp using a Covaris ME220 and then purified using Zymo RNA Clean and Concentrator 5 (Zymo) following the manufacturer’s instructions. Purified RNA was then measured for concentration using the Qubit BR RNA Assay (ThermoFisher Scientific) following the manufacturer’s instructions, and 1 µg was used for each pulldown with 2 µL of M6A antibody. Following 1 h incubation and washes, RNA was eluted and purified with MyOne Silane Beads (ThermoFisher Scientific) then prepared for reverse transcriptase in which 1 µL from pulldown or input was put in reverse transcriptase reaction. Resulting cDNA was then used for qPCR using primers designed to predict M6A sites located on BDNF-AS  and data were visualized using GraphPad Prism . Comparisons between groups were performed with Student’s 12, but similar changes in the human amygdala have not yet been examined. In order to identify potential changes that are due to adolescent drinking we evaluated both a cohort that began drinking before the age of 21 (early onset) and a cohort who began drinking after the age of 21 (late onset). We then analyzed these two cohorts to see if there was differences in drinking or other demographics. We found that our early onset group drank 234 ± 56 g (mean ± SEM) of alcohol per day at time of death versus the late onset group which drank 157 ± 28 g of alcohol per day at time of death, although this observation was not statistically significant between early onset and late onset groups  and found a significant decrease in BDNF mRNA expression in early onset AUD compared to controls (p = 0.008)   Fig. . Regulat33) Fig. . We alsoRNA Fig.  or BDNF--AS Fig.  expressiBDNF and BDNF-AS expression versus changes in several drinking variables , and standard drinks per week) to determine if there was any relationship. We first analyzed the correlation between BDNF and BDNF-AS expression and found that BDNF and BDNF-AS mRNA levels are negatively correlated . We then evaluated whether changes in BDNF-AS expression was a function of total drinking years, alcohol daily use at the time of death, and standard drinks per week. We found that BDNF-AS expression in total samples (n = 42) was positively correlated with alcohol daily use at time of death  and standard drinks per week (r = 0.466 p = 0.002) but not total drinking years , but this was not the case for controls  or in the late onset group   negatively correlated with alcohol per day use at time of death  and standard drinks per week (r = −0.374 and p = 0.015) but not total drinking years   and standard drinks per week   was not significantly different than total alcoholics . However, further analysis by separation of alcoholics by age of onset of drinking revealed a significant (p = 0.043) decrease in BDNF protein levels in subjects (n = 11) in the early onset group (0.177 ± 0.029) versus controls   differences were observed in the late onset group  versus controls   were significantly negatively correlated with both standard drinks per week  and alcohol use per day at time of death . When we determined correlation of BDNF protein expression separately in controls, early, and late of onset drinkers with standard drinks per week and alcohol use per day at time of death, we found no significant correlations similar to what was found with BDNF mRNA correlations  BDNF protein in postmortem human amygdala of AUDs and control subjects was determined via enzyme-linked immunosorbent assay. Average (mean ± SEM) BDNF protein  in controls  Fig. . Interes22) Fig. . BDNF prBDNF expression is due to changes in epigenetic modifications. Particularly, deficits in histone acetylation (H3K9/14ac) of BDNF exon IV promoter was associated with deficits in BDNF expression12. Further, BDNF-AS is known to contribute to epigenetic changes facilitated by the PRC2 complex at the BDNF exon IX promoter in HEK293 cells33. To evaluate if changes observed in BDNF expression were due to epigenetic changes, we utilized ChIP assay to determine if there were differences in enhancer of zeste homolog 2 (EZH2) occupancy and its catalytic product, repressive H3K27 trimethylation (H3K27me3), at several sites in the BDNF promoter region  and overlap region (p = 0.027) in the early onset AUD group. The overlap region 33. At the BDNF-IV promoter, though there appears to be a trend (p = 0.084) towards an increase in the early onset group  and BDNF-IX (p = 0.049) promoters and also at the overlap region (p = 0.003)  in H3K27me3 at the BDNF-IV promoter  with drinking variables. We found no significant correlations with either alcohol use per day at the time of death nor standard drinks per week for either BDNF-IX and BDNF overlap region.Previous reports in rodent models of adolescent alcohol exposure have demonstrated that changes in ion Fig.  and at aion Fig.  is the roup Fig. . We obseoup Fig. . There w03) Fig.  in the eter Fig. . We examEZH2 and suppressor of zeste 12  mRNA levels using qPCR to determine if changes in occupancy of EZH2 could be due to changes in overall levels. We found no change in either EZH2 or SUZ12 mRNA levels in early onset or late onset postmortem human amygdala   in the early onset AUD group  . We also found decreased H3K27ac  associated with the SARE site in the early onset AUD group (p = 0.009)  Fig. , but not16) Fig. . BDNF siion Fig. . H3K27meoup Fig. . We next49) Fig. . These c49) Fig. . We also09) Fig. .Fig. 3ARBDNF-AS upregulation in early onset AUD subjects. Our initial experiments focused on H3K4me3 at the BDNF-AS promoter as this mark is known to be involved in active transcription and in the regulation of lncRNAs39. However we found no significant increase in H3K4me3 in the early onset group  RIP assay to determine if there were changes in RNA methylation at predicted M6A sites in BDNF-AS. Our results indicate a decrease (p < 0.001) in BDNF-AS RNA methylation in the early onset AUD group  slowly rises to a plateau in adulthood, while the basolateral amygdala has decreased expression in adulthood relative to adolescence41 and disruption to normal BDNF expression and signaling in adolescence is likely to produce detrimental outcomes on normal brain development. Changes in BDNF signaling are likely to be involved in structural changes in the amygdala, which involve synaptic pruning and altered connectivity between the amygdala and other brain regions throughout the adolescent period8. Based on the previous finding that adolescent alcohol exposure in rodents decreases BDNF expression in the CeA12, our present findings confirm the hypothesis that there would be changes in BDNF expression in a cohort of postmortem amygdala from AUD subjects that began drinking during adolescence, demonstrating a conserved pathway that may induce increased drinking and other psychiatric problems in adulthood caused by adolescent alcohol exposure. Previous work has found that individuals with alcohol dependence have decreased circulating BDNF in blood42. Interestingly, transgenic mice harboring a homolog  of a common human polymorphism of BDNF  compulsively consume more alcohol than wild-type mice18. The Val68Met variant leads to decreased intracellular trafficking and activity-dependent BDNF release and function18. Decreasing BDNF or ARC expression in the CeA of rodent models of alcoholism in adulthood increases drinking and anxiety-like behaviors, which are measures of alcohol dependence43. These previous findings taken together with the present findings shown here suggest that disruptions to BDNF expression and ARC signaling are likely to increase the risk for alcohol dependence, and the risk is greater if these changes occur at a critical period of brain development44, such as adolescence, due to enduring effects on epigenetic programming. However, the possibility cannot be ruled out that other factors besides drinking during adolescence are involved with changes in BDNF expression that we observed in human postmortem amygdala. These factors could include a longer time period for disruption of normal neural processes by alcohol and a higher lifetime alcohol consumption at the time of death.BDNF signaling is an important modulator of CNS development through mediation of important processes such as neuronal survival, neurogenesis, neurite outgrowth, and neuronal differentiation45. However, evaluation of lncRNAs in a population of individuals with AUDs that began drinking before the age of 21 has not been explored. Previous studies have characterized the lncRNA, BDNF-AS, to be a negative regulator of BDNF expression33. Rodent studies have demonstrated that infusion of BDNF-antisense oligodeoxynucleotides into the CeA increases anxiety and alcohol consumption43. Therefore, we postulated that the naturally occurring BDNF-AS may be involved in changes observed in BDNF expression in our early onset cohort. We found increased BDNF-AS only in individuals who began drinking before the age of 21. Additionally, similar to previous findings demonstrated in vitro33, increased BDNF-AS negatively correlated with BDNF exon IX expression. Intriguingly, we also found that BDNF-AS expression positively correlated with the amount of alcohol consumed using two different metrics of alcohol consumption (grams consumed per day at time of death and standard drinks per week). One possibility is that earlier age of alcohol use may interact with later alcohol consumption to induce BDNF-AS expression. Our results suggest that BDNF-AS may also be important in epigenetic programming during development, and expression of this transcript is dysregulated after drinking in adolescence and associated with AUDs later in life.LncRNAs have recently emerged as a potential molecular target in AUDs by the use of RNA sequencingBDNF-AS may be regulated epitranscriptomically by decreased RNA methylation at a M6A site in the amygdala of early onset AUD but not in late onset AUD. Previous reports have found that decreased M6A methylation prevents RNA turnover and decreases targeted degradation by YTH domain family 2, which is recruited to the RNA by M6A methylation46. Other roles for M6A methylation appear to either promote or inhibit recruitment of proteins that bind RNA47, a potential mechanism for the binding of SUZ12 to BDNF-AS and could be explored in future experiments. Nonetheless, the observation that adolescent alcohol exposure decreases BDNF-AS methylation in the amygdala reveals new mechanisms of RNA regulation that add another layer of complexity of epigenetic regulation of BDNF expression in AUD.Our data indicate that 30, and BDNF-AS knockdown disrupts EZH2 recruitment to BDNF promoters in cell culture models33. We tested this hypothesis by examining EZH2 occupancy and its catalytic product, H3K27me3, at regions of the BDNF promoter, including the region of BDNF exon IX that overlaps with BDNF-AS. Our results indicate that there is increased EZH2 recruited to both the BDNF overlap region and the promoter of BDNF exon IX with corresponding increases in H3K27me3 at these locations. Coupled with our data showing that SUZ12 binds directly to BDNF-AS, this suggests that BDNF-AS regulates BDNF-IX expression through direct binding and recruitment of the PRC2 complex and subsequent repression of transcription by deposition of H3K27me3.LncRNAs have been demonstrated to interact with SUZ12, a member of the PRC2 complex, via its zinc-finger domainARC SARE site. This may be due to chromatin remodeling secondary to decreased BDNF signaling and recruitment of class I histone deacetylases (HDACs)22 catalyzing removal of histone acetyl groups, which we have also observed. The recruitment of EZH2 to this site warrants further experimentation to determine if it occurs through decreased BDNF signaling, subsequently decreased occupancy of CREB-binding protein, and increased occupancy of class I HDACs at the SARE site of ARC gene. EZH2 is also heavily implicated in brain development, where it has a role in regulating self-renewal and differentiation in the cerebral cortex48. Since there is significant pruning and differentiation of neurons during adolescence44, EZH2 dysregulation and an increase in repressive H3K27me3 marks at the BDNF and ARC regulatory regions by alcohol drinking during this critical period could contribute to an increased risk of AUDs and higher overall alcohol consumption later in life (Fig. In this study, we also found increases of EZH2 and H3K27me3 at the ife Fig. .BDNF-AS is upregulated most likely via decreased RNA methylation in the amygdala of adult AUD population who began drinking during adolescence, but not in AUD subjects who began drinking in adulthood. However we are unable to determine if these effects are causative without further experimentation, and indeed changes that we observed could be due to other factors and interactions. Future experiments will have to consider these possibilities in larger cohorts of human postmortem brain and through the use of reverse translational approach in animal models. We demonstrate that increased BDNF-AS is associated with decrease in BDNF expression through an epigenetic mechanism involving the recruitment of PRC2 and the repression of gene promoters. Downstream signaling targets of BDNF such as ARC are also decreased through recruitment of EZH2 and the deposition of repressive H3K27me3 marks. Our results provide translational evidence for a mechanism for decreased BDNF in the amygdala previously shown in rodent models of adolescent alcohol exposure: namely, similar decreases in BDNF and ARC that are associated with higher alcohol intake12. These results suggest the possible role for developmentally sensitive lncRNAs in early onset AUD, and that EZH2 inhibitors may prove useful in the treatment of adult psychopathology after adolescent alcohol drinking.Our results appear to suggest that Supplemental Materials


Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance. Gene dosage anomalies such as those caused by aneuploidy underlie diseases including Down syndrome. Here, the authors perform allele-specific single cell transcriptome analysis to investigate the mechanisms of gene dosage imbalance in fibroblasts with trisomies T21, T18, T13 and T8. Protein production is inherently connected with gene expression level, which, in turn, is regulated by several factors of genetic and epigenetic nature2. Perturbation of this equilibrium may induce severe cellular and organismal phenotypes. Genomic copy number alterations (CNA) such as duplications and deletions, result in gene expression imbalance3 and is associated with reproducible phenotypes as it is the case in aneuploidies. However the respective functional mechanisms are not well understood.The biochemical processes underlying complex cellular functions rely on a precise and timely dosage of their constitutive elements and, in particular, of protein stoichiometry8. Trisomy 21 (T21—Down syndrome) is the most common human aneuploidy compatible with postnatal survival, and has been extensively used as a model to study trisomies9. Other common trisomies include Trisomy 18 (T18—Edwards’s syndrome) and Trisomy 13 (T13—Patau syndrome)13. Phenotypes observed in trisomies have been attributed from bulk RNA-seq studies to the gene dosage imbalance, ~1.5 fold higher for the trisomic genes as compared to their diploid counterparts16. However the causative links between altered gene expression and phenotypes in aneuploidies are not known. To understand the molecular basis of trisomy phenotypes, we explored gene expression profiles in single trisomic cells. Hitherto, single-cell RNA-seq (scRNAseq) studies have revealed pervasive genome-wide skewed monoallelic gene expression in diploid cells17, but also variability and gradation of gene expression for different genomic phenomena/processes, such as imprinting18 and X-inactivation19. These phenomena are likely the outcomes of the discrete and stochastic nature of RNA transcription with each gene bearing his own specific regulation21. In diploid cells, it has been already shown that the large majority of genes respond to a 2-state (ON-OFF) burst-like model of transcription22 and core promoter elements and enhancers regulate transcriptional burst size and frequency, respectively23. Moreover the transcriptional kinetics of the two alleles of a gene are uncoupled and the alleles are transcribed in two substantially stochastic24 and independent processes23. It is still unclear how, in CNA, the presence of additional alleles impacts the transcriptional activity and causes gene dosage imbalance. Here we present the comparative analysis of scRNAseq from trisomic and matched isogenic diploid fibroblasts. We show that burst-like transcription shapes gene dosage imbalance in trisomies. In agreement with the 2-state burst-like model, we provide evidence that, in trisomic cells, the additional allele is independently transcribed, leading to an increased monoallelic expression with respect to the diploid cells, and a significant fraction of trisomic cells simultaneously activating gene expression as compared to diploid controls.Aneuploidy is a well-known source of gene dosage imbalance through CNA. In particular trisomies are considered to be disorders of altered gene expression of the majority of genes on the supernumerary chromosomes (gene dosage sensitive genes)25; four were from individuals mosaics for T21: CM05287, T13: GM00503, T18: AG13074, T8: GM02596  at RPSM (Reads Per Site per Million) = 20, similarly to what previously observed26  using two metrics: the average cellular gene expression and ASE at heterozygous sites, both measured from the genes located on the supernumerary chromosome (details in Methods). Briefly, after quality control  at a given time. We observed the same phenomenon in our normal and trisomic cell populations with around 20–22% of genes presenting a monoallelic expression , in line with what previously reported26. In the diploid fraction of the genome, in twins’ fibroblasts discordant for T21, 60.1% of the heterozygous sites showed monoallelic coverage  in the diploid cells and 70.3% in T21 cells . MEP represents the fraction of cells per gene with ASE ≤ 0.1 or ASE ≥ 0.9 at the heterozygous site with the highest number of cellular ASE observations  between ASE and the level of gene expression of the corresponding triplicated gene  was also observed for diploid genes in both the diploid and the trisomic cells  transcription from the two alleles of highly expressed genes at a given time point. Conversely, low expressed genes have a low transcriptional bursting frequency and therefore the event of a biallelic simultaneous transcription is rare27.We observed a significant negative correlation  and across the single cells (mean(FC) = 0.97) conversely, ATP5O has a FC of 1.55 in bulk and 1.47 in single cells , we derived the hyperbolic equation . This observation has been attributed to the increased amount of transcripts produced by the triplicated genes. Our study allows a more detailed interpretation of gene dosage imbalance in aneuploidies. As an example, considering the previous bulk study on fibroblasts from the discordant twins for T21lls Fig. . In genells Fig. . The expThe mathematical model shows that genes in three copies with low expression level in single cells tend to be expressed in more trisomic cells than diploid cells. This theoretical result, derived from the 2-state burst-like model of transcription applied to 3 alleles, supports the general hypothesis that a component of bulk gene dosage imbalance of copy altered genes is generated by the increased number of cells expressing these genes at a given time point.R3, and compared to the fraction of cells of the corresponding diploid sample R2. We consider a gene as expressed if: (i) the total number of cells expressing the site within the gene is ≥10, (ii) each cellular ASE observation has an RPSM score ≥ 20 (Reads Per Site Per Million17). The genes were classified according to their prevalence of monoallelic expression  as previously defined. Biallelic or intermediate genes did not show statistically significant differences in the ratio of the fractions of expressing cells −4, 7 × 10−6, 1 × 10−8, 3 × 10−25, 3 × 10−5  and normalization with respect to the total number of UMIs per cell, for each chr21 gene we calculated the expression distribution in normal and trisomic cell, respectively  ely Fig. . We compely Fig. . Additio39. The investigation of this phenomenon at single-cell level opened a more complex scenario. As a first observation, we detected reduced or no dosage effect at all for some genes on a single-cell level, compared to the expected fold change from our previous bulk study in T21. Moreover, ASE exploration of the supernumerary chromosome genes in our isogenic models of trisomies showed clear random monoallelic patterns of expression as already observed in diploid cells28. We confirmed that these patterns follow a random allelic selection model by observing that the number of observations expressing the duplicated allele was indeed twice the number of those expressing the single allele. As previously shown26, we observed that the monoallelic prevalence of expression  is negatively correlated with the level of expression of the respective genes. Finally, we observed that the increased fraction of trisomic cells vs. diploid presenting with active expression of supernumerary chromosome genes is contributing to the average dosage imbalance of all the trisomies analyzed in this study. This effect is more evident for low expressed/monoallelic genes.Gene dosage imbalance caused by CNA is generally interpreted as the altered production of transcripts per cell. The typical example is represented by trisomies where several studies on mouse and human samples reported the classical 1.5 average gene expression fold changeThe theoretical application of the 2-state burst-like transcriptional model to trisomies provides an explanation of these observations (Eq. ). For av40. TF dosage imbalance leading to an increased number of a activated cells could be crucial in the following (the list is not exhaustive): (1) different fractions of cells producing increased level of subunits of multimeric proteins may result in abnormal stoichiometry42; (2) abnormal number of cells with cell surface receptors and ligands that may results in a disturbed developmental fate44; (3) abnormal number of transporter molecules in the tissue resulting in metabolic disturbances45; (4) excess of cell adhesion molecules that may increase cellular adhesiveness and differential fate of a tissue46; (5) alteration in the production, concentration and diffusion of morphogens in the tissue and consequent abnormal cellular proliferation and development of aberrant cellular and tissue structures47. Furthermore, the unbalanced expression of low expressed copy altered regulatory long non-coding RNAs and microRNAs in a fraction of cells may also contribute to the disturbance of the regulatory repertoire of other cells, particularly during embryogenesis48. Indeed many of these phenotypes may manifest during the early embryonic development stages where a precise and delicate balance among gene pathways dedicated to coordinate cell-to-cell interactions as well a as specific partition among cell types must be maintained49. Additionally this effect can be mediated by the duplication of regulatory regions that modulate gene expression through specific regulatory variants. eQTLs in trisomic regions have 4 possible states  instead of the canonical  in the diploid genome. This additional degree of freedom might modulate the spectrum of gene dosage imbalance in term of RNA accumulation and fraction of expressing cells and contribute to the considerable phenotypic variability among affected individuals. More generally, we propose that the spectrum of gene dosage may contribute to phenotypes related to Copy Number Alteration, including Copy Number Variants (CNVs) and somatic partial aneuploidies typical of cancer cells50. Based on this considerations, it is straightforward to hypothesize that strong regulatory variants on low expressed genes may induce gene dosage diversity in general population. Time-series single-cell RNAseq studies in normal and aneuploid differentiating embryos are needed to reveal how the spectrum of gene dosage imbalance determines individual phenotypic features.This observation may have a significant impact on the understanding the molecular pathophysiology of aneuploidies. As an example, transcription factors (TF) have in general a lower expression level than non transcription factor genesThe study was approved by the ethics committee of the University Hospitals of Geneva, and written informed consent was obtained from both parents of the twins.25; four were cell lines obtained from Coriell and derived from individuals mosaics for T21: CM05287, T13: GM00503, T18: AG13074, T8: GM02596 (https://www.coriell.org/). DNA samples from peripheral blood were obtained from the parents of the monozygotic twins. Cell lines from mosaic individuals T8, mosaic T13, mosaic T18, were purchased from Corriel, and sample from mosaic T21 was kindly provided by Prof. Dean Nizetic. We captured in total 928 single-cell fibroblasts (484 diploid and 444 Trisomic) using the Fluidigm C1 technology. In addition we employed an alternative single-cell RNA-seq protocol based on 10X Genomics technology (Chromium Single cell 3’ Solution protocol32) to capture 8740 single cells (3801 diploid and 4939 trisomic single cells)  and by using single-cell fibroblasts from monozygotic twins discordant for DS (T21)  supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin/fungizone mix  at 37 °C in a 5% CO2 atmosphere. The day before the single-cell capture experiment; cells were trypsinized  and replated at a density of 0.3 × 101;13q14 locus) and chromosome 21 , and two alpha satellite centromere probes for chromosome 8  and chromosome 18 . The experiments were carried out according to manufacturer’s instructions . For each sample 150 interphase nuclei were examined to evaluate the mosaic rate.Fluorescent in situ Hybridization (FISH) analysis was performed on cultured-interphase nuclei with 2 set of probes: two locus specific probes on chromosome 13  to align the sequencing reads (fastq) to the human reference genome (GRCh37/hg19). We used SAMtools v.1.451 to remove paired-end duplicates and pile up the remaining reads. BCFtools v.1.4 was used to call the SNVs and Annovar (2016Feb01)52 for the annotation. SNVs with quality score <100 where excluded from the analysis. All putatively biased sites with low mappability  were removed from the analysis as suggested by Panousis et al.53. Similarly to19, we only used uniquely mapped reads for SNV calling.Genomic DNA was extracted for five individuals using a QIAamp DNA Blood Mini Kit (Qiagen) and fragmented by Covaris to peak sizes of 300–400 bp. Libraries were prepared with TruSeq DNA kit (Illumina) using 1 µg of gDNA and sequenced on an Illumina HiSeq 2000 machine with 2 × 100-bp17. The microfluidics circuit used was the C1™ Single-Cell mRNA-seq IFC, 17–25 µm. All 96 chambers were inspected under an inverted phase contrast microscope; only chambers containing a non-damaged single cell were considered for downstream analysis. For the cell lysis and cDNA synthesis, we used the SMARTer Ultra Low RNA kit for Illumina Sequencing  and a C1 Auto Prep System instrument (Fluidigm) with the original mRNA Seq Prep script provided by the manufacturer . We assessed cDNA quality on 2100 Bioanalyzer (Agilent) with the high sensitivity DNA chips (Agilent) and quantified the cDNA using Qubit dsDNA BR assay kit (Invitrogen). Sequencing libraries were prepared with 0.3 ng of pre-amplified cDNA using Nextera XT DNA kit (Illumina) according to manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq2000 machine as 100 bp reads single-end.Single-cell capture was performed by C1 single-cell auto prep system (Fluidigm) following the manufacturer’s instructionsWe captured in total 3801 diploid and 4939 trisomic single-cell fibroblasts from the monozygotic twin pair using the Chromium System powered by GemCode Technology (10x Genomics). Single-cell RNA-seq libraries were generated using the Chromium Single Cell 3' Reagent Kit version 2 (10x Genomics) according to the manufacturer’s instructions. Briefly, the concentration of trypsin dissociated fibroblasts was set to 1500 cells/µl of culture medium (Dulbecco’s Modified Eagle Medium (DMEM), 10% FBS) and 5000 individualized cells were flown per channel following the recommendation of the manufacturer. All libraries were quantified by Qubit (Invitrogen) and by quantitative real-time PCR using the PCR-based KAPA Library Quantification Kits for Illumina platforms (Kapabiosystems). Size profiles of the pre-amplified cDNA and sequencing libraries were assessed using a 2100 BioAnalyzer (Agilent) with a High Sensitivity DNA chip kit (Agilent). Barcoded libraries were sequenced with an HiSeq 4000 (Illumina) as paired-end 100 bp reads as recommended by 10x Genomics. The proprietary software CellRanger (10x Genomics) with default parameters was used in order to demultiplex the samples and quantify the abundance of mRNA molecules (UMI - Unique Molecular Identifier). Processed data were analyzed using custom R scripts.17. Libraries were sequenced on an Illumina HiSeq2000 sequencer as 100 bp single-ended reads. RNA sequences were mapped with GEM54. Uniquely mapping reads were extracted by filtering for mapping quality ≥ (MQ ≥ 150). For FPKM expression quantification an in-house algorithm was used with GENCODE v19 as reference. Cells with less than 1o million reads and/or cells with <10% of expressed genes  were excluded from the analyses. For each individual, ASE of each heterozygous SNP identified by WGS has been calculated using in-house developed Python scripts. Data have been analyzed using custom R scripts.For single cells capture with the Fluidigm C1 microfluidics system, SMARTer Ultra Low RNA kit for Illumina sequencing  was used for cell lysis and cDNA synthesis. 0.3 ng of pre-amplified cDNA, was used for the library preparation with the Nextera XT DNA kit (Illumina) as describedCellular Allelic Specific Expression (ASE) of each heterozygous site was calculated in the diploid and trisomic fraction of the genome of each single cell per individual using two different formulas.nreads is an operator giving the number of reads covering the site i, mapped according to the REFerence or the ALTernative allele (euploid) or to the Double or Single Allele (trisomic).Diploid fraction:In both cases, ASE values range from 0 to 1  approach, we combined ASE profiling and expression data of every expressed site in the supernumerary chromosomes to classify each single cell as diploid or trisomic. At the beginning the algorithm applies a k(=2)-means clustering to obtain the first partition of normal and trisomic cells using as features average minmax normalized gene expression x cell and average ASE x cell. The rationale is that average gene expression of genes in the supernumerary chromosome is on average higher than the corresponding gene in the diploid cell with the same genetic background . Moreover, as previously shown, average ASE calculated with respect to the double allele in trisomic cells (see Methods) is higher than the corresponding ASE in diploid cells. However, since we do not know a priori the allele in two copies (double) the accuracy of the first partition is expected to be quite poor. Therefore the next step is to estimate for each heterozygous site the double allele using ASE imbalance of single cells. More specifically, the average ASE x site is calculated across all single cells tagged as trisomic in the previous iteration and the double allele assigned to each site as the max expressed allele. Once this estimation is done, the improved ASE prediction is used in a new k(=2)-means iteration to improve the partition of trisomic and diploid cells (the second feature is again the average gene expression). K-means cell clustering and double allele estimation are repeated until convergence is reached   are identified and eliminated. First, during the capturing procedure, doublets are identified by visual inspection under the microscope, and eliminated from further analysis. Second, after RNA sequencing and ASE analysis, potential double cells of female individuals are eliminated based on the study of X chromosome haplotype expression. For each cell, the expressed haplotype is estimated by calculating the allelic ratio of each heterozygous site in the X chromosome as identified by whole genome sequencing. Sites in the pseudoautosomal regions  and known escapee genes are a priori excluded. The estimated haplotype of each cell was compared to all the others through correlation based hierarchical clustering. Cells expressing concordant and discordant haplotypes results in a correlation near 1 and −1 respectively. Doublets simultaneously expressing both discordant haplotypes cluster around the absolute correlation of 0.5 and are excluded from further analysis 17, we identified the threshold RPSM = 20 to drastically reduce false positive monoallelic ASE calls Further information on research design is available in the Supplementary InformationReporting SummarySource Data


Raised lipoprotein(a) [Lp(a)] is an important independent cardiovascular risk factor and predictor of adverse outcomes. Challenges remain with regards to the screening, diagnosis and management of this condition. Although further prospective randomised controlled data is required, there is growing evidence suggesting that lowering Lp(a) may reduce the risk of cardiovascular events and ameliorate symptoms. Large scale epidemiological studies have demonstrated that elevated Lp(a) level (>600 mg/L) is an important independent cardiovascular risk factor and predictor of adverse outcomes in atherosclerotic disease2,3. However, the subject of Lp(a) remains a dilemma in contemporary cardiovascular medicine in terms of whom we should screen and subsequently treat, the heterogeneity of available assays and ongoing challenges in management. With new therapeutic options on the horizon, we need to establish some clarity on whom we should screen and treat and determine which therapies will offer the best chance of lowering cardiovascular risk amongst those with raised Lp(a).Although lipoprotein(a) [Lp(a)] was first discovered in the 1960s by Berg4.Large epidemiological studies have erased any doubt that elevated Lp(a) represents a significant independent cardiovascular risk factor. A very large epidemiological multi-centre study on Lp(a) assessed individual records of 126,634 participants in 36 prospective studies4. This suggests that the association is only minimally confounded by conventional risk factors.The association of Lp(a) with coronary heart disease (CHD) was broadly continuous in shape and curvilinear, with no evidence of a threshold. The relative risk of CHD per 3.5-fold higher Lp(a) level adjusted for age and sex only was 1.16 and 1.13 (95% CI [1.09–1.18]) following further adjustment for systolic blood pressure, smoking, history of diabetes and total cholesterol5. Accordingly, amongst 18,720 participants from the European Prospective Investigation of Cancer (EPIC)-Norfolk cohort, Lp(a) levels were associated with future peripheral artery disease (PAD) and coronary artery disease (CAD) events6, and the association between Lp(a) and cardiovascular disease was not modified by LDL cholesterol levels.Similarly, a recent prospective study found that the Lp(a)/CHD risk association did not depend on levels of other CVD risk factors, including LDL cholesterol levels7. This implies that despite aggressive LDL cholesterol reduction, raised Lp(a) still confers residual risk which deserves further attention. Further prospective studies will help to assess the impact of specifically lowering Lp(a) concentrations to potentially reduce this residual risk. Raised Lp(a) is also prevalent amongst patient with refractory angina and was recently demonstrated in 60% of a cohort of patients with refractory angina8, suggesting a potential causal role in this challenging condition.More recently Khera et al. demonstrated that in a cohort of 9612 JUPITER participants with low LDL cholesterol and elevated hsCRP, Lp(a) was a significant determinant of residual cardiovascular risk9). It has been shown that apolipoprotein(a) [apo(a)] size heterogeneity affects the outcome of the immunochemical methods that are still abundantly used to measure Lp(a)10; and that the inaccuracy of Lp(a) values determined by methods sensitive to apo(a) size significantly affects the assessment of individual risk status for coronary artery disease10.The heterogeneity of available Lp(a) assays remains a source of confusion amongst clinicians as well as a confounding factor in clinical research studies. Lp(a) is a genetically determined plasma lipoprotein consisting of a cholesterol-rich LDL particle with one molecule of apolipoprotein B100 and an additional protein, apolipoprotein(a), attached via a disulphide bond, which is proatherogenic and prothrombotic via numerous mechanisms 9. It has11. A monoclonal enzyme-linked immunosorbent assay (ELISA) method that is unaffected by apo(a) size variation was designated as the internationally recommended reference method11. However, much work remains in terms of establishing widespread standardised use of the reference method across centres worldwide.The first World Health Organization/International Federation of Clinical Chemistry and Laboratory Medicine (WHO/IFCC) International Reference Reagent for Lp(a) immunoassays was approved in 2004 with an accuracy-based value assigned in molar concentration (nmol/L) of Lp(a) particles12 recommends Lp(a) screening in individuals with:The 2016 ESC guidelines for the management of dyslipidaemias •Premature CVD (<55 years in men and <65 years women). •Familial hypercholesterolemia. •A family history of premature CVD and/or elevated Lp(a). •Recurrent CVD despite optimal statin treatment. •≥5% 10-year risk of fatal CVD according to SCORE. •Subjects with borderline risk for reclassification of risk.13, raises a plethora of complex questions surrounding cascade screening and thereby the contentious issue of paediatric testing and the optimal timing for screening.The uptake of screening for Lp(a) and awareness of its significance as an important cardiovascular risk factor remains poor, and the vast majority of cardiac and primary care centres are not currently following these recommendations, leaving a substantial proportion of affected individuals undiagnosed. Furthermore, the fact that Lp(a) levels are co-dominantly inherited14, either because Lp(a) is catabolized by alternative mechanisms to the LDL receptor pathway, or because Lp(a) transports PCSK9, which counteracts the statin effect. Nicotinic acid lowers Lp(a) levels by about 25%, most likely by interfering with its production15. However, despite its positive effects on Lp(a) as well as the conventional lipoprotein traits LDL-C, HDLC, and triglycerides; nicotinic acid was not found to reduce cardiovascular risk over and above statin therapy16, hence it is not recommended for preventive treatment.One may argue that we should only screen for a condition if we are prepared to treat it and certainly challenges remain in terms of available therapies for raised Lp(a). To date, therapeutic options to lower Lp(a) levels have been limited. Statins do not have any significant impact on Lp(a) levels17. However, it remains unknown whether this effect contributes to the lowering of cardiovascular risk by PCSK9 inhibitors; which could be resolved by conducting a large phase III clinical outcomes trial assessing the impact of PCSK9 inhibitors in patients with exclusively raised Lp(a), given most patients assessed in studies to date had concomitant raised levels of LDL cholesterol17. Currently lipoprotein apheresis, a lipid-lowering extracorporeal treatment by which atherogenic ApoB100-containing lipoproteins including Lp(a) and LDL cholesterol are removed from blood or plasma (18. Lipoprotein apheresis can acutely decrease Lp(a) by approximately 60–75%19 and is increasingly recognised as the gold standard of treatment for elevated Lp(a). However, some consider lipoprotein apheresis to be cumbersome and prohibitive in terms of the cost, training and infrastructure required to deliver the service.Inhibitors of PCSK9, which are increasingly used for statin resistant hypercholesterolaemia, were found to lower Lp(a) levels by approximately 25%r plasma ; remains20. Similar findings were observed in another retrospective observational study21. In a single-blinded randomised controlled trial in patients with refractory angina and raised lipoprotein(a), we recently demonstrated that lipoprotein apheresis led to significant improvement in myocardial perfusion, atheroma burden, exercise capacity, angina symptoms and quality of life22. This suggests that lipoprotein apheresis should be considered as a therapeutic option in patients with refractory angina and raised Lp(a), especially given the relatively high prevalence of raised Lp(a) amongst patients with this problematic condition8, for whom treatment options remain limited. However, more randomised controlled prospective trial data is required to globally convince clinicians to aggressively treat raised Lp(a).There remains a paucity of prospective randomised controlled trial data which aims to examine the impact of aggressively treating raised Lp(a). An observational cohort study demonstrated that patients who commenced lipoprotein apheresis because of elevated Lp(a) and progressive cardiovascular disease showed a reduction in major adverse coronary events from 0.41 to 0.09 per year23. The most recent generation of antisense oligonucleotides directed against apo(a) mRNA lowers Lp(a) levels dose dependently, by 30 to ≥80% in a single-dose approach, and by 60% to ≥90% in a multi-dose approach24. However, further phase II and III trial data is required to establish the safety of this treatment and the impact on cardiovascular events before it can be established for widespread use.Most recently, antisense technology offers hope and has been used to develop a specific therapy to lower Lp(a) plasma levelsTo conclude, although there is little doubt that raised Lp(a) is an important independent cardiovascular risk factor and predictor of adverse outcomes, challenges remain in terms of screening, testing methods, treatment decisions and access to effective therapy. Lipoprotein apheresis, albeit a cost intensive procedure, is increasingly recognised as the most efficient treatment and the gold standard for management of elevated Lp(a) and there is growing evidence to suggest that it should be utilised more widely to reduce the risk of cardiovascular events and ameliorate symptoms. Although emerging therapies offer hope for the future, further prospective randomised controlled data is required to confirm whether there should be global implementation of aggressive Lp(a) lowering therapy.All authors confirm that they have no competing interests to declare.