Deciphering the physiopathology of neurodevelopmental disorders using brain organoids.

Neurodevelopmental disorders (NDD) encompass a range of conditions marked by abnormal brain development in conjunction with impaired cognitive, emotional, and behavioural functions. Transgenic animal models, mainly rodents, traditionally served as key tools for deciphering the molecular mechanisms driving NDD physiopathology, and significantly contributed to the development of pharmacological interventions aimed at treating these disorders. However, the efficacy of these treatments in humans has proven to be limited, due in part to the intrinsic constraint of animal models to recapitulate the complex development and structure of the human brain but also to the phenotypic heterogeneity found between affected individuals. Significant advancements in the field of induced pluripotent stem cells (iPSC) offer a promising avenue for overcoming these challenges. Indeed, the development of advanced differentiation protocols for generating iPSC-derived brain organoids gives the unprecedented opportunity to explore the human neurodevelopment. This review provides an overview of how 3D brain organoids have been used to investigate various NDD (i.e., Fragile X syndrome, Rett syndrome, Angelman syndrome, microlissencephaly, Prader-Willi syndrome, Timothy Syndrome, tuberous sclerosis syndrome), and elucidate their pathophysiology. We also discuss the benefits and limitations of employing such innovative 3D models compared to animal models and 2D cell culture systems, in the realm of personalized medicine.

Exploring the Alternative Splicing of Long Noncoding RNAs.

Like protein-coding genes, long noncoding RNA (lncRNA) genes are composed of introns and exons. After their transcription, lncRNAs are subject to constitutive and/or alternative splicing. Here, we describe the current knowledge on lncRNA splice variants and their functional implications in cell biology.

MEIS-WNT5A axis regulates development of fourth ventricle choroid plexus.

The choroid plexus (ChP) produces cerebrospinal fluid and forms an essential brain barrier. ChP tissues form in each brain ventricle, each one adopting a distinct shape, but remarkably little is known about the mechanisms underlying ChP development. Here, we show that epithelial WNT5A is crucial for determining fourth ventricle (4V) ChP morphogenesis and size in mouse. Systemic Wnt5a knockout, or forced Wnt5a overexpression beginning at embryonic day 10.5, profoundly reduced ChP size and development. However, Wnt5a expression was enriched in Foxj1-positive epithelial cells of 4V ChP plexus, and its conditional deletion in these cells affected the branched, villous morphology of the 4V ChP. We found that WNT5A was enriched in epithelial cells localized to the distal tips of 4V ChP villi, where WNT5A acted locally to activate non-canonical WNT signaling via ROR1 and ROR2 receptors. During 4V ChP development, MEIS1 bound to the proximal Wnt5a promoter, and gain- and loss-of-function approaches demonstrated that MEIS1 regulated Wnt5a expression. Collectively, our findings demonstrate a dual function of WNT5A in ChP development and identify MEIS transcription factors as upstream regulators of Wnt5a in the 4V ChP epithelium.

RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Corrigendum: RNA m6A methylation regulates the ultraviolet-induced DNA damage response.

This corrects the article DOI: 10.1038/nature21671.

A specific LSD1/KDM1A isoform regulates neuronal differentiation through H3K9 demethylation.

Lysine-specific demethylase 1 (LSD1) has been reported to repress and activate transcription by mediating histone H3K4me1/2 and H3K9me1/2 demethylation, respectively. The molecular mechanism that underlies this dual substrate specificity has remained unknown. Here we report that an isoform of LSD1, LSD1+8a, does not have the intrinsic capability to demethylate H3K4me2. Instead, LSD1+8a mediates H3K9me2 demethylation in collaboration with supervillin (SVIL), a new LSD1+8a interacting protein. LSD1+8a knockdown increases H3K9me2, but not H3K4me2, levels at its target promoters and compromises neuronal differentiation. Importantly, SVIL co-localizes to LSD1+8a-bound promoters, and its knockdown mimics the impact of LSD1+8a loss, supporting SVIL as a cofactor for LSD1+8a in neuronal cells. These findings provide insight into mechanisms by which LSD1 mediates H3K9me demethylation and highlight alternative splicing as a means by which LSD1 acquires selective substrate specificities (H3K9 versus H3K4) to differentially control specific gene expression programs in neurons.

JMJD1C is required for the survival of acute myeloid leukemia by functioning as a coactivator for key transcription factors.

RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program. JMJD1C is directly recruited by RUNX1-RUNX1T1 to its target genes and regulates their expression by maintaining low H3K9 dimethyl (H3K9me2) levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for RUNX1-RUNX1T1's ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors.

LAMP for in-field quantitative assessments of airborne grapevine downy mildew inoculum.

AIMS: Cheap, rapid tools for measuring emissions of Plasmopara viticola sporangia directly in the field are required to protect grapevines efficiently and sustainably against downy mildew. To this end, we adapted an existing loop-mediated isothermal amplification (LAMP) protocol based on ITS2 sequences, coupled with a rotating-arm sampler and simple cell lysis, for the in-field measurement of airborne sporangia of P. viticola. METHODS AND RESULTS: We estimated the sensitivity and specificity of the molecular reaction with an unpurified DNA template in controlled conditions, using the droplet digital PCR (ddPCR) as a reference. We show that the LAMP lower limit of quantification is 3.3 sporangia.m-3 air sampled. Cell lysis in KOH solution was less efficient than CTAB for DNA extraction, but the repeatability of the method was good. We tested this protocol directly in a plot at Chateau Dillon (Blanquefort, France) in which we monitored P. viticola sporangia concentrations from March to October 2020 (88 samples which revealed concentrations ranging from 0 to 243 sporangia.m-3 ). There was a significant quantitative correlation (R2  = 0.52) between ddPCR and LAMP results. CONCLUSION: LAMP analysis of an unpurified DNA matrix is a simple and reliable method for in-field estimations of the concentration of airborne P. viticola sporangia. SIGNIFICANCE AND IMPACT OF THE STUDY: This study constitutes a first step towards the development of a regional grapevine downy mildew monitoring network in the vineyards of Bordeaux.

Transcriptomic modulation in response to high-intensity interval training in monocytes of older women with type 2 diabetes.

PURPOSE: Type 2 diabetes is associated with a higher risk of cardiovascular diseases, lowering the quality of life and increasing mortality rates of affected individuals. Circulating monocytes are tightly involved in the atherosclerosis process leading to cardiovascular diseases (CVD), and their inflammatory profile can be modified by exercise. The objective was to exploratory identify genes associated with CVD that could be regulated by high-intensity interval training (HIIT) in monocytes of type 2 diabetes patients. METHODS: Next-generation RNA sequencing (RNA-seq) analyses were conducted on isolated circulating monocytes (CD14+) of six women aged 60 and over with type 2 diabetes who completed a 12-week supervised HIIT intervention on a treadmill. RESULTS: Following the intervention, a reduction of resting diastolic blood pressure was observed. Concomitant with this result, 56 genes were found to be downregulated following HIIT intervention in isolated monocytes. A large proportion of the regulated genes was involved in cellular adhesion, migration and differentiation into an "atherosclerosis-specific" macrophage phenotype. CONCLUSION: The downregulation of transcripts in monocytes globally suggests a favorable cardiovascular effect of the HIIT in older women with type 2 diabetes. In the context of precision medicine and personalized exercise prescription, shedding light on the fundamental mechanisms underlying HIIT effects on the gene profile of immune cells is essential to develop efficient nonpharmacological strategies to prevent CVD in high-risk population.

QTL mapping in Fusarium graminearum identified an allele of FgVe1 involved in reduced aggressiveness.

Fusarium graminearum is one of the most frequent causal agents of the Fusarium Head Blight, a cereal disease spread throughout the world, reducing grain production and quality. F. graminearum isolates are genetically and phenotypically highly diverse. Notably, remarkable variations of aggressiveness between isolates have been observed, which could reflect an adaptive potential of this pathogen. In this study, we aimed to characterize the genetic basis of aggressiveness variation observed in an F1 population (n = 94), for which genome sequences of both parental strains are available. Aggressiveness was assessed by a panel of in planta and in vitro proxies during two phenotyping trials including, among others, disease severity and mycotoxin accumulation in wheat spike. One major and single QTL was mapped for all the traits measured, on chromosome I, that explained up to 90% of the variance for disease severity. The confidence interval at the QTL spanned 1.2 Mb and contained 428 genes on the reference genome. Of these, four candidates were selected based on the postulate that a non-synonymous mutation affecting protein function may be responsible for phenotypic differences. Finally, a new mutation was identified and functionally validated in the gene FgVe1, coding for a velvet protein known to be involved in pathogenicity and secondary metabolism production in several fungi.

Hyperactivation of monocytes and macrophages in MCI patients contributes to the progression of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease ultimately manifesting as clinical dementia. Despite considerable effort and ample experimental data, the role of neuroinflammation related to systemic inflammation is still unsettled. While the implication of microglia is well recognized, the exact contribution of peripheral monocytes/macrophages is still largely unknown, especially concerning their role in the various stages of AD. OBJECTIVES: AD develops over decades and its clinical manifestation is preceded by subjective memory complaints (SMC) and mild cognitive impairment (MCI); thus, the question arises how the peripheral innate immune response changes with the progression of the disease. Therefore, to further investigate the roles of monocytes/macrophages in the progression of AD we assessed their phenotypes and functions in patients at SMC, MCI and AD stages and compared them with cognitively healthy controls. We also conceptualised an idealised mathematical model to explain the functionality of monocytes/macrophages along the progression of the disease. RESULTS: We show that there are distinct phenotypic and functional changes in monocyte and macrophage populations as the disease progresses. Higher free radical production upon stimulation could already be observed for the monocytes of SMC patients. The most striking results show that activation of peripheral monocytes (hyperactivation) is the strongest in the MCI group, at the prodromal stage of the disease. Monocytes exhibit significantly increased chemotaxis, free radical production, and cytokine production in response to TLR2 and TLR4 stimulation. CONCLUSION: Our data suggest that the peripheral innate immune system is activated during the progression from SMC through MCI to AD, with the highest levels of activation being in MCI subjects and the lowest in AD patients. Some of these parameters may be used as biomarkers, but more holistic immune studies are needed to find the best period of the disease for clinical intervention.

Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network.

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.

Editorial Commentary: Taming Hard Problems With Soft Tissues? Anterior Shoulder Instability After Prior Bone Block Stabilization.

Recurrent anterior shoulder instability after prior bone block stabilization is a therapeutically challenging condition. Historically, repeated glenoid bone grafting has been advocated in such cases to achieve lasting shoulder stability. However, recent insights into the pathomechanics of shoulder instability, especially regarding bipolar bony lesions, have renewed our interest in the arthroscopic treatment of Hill-Sachs lesions and the role of soft-tissue interventions after bone block procedures.

Landscape of genomic diversity and host adaptation in Fusarium graminearum.

BACKGROUND: Fusarium graminearum is one of the main causal agents of the Fusarium Head Blight, a worldwide disease affecting cereal cultures, whose presence can lead to contaminated grains with chemically stable and harmful mycotoxins. Resistant cultivars and fungicides are frequently used to control this pathogen, and several observations suggest an adaptation of F. graminearum that raises concerns regarding the future of current plant disease management strategies. To understand the genetic basis as well as the extent of its adaptive potential, we investigated the landscape of genomic diversity among six French isolates of F. graminearum, at single-nucleotide resolution using whole-genome re-sequencing. RESULTS: A total of 242,756 high-confidence genetic variants were detected when compared to the reference genome, among which 96% are single nucleotides polymorphisms. One third of these variants were observed in all isolates. Seventy-seven percent of the total polymorphism is located in 32% of the total length of the genome, comprising telomeric/subtelomeric regions as well as discrete interstitial sections, delineating clear variant enriched genomic regions- 7.5 times in average. About 80% of all the F. graminearum protein-coding genes were found polymorphic. Biological functions are not equally affected: genes potentially involved in host adaptation are preferentially located within polymorphic islands and show greater diversification rate than genes fulfilling basal functions. We further identified 29 putative effector genes enriched with non-synonymous effect mutation. CONCLUSIONS: Our results highlight a remarkable level of polymorphism in the genome of F. graminearum distributed in a specific pattern. Indeed, the landscape of genomic diversity follows a bi-partite organization of the genome according to polymorphism and biological functions. We measured, for the first time, the level of sequence diversity for the entire gene repertoire of F. graminearum and revealed that the majority are polymorphic. Those assumed to play a role in host-pathogen interaction are discussed, in the light of the subsequent consequences for host adaptation. The annotated genetic variants discovered for this major pathogen are valuable resources for further genetic and genomic studies.

Mutations in the intellectual disability gene KDM5C reduce protein stability and demethylase activity.

Mutations in KDM5C are an important cause of X-linked intellectual disability in males. KDM5C encodes a histone demethylase, suggesting that alterations in chromatin landscape may contribute to disease. We used primary patient cells and biochemical approaches to investigate the effects of patient mutations on KDM5C expression, stability and catalytic activity. We report and characterize a novel nonsense mutation, c.3223delG (p.V1075Yfs*2), which leads to loss of KDM5C protein. We also characterize two KDM5C missense mutations, c.1439C>T (p.P480L) and c.1204G>T (p.D402Y) that are compatible with protein production, but compromise stability and enzymatic activity. Finally, we demonstrate that a c.2T>C mutation in the translation initiation codon of KDM5C results in translation re-start and production of a N-terminally truncated protein (p.M1_E165del) that is unstable and lacks detectable demethylase activity. Patient fibroblasts do not show global changes in histone methylation but we identify several up-regulated genes, suggesting local changes in chromatin conformation and gene expression. This thorough examination of KDM5C patient mutations demonstrates the utility of examining the molecular consequences of patient mutations on several levels, ranging from enzyme production to catalytic activity, when assessing the functional outcomes of intellectual disability mutations.

A short Gfi-1B isoform controls erythroid differentiation by recruiting the LSD1-CoREST complex through the dimethylation of its SNAG domain.

Gfi-1B is a transcriptional repressor essential for the regulation of erythropoiesis and megakaryopoiesis. Here we identify Gfi-1B p32, a Gfi-1B isoform, as essential for erythroid differentiation. Gfi-1B p32 is generated by alternative splicing and lacks the two first zinc finger domains of the protein. Selective knock down of Gfi-1B p32 compromises erythroid differentiation, whereas its ectopic expression induces erythropoiesis in the absence of erythropoietin. Gfi-1B p32 isoform binds to Gfi-1B target gene promoters and associates with the LSD1-CoREST repressor complex more efficiently than the major Gfi-1B p37 isoform. Furthermore, we show that Gfi-1B includes a KSKK motif in its SNAG domain, which recruits the repressor complex only when dimethylated on lysine 8. Mutation of lysine 8 prevents Gfi-1B p32-induced erythroid development. Our results thus highlight a key role for the alternatively spliced Gfi-1B p32 isoform in erythroid development.

Assessing tissue mechanical properties: Development of a custom-made tensile device and application on rodents sciatic nerves.

The development of biomaterials such as synthetic scaffolds for peripheral nerve regeneration requires a precise knowledge of the mechanical properties of the nerve in physiological-like conditions. Mechanical properties (Young's modulus, maximum stress and strain at break) for peripheral nerves are scarce and large discrepancies are observed in between reports. This is due in part to the absence of a robust testing device for nerves. To overcome this limitation, a custom-made tensile device (CMTD) has been built. To evaluate its reproducibility and accuracy, the imposed speed and distance over measured speed and distance was performed, followed by a validation using poly(dimethylsiloxane) (PDMS), a commercial polymer with established mechanical properties. Finally, the mechanical characterization of rodents (mice and rats) sciatic nerves using the CMTD was performed. Mouse and rat sciatic nerves Young's modulus were 4.57 ± 2.04 and 19.2 ± 0.86 MPa respectively. Maximum stress was 1.26 ± 0.56 MPa for mice and 3.81 ± 1.84 MPa for rats. Strain at break was 53 ± 17% for mice and 32 ± 12% for rats. The number of axons per sciatic nerve was found to be twice higher for rats. Statistical analysis of the measured mechanical properties revealed no sex-related trends, for both mice and rats (except for mouse maximum stress with p=0.03). Histological evaluation of rat sciatic nerve corroborated these findings. By developing a robust CMTD to establish the key mechanical properties (Young's modulus, maximum stress and strain at break) values for rodents sciatic nerves, our work represent an essential step toward the development of better synthetic scaffolds for peripheral nerve regeneration.

Re-evaluating the impact of alternative RNA splicing on proteomic diversity.

Alternative splicing (AS) constitutes a mechanism by which protein-coding genes and long non-coding RNA (lncRNA) genes produce more than a single mature transcript. From plants to humans, AS is a powerful process that increases transcriptome complexity. Importantly, splice variants produced from AS can potentially encode for distinct protein isoforms which can lose or gain specific domains and, hence, differ in their functional properties. Advances in proteomics have shown that the proteome is indeed diverse due to the presence of numerous protein isoforms. For the past decades, with the help of advanced high-throughput technologies, numerous alternatively spliced transcripts have been identified. However, the low detection rate of protein isoforms in proteomic studies raised debatable questions on whether AS contributes to proteomic diversity and on how many AS events are really functional. We propose here to assess and discuss the impact of AS on proteomic complexity in the light of the technological progress, updated genome annotation, and current scientific knowledge.

Distinct regulatory functions and biological roles of lncRNA splice variants.

Alternative splicing (AS) of RNA molecules is a key contributor to transcriptome diversity. In humans, 90%-95% of multi-exon genes produce alternatively spliced RNA transcripts. Therefore, every single gene has the opportunity of producing multiple splice variants, including long non-coding RNA (lncRNA) genes that undergo RNA maturation steps such as conventional and alternative splicing. Emerging evidence suggests significant roles for these lncRNA splice variants in many aspects of cell biology. Differential changes in expression of specific lncRNA splice variants have also been associated with many diseases including cancer. This review covers the current knowledge on this emerging topic of investigation. We provide exclusive insights on the AS landscape of lncRNAs and also describe at the molecular level the functional relevance of lncRNA splice variants, i.e., RNA-based differential functions, production of micropeptides, and generation of circular RNAs. Finally, we discuss exciting perspectives for this emerging field and outline the work required to further develop research endeavors in this field.