The molecular programs coordinating inhibitory interneuron migration, maturation, and survival during forebrain development remain incompletely understood. Here we investigate ARHGEF6, a RAC1/CDC42 guanine nucleotide exchange factor linked to X-linked intellectual disability (XLID46) and previously studied only at postsynaptic compartments, and reveal an earlier, conserved role in forebrain interneuron development. ARHGEF6 is selectively enriched in the inhibitory lineage during the peak of interneuron generation and migration. Its loss in mice reduces the number of cortical and hippocampal interneurons, disrupts tangential migration, increases developmental cell death, and impairs morphological and electrophysiological maturation. Strikingly, ARHGEF6 -knockout human iPSC-derived organoids and assembloids mirror these deficits exhibiting increased apoptosis, reduced neuronal output, disorganized growth cones, impaired neurite branching, and disrupted migratory dynamics. These cross-species findings reframe ARHGEF6 as an early, essential orchestrator of inhibitory circuit assembly and reveal a conserved cytoskeletal program whose disruption produces the excitatory-inhibitory imbalance linked to cognitive dysfunction.

Summary Psychedelic compounds such as lysergic acid diethylamide (LSD) are increasingly studied for their neuroplastic effects and potential relevance to brain aging and neurodegeneration. However, the molecular mechanisms linking psychedelic-induced plasticity to age-associated cognitive decline remain unclear. Brain aging and dementia are characterized by coordinated transcriptional programs that underlie synaptic dysfunction and altered neuron-glia interactions. If psychedelic-induced plasticity engages opposing molecular programs, it could counteract these conserved trajectories. In computational drug discovery, this concept has been formalized as the principle of transcriptional signature reversal, whereby compounds inducing gene expression states opposite to disease-associated programs may exert a therapeutic effect by counteracting disease-associated phenotypes. Here, we combine cross-species transcriptomic analyses with experimental validation to test whether LSD opposes conserved signatures of brain aging and dementia. By comparing transcriptional profiles induced by chronic LSD treatment in rodents with age- and dementia-associated gene expression changes in the human prefrontal cortex, we show that LSD induces gene expression patterns strongly anti-correlated with aging and neurodegeneration programs. This reversal is specific compared to other pharmacological perturbations and is reproducible across datasets and species. Moreover, LSD counteracts amyloid-β-induced structural and molecular alterations in primary cortical neurons, linking transcriptomic opposition to functional rescue under neurodegenerative stress. Together, our findings suggest that LSD modulates molecular and cellular pathways associated with brain aging and neurodegeneration, linking systems-level gene expression changes to structural and functional resilience in neurodegeneration-relevant contexts.

Background Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. Methods We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt’s and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment’ synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. Results We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. Conclusions The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Functional genomics screens in human induced pluripotent stem cells (hiPSCs) remain challenging despite their transformative potential. We developed iPS2-seq: an inducible, clone-aware screening platform that enables phenotype-agnostic, single-cell resolved dissection of loss-of-function effects in hiPSC derivatives, including complex multicellular models such as organoids. iPS2-seq distinguishes true perturbation effects from genetic and epigenetic variability. It supports pooled and arrayed formats, integrates with microfluidic or split-pool single-cell RNA sequencing, and extends to multi-omic profiling of chromatin and proteins. A dedicated pipeline, catcheR, streamlines design and analysis. The platform enables stage-specific follow-up dissection of screen hits. We demonstrate this by targeting congenital heart disease-associated genes in monolayer cardiomyocytes and organoids. This reveals that epigenetic neuroectodermal priming interferes with germ layer differentiation in specific clones. Accounting for this bias, we show that SMAD2 controls cardiac progenitor specification, with knockdown redirecting cells toward fibroblast and epicardial fates. iPS2-seq unlocks rigorous functional genomics in hiPSC-based models.

The impairment of development/migration of hypothalamic gonadotropin-releasing hormone (GnRH) neurons is the main cause of Kallmann's syndrome (KS), an inherited disorder characterized by hypogonadism, anosmia, and other developmental defects. Olfactorin is an extracellular matrix protein encoded by the UMODL1 (uromodulin-like 1) gene expressed in the mouse olfactory region along the migratory route of GnRH neurons. It shares a combination of WAP and FNIII repeats, expressed in complementary domains, with anosmin-1, the product of the ANOS1 gene, identified as the causative of KS. In the present study, we have investigated the effects of olfactorin in vitro and in vivo models. The results show that olfactorin exerts an anosmin-1-like strong chemoattractant effect on mouse-immortalized GnRH neurons (GN11 cells) through the activation of the FGFR and MAPK pathways. In silico analysis of olfactorin and anosmin-1 reveals a satisfactory similarity at the N-terminal region for the overall arrangement of corresponding WAP and FNIII domains and marked similarities between WAP domains' binding modes of interaction with the resolved FGFR1-FGF2 complex. Finally, in vivo experiments show that the down-modulation of the zebrafish z-umodl1 gene (orthologous of UMODL1) in both GnRH3:GFP and omp(2k):gap-CFPrw034 transgenic zebrafish strains leads to a clear disorganization and altered fasciculation of the neurites of GnRH3:GFP neurons crossing at the anterior commissure and a significant increase in olfactory CFP + fibers with altered trajectory. Thus, our study shows olfactorin as an additional factor involved in the development of olfactory and GnRH systems and proposes UMODL1 as a gene worthy of diagnostic investigation in KS.

In the human brain, long non-coding RNAs (lncRNAs) are widely expressed in an exquisitely temporally and spatially regulated manner, thus suggesting their contribution to normal brain development and their probable involvement in the molecular pathology of neurodevelopmental disorders (NDD). Bypassing the classic protein-centric conception of disease mechanisms, some studies have been conducted to identify and characterize the putative roles of non-coding sequences in the genetic pathogenesis and diagnosis of complex diseases. However, their involvement in NDD, and more specifically in intellectual disability (ID), is still poorly documented and only a few genomic alterations affecting the lncRNAs function and/or expression have been causally linked to the disease endophenotype. Considering that a significant fraction of patients still lacks a genetic or molecular explanation, we expect that a deeper investigation of the non-coding genome will unravel novel pathogenic mechanisms, opening new translational opportunities. Here, we present evidence of the possible involvement of many lncRNAs in the etiology of different forms of ID and NDD, grouping the candidate disease-genes in the most frequently affected cellular processes in which ID-risk genes were previously collected. We also illustrate new approaches for the identification and prioritization of NDD-risk lncRNAs, together with the current strategies to exploit them in diagnosis.

Delayed wound healing and chronic skin lesions represent a major health problem. Over the past years, growth factors mediated by platelet-rich plasma (PRP) and cell-based therapies were developed as effective and affordable treatment able to improve wound healing capacity. We have advanced existing concepts to develop a highly efficient high-throughput protocol with proven application for the isolation of PRP and pro-angiogenic cells (Angio PRP ). This protocol outlines the effectiveness of Angio PRP in promoting the critical healing process including wound closure, re-epithelialization, granulation tissue growth, and blood vessel regeneration. We coupled this effect with normalization of mechanical properties of rescued mouse wounds, which is sustained by a correct arrangement of elastin and collagen fibers. Proteomic analysis of treated wounds demonstrated a fingerprint of Angio PRP based on the up-regulation of detoxification pathway of glutathione metabolism, correlated to a decrease in inflammatory response. Overall, these results have enabled us to provide a framework for how Angio PRP supports wound healing, opening avenues for further clinical advances.

GTPases of the Rho family are components of signaling pathways linking extracellular signals to the control of cytoskeleton dynamics. Among these, RAC1 plays key roles during brain development, ranging from neuronal migration to neuritogenesis, synaptogenesis, and plasticity. RAC1 activity is positively and negatively controlled by guanine nucleotide exchange factors (GEFs), guanosine nucleotide dissociation inhibitors (GDIs), and GTPase-activating proteins (GAPs), but the specific role of each regulator in vivo is poorly known. ARHGAP15 is a RAC1-specific GAP expressed during development in a fraction of migrating cortical interneurons (CINs) and in the majority of adult CINs. During development, loss of ARHGAP15 causes altered directionality of the leading process of tangentially migrating CINs, along with altered morphology in vitro. Likewise, time-lapse imaging of embryonic CINs revealed a poorly coordinated directional control during radial migration, possibly due to a hyper-exploratory behavior. In the adult cortex, the observed defects lead to subtle alteration in the distribution of CALB2-, SST-, and VIP-positive interneurons. Adult Arhgap15-knock-out mice also show reduced CINs intrinsic excitability, spontaneous subclinical seizures, and increased susceptibility to the pro-epileptic drug pilocarpine. These results indicate that ARHGAP15 imposes a fine negative regulation on RAC1 that is required for morphological maturation and directional control during CIN migration, with consequences on their laminar distribution and inhibitory function.

p140Cap, encoded by the gene SRCIN1 (SRC kinase signaling inhibitor 1), is an adaptor/scaffold protein highly expressed in the mouse brain, participating in several pre- and post-synaptic mechanisms. p140Cap knock-out (KO) female mice show severe hypofertility, delayed puberty onset, altered estrus cycle, reduced ovulation, and defective production of luteinizing hormone and estradiol during proestrus. We investigated the role of p140Cap in the development and maturation of the hypothalamic gonadotropic system. During embryonic development, migration of Gonadotropin-Releasing Hormone (GnRH) neurons from the nasal placode to the forebrain in p140Cap KO mice appeared normal, and young p140Cap KO animals showed a normal number of GnRH-immunoreactive (-ir) neurons. In contrast, adult p140Cap KO mice showed a significant loss of GnRH-ir neurons and a decreased density of GnRH-ir projections in the median eminence, accompanied by reduced levels of GnRH and LH mRNAs in the hypothalamus and pituitary gland, respectively. We examined the number of kisspeptin (KP) neurons in the rostral periventricular region of the third ventricle, the number of KP-ir fibers in the arcuate nucleus, and the number of KP-ir punctae on GnRH neurons but we found no significant changes. Consistently, the responsiveness to exogenous KP in vivo was unchanged, excluding a cell-autonomous defect on the GnRH neurons at the level of KP receptor or its signal transduction. Since glutamatergic signaling in the hypothalamus is critical for both puberty onset and modulation of GnRH secretion, we examined the density of glutamatergic synapses in p140Cap KO mice and observed a significant reduction in the density of VGLUT-ir punctae both in the preoptic area and on GnRH neurons. Our data suggest that the glutamatergic circuitry in the hypothalamus is altered in the absence of p140Cap and is required for female fertility.

Intellectual disability (ID) is a pathological condition characterized by limited intellectual functioning and adaptive behaviors. It affects 1–3% of the worldwide population, and no pharmacological therapies are currently available. More than 1000 genes have been found mutated in ID patients pointing out that, despite the common phenotype, the genetic bases are highly heterogeneous and apparently unrelated. Bibliomic analysis reveals that ID genes converge onto a few biological modules, including cytoskeleton dynamics, whose regulation depends on Rho GTPases transduction. Genetic variants exert their effects at different levels in a hierarchical arrangement, starting from the molecular level and moving toward higher levels of organization, i.e., cell compartment and functions, circuits, cognition, and behavior. Thus, cytoskeleton alterations that have an impact on cell processes such as neuronal migration, neuritogenesis, and synaptic plasticity rebound on the overall establishment of an effective network and consequently on the cognitive phenotype. Systems biology (SB) approaches are more focused on the overall interconnected network rather than on individual genes, thus encouraging the design of therapies that aim to correct common dysregulated biological processes. This review summarizes current knowledge about cytoskeleton control in neurons and its relevance for the ID pathogenesis, exploiting in silico modeling and translating the implications of those findings into biomedical research.

BackgroundDelayed wound healing and chronic skin lesions represent a major health problem. Over the past years, growth factors mediated by platelet-rich plasma (PRP) and cell-based therapies were developed as effective and affordable treatment able to improve wound healing capacity. However, the precise molecular mechanism through which PRP exhibits its beneficial effects remains unrevealed. Herein we show that a combination of PRP and pro-angiogenic cells (Angio PRP ) could exert a synergistic positive effect on keratinocyte proliferation and angiogenesis accelerating wound healing.MethodWe designed a sterile and closed class IIa device (NovySep) for single use only to collect blood-derived mononucleated cells and the plasma phase after centrifugation without opening the system. We performed in vitro and in vivo wound healing experiments to assess the angiogenic potential of Angio PRP ; we evaluated the extracellular matrix remodeling and the physical properties of treated wounds through mechanical (stress-strain test) and proteomics analysis.ResultAngio PRP enhanced wound healing by promoting uniform regeneration of the basal and granular layers and vessel remodeling. We coupled this effect with normalization of mechanical properties of rescued mouse wounds, which is sustained by a correct arrangement of elastin and collagen fibers. The network analysis-based protein–protein interactions of Angio PRP -treated wounds demonstrated a fingerprint of Angio PRP -related proteins that may provide the signal for a faster wound healing response which include Caveolin and EGFR/TGFβ/β-catenin pathways.ConclusionA combined treatment composed of PRP and a pool of pro-angiogenic/keratogenic cells (Angio PRP ) may provide a more integrated method to supports wound healing, by promoting a cascade of events leading to the reduction of TGFβ1/β-catenin up-downstream signaling pathways. The molecular mechanism undergoing the support of Angio PRP to wound healing opens new perspectives in the treatment of skin injuries.

ESCRTing lenses away from senescence ESCRT proteins control membrane fusion in various key cellular processes, but the mechanisms involved are still incompletely understood. Gulluni et al . report that ESCRT recruitment at the cytokinetic bridge is mediated by the binding of an ESCRT-II subunit to the signaling lipid phosphatidylinositol 3,4-bisphosphate (see the Perspective by Brill and Wilde). This pathway acts in parallel to a known cascade driven by a protein called ALIX, but its failure is sufficient to lead to premature senescence in the lens of fish, mouse, and human eyes, where ALIX is expressed at lower levels. These results point to an evolutionarily conserved pathway for the cell-specific control of cytokinesis that serves to protect from senescence and the early onset of cataracts. —SMH

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron’s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

Alternative polyadenylation (APA) is a widespread mechanism involving about half of the expressed genes, resulting in varying lengths of the 3'untranslated region (3'UTR). Variations in length and sequence of the 3'UTR may underlie changes of post-transcriptional processing, localization, miRNA targeting and stability of mRNAs. During embryonic development a large array of mRNAs exhibit APA, with a prevalence of the longer 3'UTR versions in differentiating cells. Little is known about polyA+ site usage during differentiation of mammalian neural progenitors. Here we exploit a model of adherent neural stem (ANS) cells, which homogeneously and efficiently differentiate into GABAergic neurons. RNAseq data shows a global trend towards lengthening of the 3'UTRs during differentiation. Enriched expression of the longer 3'UTR variants of Pes1 and Gng2 was detected in the mouse brain in areas of cortical and subcortical neuronal differentiation, respectively, by two-probes fluorescent in situ hybridization (FISH). Among the coding genes upregulated during differentiation of ANS cells we found Elavl3, a neural-specific RNA-binding protein homologous to Drosophila Elav. In the insect, Elav regulates polyA+ site choice while interacting with paused Pol-II promoters. We tested the role of Elavl3 in ANS cells, by silencing Elavl3 and observed consistent changes in 3'UTR length and delayed neuronal differentiation. These results indicate that choice of the polyA+ site and lengthening of 3'UTRs is a possible additional mechanism of posttranscriptional RNA modification involved in neuronal differentiation.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 x 10(-6)). Three heterozygous PTVs (p.Lys62*, p.Tyr128Thrfs*55, and p.Trp469*, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.

Forebrain GABAergic neurons, the main inhibitory type of neuron in the cortex and hippocampus, represent a highly heterogeneous cell population that has been implicated in the predisposition to epilepsy and the onset of seizure.