Biology subjects

Di Donato, N.

Publications and source records attributed to Di Donato, N..

9 recordsLinked to original sources

bioRxiv2026

The distinct role of actin isoforms on mechanosensing-based maturation of hiPSC-derived neurons unveiled by isoform-specific mutations

Neuronal maturation is governed by the integration of intrinsic programs, such as gene regulation and cellular metabolism, with external mechanical cues. The conversion of mechanical forces into developmental responses (mechanotransduction) requires mechanical coupling of neurons to the extracellular matrix and neighboring cells. This coupling is mediated by the actin cytoskeleton and associated transmembrane protein complexes. However, the specific role of the {beta}- and {gamma}-actin isoforms in mechanotransduction, and the functional consequences of their pathogenic mutations, remain poorly understood. To address this question, we combined optical tweezers mechanics with immunofluorescence in human iPSC-derived neural progenitor cells (NPCs) and postmitotic neurons (NCs), both wild-type (WT) and carrying pathogenic mutations in {beta}-actin (R196H) or {gamma}-actin (T203M) associated with Baraitser-Winter cerebrofrontofacial (BWCFF) syndrome. Using membrane tether elongation as a proxy for early protrusion formation, we found that {gamma}-T203M NPCs require a fourfold lower force than WT NPCs and, upon differentiation, fail to develop the neurite-filopodia architecture retaining abundant immature protrusions In contrast, {beta}-R196H NPCs exhibit only reduction of cell surface tension associated with increased neuron fragility, without pronounced protrusion abnormalities. These results reveal non-redundant roles of {beta}- and {gamma}-actin isoforms in neuronal mechanotransduction and provide mechanistic insight into the pathogenesis of BWCFF syndrome.

neuroscience↗

bioRxiv2026

Coordinated neural progenitor adaptations drive primate neocortical downscaling

Most primates possess a large, highly folded (gyrencephalic) neocortex, a feature present in the primate common ancestor. In contrast, several New World monkey species display a comparatively small and largely smooth, unfolded (lissencephalic) neocortex. One prominent example is the common marmoset, an increasingly popular model in neuroscience. This phenotype likely reflects an evolutionary reduction from the ancestral primate condition, implying modifications in neurodevelopmental programs. One essential basis for neocortical development is the activity and behavior of neural progenitor cells (NPCs). Here, we identify coordinated adaptations in NPC biology that bias neurodevelopmental trajectories toward neocortical downscaling. By combining marmoset and human cerebral organoids with analyses of fetal marmoset neocortical tissue and previously published histological data, we uncover multiple adaptations in apical and basal progenitors that converge on reduced progenitor capacity, alter early progenitor dynamics, and likely constrain neuronal output, thereby limiting the size and folding of the marmoset neocortex.

evolutionary biology↗

bioRxiv2026

Non-muscle actinopathy-associated loss-of-function actin variants modulate cytoskeletal reorganization

Variants in ACTB gene encoding for cytoplasmic {beta}-actin result in a group of rare disorders called non-muscle actinopathies (NMA). We investigated the cellular effects of a missense variant, G302A, and a four-amino-acid deletion, S338-I341, associated with the subgroup of NMA - ACTB pLoF (predicted loss-of-function) disorder in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the mechanically-induced reorganization of the actin cytoskeleton, we found that G302A mutant cells showed more dense actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of the deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was drastically reduced. We could partially rescue this fascinating effect by overexpressing the active form of the formin mDia. Our findings open the possibility to validate the cellular phenotype in the most affected patients cells, in neurons.

biophysics↗

bioRxiv2025

The Baraitser-Winter Cerebrofrontofacial Syndrome recurrent R196H variant in cytoplasmic β-actin impairs its cellular polymerization and stability

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non-muscle actinopathies. Among them, Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. The exact consequences of the mutation on actins properties, however, are not fully understood. Here we explored the cellular effects of the R196H mutation in patient-derived fibroblasts. We show that the heterozygous mutation causes an actin polymerization defect in cells, leading to a fifty percent decrease in filamentous (F-) actin content. This effect can be rescued by the addition of the actin-polymerizing and stabilizing drug, jasplakinolide. We observed no significant defects either in the organization of the cellular actin cytoskeleton, analyzed by superresolution (STED) microscopy, or in the structure of purified filaments stabilized with phalloidin, explored with atomic force microscopy (AFM). The reduced F-actin content correlated with an approximately fourfold reduction in the stiffness of patient-derived cells probed with AFM. Manipulating the cells by mechanical forces through the application of the Dual Laser Optical Tweezers (DLOT) technique suggests that the mutation weakens the attachment of cytoskeletal actin to the plasma membrane. Inducing dynamical reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Based on the existing cofilin-actin structures, the binding of cofilin may weaken the interaction of the neighboring residue E195 with K113, one of the lateral contacts stabilizing the filament. Thus, the mutation possibly exerts its effect through the destabilization of the interfilament interactions, potentially interfering allosterically with cofilin binding during actin depolymerization.

biophysics↗

bioRxiv2024

Classification of human actin pathological variants using C. elegans CRISPR-generated models

Actin plays a crucial role in diverse physiological processes by forming dynamic networks that determine cellular shape and mechanical properties. Non-Muscle Actinopathies (NMA) are rare diseases caused by de novo variants in human cytoskeletal {beta}-actin (ACTB) and {gamma}-actin (ACTG1) genes, ranging from missense mutations to whole gene deletions. Currently, the high clinical variability and genotype-phenotype correlations in NMA remain largely unresolved. To address this concern, we inserted nine mutations identified in patients in the C. elegans cytoplasmic actin orthologue act-2 and performed a quantitative multiscale characterization of these animal models. We uncovered various perturbations including micro-scale actin network defects, cell-scale abnormalities, morphogenesis failure, and weaker behavioural phenotypes. Notably, the severity of the observed defects correlates with the severity of patients symptoms. Thus, we provide evidence that such C. elegans models are relevant to investigate the mechanisms underlying NMA physiopathology and could ultimately be used to screen for therapeutic strategies.

cell biology↗

bioRxiv2022

Cerebral organoids expressing mutant actin genes reveal cellular mechanism underlying microcephalic cortical malformation

Actins are structural cytoskeletal proteins playing crucial roles in multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma- cytoskeletal actin isoforms, respectively, cause a broad spectrum of neurodevelopmental disorders, with microcephaly as the most frequent one. Here we used patient-derived cerebral organoids to gain insight into the pathogenesis underlying this cortical malformation. Cerebral organoids from induced pluripotent stem cells (iPSCs) of patients with the Baraitser-Winter- CerebroFrontoFacial syndrome (BWCFF-S), expressing either an ACTB or an ACTG1 missense mutation, are reduced in size, showing a thinner ventricular zone (VZ). This decrease in VZ progenitors is in turn associated with a striking change in the orientation of their cleavage plane from predominantly vertical (control) to predominantly horizontal (BWCFF-S), which is incompatible with increasing VZ progenitor abundance. Various cytoskeletal and morphological irregularities of BWCFF-S VZ progenitors, notably in the apical region of these cells, seemingly contribute to their predominantly horizontal cleavage plane orientation. Our results provide insight into the cell biological basis of the microcephaly associated with BWCFF-S caused by actin mutations.

neuroscience↗

bioRxiv2019

Scientific evaluation of negative exome sequencing followed by systematic scoring of candidate genes to decipher the genetics of neurodevelopmental disorders

BackgroundDeciphering the monogenetic causes of neurodevelopmental disorders (NDD) is an important milestone to offer personalized care. But the plausibility of reported candidate genes in exome studies often remains unclear, which slows down progress in the field.\n\nMethodsWe performed exome sequencing (ES) in 198 cases of NDD. Cases that remained unresolved (n=135) were re-investigated in a research setting. We established a candidate scoring system (CaSc) based on 12 different parameters reflecting variant and gene attributes as well as current literature to rank and prioritize candidate genes.\n\nResultsIn this cohort, we identified 158 candidate variants in 148 genes with CaSc ranging from 2 to 11.7. Only considering the top 15% of candidates, 14 genes were already published or funneled into promising validation studies.\n\nConclusionsWe promote that in an approach of case by case re-evaluation of primarily negative ES, systematic and standardized scoring of candidate genes can and should be applied. This simple framework enables better comparison, prioritization, and communication of candidate genes within the scientific community. This would represent an enormous benefit if applied to the tens of thousands of negative ES performed in routine diagnostics worldwide and speed up deciphering the monogenetic causes of NDD.

genetics↗

bioRxiv2018

Pathogenic DDX3X mutations impair RNA metabolism and neurogenesis during fetal cortical development

De novo germline mutations in the RNA helicase DDX3X account for 1-3% of unexplained intellectual disability (ID) cases in females, and are associated with autism, brain malformations, and epilepsy. Yet, the developmental and molecular mechanisms by which DDX3X mutations impair brain function are unknown. Here we use human and mouse genetics, and cell biological and biochemical approaches to elucidate mechanisms by which pathogenic DDX3X variants disrupt brain development. We report the largest clinical cohort to date with DDX3X mutations (n=78), demonstrating a striking correlation between recurrent dominant missense mutations, polymicrogyria, and the most severe clinical outcomes. We show that Ddx3x controls cortical development by regulating neuronal generation and migration. Severe DDX3X missense mutations profoundly disrupt RNA helicase activity and induce ectopic RNA-protein granules and aberrant translation in neural progenitors and neurons. Together, our study demonstrates novel mechanisms underlying DDX3X syndrome, and highlights roles for RNA-protein aggregates in the pathogenesis of neurodevelopmental disease.

genetics↗

bioRxiv2018

Thrombocytopenia Microcephaly Syndrome - a novel phenotype associated with ACTB mutations

Introductory paragraphUntil recently missense germ-line mutations in ACTB, encoding the ubiquitously expressed {beta}-cytoplasmic actin (CYA), were exclusively associated with Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF), a complex developmental disorder1,2. Here, we report six patients with previously undescribed heterozygous variants clustered in the 3-coding region of ACTB. These patients present with clinical features different from BWCFF, including thrombocytopenia, microcephaly, and mild developmental disability. Patient derived cells are morphologically and functionally distinct from controls. Assessment of cytoskeletal constituents identified a discrete filament population altered in these cells, which comprises force generating and transmitting actin binding proteins (ABP) known to be associated with thrombocytopenia3-8. In silico modelling and molecular dynamics (MD)-simulations support altered interactions between these ABP and mutant {beta}-CYA. Our results describe a new clinical syndrome associated with ACTB mutations with a distinct genotype-phenotype correlation, identify a cytoskeletal protein interaction network crucial for thrombopoiesis, and provide support for the hypomorphic nature of these actinopathy mutations.

genetics↗