Biology subjects

Dottori, M.

Publications and source records attributed to Dottori, M..

12 recordsLinked to original sources

bioRxiv2026

Autonomic neurons are intrinsically hyperexcitable in a Friedreich ataxia iPSC model

Friedreich ataxia (FRDA) is an inherited multisystem disorder caused by a GAA repeat expansion in the FXN gene and consequent frataxin deficiency, causing progressive neurodegeneration and heart disease. Clinical studies have documented autonomic dysfunction in FRDA, including elevated resting heart rate, raised plasma catecholamines and a high burden of urinary, bowel and sexual symptoms. Consistent with this, autopsy studies show degeneration of the cardiac conduction system and its associated ganglia in FRDA. Whether these features are intrinsic to FRDA autonomic neurons has been untestable without a human cellular model. Here, a human model of FRDA autonomic neurons was established by differentiating induced pluripotent stem cells (iPSCs) from individuals with FRDA and from their CRISPR/Cas9-corrected isogenic controls. FRDA autonomic neurons expressed canonical autonomic lineage markers and retained reduced FXN mRNA and frataxin protein levels. Electrophysiological analysis revealed pronounced hyperexcitability, with an approximately five-fold increase in spike number, shortened interspike intervals, and longer bursts. FRDA autonomic neurons also exhibited increased cell death, elevated mitochondrial reactive oxygen species, and a shift from networked to tubular mitochondrial morphology. Bulk RNA sequencing of all three isogenic pairs revealed loss of extracellular matrix programs and activation of stress response pathways. The top differentially expressed gene was MEG3, a maternally imprinted long noncoding RNA, which was consistently upregulated across all three isogenic pairs. MEG3 sits within the imprinted 14q32 DLK1-DIO3 locus, whose maternally expressed noncoding transcripts were co-ordinately de-repressed in FRDA autonomic neurons. Competitive gene-set testing of 43 imprinted genes across 20 domains showed this to be specific to 14q32: no other imprinted domain was co-ordinately altered. Together, these findings establish hyperexcitability of autonomic neurons as a cell autonomous consequence of frataxin deficiency, accompanied by mitochondrial dysfunction. This provides a cellular basis for the increased sympathetic tone observed in clinical studies.

neuroscience↗

bioRxiv2026

Friedreich ataxia transcriptomic dysregulation and identification of cell type-specific biomarkers: A systematic review and meta-analysis

Friedreich ataxia (FRDA) is a progressive multisystem neurodegenerative disease mostly caused by a homozygous GAA repeat expansion in the FXN gene, leading to deficiency of the protein frataxin. Despite ubiquitous frataxin expression, FRDA pathology is tissue-specific, disproportionately affecting dorsal root ganglia sensory neurons, dentate nuclei of the cerebellum, corticospinal tracts and cardiomyocytes. The molecular basis for this selective vulnerability remains unresolved, suggesting that cell-type specific responses to frataxin deficiency shape disease susceptibility. This incomplete understanding is compounded by the lack of molecular biomarkers that capture FRDA biology beyond frataxin deficiency, thereby limiting therapeutic development and evaluation. Here, we integrated all available human bulk RNA-seq datasets in FRDA (23 datasets across 10 cell types), spanning disease-related (cardiomyocytes, sensory neurons) and relatively FRDA-spared cell types (fibroblasts, lymphoblastoid cells) under a unified analytical framework to identify transcriptional dysregulation underlying selective vulnerability and candidate biomarkers. Meta-analysis revealed recurrent transcriptional perturbations beyond FXN, involving long non-coding RNAs, translational control and cytoskeletal organisation. While shared transcriptional themes were observed, the specific biological programmes engaged were strongly cell-type dependent. The top candidate biomarkers, MYH14, MEG9, and MEG8 showed preferential upregulation in disease-relevant cell types including sensory neurons and cardiomyocytes, supporting their potential relevance to selective vulnerability. Therapeutic responsiveness to these candidates were assessed across RNA-seq datasets from FRDA models exposed to diverse therapeutic strategies, including epigenetic modulation and FXN-targeting approaches, revealing that transcriptional alterations in FRDA are pharmacologically modifiable. To facilitate transparent exploration and reuse of these findings, we developed an interactive FRDA Transcriptomic Atlas, providing a community-accessible resource for investigating gene and pathway-level dysregulation across FRDA studies: https://marniemaddock.github.io/FRDATranscriptomicAtlas/. Together, these findings implicate cell type specific transcriptional programs as potential drivers of selective vulnerability and establish a framework for prioritising biomarkers in FRDA. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712785v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@26138org.highwire.dtl.DTLVardef@3cbc27org.highwire.dtl.DTLVardef@164dbf5org.highwire.dtl.DTLVardef@a4b59d_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

A novel protocol for the efficient generation of all three major hippocampal neuronal sub-populations from human pluripotent stem cells

The diverse computational functions of the human hippocampus rely on coordinated interactions among dentate gyrus (DG), CA3, and CA1 subfields, yet generating all three neuronal identities in vitro - particularly CA1 - has remained challenging. Here we establish a reproducible and modular differentiation protocol that directs human pluripotent stem cells (hPSCs) through dorsomedial telencephalic progenitors to yield DG, CA3, and CA1 neuronal subtypes together with hippocampal regionally specified astrocytes. Early tri-inhibition combined with Sonic hedgehog suppression produced dorsal forebrain progenitors (FOXG1+, PAX6+), while FGF2 treatment supported progenitor maintenance and induced TBR2+ intermediate progenitors. Controlled WNT activation using CHIR99021 drove progressive enrichment of PROX1 hippocampal progenitors across two independent donor lines. Terminal differentiation produced MAP2+/TAU+ neurons that expressed DG (PROX1), CA3 (GRIK4), and CA1 (WFS1, OCT6) markers, with maturing synaptic puncta. Defined progenitors generated long-lived (>400 days) hippocampal organoids exhibiting mixed neuronal-glial populations and spontaneous activity characterized by increased firing rates, high information entropy, and hub-like causal connectivity relative to monolayers, whereas astrocytes-supplemented monolayers displayed intermediate maturation. Population level electrophysiological analysis was also conducted to explore the dynamics of these different cultures. This platform enables systematic experimental control over neuron-astrocyte ratios, culture geometry, and developmental timing, providing a foundation for mechanistic studies of human hippocampal development, circuit function, and disease. Note on figure qualityThis is the preprint version of the manuscript. Figures are included adjacent to described results for the convenience of the reader but may be lower resolution due to file size restrictions on bioRxiv. High resolution figures are included as separate .tiff files for download.

neuroscience↗

bioRxiv2025

Frataxin deficiency drives cardiac dysfunction and transcriptional dysregulation in Friedreich ataxia iPSC model

Friedreich ataxia (FRDA) is a progressive neuromuscular degenerative disorder caused by GAA repeat expansions in the FXN gene, leading to frataxin deficiency and multisystem pathology. Cardiomyopathy is the leading cause of mortality in individuals with FRDA. To investigate the cellular and molecular mechanisms underlying FRDA-associated cardiac dysfunction, we employed induced pluripotent stem cell (iPSC) lines derived from three individuals with FRDA, each paired with an isogenic control line generated through CRISPR/Cas9-mediated excision of the pathogenic GAA repeat expansion. Correction of the mutation restored FXN expression to levels comparable to healthy donor iPSCs, and all lines differentiated efficiently into cardiomyocytes. Functional analysis revealed significant contractile abnormalities in FRDA cardiomyocytes and multicellular cardiac microtissues, including prolonged contraction and relaxation times and faster beating rates, consistent with clinical observations of cardiac contractile dysfunction. FRDA cardiomyocytes also exhibited pathological features such as increased cell size, irregular calcium transients, elevated mitochondrial reactive oxygen species levels, increased mitochondrial fission and increased cell death. These phenotypes were exacerbated by pathological levels of iron supplementation in culture media, highlighting the heightened sensitivity of frataxin-deficient cardiomyocytes to iron-induced metabolic stress. RNA sequencing revealed a distinct transcriptional profile associated with frataxin deficiency. MEG3 and PCDHGA10 were consistently dysregulated across all three FRDA-iPSC lines and may represent early molecular markers of FRDA cardiomyopathy. Together, these findings establish a robust human iPSC model of FRDA cardiomyopathy that captures early disease phenotypes and reveals novel molecular targets. This preclinical human model provides valuable insight into the pathogenesis of FRDA and provides a platform for developing early-stage therapeutic interventions.

pathology↗

bioRxiv2025

Association of avian biodiversity and West Nile virus circulation in Culex mosquitoes in Emilia-Romagna, Italy

West Nile Virus (WNV) is a zoonotic arbovirus maintained in a transmission cycle between Culex mosquitoes and birds, occasionally spilling over into humans. The impact of avian biodiversity on WNV circulation remains debated, with studies reporting both negative and positive correlations (dilution and amplification effects respectively) across different settings. In Europe, this relationship remains largely unexplored, particularly in regions with high WNV transmission, such as Emilia-Romagna in Northern Italy. We explored the association between avian biodiversity and WNV circulation in Culex mosquitoes in Emilia-Romagna using 11 consecutive years (2013-2023) of entomological surveillance data paired with two avian data sources. We calculated avian biodiversity indices (Shannons, Simpsons, and Chao2) from observation records from the Farmland Bird Index project and applied linear regression models to assess their relationship with WNV circulation levels. Moreover, we used Bayesian spatiotemporal regression models and gridded weekly avian abundance estimates from the eBird project, to analyse the associations between avian species richness indices and WNV-positivity rates at 68 geolocated mosquito traps across the region. We observed consistent negative associations between WNV circulation in the Culex population and avian biodiversity indices, supporting the dilution effect hypothesis (DEH). We found that non-passerine species richness was negatively associated with WNV mosquito circulation, whereas passerine species richness exhibited a positive association after adjusting for environmental factors and spatial random effects. These findings suggest that passerines may amplify WNV transmission, while the presence of non-passerine species is associated with reductions in WNV circulation. This study provides the first empirical evidence supporting the DEH for WNV in Europe. These findings have important implications for avian surveillance and future WNV modelling studies, and can inform regional, national and European policies on biodiversity conservation and public health planning. Author summaryWest Nile Virus (WNV) is a zoonotic, vector-borne disease maintained in a mosquito-bird-mosquito transmission cycle. The role of avian biodiversity in WNV transmission remains debated, with studies reporting both dilution effects (negative associations) and amplification effects (positive associations) across different settings. Here, we examined the impact of avian biodiversity on WNV circulation in Culex mosquitoes in Emilia-Romagna, Italy, one of Europes WNV hotspots. We analysed 11 years (2013-2023) of entomological surveillance data alongside two ornithological datasets, respectively collected by the Farmland Bird Index project and the citizen science project eBird. These different datasets allowed us to test the dilution effect hypothesis (DEH) at broad and fine spatiotemporal resolutions. Our findings provide support for the DEH for WNV, revealing that higher bird community diversity is associated with reduced WNV circulation. Additionally, we found that non-passerine species richness is linked to lower levels of WNV circulation in the vector, whereas passerine species richness is positively associated with WNV transmission from entomological surveillance. These results point towards the potential amplifying role of passerine species and the protective role of non-passerine species in WNV transmission dynamics, which has important implications for species conservation, public health policy and future modelling studies.

ecology↗

bioRxiv2025

Optimizing the In Vitro Neuronal Microenvironment to Mitigate Phototoxicity in Live-cell Imaging

Long-term imaging formats are ideal for capturing dynamic neuronal network formation in vitro, yet fluorescent techniques are often constrained by the impact of phototoxicity on cell survival. Here we present a live-imaging protocol that was optimised via quantitative analysis of 3 target culturing conditions on neuromorphological health: extracellular matrix (human- versus murine-derived laminin), culture media (Neurobasal versus Brainphys Imaging media), and seeding density (1x105 versus 2x105 cells/cm2). A cortical neuron reporter line was differentiated from human embryonic stem cells by transduction of Neurogenin-2 and green fluorescent protein, then fluorescently imaged in 8 different microenvironments daily for 33 days. Alongside viability analysis by PrestoBlue assay and gene quantification by digital polymerase chain reaction, an automated image analysis pipeline was developed to characterise network morphology and organisation over time. Brainphys Imaging medium was observed to support neuron viability, outgrowth, and self-organisation to a greater extent than Neurobasal medium with either laminin type, while the combination of Neurobasal medium and human laminin reduced cell survival. Further, a higher seeding density fostered somata clustering, but did not significantly extend viability compared to low density. These findings suggest a synergistic relationship between species-specific laminin and culture media in phototoxic environments, which is positively mediated by light-protective compounds found in Brainphys Imaging medium.

cell biology↗

bioRxiv2025

THE EFFECT OF SERTRALINE AND VOLUNTARY EXERCISE DURING PREGNANCY ON LITTER CHARACTERISTICS AND POSTNATAL AFFECTIVE BEHAVIOUR IN RAT DAMS

IntroductionSertraline is the frontline pharmacotherapy for the treatment of depression and anxiety during pregnancy. However, there is little evidence regarding the effects of sertraline on maternal behaviour or the maternal brain. Furthermore, the efficacy of non-pharmacological approaches to treatment in pregnancy, such as exercise, are unclear. Therefore, the aim of this study was to examine the effects of sertraline and exercise during pregnancy on maternal postnatal depressive-like, anxiety-like and associated behaviours, as well as litter characteristics, in a rat model of depression. We also investigated the effects of these treatments on the maternal brain, focusing initially on DNA methylation and glutamatergic markers, which have been implicated in depression. MethodTwenty-four female Wistar-Kyoto (WKY; strain that models depression and anxiety) rats were divided into three groups: 1. WKY-Sertraline; 2. WKY-Exercise, 3. WKY-Vehicle; Six female Wistar rats were included as controls. Rats were treated with sertraline (10mg/kg) or vehicle (33% propylene glycol) twice/day, from gestational day (GD) 1 to postnatal day (PN) 14. The WKY-Exercise group were provided access to a running wheel during pregnancy for 3 hours/day from GD1-18. Dam and litter characteristics, as well as pup ultrasonic vocalisations (USVs), were measured. Dams underwent behavioural testing at 5-weeks postnatal to assess depressive-, anxiety- and cognitive-like behaviours. Gene expression of DNA methylation markers (Dnmt1, Dnmt3a) and glutamate receptors (Grin1, Grin2a, Grin2b) were measured in the prefrontal cortex (PFC), using RT-qPCR. ResultThe WKY-Sertraline group gained 39% less weight in their first pregnancy week compared to all other groups (p<0.05) and produced smaller litters compared to Wistar controls (-43%; p=0.003) and WKY-Exercise (-38%; p=0.012), and WKY-Sertraline pups had slightly smaller brain weights (p=0.031 compared to WKY-Vehicle). The WKY-Exercise pups produced increased number of USVs at PN7 compared to WKY-Vehicle, with no treatment differences at PN14. The WKY strain however, did showed reduced average mean amplitude of calls and reduced average duration of calls at PN7 compared to WIS (p<0.01) and reduced number of calls at PN14 (p<0.01). Maternal sertraline treatment did not significantly affect dam behavioural measures, all maternal cortical gene expression. The WKY-Exercise group however showed reduced anxiety-like behaviours, spending more time in the open arms (620%; p=0.027) and less time in the closed arms (-22%; p=0.047) of the elevated plus maze (EPM) compared to WKY-Vehicle, and more time in the centre of the open field test (OFT) compared to WKY-Vehicle (132%; p=0.057). Furthermore, WKY-Exercise dams showed a 64% increase in Dnmt3a mRNA levels in the PFC compared to WKY-Vehicle (p=0.019). ConclusionVoluntary exercise during pregnancy in the WKY rat model, reduced postnatal anxiety-like behaviour in the dam. This was accompanied by elevated DNMT3a gene expression in the PFC, suggesting this region may be sensitive to DNA methylation changes following maternal exercise. In contrast, maternal sertraline did not impact these behaviours or genes. Maintaining sertraline treatment beyond PN14, may have resulted in broader effects on dam behaviour, which should be explored further. Maternal sertraline did appear to have some adverse effects on the in utero environment, evidenced by smaller litters, with slightly smaller pup brain weights, which should be investigated further. Our findings suggest a long-term beneficial effect of exercise during pregnancy and supports future studies examining the effects of exercise in antenatal depression in the human population.

neuroscience↗

bioRxiv2024

Astrocytic glutamate regulation is implicated in the development of stress-related psychiatric disorders

Astrocytes are a brain cell type vulnerable to the effects of stress and the development of psychiatric-like phenotypes in animals, yet how this translates to humans is unclear. Here, we probed the diversity of [~]145,000 total human cortical astrocytes with single nucleus and spatial transcriptomics, showing that human astrocytes comprise a molecularly and anatomically diverse cell population. In individuals with psychiatric disorders and high adversity exposure, we identified distinct alterations to glutamate-related synaptic functions, supported by histological quantification of >20,000 astrocytes. Early-life adversity exposure produced more pronounced cellular changes than adversity experienced later in life, and female cases displayed stronger transcriptomic associations than males with adversity exposure. Human pluripotent stem cell-derived astrocytes from both two- and three-dimensional models confirmed that glutamate signalling is directly impacted by glucocorticoid activation. Our findings highlight astrocytes as crucial players in how exposure to severe adversity raises risk to psychopathology and position them as strategic pharmacological targets for future intervention strategies.

neuroscience↗

bioRxiv2024

Distinct molecular and functional properties of human induced-proprioceptor and low-threshold mechanoreceptor neurons

Sensing mechanical stimuli is crucial for the function of internal and external tissues, such as the skin and muscles. Much of our understanding of mechanosensory physiology relies on rodent studies, which may not directly translate to humans. To address the knowledge gap in human mechanosensation, we developed distinct populations of human mechanosensory neuronal subtypes from human pluripotent stem cells (hPSC). By inducing co-expression of NGN2/RUNX3 or NGN2/SHOX2 in hPSC-derived migrating neural crest cells we directed their specification to proprioceptor and low-threshold mechanoreceptor neuronal subtypes, respectively. The induced neurons exhibited transcriptional profiles consistent with mechanosensory neurons and displayed functional responses to mechanical stimuli, such as stretch and submicrometer probe indentation to the soma. Notably, each subtype displayed unique mechanical thresholds and desensitization properties akin to proprioceptors and low-threshold mechanoreceptors and both induced neuronal subtypes fired action potentials in response to minute mechanical stimuli, predominantly relying on PIEZO2 for mechanosensory function. Collectively, this study provides a foundational model for exploring human neuronal mechanosensory biology.

neuroscience↗

bioRxiv2024

Alzheimer's disease induced neurons bearing PSEN1 mutations exhibit reduced excitability

Alzheimers disease (AD) is a devastating neurodegenerative condition that affects memory and cognition, characterized by neuronal loss and currently lacking a cure. Mutations in PSEN1 (Presenilin 1) are among the most common causes of early-onset familial AD (fAD). While changes in neuronal excitability are believed to be early indicators of AD progression, the link between PSEN1 mutations and neuronal excitability remains to be fully elucidated. This study examined induced pluripotent stem cell (iPSC)-derived NGN2 induced neurons (iNs) from fAD patients with PSEN1 mutations S290C or A246E, alongside CRISPR-corrected isogenic cell lines, to investigate early changes in excitability. Electrophysiological profiling revealed reduced excitability in both PSEN1 mutant iNs compared to their isogenic controls. Neurons bearing S290C and A246E mutations exhibited divergent passive membrane properties compared to isogenic controls, suggesting distinct effects of PSEN1 mutations on neuronal excitability. Additionally, both PSEN1 backgrounds exhibited higher current density of voltage-gated potassium (Kv) channels relative to their isogenic iNs, while displaying comparable voltage-gated sodium (Nav) channel current density. This suggests that the Nav/Kv imbalance contributes to impaired neuronal firing in fAD iNs. Deciphering these early cellular and molecular changes in AD is crucial for understanding the disease pathogenesis.

cell biology↗

bioRxiv2024

REST and RCOR genes display distinct expression profiles in neurons and astrocytes using 2D and 3D human pluripotent stem cell models

Repressor element-1 silencing transcription factor (REST) is a transcriptional repressor involved in neurodevelopment and neuroprotection. REST forms a complex with the REST corepressors, CoREST1, CoREST2, or CoREST3 (encoded by RCOR1, RCOR2, and RCOR3, respectively). Emerging evidence suggests that the CoREST family can target unique genes independently of REST, in various neural and glial cell types during different developmental stages. However, there is limited knowledge regarding the expression and function of the CoREST family in human neurodevelopment. To address this gap, we employed 2D and 3D human pluripotent stem cell (hPSC) models to investigate REST and RCOR gene expression levels. Our study revealed a significant increase in RCOR3 expression in glutamatergic cortical and GABAergic ventral forebrain neurons, as well as mature functional NGN2-induced neurons. Additionally, a simplified astrocyte transdifferentiation protocol resulted in a significant decrease in RCOR2 expression following differentiation. REST expression was notably reduced in mature neurons and cerebral organoids, along with RCOR2 in the latter. In summary, our findings provide the first insights into the cell-type-specific expression patterns of RCOR genes in human neuronal and glial differentiation. Specifically, RCOR3 expression increases in neurons, while RCOR2 levels decrease in astrocytes. The dynamic expression patterns of REST and RCOR genes during hPSC neuronal and glial differentiation underscore the potential distinct roles played by REST and CoREST proteins in regulating the development of these cell types in humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/584254v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@7be30borg.highwire.dtl.DTLVardef@1771388org.highwire.dtl.DTLVardef@a376aforg.highwire.dtl.DTLVardef@1c4dc83_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIREST and RCOR genes display cell-type specific expression patterns in neural cells C_LIO_LIRCOR3 (encodes CoREST3) is upregulated during neuronal and astrocyte differentiation C_LIO_LIRCOR2 (encodes CoREST2) is downregulated during differentiation of astrocytes C_LIO_LIEvidence of potential cell-type specific functions of the CoREST family C_LI

neuroscience↗

bioRxiv2023

Parsing the effect of co-culture with brain organoids on Diffuse Intrinsic Pontine Glioma (DIPG) using quantitative proteomics

Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly brain cancers in children for which there is currently no effective treatment. This can partly be attributed to preclinical models that lack essential elements of the in vivo tissue environment, resulting in treatments that appear promising preclinically, but fail to result in effective cures. Recently developed co-culture models combining stem cell-derived brain organoids with brain cancer cells provide tissue dimensionality and a human-relevant tissue-like microenvironment. As these models are technically challenging and time consuming it is imperative to establish whether interaction with the organoid influences DIPG biology and thus warrants their use. To address this question, we cultured DIPG cells with cortical organoids. We created "mosaic" co-cultures enriched for tumour cell-neuronal cell interactions versus "assembloid" co-cultures enriched for tumour cell-tumour cell interactions. Sequential window acquisition of all theoretical mass spectra (SWATH-MS) was used to analyse the proteomes of DIPG fractions isolated by flow-assisted cell sorting. Control proteomes from DIPG spheroids were compared with DIPG cells isolated from mosaic and assembloid co-cultures. This revealed that tumour cell adhesion was reduced, and DNA synthesis and replication were increased, in DIPG cells under either co-culture condition. By contrast, the mosaic co-culture was associated with pathways implicated in dendrite growth. We propose that co-culture with brain organoids is a valuable tool to parse the contribution of the brain microenvironment to DIPG tumour biology.

cancer biology↗