Biology subjects

Prigione, A.

Publications and source records attributed to Prigione, A..

8 recordsLinked to original sources

bioRxiv2026

Mitochondrial genome editing reveals the impact of complex I heteroplasmic variants on human neurogenesis

Mitochondrial DNA (mtDNA) variants, whether maternally inherited or acquired somatically during aging, can be particularly detrimental to cells that rely on mitochondrial metabolism such as neurons. The functional consequences of mtDNA variants are thought to depend on their heteroplasmy, defined as the proportion of mtDNA molecules carrying that variant. However, investigating the specific effects of heteroplasmic mtDNA variants on human neuronal function has been challenging. Studies using patient-derived cells cannot conclusively attribute observed phenotypes to a single mtDNA variant, while conventional genome editing approaches are not applicable to mtDNA. Here, we use DddA-derived cytosine base editor (DdCBE) to introduce pathogenic heteroplasmic mtDNA variants into Neurogenin 2 (NGN2)-inducible human induced pluripotent stem cells (iPSCs). We focus on two variants in the genes MT-ND1 and MT-ND5 belonging to complex I (CI), the largest component of the mitochondrial respiratory chain and a frequent contributor to human disease, including the incurable neurodevelopmental disorder Leigh syndrome. mtDNA-edited iPSC lines exhibit low off-target editing and maintain similar heteroplasmy following subclonal isolation and differentiation into NGN2-induced neurons (iNs). Although the CI variants do not substantially affect mitochondrial energy metabolism in undifferentiated iPSCs, they reduce mitochondrial membrane potential in iNs where they drive a transcriptional reprogramming indicative of impaired neuronal commitment. These findings suggest that heteroplasmic CI variants are sufficient to disrupt neural fate specification even at relatively low heteroplasmy. More broadly, mtDNA editing in human iPSCs provides a powerful platform to investigate how mitochondrial fitness and heteroplasmy influence development and disease.

bioengineering↗

bioRxiv2026

Defining early human developmental identity: A curated, cross-platform marker framework

Early human development involves dynamic transitions in cell identity, including transient transcriptional modulation and stable lineage commitment. Distinguishing these types of gene expression changes is challenging and can be further exacerbated by genetic and experimental heterogeneity in the context of human pluripotent stem cell (hPSC) research. To address this challenge and help uncover transcriptional changes indicative of true developmental state, we establish a curated, cross-platform marker framework for robust identification of pluripotency and early germ-layer identity. Starting from an unbiased RNA-seq discovery set, we systematically validate candidate markers across qPCR, bulk and single-cell RNA sequencing, and quantitative proteomics platforms, yielding a refined panel of 67 markers (20 for the undifferentiated state, 17 for endoderm, 15 for ectoderm, and 15 for mesoderm). We show that this framework reliably identifies early developmental states across heterogeneous datasets, generalizes to in vivo human embryo cell types, and preserves lineage identity despite substantial transcriptional variability. Furthermore, we demonstrate concordant protein-level expression for a subset of markers, supported by deep proteomic profiling of the reference line KOLF2.1J. To enable broad application, we introduce DeepDiff, a web-based resource integrating the validated markers, allowing automated fate classification in a user-friendly interface. Together, this work provides a standardized framework for defining early human developmental identity and disentangling lineage commitment from context-dependent modulation.

developmental biology↗

bioRxiv2025

Canonical autophagy remains inactive in induced pluripotent stem cells and neuronal progenitor cells following DNA damage induced by BPDE or etoposide.

(Macro-)Autophagy is a key cellular stress response mediating the recycling of long-lived or damaged proteins and organelles. In stem cells, autophagy is essential for the decision between quiescence, self-renewal and differentiation. We observed that induced pluripotent stem cells (iPSCs) and thereof derived neural progenitor cells (NPCs) have a functional autophagy machinery, as shown by starvation-induced autophagic flux and ULK1 activation. Using the human iPSC line iPS11 and thereof derived NPCs (niPS11), we investigated whether genotoxic stress induced by benzo[a]pyrene diolepoxide (BPDE) or etoposide can similarly activate autophagy, as previously reported for cancer cell lines. While both BPDE and etoposide induced the DNA damage markers phospho-p53 Ser15 and {gamma}H2AX and slightly altered the expression of DNA repair proteins such as XPC, they did not trigger autophagic flux in either iPSCs or NPCs. After genotoxin treatment, ULK1 activation was only observed in NPCs, but this was not sufficient to trigger a significant downstream autophagic response. Mass spectrometry revealed minimal proteomic changes in iPSCs and moderate changes in NPCs, mainly involving mitotic regulators. These results suggest that genotoxic agents do not strongly affect canonical autophagy in pluripotent stem cells or their neural derivatives despite an otherwise responsive autophagic system.

cell biology↗

bioRxiv2024

Deep learning-driven neuromorphogenesis screenings identify repurposable drugs for mitochondrial disease

Mitochondrial disease encompasses untreatable conditions affecting tissues with high energy demands. A severe manifestation of mitochondrial disease is Leigh syndrome (Leigh), which causes defects in basal ganglia and midbrain regions, psychomotor regression, lactic acidosis, and early death. We previously generated isogenic pairs of Leigh cerebral organoids and uncovered defects in neuromorphogenesis. Here, we leveraged on this disease feature to devise drug discovery pipelines. We developed a deep learning algorithm tailored for cell type-specific drug repurposing to identify drugs capable of promoting neuronal commitment. In parallel, we performed a survival drug screen in yeast and validated the repurposable hits on branching capacity in Leigh neurons. The two approaches independently highlighted azole compounds, Talarozole and Sertaconazole, both of which lowered lactate release and improved neurogenesis and neurite organization in Leigh midbrain organoids. Hence, targeting neuromorphogenesis has led to identify potential new drugs for mitochondrial disease and could prove an effective strategy for further drug discovery.

neuroscience↗

bioRxiv2023

Mutant Huntingtin impairs neurodevelopment in human brain organoids through CHCHD2-mediated neurometabolic failure

Expansion of the glutamine tract (poly-Q) in the protein Huntingtin (HTT) causes the neurodegenerative disorder Huntingtons disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT, we engineered induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. 70Q introduction caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2), a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics and elevated resting energy expenditure. Elimination of the poly-Q tract of HTT normalized CHCHD2 expression and mitochondrial defects. Hence, mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2, which could then serve as an early intervention target for HD.

neuroscience↗

bioRxiv2021

PiggyBac Transposable Element-derived 1 controls Neuronal Progenitor Identity, Stress Sensing and mammal-specific paraspeckles

The evolution and functional integration of new genes, especially those that become core to key functions, remains enigmatic. We consider the mammal-specific gene, piggyBac transposable element derived 1 (PGBD1), implicated in neuronal disorders. While it no longer recognises piggyBac transposon-like inverted repeats and transposase functionality having been lost, it has evolved a core role in neural homeostasis. Depletion of PGBD1 triggers accumulation of mammal-specific paraspeckles and neural differentiation. It acts by two modalities, DNA binding and protein-protein interaction. As a transcriptional repressor of (lnc)NEAT1, the backbone of paraspeckles, it inhibits paraspeckle formation in neural progenitor cells (NPCs). At the protein level it is associated with the stress response system, a function partially shared with (lnc)NEAT1. PGBD1 thus presents as an unusual exemplar of new gene creation, being a recently acquired multi-function, multi-modal gene. Mammalian specificity associated with control of a mammal-specific structure implies coevolution of new genes with new functions.

molecular biology↗

bioRxiv2020

Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human dopaminergic (DA) neurons, which are crucial for the development and maintenance of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human DA neurons from induced pluripotent stem cells (iPSCs). The assay, dubbed mitochondrial neuronal health (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DA neurons decreases MMP and branching complexity in a dose-dependent manner. The toxic effect of ethanol on DA neurons was already detectable after 1 hour of exposure, and occurred similarly in DA neurons derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate drugs modulating acute ethanol toxicity in human DA neurons. Among these drugs, flavoxate and disulfiram influenced mitochondrial neuronal health independently from ethanol, leading to amelioration and worsening, respectively. Altogether, we developed an HT assay to probe human mitochondrial neuronal health and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of mitochondrial neuronal health and toxicity in live human neurons.

cell biology↗

bioRxiv2019

SURF1 mutations causative of Leigh syndrome impair human neurogenesis

Mutations in the mitochondrial complex IV assembly factor SURF1 represent a major cause of Leigh syndrome (LS), a rare fatal neurological disorder. SURF1-deficient animals have failed to recapitulate the neuronal pathology of human LS, hindering our understanding of the disease mechanisms. We generated induced pluripotent stem cells from LS patients carrying homozygous SURF1 mutations (SURF1 iPS) and performed biallelic correction via CRISPR/Cas9. In contrast to corrected cells, SURF1 iPS showed impaired neuronal differentiation. Aberrant bioenergetics in SURF1 iPS occurred already in neural progenitor cells (NPCs), disrupting their neurogenic potency. Cerebral organoids from SURF1 iPS were smaller and recapitulated the neurogenesis defects. Our data imply that SURF1 mutations cause a failure in the development of maturing neurons. Using NPC function as an interventional target, we identified SURF1 gene augmentation as a potential strategy for restoring neurogenesis in LS patients carrying SURF1 mutations.

neuroscience↗