Biology subjects

Tigano, M.

Publications and source records attributed to Tigano, M..

8 recordsLinked to original sources

bioRxiv2026

A Human Neuronal Cell Model of Endogenous TDP-43 A315T Reveals Altered Protein Dynamics and Disease-Relevant Cellular Dysfunction

TAR DNA-binding protein 43 (TDP-43) aggregation is the defining pathological hallmark of nearly all cases of amyotrophic lateral sclerosis (ALS), yet physiologically relevant human models that faithfully recapitulate disease-associated TDP-43 proteinopathy and dysfunction remain limited. To cover this gap, we generated a novel human-based model of cortical neurons carrying the endogenous ALS-linked TDP-43 A315T mutation together with an in-frame Dendra2 fluorescent reporter, enabling temporal and spatial monitoring of the protein. Neurons expressing TDP-43 A315T exhibited progressive neurite degeneration, altered neuronal activity, and impaired mitochondrial respiration, recapitulating several ALS-associated phenotypes. Our model also displays autophagy-dependent accumulation of cytoplasmic aggregates of mutant TDP-43 without overt loss of nuclear function, maintaining normal processing of canonical cryptic exon targets. In contrast, experimental induction of TDP-43 nuclear exclusion readily triggered cryptic exon incorporation, demonstrating that the model faithfully reproduces loss-of-function phenotypes under stress conditions. In line with perturbed protein solubility, mutant TDP-43 neurons show increased stress granule (SG) formation at baseline and under oxidative stress condition. Finally, treatment with the RNA chaperone Clip34 significantly reduced TDP-43 aggregation under both basal and oxidative stress conditions as well as its localization to SGs. Taken together, these findings establish a physiologically relevant human model that separates early TDP-43 toxic gain-of-function from basal loss-of-function while providing a robust platform for investigating TDP-43 biology and accelerating therapeutic discovery in ALS.

neuroscience↗

bioRxiv2026

Cell free-mtDNA release drives pyroptosis and inflammation in cell models carrying the mitochondrial m.3243A>G pathogenic variant

Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) syndrome is primarily caused by the heteroplasmic m.3243A>G/MT-TL1 pathogenic variant. Patients exhibit elevated circulating cell-free mtDNA (cf-mtDNA) in plasma, which acts as a damage-associated molecular pattern. Using patient-derived fibroblasts and neuronal progenitors, as well as transmitochondrial cytoplasmic hybrids (cybrids), we show that mutant cells release higher levels of cf-mtDNA than wild-type controls, demonstrating that the m.3243A>G pathogenic variant drives mtDNA release. Mechanistically, increased mitochondrial oxidative stress promotes mtDNA oxidation and fragmentation, leading to Ca2+ overload and subsequent mtDNA extrusion. This, in turn, triggers inflammasome activation and pyroptosis, resulting in the secretion of pro-inflammatory cytokines and the activation of innate immune pathways. Pharmacological inhibition of the Mitochondrial Calcium Uniporter (MCU) or Voltage-Dependent Anion Channel (VDAC) reduced mtDNA release, confirming their involvement. Overall, our findings reveal a previously unrecognized mechanism in MELAS linking mitochondrial dysfunction to innate immune activation, with potential implications for therapeutic intervention.

cell biology↗

bioRxiv2026

Nuclear DNA Damage Response Triggers Reorganization of Mitochondrial Nucleic Acids

Mitochondria are subcellular organelles responsible for energy production, and a hub for several cellular signaling pathways that ultimately control cellular processes ranging from cell death to innate immunity. Genotoxic stress, including cellular irradiation, has been shown to cause the mitochondrial-dependent activation of innate immunity via release of mitochondrial nucleic acids in the cytosol. Yet, how the other cellular events triggered by genotoxic stress affects mitochondria and mitochondrial immunity is largely unexplored. Nuclear DNA damage responses (DDR) are a set of well-described responses to genotoxic stressors that allow the cells to, through characterized mechanisms including transcriptional responses and checkpoint activation, survive or die. Whether canonical DDR directly influence mitochondrial structure and activation of downstream pathways of immunity remains unclear. Here, we identify mito-blobs: enlarged TOMM20-positive mitochondrial structures induced by genotoxic stress that are enriched for TFAM-marked mitochondrial DNA nucleoids, FASTKD2-positive mitochondrial RNA granules, and immunogenic double-stranded RNA. While structurally resembling other mitochondria stress responsive rearrangements, mito-blobs carry the distinctive feature of being induced by nuclear DNA double-stranded breaks alone. Strikingly, mitochondrial double-stranded breaks failed to induce mito-blobs, indicating nuclear-to-mitochondrial signaling rather than an autonomous response to mitochondrial genome damage. Mechanistically, we show that mito-blobs formation strictly requires MFN1/2- and OPA1-dependent fusion machinery, while nuclear DNA damage invokes a classical ATM-p53 response, which lead to cell cycle block in the G1 phase that reduce DRP1 S616 phosphorylation and shifting mitochondrial morphology toward a low-fission pro-fusion state. Strikingly, the use of the standard of care CDK4/6 inhibitor palbociclib, was sufficient to trigger mito-blobs without nuclear DNA damage. Considering the mito-blobs high content in nucleic acids, we additionally investigated if affecting their life cycle could perturb inflammatory and interferon-associated gene expression downstream of genotoxic stress. We describe how autophagic-lysosomal clearance triggered upon cell cycle block limited mito-blob persistence, and blocking autophagy disposal unmasked a strong inflammatory response. Taken together, these findings suggest mito-blobs are the product of an active mitochondrial remodeling process elicited in response to nuclear genotoxic stress and that concentrate immunogenic mitochondrial nucleic acids in defined structures for their correct disposal via autophagy and avoid aberrant activation of innate immunity. HighlightsO_LIGenotoxic stress induces enlarged mitochondrial structures called mito-blobs. C_LIO_LIMito-blobs concentrate mitochondrial DNA, RNA granules, and double-stranded RNA. C_LIO_LINuclear DNA damage, but not mitochondrial, is sufficient to induce mito-blobs. C_LIO_LICDK4 Inhibitors Trigger Mito-blob Formation. C_LIO_LIMito-blob formation and clearance alter inflammatory gene expression. C_LI eTOC blurbYu et al. identify mito-blobs as enlarged mitochondrial structures that form after genotoxic stress. They show that nuclear DNA breaks - as opposed to mitochondrial DNA breaks - trigger mito-blob formation through nuclear-to-mitochondrial signaling involving altered DRP1 phosphorylation and mitochondrial fusion machinery. Mito-blob persistence is limited by autophagic clearance and is associated with inflammatory and interferon-associated gene expression after stress.

cell biology↗

bioRxiv2026

Mutations Causative of CPEO Differentially Engage Innate Immunity Sensors

Chronic Progressive External Ophthalmoplegia (CPEO) is a primary mitochondrial disorder (PMD) caused by mutations in nuclear genes encoding mitochondrial DNA (mtDNA) maintenance proteins. CPEO is characterized by mtDNA depletion and deletions, and patients primarily present with ocular and muscular features (isolated CPEO). However, additional encephalomyopathy, neurological complications, and Parkinsonism can drive a more severe disease form, CPEO-plus. The evolution from isolated CPEO to CPEO-plus remains poorly understood. Inflammatory and innate immune processes are emerging as strong disease modifiers and may underlie this heterogeneity. Instability of mitochondrial DNA is a major driver of organellar stress and release of mitochondrial contents into the cytosol. Mutations in several genes involved in mtDNA replication and maintenance have been implicated in triggering the escape of mitochondrial nucleic acids from the mitochondrial matrix. Once exposed to cytosolic innate immune sensors, mtDNA and mitochondrial double-stranded RNA (mt-dsRNA) act as potent immunogens, with more than 10 innate immune sensors capable of recognizing them. Therefore, mtDNA and mt-dsRNA release are likely pathological mechanisms in CPEO, yet the list of CPEO-related genes that can trigger inflammatory processes is far from complete. Here, we use patient-derived fibroblasts from individuals with CPEO carrying mutations in RNASEH1 and Twinkle, and provide - for the first time - evidence that their mutations drive innate immune activation through the release of different mitochondrial nucleic acids. RNASEH1 mutations lead to the accumulation and subsequent release of mt-dsRNA, while mtDNA remains protected. On the other hand, mutations in Twinkle cause the release of mtDNA without triggering mt-dsRNA production, or leakage. Supporting this notion, the POLRMT inhibitor IMT-1, and the STING inhibitor H-151, reduced interferon stimulated genes expression downstream of RNASEH1 and Twinkle mutations, respectively. Further, when we analyzed a unique compound patient line carrying mutations in both genes simultaneously, we detect both species of nucleic acids in its cytosol, indicating that both pathways can be engaged simultaneously in the same cell. Lastly, we show that cytosolic sensing triggers paracrine signaling to activate bystander microglia - the resident macrophages of the retina and brain - with potential implications to the neurological progression of CPEO. Overall, our findings reveal a new role for RNASEH1 and Twinkle in driving aberrant innate immunity and paracrine inflammation in CPEO. Our data support a model in which innate immunity is a universal feature of mutations causing mtDNA instability; yet different mutations engage distinct sensing pathways, and in complex scenarios multiple pathways can be triggered at the same time. Given the clinical heterogeneity observed in patients with PMDs, our findings that different signaling pathways are triggered in patient-specific manners might have direct implications for precision medicine approaches aimed at targeting specific innate immunity.

cell biology↗

bioRxiv2024

Peri-mitochondrial actin filaments inhibit Parkin assembly via disruption of ER-mitochondrial contact

Mitochondrial damage represents a dramatic change in cellular homeostasis, necessitating metabolic adaptation as well as clearance of the damaged organelle. One rapid response to mitochondrial damage is peri-mitochondrial actin polymerization within 2 mins, which we term ADA (Acute Damaged-induced Actin). ADA is vital for a metabolic shift from oxidative phosphorylation to glycolysis upon mitochondrial dysfunction. In the current study we investigated the effect of ADA on Pink1/Parkin mediated mitochondrial quality control. We show that inhibition of proteins involved in the ADA pathway significantly accelerates Parkin recruitment onto depolarized mitochondria. Addressing the mechanism by which ADA resists Parkin recruitment onto depolarized mitochondria, we found that ADA disrupts ER- mitochondrial contacts in an Arp2/3 complex-dependent manner. Interestingly, over-expression of ER-mitochondrial tethers overrides the effect of ADA, allowing rapid recruitment of not only Parkin but also LC3 after mitochondrial depolarization. During chronic mitochondrial dysfunction, Parkin and LC3 recruitment are completely blocked, which is reversed rapidly by inhibiting ADA. Taken together we show that ADA acts as a protective mechanism, delaying mitophagy following acute damage, and blocking mitophagy during chronic mitochondrial damage.

cell biology↗

bioRxiv2024

Co-option of mitochondrial nucleic acid sensing pathways by HSV-1 UL12.5 for reactivation from latent Infection

Although viruses subvert innate immune pathways for their replication, there is evidence they can also co-opt anti-viral responses for their benefit. The ubiquitous human pathogen, Herpes Simplex Virus-1 (HSV-1), encodes a protein (UL12.5) that induces the release of mitochondrial nucleic acid into the cytosol, which activates immune sensing pathways and reduces productive replication in non-neuronal cells. HSV-1 establishes latency in neurons and can reactivate to cause disease. We found that UL12.5 is required for HSV-1 reactivation in neurons and acts to directly promote viral lytic gene expression during initial exit from latency. Further, the direct activation of innate immune sensing pathways triggered HSV reactivation and compensated for a lack of UL12.5. Finally, we found that the induction of HSV-1 lytic genes during reactivation required intact RNA and DNA sensing pathways, demonstrating that HSV-1 can both respond to and active antiviral nucleic acid sensing pathways to reactivate from a latent infection.

microbiology↗

bioRxiv2024

Transcriptional regulation in the absence of inositol trisphosphate receptor calcium signaling

The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that regulate a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB, AP-1 and NF{kappa}b) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition the role of protein kinase C (PKC) was investigated with inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression (DEG) of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. In summary three main adaptations in TKO cells are identified in this study: 1) increased basal activity of NFAT, CREB, AP-1 and NF{kappa}b; 2) an increased reliance on Ca2+-insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g. PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.

cell biology↗

bioRxiv2020

Nuclear sensing of mitochondrial DNA breaks enhances immune surveillance

Mitochondrial double-strand breaks (mtDSBs) are toxic lesions that compromise mitochondrial function. Mito-nuclear communication is essential to maintain cellular homeostasis, however, the nuclear response to mtDSBs remains unknown. Using mitochondrial-targeted TALENs, we show that mtDSBs activate a type I interferon response evidenced by phosphorylation of STAT1 and activation of interferon stimulated genes (ISG). Following mtDNA break formation, BAK-BAX mediated herniation releases mitochondrial RNA to the cytoplasm and trigger a RIG-I/MAVS-dependent immune response. In an independent set of experiments, we investigate the role of mtDSBs in interferon signaling due to genotoxic stress. Our data reveal that activation of ISGs is greatly diminished when cells lacking mtDNA are exposed to ionizing radiation. Furthermore, we show that mtDNA breaks synergize with nuclear DNA damage to mount a robust interferon response. In conclusion, cytoplasmic accumulation of mitochondrial RNA is as an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including ones inflicted by genotoxic agents.

molecular biology↗