Biology subjects

Horvath, R.

Publications and source records attributed to Horvath, R..

At least 19 recordsLinked to original sources

bioRxiv2025

Cell-type-specific adaptations to mitochondrial stress underly the neurological presentations of MTRFR mutations

Mitochondrial diseases are a group of heterogeneous genetic disorders that exhibit striking tissue specificity. Neurological involvement is among the most consistent features, yet the mechanisms that determine why selective neuronal populations are particularly vulnerable to mitochondrial dysfunction remain poorly understood. Mutations in MTRFR, a mitochondrial ribosome rescue factor, cause a progressive neuromuscular phenotype, but no relevant disease model exists to explain its cell-type-specific pathology. Here, we established the first human iPSC-derived neuronal model of MTRFR loss and identified mechanisms driving differential vulnerability between cortical and motor neurons. Although knockdown led to comparable deficits in mitochondrial translation and OXPHOS across both subtypes, cortical neurons engaged adaptive programs, including dendritic mitochondrial remodelling and heat-shock response activation, that preserved survival. Motor neurons failed to mount these responses and instead displayed apoptotic and inflammatory priming. Pharmacological enhancement of stress adaptation rescued motor neuron survival, indicating that resilience is programmable. These findings provide the first mechanistic evidence that neuronal susceptibility to mitochondrial translation defects is defined by the capacity to activate mitochondrial and cytoprotective stress-response pathways.

neuroscience↗

bioRxiv2025

Endothelial tight junctions and cell-matrix adhesions reciprocally control blood-brain barrier integrity

Brain endothelial cells (ECs) rely on mechanical cues to provide a physical barrier that protects the brain. Yet how ECs integrate forces to establish and maintain the blood-brain barrier (BBB) remains poorly understood. Here, we show that the two main endothelial force-bearing systems, tight junctions and cell-matrix adhesions, reciprocally control BBB integrity. Using a combination of super-resolution imaging and biophysical techniques, we reveal increasing mechanical loads on cell-cell junctions vs. cell-matrix adhesions in human stem cell-derived ECs during BBB maturation. This force redistribution is enabled by cytoskeletal remodeling, a compacted pattern of the tight junction protein claudin-5, and the emergence of specialised perinuclear cell-matrix adhesions. Mechanistically, we find an inverse relationship between claudin-5 levels and the expression of key cell-matrix adhesion proteins zyxin and vinculin in vitro and in mice. Finally, we demonstrate that this mechanobiological signature associated with BBB maturation is reversed upon BBB dysfunction after seizures in mice and in human patients with temporal lobe epilepsy. Collectively, our findings establish a novel interplay between mechanoresponsive elements in brain ECs, with implications for BBB stabilisation therapy in epilepsy.

neuroscience↗

bioRxiv2025

Disease-causing MFN2 mutants impair mitochondrial fission dynamics by distinct DRP1 dysregulation

Mitochondria undergo fusion and fission. While DRP1 regulates fission, fusion is controlled by OPA1, MFN1, and MFN2. The balance between these processes and the crosstalk between machineries remains poorly understood. MFN2 mutations cause Charcot-Marie-Tooth disease type 2A (CMT2A), affecting mitochondrial fusion and morphology. However, their role in fission is unclear. Using skin fibroblasts from CMT2A patients (L248H and M376V MFN2 mutations) and wild-type mouse embryonic fibroblasts expressing these variants we studied how MFN2 mutations impact mitochondrial dynamics beyond fusion. We analysed mitochondrial morphology and dynamics, by live-cell confocal microscopy, and tested fusion/fission protein levels, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and oxidative phosphorylation complex subunits. MFN2 mutations impaired mitochondrial fusion and displayed distinct effects on fission and cellular metabolism. L248H-expressing cells showed hyper-elongated mitochondria, impaired fission, and increased OCR, while, M376V cells exhibited fragmentation, enhanced fission, and elevated ECAR. These effects correlated with differential Drp1 phosphorylation. Our findings demonstrate that MFN2 mutants differentially influences fission and metabolism, highlighting the need to consider these effects in therapies aimed at modulating mitochondrial dynamics.

cell biology↗

bioRxiv2025

Mitochondrial DNA heteroplasmy drives cortical neuronal disturbances in human organoids harbouring the common m.3243A>G mutation

Mitochondrial diseases frequently affect the brain leading to severe and disabling neurological symptoms. The heteroplasmic m.3243A>G mutation in MT-TL1, encoding mt-tRNALeu, is responsible for [~]80% of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), which is one of the most characteristic mitochondrial syndromes, leading to disability and early death. There are no animal models harbouring this mutation to provide precise mechanistic insights informing therapeutic interventions. Here, we generated a human iPSC-derived cerebral organoid slice model that recapitulates cortical architecture and mitochondrial pathology. Using biological assays and single-cell RNA sequencing, we uncovered heteroplasmy-dependent transcriptional shifts and changes in key cellular processes in cortical neurons. Organoids with high heteroplasmy showed a predominant impairment of deep-layer neurons triggered by mitochondrial stress, leading to axonal degeneration and apoptosis, similar to brain autopsy of a MELAS patient. Our findings provide insights into the vulnerability of long-range projection neurons in mitochondrial diseases, advancing our understanding of disease mechanisms with a view to potential therapeutic strategies.

neuroscience↗

bioRxiv2024

Mitochondrial leakage and mtDNA damage trigger early immune response in Inclusion Body Myositis.

Polymyositis with mitochondrial pathology (PM-Mito) was first identified in 1997 as a subtype of idiopathic inflammatory myopathy. Recent findings demonstrated significant molecular similarities between PM-Mito and Inclusion Body Myositis (IBM), suggesting a trajectory from early to late IBM and prompting the inclusion of PM-Mito as an IBM precursor (early IBM) within the IBM spectrum. Both PM-Mito and IBM show mitochondrial abnormalities, suggesting mitochondrial disturbance is a critical element of IBM pathogenesis. The primary objective of this cross-sectional study was to characterize the mitochondrial phenotype in PM-Mito at histological, ultrastructural, and molecular levels and to study the interplay between mitochondrial dysfunction and inflammation. Skeletal muscle biopsies of 27 patients with PM-Mito and 27 with typical IBM were included for morphological and ultrastructural analysis. Mitochondrial DNA (mtDNA) copy number and deletions were assessed by qPCR and long-range PCR, respectively. In addition, full-length single-molecule sequencing of the mtDNA enabled precise mapping of deletions. Protein and RNA levels were studied using unbiased proteomic profiling, immunoblotting, and bulk RNA sequencing. Cell-free mtDNA (cfmtDNA) was measured in the serum of IBM patients. We found widespread mitochondrial abnormalities in both PM-Mito and IBM, illustrated by elevated numbers of COX-negative and SDH-positive fibers and prominent ultrastructural abnormalities with disorganized and concentric cristae within enlarged and dysmorphic mitochondria. MtDNA copy numbers were significantly reduced, and multiple large-scale mtDNA deletions were already evident in PM-Mito, compared to healthy age-matched controls, similar to the IBM group. The activation of the canonical cGAS/STING inflammatory pathway, possibly triggered by the intracellular leakage of mitochondrial DNA, was evident in PM-Mito and IBM. Elevated levels of circulating cfmtDNA also indicated leakage of mtDNA as a likely inflammatory trigger. In PM-Mito and IBM, these findings were accompanied by dysregulation of proteins and transcripts linked to the mitochondrial membranes. In summary, we identified that mitochondrial dysfunction with multiple mtDNA deletions and depletion, disturbed mitochondrial ultrastructure, and defects of the inner mitochondrial membrane are features of PM-Mito and IBM, underlining the concept of an IBM-spectrum disease (IBM-SD). The activation of inflammatory pathways related to mtDNA release indicates a significant role of mitochondria-associated inflammation in the pathogenesis of IBM-SD. Thus, mitochondrial abnormalities precede tissue remodeling and infiltration by specific T-cell subpopulations (e.g., KLRG1+) characteristic of late IBM. This study highlights the critical role of early mitochondrial abnormalities in the pathomechanism of IBM, which may lead to new approaches to therapy.

neuroscience↗

bioRxiv2024

CBP/p300 lysine acetyltransferases inhibit HIV-1 expression in latently infected T-cells

HIV-1 latency is regulated by chromatin modifying enzymes, and histone deacetylase inhibitors (HDACi) were previously found to reactivate provirus expression. We report that inhibitors of CBP/p300 acetyltransferases also cause reversal of latency in T-cells. CBP/p300 inhibitors synergize with mechanistically diverse latency reversing agents to cause HIV-1 reactivation. In contrast, inhibition of CBP/p300 impaired the latency reversal by the HDACi SAHA, indicating that CBP/p300 contribute to acetylation on the HIV-1 LTR associated with HDACi-mediated latency reversal. CBP/p300 inhibition caused loss of H3K27ac and H3K4me3 from the LTR, but did not affect association of the inhibitor protein BRD4. Furthermore, inhibition of the additional lysine acetyl transferases PCAF/GCN5 or KAT6A/KAT6B also caused reversal of latency, suggesting that protein acetylation has an inhibitory effect on HIV-1 expression. Collectively, these observations indicate that transcription from the HIV-1 LTR is controlled both positively and negatively by protein acetylation, likely including both histone and non-histone regulatory targets.

molecular biology↗

bioRxiv2024

OPA1 and disease-causing mutants perturb mitochondrial nucleoid distribution

Optic atrophy protein 1 (OPA1) mediates inner mitochondrial membrane (IMM) fusion and cristae organization. Mutations in OPA1 cause autosomal dominant optic atrophy (ADOA), a leading cause of blindness. Cells from ADOA patients show impaired mitochondrial fusion, cristae structure, bioenergetic function, and mitochondrial DNA (mtDNA) integrity. The mtDNA encodes electron transport chain subunits and is packaged into nucleoids spread within the mitochondrial population. Nucleoids interact with the IMM, and their distribution is tightly linked to mitochondrial fusion and cristae shaping. Yet, little is known about the physio-pathological relevance of nucleoid distribution. We studied the effect of OPA1 and ADOA-associated mutants on nucleoid distribution using high-resolution confocal microscopy. We applied a novel model incorporating the mitochondrial context, separating nucleoid distribution into the array in the mitochondrial population and intramitochondrial longitudinal distribution. Opa1-null cells showed decreased mtDNA levels and nucleoid abundance. Also, loss of Opa1 lead to an altered distribution of nucleoids in the mitochondrial population, loss of cristae periodicity, and altered nucleoids to cristae proximity partly rescued by OPA1 isoform 1. Overexpression of WT OPA1 or ADOA-causing mutants c.870+5G>A or c.2713C>T in WT cells, showed perturbed nucleoid array in the mitochondria population associated with cristae disorganization. Opa1-null and cells overexpressing ADOA mutants accumulated mitochondria without nucleoids. Interestingly, intramitochondrial nucleoid distribution was only altered in Opa1-null cells. Altogether, our results highlight the relevance of OPA1 in nucleoid distribution in the mitochondrial landscape and at a single-organelle level and shed light on new components of ADOA etiology.

cell biology↗

bioRxiv2023

The evolution of transposable elements in Brachypodium distachyon is governed by purifying selection, while neutral and adaptive processes play a minor role

Understanding how plants adapt to changing environments and the potential contribution of transposable elements (TEs) to this process is a key question in evolutionary genomics. While TEs have recently been put forward as active players in the context of adaptation, few studies have thoroughly investigated their precise role in plant evolution. Here we used the wild Mediterranean grass Brachypodium distachyon as a model species to identify and quantify the forces acting on TEs during the adaptation of this species to various conditions, across its entire geographic range. Using sequencing data from more than 320 natural B. distachyon accessions and a suite of population genomics approaches, we reveal that putatively adaptive TE polymorphisms are rare in wild B. distachyon populations. After accounting for changes in past TE activity, we show that only a small proportion of TE polymorphisms evolved neutrally (< 10%), while the vast majority of them are under moderate purifying selection regardless of their distance to genes. TE polymorphisms should not be ignored when conducting evolutionary studies, as they can be linked to adaptation. However, our study clearly shows that while they have a large potential to cause phenotypic variation in B. distachyon, they are not favored during evolution and adaptation over other types of mutations (such as point mutations) in this species.

evolutionary biology↗

bioRxiv2023

Small molecule inhibitors of transcriptional Cyclin Dependent Kinases impose HIV-1 latency, presenting "block and lock" treatment strategies

Antiretroviral therapy is not a cure for HIV-1 as viral rebound ensues immediately following discontinuation. The block and lock therapeutic strategy seeks to enforce proviral latency and durably suppress viremic reemergence in the absence of antiretroviral therapy. Transcriptional Cyclin Dependent Kinase activity regulates LTR transcription, however, the effect and therapeutic potential of inhibiting these kinases for enforcing HIV-1 latency remains unrecognized. Using newly developed small molecule inhibitors that are highly selective for either CDK7 (YKL-5-124), CDK9 (LDC000067), or CDK8/19 (Senexin A), we found that targeting any one of these kinases prevented HIV-1 expression at concentrations that showed no toxicity. Furthermore, although CDK7 inhibition induced cell cycle arrest, inhibition of CDK9 and/or CDK8/19 did not. Of particular interest, proviral latency as induced by CDK8/19 inhibition was maintained following drug removal while CDK9 inhibitor induced latency rebounded within 24 hrs of discontinuation. Our results indicate that the Mediator complex kinases, CDK8/CDK19, are attractive block and lock targets while sole disruption of P-TEFb is unlikely to be efficacious.

microbiology↗

bioRxiv2023

CDK8 inhibitors antagonize HIV-1 reactivation and promote provirus latency in T cells

Latent HIV-1 provirus represents a barrier towards a cure for infection, but is dependent upon the host RNA Pol II machinery for expression. We find that inhibitors of the RNA Pol II mediator kinases CDK8/19, Senexin A and BRD6989, inhibit induction of HIV-1 expression in response to latency reversing agents and T cell signaling agonists. These inhibitors were found to impair recruitment of RNA Pol II to HIV-1 LTR. HIV-1 expression in response to several latency reversal agents was impaired upon disruption of CDK8 by shRNA or gene knockout. However, the effects of CDK8 depletion did not entirely mimic CDK8/19 kinase inhibition suggesting that the mediator kinases are not functionally redundant. Furthermore, treatment of CD4+ PBMCs isolated from people living with HIV-1 and who are receiving ART with Senexin A inhibited induction of virus replication in response to T cell stimulation by PMA and ionomycin. These observations indicate that the mediator kinases CDK8 and CDK19, play a significant role for regulation of HIV-1 transcription, and that small molecule inhibitors of these enzymes may contribute to therapies designed to promote deep latency involving the durable suppression of provirus expression.

microbiology↗

bioRxiv2023

New resources for environmental genomics in the wild Mediterranean grass B. distachyon

AO_SCPLOWBSTRACTC_SCPLOWClosely related to economically important crops, the grass Brachypodium distachyon has been originally established as a pivotal species for grass genomics but more recently flourished as a model for developmental biology. Grasses encompass more than 10,000 species and cover more than 40% of the world land area from tropical to temperate regions. Given that grasses also supply about a fifth of the worlds dietary protein as cereal grains, unlocking the sources of phenotypic variation in B. distachyon is hence of prime interest in fundamental and applied research in agronomy, ecology and evolution. We present here the B. distachyon diversity panel, which encompasses 332 fully sequenced accessions covering the whole species distribution from Spain to Iraq. By combining population genetics, niche modeling and landscape genomics, we suggest that B. distachyon recolonized Europe and the Middle East following the last glacial maximum. Consequently, the species faced new environmental conditions which led to clear associations between bioclimatic variables and genetic factors as well as footprints of positive selection in the genome. Altogether, this genomic resource offers a powerful alternative to Arabidopsis thaliana to investigate the genetic bases of adaptation and phenotypic plasticity in plants and more specifically in monocots.

ecology↗

bioRxiv2023

Upstream Stimulatory Factors regulate HIV-1 latency and are required for robust T cell activation

RBF-2, composed minimally of the factors USF1, USF2, and TFII-I, is the cognate binding complex of stringently conserved cis-elements on the HIV-1 LTR, designated RBE3 and RBE1. Mutations of these elements prevent induction of provirus in response to T cell signaling. However, the function of USF1 and USF2 for this effect are relatively uncharacterized. Here, we find that deletion of the USF2 but not USF1 gene in T cells abrogates HIV-1 expression. Loss of USF2 caused a reduction in expression of USF1 protein, an effect that was not associated with decreased USF1 mRNA abundance. USF1 and USF2 were previously shown to exist predominately as heterodimers on DNA in vivo and cooperatively regulate target genes. To examine this, we performed RNA-seq analysis of T cell lines bearing knockouts of genes encoding these factors. In untreated cells we found limited evidence of coordinated global gene regulation between USF1 and USF2. In contrast we observed a high degree of genome-wide cooperative regulation of RNA expression between these factors in cells stimulated with the combination of PMA and ionomycin. In particular, we found that USF1 deletion resulted in a restricted T cell activation response, and this phenotype was exaggerated in USF2 knockout cells. These observations indicate that USF2, but not USF1, is crucial for HIV-1 expression, but the combined function of these factors is required for full T cell response.

molecular biology↗

bioRxiv2021

TRIM24 controls induction of latent HIV-1 by stimulating transcriptional elongation

The conserved HIV-1 LTR cis elements RBE1/3 bind the factor RBF2, consisting of USF1/2 and TFII-I, and are essential for reactivation of HIV-1 by T cell signaling. We determined that TFII-I recruits the tripartite motif protein TRIM24 to the LTR, and this interaction is required for efficient reactivation of HIV-1 expression in response to T cell signaling, similar to the effect of TFII-I depletion. Knockout of TRIM24 did not affect recruitment of RNA Pol II to the LTR promoter, but inhibited transcriptional elongation, an effect that was associated with decreased RNA Pol II CTD S2 phosphorylation and impaired recruitment of CDK9 to the LTR. These results demonstrate that TFII-I promotes transcriptional elongation in response to T cell activation through recruitment of the co-factor TRIM24, which is necessary for efficient recruitment of the elongation factor P-TEFb.

molecular biology↗

bioRxiv2021

Genomic signatures of sexual selection on pollen-expressed genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

evolutionary biology↗

bioRxiv2021

Controlling for variable transposition rate with an age-adjusted site frequency spectrum

Recognition of the important role of transposable elements (TEs) in eukaryotic genomes quickly led to a burgeoning literature modeling and estimating the effects of selection on TEs. Much of the empirical work on selection has focused on analyzing the site frequency spectrum (SFS) of TEs. But TEs differ from standard evolutionary models in a number of ways that can impact the power and interpretation of the SFS. For example, rather than mutating under a clock-like model, transposition often occurs in bursts which can inflate particular frequency categories compared to expectations under a standard neutral model. If a TE burst has been recent, the excess of low frequency polymorphisms can mimic the effect of purifying selection. Here, we investigate how transposition bursts affect the frequency distribution of TEs and the correlation between age and allele frequency. Using information on the TE age distribution, we propose an age-adjusted site frequency spectrum to compare TEs and neutral polymorphisms to more effectively evaluate whether TEs are under selective constraints. We show that our approach can minimize instances of false inference of selective constraint, but also allows for a correct identification of even weak selection affecting TEs which experienced a transposition burst and is robust to at least simple demographic changes. The results presented here will help researchers working on TEs to more reliably identify the effects of selection on TEs without having to rely on the assumption of a constant transposition rate.

genetics↗

bioRxiv2021

Microsecond resolved infrared spectroscopy on non-repetitive protein reactions by applying caged-compounds and quantum cascade laser frequency combs

Infrared spectroscopy is ideally suited for the investigation of protein reactions at the atomic level. Many systems were investigated successfully by applying Fourier transform infrared (FTIR) spectroscopy. While rapid-scan FTIR spectroscopy is limited by time resolution (about10 ms with 16 cm-1 resolution), step-scan FTIR spectroscopy reaches a time-resolution of about 10 ns but is limited to cyclic reactions that can be repeated hundreds of times under identical conditions. Consequently, FTIR with high time resolution was only possible with photoactivable proteins that undergo a photocycle. The huge number of non-repetitive reactions, e.g. induced by caged compounds, were limited to the ms time domain. The advent of dual comb quantum cascade laser allows now for a rapid reaction monitoring in the s time domain. Here we investigate the potential to apply such an instrument to the huge class of G-proteins. We compare caged-compound induced reactions monitored by FTIR and dual comb spectroscopy, respectively, by applying the new technique to the subunit of the inhibiting Gi protein and to the larger protein-protein complex of Gi with its cognate regulator of G-protein signaling (RGS). We observe good data quality with 4 s time resolution with a wavelength resolution comparable to FTIR. This is more than three orders of magnitude faster than any FTIR measurement on G-proteins in the literature. This study paves the way for infrared spectroscopic studies in the so far unresolvable s time regime for non-repetitive biological systems including all GTPases and ATPases.

biophysics↗

bioRxiv2020

TOR signaling modulates Cdk8-dependent GAL gene expression in Saccharomyces cerevisiae

Cdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth of gal3 yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

genetics↗

bioRxiv2018

Pervasive population genomic consequences of genome duplication in Arabidopsis arenosa

Ploidy-variable species allow direct inference of the effects of chromosome copy number on fundamental evolutionary processes. While an abundance of theoretical work suggests polyploidy should leave distinct population genomic signatures, empirical data remains sparse. We sequenced [~]300 individuals from 39 populations of Arabidopsis arenosa, a naturally diploid-autotetraploid species. We find the impacts of polyploidy on population genomic processes are subtle yet pervasive, including reduced efficiency on linked and purifying selection as well as rampant gene flow from diploids. Initial masking of deleterious mutations, faster rates of nucleotide substitution, and interploidy introgression all conspire to shape the evolutionary potential of polyploids.

evolutionary biology↗