Biology subjects

Kovalchuk, I.

Publications and source records attributed to Kovalchuk, I..

8 recordsLinked to original sources

bioRxiv2026

Differential adenosine to inosine RNA editing of SINE B2 non-coding RNAs in mouse unveils a novel type of epi-transcriptome response to amyloid beta neuro-toxicity

Alzheimers disease (AD) is characterized by early molecular responses to amyloid beta neuro-toxicity that remain poorly defined. RNA editing by adenosine-to-inosine (A-to-I) conversion is a major epitranscriptomic mechanism, yet its contribution to non-coding RNA regulation during neurodegeneration is largely unknown. Short Interspersed Nuclear Element (SINE)-derived RNAs, including mouse B2 RNAs, represent the dominant substrates of A-to-I editing and have recently been shown to regulate gene expression through ribozyme-mediated processing. Here, we introduce and validate a repeat-aware bioinformatics framework that enables position-specific quantification of A-to-I editing within highly repetitive SINE RNAs--an analysis that has been challenging using standard genome-based pipelines. Applying this approach, we uncover discrete editing hotspots in B2 RNAs whose modification is selectively increased during the earliest stages of amyloid beta pathology. Elevated B2 RNA editing is consistently observed across hippocampal or neocortical tissue from two independent mouse models of amyloid beta accumulation and in hippocampal neurons exposed to acute amyloid beta toxicity. Functional perturbation of ADAR activity alters both B2 RNA editing levels and the expression of B2 RNA regulated stress-response genes, directly linking RNA editing to SINE-mediated transcriptional control. Independent validation using Nanopore direct RNA sequencing indicates increased RNA modification signals at the same B2 RNA regions identified by short-read Illumina sequencing. Together, our findings establish a previously unrecognized epitranscriptomic response to amyloid beta neurotoxicity mediated by site-specific A-to-I editing of SINE RNAs. This work defines a new analytical paradigm for studying RNA editing in RNAs from repetitive elements and reveals a regulatory axis connecting amyloid beta toxicity, ADAR activity, SINE RNA editing, and stress-responsive gene expression, with implications for conserved mechanisms underlying Alzheimers disease and related neurodegenerative disorders.

bioinformatics↗

bioRxiv2024

Glycolytic flux controls retinal progenitor cell differentiation via regulating Wnt signaling

Metabolic pathways are remodeled in response to energy and other homeostatic demands and are dynamically regulated during embryonic development, suggesting a role in guiding cellular differentiation. Here, we show that glycolytic flux is required and sufficient to bias multipotent retinal progenitor cells (RPCs) to acquire a rod photoreceptor fate in the murine retina. In RPC-specific Phosphatase and tensin homolog conditional knock-out (Pten-cKO) and RPC-specific conditional gain-of-function of dominant active PFKB3 (cytoPFKB3) mice, glycolytic gene expression and activity are elevated, correlating with precocious rod photoreceptor differentiation and outer segment maturation. Conversely, glycolytic inhibition in retinal explants suppresses RPC proliferation and photoreceptor differentiation, achieved either with 2-deoxy-d-glucose, a competitive inhibitor of glucose metabolism, by lowering media pH, which disables PKM2, a rate-limiting enzyme, or by inhibiting lactate/H+ symporters, which lowers intracellular pH. Mechanistically, we show that Wnt signaling, the top-upregulated pathway in Pten-cKO retinas, is a glycolysis-dependent pathway. Pharmacological and genetic perturbation of Wnt signaling by knocking-out Ctnnb1, encoding {beta}-catenin, phenocopies glycolytic inhibition, suppressing RPC proliferation, photoreceptor differentiation and outer segment maturation. Thus, developmental rewiring of glycolytic flux modulates Wnt signaling to drive rod photoreceptor differentiation and maturation, an instructive role that may be exploited therapeutically for cell replacement strategies. IMPACT STATEMENTTransgenic and pharmacological approaches reveal developmental elevations in glycolytic flux have an instructive role in promoting rod photoreceptor differentiation and maturation via activation of Wnt signaling.

developmental biology↗

bioRxiv2022

Pten regulates endocytic trafficking of cell adhesion and signaling molecules to pattern the retina

The retina is an exquisitely patterned tissue, with neuronal somata positioned at regular intervals to completely sample the visual field. Cholinergic amacrine cells are spectacular exemplars of precision, distributing in two radial layers and tangentially, forming regular mosaics. Here, we investigated how the intracellular phosphatase Pten and the cell adhesion molecule Dscam cooperate to regulate amacrine cell patterning. Using double mutants to test epistasis, we found that Pten and Dscam function in parallel pathways to regulate amacrine cell positioning. Mechanistically, Pten regulates endocytic remodeling of cell adhesion molecules (Dscam, Megf10, Fat3), which are aberrantly redistributed in Pten conditional-knock-out (cKO) amacrine cells. Furthermore, extracellular vesicles derived from multivesicular endosomes have altered proteomes in PtencKO retinas. Consequently, Wnt signaling is elevated in PtencKO retinal amacrine cells, the pharmacological disruption of which phenocopies PtencKO patterning defects. Pten thus controls endocytic trafficking of critical cell adhesion/signaling molecules to control amacrine cell spacing. HIGHLIGHTSO_LIPten and Dscam act in parallel pathways to regulate amacrine cell spacing C_LIO_LIEndocytic remodeling of cell adhesion molecules is perturbed in PtencKO retinas C_LIO_LIExtracellular vesicle content is altered in PtencKO retinas C_LIO_LIPerturbation of Wnt signaling phenocopies defects in amacrine cell positioning C_LI eTOC BLURBPatterns in nature range from stereotyped distributions of colored patches on butterfly wings to precise neuronal spacing in the nervous system. Waddington proposed that built-in constraints canalize developmental patterns. Touahri et al. identified Pten-mediated endocytic trafficking of cell adhesion/signaling molecules as a novel constraint measure controlling retinal amacrine cell patterning.

neuroscience↗

bioRxiv2021

Plagl1 is part of the mammalian retinal injury response and a critical regulator of Muller glial cell quiescence

Retinal damage triggers reactive gliosis in Muller glia across vertebrate species, but only in regenerative animals, such as teleost fish, do Muller glia initiate repair; proliferating and undergoing neurogenesis to replace lost cells. By mining scRNA-seq and bulk RNA-seq datasets, we found that Plagl1, a maternally imprinted gene, is dynamically regulated in reactive Muller glia post-insult, with transcript levels transiently increasing before stably declining. To study Plagl1 retinal function, we examined Plagl1+/-pat null mutants postnatally, revealing defects in retinal architecture, visual signal processing and a reactive gliotic phenotype. Plagl1+/-pat Muller glia proliferate ectopically and give rise to inner retinal neurons and photoreceptors. Transcriptomic and ATAC-seq profiles revealed similarities between Plagl1+/-pat retinas and neurodegenerative and injury models, including an upregulation of pro-gliogenic and pro-proliferative pathways, such as Notch, not observed in wild-type retinas Plagl1 is thus an essential component of the transcriptional regulatory networks that retain mammalian Muller glia in quiescence.

neuroscience↗

bioRxiv2020

Multigenerational exposure to heat stress induces phenotypic resilience, and genetic and epigenetic variations in Arabidopsis thaliana offspring

Plants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. 25 generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations (SNPs and INDELs) as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.

plant biology↗

bioRxiv2020

Proneural genes define ground state rules to regulate neurogenic patterning and cortical folding

Transition from smooth, lissencephalic brains to highly-folded, gyrencephalic structures is associated with neuronal expansion and breaks in neurogenic symmetry. Here we show that Neurog2 and Ascl1 proneural genes regulate cortical progenitor cell differentiation through cross-repressive interactions to sustain neurogenic continuity in a lissencephalic rodent brain. Using in vivo lineage tracing, we found that Neurog2 and Ascl1 expression defines a lineage continuum of four progenitor pools, with double+ progenitors displaying several unique features (least lineage-restricted, complex gene regulatory network, G2 pausing). Strikingly, selective killing of double+ progenitors using split-Cre;Rosa-DTA transgenics breaks neurogenic symmetry by locally disrupting Notch signaling, leading to cortical folding. Finally, consistent with NEUROG2 and ASCL1 driving discontinuous neurogenesis and folding in gyrencephalic species, their transcripts are modular in folded macaque cortices and pseudo-folded human cerebral organoids. Neurog2/Ascl1 double+ progenitors are thus Notch-ligand expressing niche cells that control neurogenic periodicity to determine cortical gyrification. HIGHLIGHTSO_LINeurog2 and Ascl1 expression defines four distinct transitional progenitor states C_LIO_LIDouble+ NPCs are transcriptionally complex and mark a lineage branch point C_LIO_LIDouble+ NPCs control neurogenic patterning and cortical folding via Notch signaling C_LIO_LINeurog2 and Ascl1 expression is modular in folded and not lissencephalic cortices C_LI eTOC BLURBEmergence of a gyrencephalic cortex is associated with a break in neurogenic continuity across the cortical germinal zone. Han et al. identify a pool of unbiased neural progenitors at a lineage bifurcation point that co-express Neurog2 and Ascl1 and produce Notch ligands to control neurogenic periodicity and cortical folding.

developmental biology↗

bioRxiv2020

Development and optimization of a germination assay and long-term storage for Cannabis sativa pollen

Pollen viability and storage is of great interest to cannabis breeders and researchers to maintain desirable germplasm for future use in breeding or for biotechnological and gene editing applications. Here, we report a simple and efficient cryopreservation method for long-term storage of Cannabis sativa pollen. Additionally, we have deciphered the bicellular nature of cannabis pollen using DAPI staining. We have also standardized a pollen germination assay to assess the viability of cannabis pollen, and found pollen collected from different principal growth stages exhibits different longevity. Finally, we developed a long-term storage method which includes pollen combination with baked whole wheat flower and desiccation under vacuum for cryopreservation. By using this method, we were able to maintain germination viability in liquid nitrogen after 4 months, suggesting potentially indefinite preservation of cannabis pollen.

plant biology↗

bioRxiv2020

Development of transient expression assay for Cannabis sativa which revealed differential Agrobacterium susceptibility among cannabis cultivars

In plant biology, transient expression analysis plays a vital role to provide a fast method to study the gene of interest and subsequently leads the path to develop an improved crop variety with better agronomic traits. In this study, we have reported a rapid and efficient method for transient expression in Cannabis sativa seedlings using Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 carrying the pCAMBIA1301 construct with uidA gene was used to transform cannabis seedlings and the GUS assay was used to detect the uidA expression. A 1% hydrogen peroxide (H2O2) solution was used for both seed sterilization and rapid germination steps. Transient transformation revealed that both cotyledons and young true leaves are amenable to transformation. Comparison to Nicotiana tabacum (tobacco) showed that cannabis seedlings were less susceptible to transformation with Agrobacterium tumefaciens. The susceptibility to Agrobacterium infection also varied with the different cannabis cultivars. The method established in this study has potential to be an important tool for gene-function studies and genetic improvement in cannabis.

molecular biology↗