Biology subjects

Podlesniy, P.

Publications and source records attributed to Podlesniy, P..

3 recordsLinked to original sources

bioRxiv2026

Unscheduled DNA synthesis reveals a DNA repair hotspot and biomarker of somatic instability at the expanded-CAG repeat tract in the huntingtin gene

Somatic instability (SI) of expanded DNA repeats is a hallmark of repeat expansion disorder (REDs) and drives onset and progression in Huntingtons disease (HD) yet the absence of target engagement (TE) biomarkers for SI-modulating therapies represents a critical gap to clinical development. Here, we describe the development of the unscheduled repair synthesis assay (URSA)--combining 5-ethynyl-2-deoxyuridine (EdU) pulse-labeling with digital PCR or sequencing--and show that the CAG-expanded huntingtin (HTT) exon 1 allele is a highly active DNA repair hotspot in cells from people with HD (PwHD). Repair activity increases with repeat length, is allele-specific, and depends strongly on MSH3, a central driver of somatic expansion. URSA robustly quantifies MSH3 modulation in preclinical models within days compared to weeks or months required by conventional repeat-length measurements. Critically, substantial repair activity is detectable in peripheral blood mononuclear cells (PBMCs) from PwHD, where signal correlates with CAG length and improves predictive models of somatic expansion (SE) beyond age and CAG length alone. Unlike repeat-length changes, which require years to accumulate in blood, URSA signal is measurable within days. These findings establish DNA repair activity at the mutant HTT locus as a mechanistically grounded pharmacodynamic biomarker, enabling TE monitoring on a clinically actionable timescale, and with broad applicability to REDs and other diseases where modulation of the DNA damage response is therapeutically targeted.

neuroscience↗

bioRxiv2025

Alpha-synuclein overexpression triggers divergent cellular responses and post-translational modifications in SH-SY5Y and ReNcell VM models

Alpha-synuclein (-syn) overexpression models are widely used to unravel the molecular mechanisms of Parkinsons disease (PD), particularly in light of the dose-dependent transition between its physiological and toxic roles. However, existing systems rely on inducible expression, lack robust dose stratification and comparative cellular contexts. Here, we developed and characterized a panel of stable neuronal cell lines in two human cellular models (SH-SY5Y neuroblastoma cells and ReNcell VM neural progenitors) overexpressing GFP-tagged wild-type (WT) or A53T mutant -syn at low and high overexpression levels. Utilizing this framework, we demonstrated that A53T consistently induces cytotoxicity, oxidative stress and mitochondrial dysfunction in both cell types. In contrast, WT -syn had divergent effects depending on the cellular context. In SH-SY5Y cells, it enhanced mitochondrial function and viability, whereas in ReNcell VM cells, the same protein triggered mitochondrial impairment and elevated oxidative stress. This opposing metabolic response was reflected in increased respiratory activity in SH-SY5Y cells and a marked decline across WT -syn overexpressing ReNcell VM. Importantly, post-translational modification (PTM) landscape of overexpressed WT -syn varied dramatically by cell type. ReNcell VM cells exhibited more robust modifications signatures, even in the absence of overt aggregation, which highlights a cell-type-specific PTM landscape that may contribute to differential vulnerability. Our findings underscore a complex interplay between -syn dosage, mutational status, cellular environment, and PTM profiles highlighting that neuronal vulnerability in PD is context-dependent. This work establishes a modular in vitro platform for dissecting -syn pathology and testing targeted therapeutic strategies grounded in cell-type specificity.

neuroscience↗

bioRxiv2023

Molecular Basis for Maternal Inheritance of Human Mitochondrial DNA

Uniparental inheritance of mitochondrial DNA (mtDNA) is an evolutionary trait found in nearly all eukaryotes. In many species, including humans, the sperm mitochondria are introduced to the oocyte during fertilization1, 2. The mechanisms hypothesized to prevent paternal mtDNA transmission include ubiquitination of the sperm mitochondria and mitophagy3, 4. However, whether these mechanisms play a decisive role in paternal mtDNA elimination has been disputed5, 6. We found that mitochondria in human spermatozoa are devoid of mtDNA and lack mitochondrial transcription factor A (TFAM), the major nucleoid protein required to protect, maintain, and transcribe mtDNA. During spermatogenesis, sperm cells express an isoform of TFAM, which retains the mitochondrial pre-sequence, ordinarily removed upon mitochondrial import. Phosphorylation of this pre-sequence prevents mitochondrial import and directs TFAM to the spermatozoon nucleus. TFAM re-localization from the mitochondria of spermatogonia to the spermatozoa nucleus directly correlates with the elimination of mitochondrial DNA, thereby explaining maternal inheritance in this species.

molecular biology↗