Biology subjects

Pokhilko, A.

Publications and source records attributed to Pokhilko, A..

6 recordsLinked to original sources

bioRxiv2025

Prominent white matter related abnormalities associated with diverse molecular alterations in a mouse model of Col4a1 cerebral small vessel disease.

White matter abnormalities are a hallmark of cerebral small vessel disease (cSVD) and are closely linked to cognitive decline and dementia. However, despite their clinical importance, underlying mechanisms remain poorly understood. Collagen IV, encoded by genes COL4A1/COL4A2, is a major component of the basement membrane, a specialised extracellular matrix (ECM) structure. Mutations in these genes cause a genetic form of cSVD. We tested the hypothesis that ECM defects caused by a Col4a1 mutation lead to white matter pathology using an established mouse model of cSVD (Col4a1+/Svc). Behavioural testing with magnetic resonance diffusion tensor imaging, pathology and ultrastructural investigations of white matter were studied. The studies revealed that Col4a1+/Svc mice have cognitive impairments, reduced myelinating oligodendrocyte pools, axonal myelination defects, and altered white matter structural integrity. Proteomic analysis, of isolated white matter from Col4a1+/Svcmice, identified extensive changes to ECM composition and endoplasmic reticulum (ER) biology including ER stress induction. We also demonstrated that targeting protein folding to promote collagen secretion and reduce ER stress, increased myelinating oligodendrocytes and axon-glial integrity in Col4a1+/Svc mice. These data provide novel insight into the pathomolecular mechanisms of white matter abnormalities in cSVD and identify a modifiable pathway as a putative therapeutic target for cSVD.

neuroscience↗

bioRxiv2025

An Nlrp5-null mutation leads to attenuated de novo methylation in oocytes, accompanied by a significant reduction in DNMT3L

Nlrp5 encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo. NLRP5 mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC gene KHDC3L had globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of an Nlrp5- null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show that de novo DNA methylation is attenuated in Nlrp5-null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.

developmental biology↗

bioRxiv2024

Protein degradation shapes developmental tempo in mouse and human neural progenitors

The pace of embryonic development differs markedly across mammalian species, yet the molecular mechanisms underlying these tempo differences remain largely unknown. Here, we systematically compared protein dynamics in mouse and human neural progenitors (NPs) and examined how protein stability influences developmental timing. We find that mouse NPs exhibit faster protein production and degradation than human NPs. Human NPs display broadly increased protein half-lives, independent of cellular compartment or protein function, and this difference persists in post-mitotic neurons. Consistent with this, proteasomal activity is lower in human embryonic spinal cord and stem cell-derived neural progenitors than mouse, correlating with reduced expression of proteasome-associated proteins. Functionally, accelerating the degradation of the key transcriptional repressor IRX3 in mouse NPs speeds the activation of its target gene, providing causal evidence that protein turnover modulates developmental tempo. These results reveal that species-specific regulation of protein degradation shapes the timing of neural development and suggest that evolutionary tuning of proteasomal activity contributes to differences in embryonic developmental pace.

developmental biology↗

bioRxiv2022

Mathematical modelling of brain mTOR activity identifies selective vulnerability of cell types and signalling pathways

The mTOR pathway is a global regulator of protein biosynthesis and cellular homeostasis. Understanding the differences in mTOR pathway activity between cell types is important for elucidating the role of mTOR in physiological and pathophysiological processes. The non-linear structure of the pathway, with multiple feedback loops and inputs complicates the interpretation of experimental data and requires mathematical modelling. We modelled mTOR activation under healthy and disease conditions using recently published single nuclei gene expression data from the human brain. The model predicts substantial variations in mTOR pathway activity between cell types, with neurons and astrocytes being highly sensitive to insulin and neuregulins, while vascular smooth muscle cells and pericytes are highly sensitive to PDGF. Two principal negative regulators of mTOR - TSC and PTEN have prominent roles in PDGF-mediated mTOR activation in endothelial cells and oligodendrocytes. In Alzheimers Disease brain we find that insulin-mediated activitation of mTOR pathway is selectively upregulated in microglia and oligodendrocytes and downregulated in other cells. Our mathematical modelling characterises ligand sensitivity of cerebrovascular cell types and provides insights into the brain mTOR dynamics in health and disease.

systems biology↗

bioRxiv2022

Cryptochrome Stabilization Ameliorates Chronic Pain

Physiological and pathological pain exhibits striking diurnal variation, but the underlying mechanisms are largely unknown. We now describe an independent molecular clock in peripheral sensory neurons and satellite glial cells of sensory ganglia. We show that it is the sensory neuron transcription-translation feedback loops (TTFLs) that are responsible for diurnal pain behaviors. This clock regulates diurnal neurophysiological responses to a range of ligands, as well as synaptic activities of primary nociceptors. Furthermore, we find that loss of Cry1 and Cry2, the repressive arm of the core TTFLs, intensifies pain responses associated with increased voltage-gated sodium channel currents. Conversely, stabilization of CRY1 and CRY2 using the small molecule KL001, reduces pain sensitivity. Our results highlight novel opportunities to address chronic pain by directly harnessing circadian mechanisms. One-Sentence SummaryA peripheral pain clock governs daily pain fluctuations, which can be harnessed for treating pain disorders.

neuroscience↗

bioRxiv2018

Mathematical modelling of serine integrase - mediated gene assembly

Site-specific recombination promoted by serine integrases can be used for ordered assembly of DNA fragments into larger arrays. When a plasmid vector is included in the assembly, the circular product DNA molecules can transform E. coli cells. A convenient \"one-pot\" method using a single integrase involves recombination between pairs of matched orthogonal attachment sites, allowing assembly of up to six DNA fragments. However, the efficiency of assembly decreases as the number of fragments increases, due to accumulation of incorrect products in which recombination has occurred between mismatched sites. Here we use mathematical modelling to analyse published experimental data for the assembly reactions and suggest potential ways to improve assembly efficiency. We assume that unproductive synaptic complexes between pairs of mismatched sites become predominant as the number and diversity of sites increase. Our modelling predicts that the proportion of correct products can be improved by raising fragment DNA concentrations and lowering plasmid vector concentration. The assembly kinetics is affected by the inactivation of integrase in vitro. The model also predicts that the precision might be improved by redesigning the location of attachment sites on fragments to reduce the formation of the wrong circular products. Our preliminary experimental explorations of assembly with {phi}C31 integrase confirmed that assembly efficiency might be improved. However, optimization of efficiency would require more experimental work on the mechanisms of wrong product formation. The use of a more efficient integrase (such as Bxb1) might be a more promising approach to assembly optimization. The model might be easily extended for different integrases or/and different assembly strategies, such as those using multiple integrases or multiple substrate structures.

synthetic biology↗