Biology subjects

Neniskyte, U.

Publications and source records attributed to Neniskyte, U..

7 recordsLinked to original sources

bioRxiv2026

Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. Here, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We also introduced specific mismatches to the gRNA: Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage kinetics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they shifted editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

biochemistry↗

bioRxiv2026

Electrophysiological properties and synaptic activity of the mouse hippocampal CA1 neurons during postnatal development

Early postnatal development is a critical period for hippocampal circuit maturation. While postnatal hippocampal development has been mostly studied in rats, less is known about the developmental trajectory of electrophysiological properties in mice, despite the wide use of these animal models for molecular and genetic studies of nervous system. In this study, we investigated the postnatal maturation of hippocampal CA1 pyramidal neurons in male and female wild-type mice. Whole-cell patch-clamp recordings were performed in acute hippocampal slices to assess passive and active membrane properties as well as spontaneous excitatory synaptic activity. We found that maturation of neuronal firing properties was associated with faster responses to stimulation, higher-amplitude and shorter-duration action potentials, and more precise control of neuronal firing. Simultaneously, synaptic activity changed across development, with decreased sEPSC inter-event intervals and stable event amplitudes, suggesting enhanced functional connectivity without major changes in synaptic strength. Sex-dependent differences in electrophysiological properties were observed primarily during the first postnatal week, indicating that sex influences the early trajectory of neuronal maturation. Together, our findings provide a comprehensive electrophysiological baseline for mouse hippocampal CA1 pyramidal neurons during postnatal development.

biophysics↗

bioRxiv2026

Improved Protein Encapsulation and Delivery by Lipid Nanoparticles with Refined Ionizable Lipid Content

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

biochemistry↗

bioRxiv2026

Maternal High-Fat Diet Induces Sex- and Estrous Cycle-Specific Glial Dysregulation in the Peripheral Offspring Retina

PurposeMaternal high-fat diet (mHFD) induces metabolic disturbances that lead to inflammatory responses in the offsprings brain. The retina, as part of the central nervous system, may be similarly affected. This study aimed to determine how mHFD affects microglial and Muller cell activity in the retinas of offspring and assess how these effects depend on sex and the female estrous cycle. MethodsFemale C57Bl/6J mice were fed a control diet (CD, 10% fat) or a high-fat diet (HFD, 60% fat) from weaning to lactation. The offspring were weaned to a normal rodent diet. Retinal structure and glial cells were assessed using immunohistochemical labeling of retinal ganglion cells, Muller glia, astrocytes, phagocytic and inflammatory markers. Observed retinal changes in female offspring were correlated with the estrous cycle stages. ResultsmHFD induced subtle retinal structural changes and sex-specific alterations in glial cells of offspring peripheral retina. Male offspring exhibited a reduced microglial area, accompanied by increased phagocytic capacity, whereas females showed the opposite pattern. Under mCD, the microglial area and its phagocytic and metabolic activity fluctuated with the female estrous cycle, while mHFD diminished the differences between phases. Additionally, mHFD reduced Muller glial reactivity in females, indicating disrupted glial communication. ConclusionsOur findings demonstrate that mHFD has a sex-specific effect on the offsprings peripheral retina, affecting the response of retinal microglia to female reproductive hormones.

neuroscience↗

bioRxiv2025

Varicose-projection astrocytes: a reactive phenotype associated with neuropathology

Glial cells are fundamental for the pathophysiology of all neurological disorders. Astrocytes, the primary home-ostatic cells of the central nervous system (CNS), exhibit species-specific characteristics, with human astrocytes specifically displaying unique structural and functional features. It is thus essential to investigate human-specific astrocytic responses to neuropathology using human-relevant models. Varicose projection (VP) astrocytes, traditionally considered specific to humans and apes, were suggested to reflect pathological burden, albeit direct evidence linking them to neurological diseases has been lacking. Here, we demonstrate for the first time that VP astrocytes are present in mice and tigers (Panthera tigris) and we provide evidence from four distinct human-based models that VP astrocytes are not a distinct physiological astrocyte subtype but rather a novel class of reactive astrocytes associated with neuropathology. Using human induced pluripotent stem cell (hiPSC)-derived astrocytes, mixed neural cultures, and cortical organoids, we showed that VP astrocytes are induced by pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor- (TNF-) or LPS. Notably, cytokine withdrawal reverses the VP phenotype of astrocytes, indicating that it is a transient, inflammation-dependent state. We characterized the distinctive components of varicosities, including markers for extracellular vesicles, mitochondria, Golgi and endoplasmic reticulum components, suggesting roles in cellular stress responses and metabolic dysregulation. We further validated the pathological relevance of VP astrocytes by documenting their significant enrichment in postmortem brain samples from patients with several neurodegenerative diseases including as Alzheimers disease, Parkinsons disease, and multiple sclerosis, as well as in surgical resections from patients with epilepsy due to hippocampal sclerosis or brain tumors, including previously unreported subcortical regions such as basal ganglia. Additionally, we identified a higher number of VP astrocytes also in mouse astrocytes upon treatment with pro-inflammatory cytokines, suggesting that formation of VP astrocytes is an evolutionarily conserved astrocytic response to neuroinflammation. Our findings point to VP astrocytes as a novel reactive astrocyte subtype closely linked to neuropathology, highlighting their potential as biomarkers and therapeutic targets in neurological diseases. This study lays the groundwork for future investigations into the mechanisms driving VP astrocyte formation and their broader implications in neuropathology.

neuroscience↗

bioRxiv2024

Integrity assay for messenger RNA in mouse and human brain samples and synaptosomal preparations

Traditionally, RNA integrity evaluation is based on ribosomal RNAs (rRNAs). Nevertheless, gene expression studies are usually focused on protein coding messenger RNAs (mRNAs). As rRNA and mRNA have significant structural and functional differences, the assumption that rRNA integrity properly represents mRNA integrity may not be accurate. Moreover, contrary to whole tissue RNA samples, subcellular preparations such as synaptosomes contain almost no rRNA, thus prohibiting the use of traditional rRNA-based methods to assess sample RNA integrity. Here we present a RT-qPCR based assay, which estimates mRNA integrity by comparing the abundance of 3 and 5 mRNA fragments in a long constitutively expressed mouse or human PGK1 mRNA. The assay was tested and validated using plasmids with cloned 3- and 5-ends of the PGK1 cDNA reflecting different ratios of 3 and 5 cDNA amplicons in partially degraded RNA samples. The accuracy of integrity score calculation was ensured by integrating a mathematical correction of qPCR results to account for the variable amplification efficiency of different primer pairs. The 5:3 assay was used to quantify RNA degradation in heat-degraded mouse and human brain tissue RNA as well as in clinical human brain RNA samples. Importantly, the expression of housekeeping genes correlated better with 5:3 integrity value than with the RIN. Finally, we were even able to use 5':3' assay to assess mRNA integrity in mouse synaptosomal preparations that lack rRNAs. We concluded that the 5:3 assay can be used as a reliable and sensitive method to evaluate mRNA integrity in mouse and human brain tissue and subcellular preparations.

molecular biology↗

bioRxiv2017

Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction

Microglia are highly motile glial cells that are proposed to mediate synaptic pruning during neuronal circuit formation. Disruption of signaling between microglia and neurons leads to an excess of immature synaptic connections, thought to be the result of impaired phagocytosis of synapses by microglia. However, until now the direct phagocytosis of synapses by microglia has not been reported and fundamental questions remain about the precise synaptic structures and phagocytic mechanisms involved. Here we used light sheet fluorescence microscopy to follow microglia-synapse interactions in developing organotypic hippocampal cultures, complemented by three-dimensional ultrastructural characterization using correlative light and electron microscopy (CLEM). Our findings define a set of dynamic microglia-synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia. These findings allow us to propose a mechanism for the facilitatory role of microglia in synaptic circuits remodeling and maturation.

neuroscience↗