Biology subjects

Prigge, M.

Publications and source records attributed to Prigge, M..

7 recordsLinked to original sources

bioRxiv2026

EOLA1 functions in nucleotide salvage through deacetylating free N4-acetylcytidine

RNA-based medicines rely on modified nucleotides to promote immune evasion and in vivo efficacy. Nucleotides generated from RNA degradation are either exported or recycled through metabolically favorable salvage pathways, though whether modified nucleotides are efficiently recycled remains unclear. N4-acetylcytidine (acC) is a naturally occurring modification in rRNA and tRNA that has shown promise in therapeutic mRNA applications. However, N4-acetylation impairs cytidine deamination, the first step in cytidine salvage. Here, we investigate the endogenous mechanisms that enable acC metabolism. Through sensitive sequence and structural analyses, we identify the uncharacterized human ASCH domain protein EOLA1 as a key acC deacetylase in nucleotide salvage. EOLA1 inactivation leads to free intracellular acC accumulation and increased cytotoxicity upon nucleotide export inhibition. While steady-state acC levels in cellular RNAs remain unchanged, EOLA1-dependent regulation of free acC is evident basally and is exacerbated by exogenous mRNA delivery. Proteomic analyses place EOLA1 in proximity to ribosomal proteins, adjacent to endogenous acC sources. In vitro assays confirm EOLA1 specificity for acC, and structural analysis reveals a narrow nucleotide-binding pocket consistent with mononucleotide selectivity. These findings identify EOLA1 as a bona fide acC eraser and uncover a previously unrecognized pathway for recycling modified nucleotides with relevance to therapeutic RNA design.

molecular biology↗

bioRxiv2026

A VTA-pontine GABA pathway biases backward locomotion via local and distal inhibition

Locomotor direction in mammals is implemented by descending circuits, yet how midbrain selection systems bias directional motor output remains unclear. Here we define a projection-defined inhibitory pathway from the ventral tegmental area to the oral pontine reticular nucleus (VTAPnO) whose activation is sufficient to drive backward locomotion. These TH- VTA neurons form monosynaptic GABAA synapses locally while projecting to PnO, establishing a dual local-projection inhibitory architecture. Somatic activation reliably induced backward locomotion, and selective stimulation of VTAPnO terminals reproduced the effect. Pathway recruitment produced a rapid transient increase in dopaminergic single-unit activity and frequency-dependent increases in dopaminergic population calcium signals in awake mice. During forced locomotion, chronically recorded VTAPnO neurons were preferentially engaged during reverse compared to forward rotations. Together, these findings reveal a projection-defined midbrain pathway that biases locomotor direction through coordinated local inhibition and distal engagement of a brainstem premotor node.

neuroscience↗

bioRxiv2023

Robust Optogenetic Inhibition with Red-light-sensitive Anion-conducting Channelrhodopsins

Channelrhodopsins (ChRs) are light-gated ion channels widely used to optically activate or silence selected electrogenic cells, such as individual brain neurons. Here, we describe identifying and characterizing a set of anion-conducting ChRs (ACRs) from diverse taxa and representing various branches of the ChR phylogenetic tree. The Mantoniella squamata ACR (MsACR1) showed high sensitivity to yellow-green light ({lambda}max at 555 nm) and was further engineered for optogenetic applications. A single amino-acid substitution that mimicked red-light sensitive rhodopsins like Chrimson shifted the photosensitivity 20 nm towards red light and accelerated photocurrent kinetics. Hence, it was named red and accelerated ACR, raACR. Both wild-type and mutant are capable optical silencers at low light intensities in mouse neurons in vitro and in vivo, while raACR offers a higher temporal resolution.

biophysics↗

bioRxiv2023

A versatile synaptotagmin-1 nanobody provides perturbation-free live synaptic imaging and low linkage-error in super-resolution microscopy

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy changes the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here we introduce a nanobody that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions in living neurons as an intrabody (iNbSyt1) and is minimally invasive, leaving synaptic transmission almost unaffected, as demonstrated by the crystal structure of the NbSyt1 bound to synaptotagmin-1 and by our physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as we showcase here by measuring spatially-localized presynaptic Ca2+ with an NbSyt1-jGCaMP8 chimera. Moreover, its small size makes the NbSyt1 ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience at a higher precision than possible in the past, over multiple spatiotemporal scales.

neuroscience↗

bioRxiv2022

Exploring volume mediated bystander-inhibition in a neuron model of the Locus coeruleus

The Locus coeruleus (LC) modulates various neuronal circuits throughout the brain. Its unique architectural organization encompasses a net of axonal innervation that spans the entire brain; while its somatic core is highly compact. Recent research revealed an unexpected cellular input specificity within the nucleus that can give rise to various network states that either broadcast norepinephrine signals throughout the brain or pointedly modulate specific brain areas. Such adaptive input-output functions likely surpass our existing network models that build upon a given synaptic wiring configuration between neurons. As the distances between noradrenergic neurons in the core of the LC are unusually small, neighboring neurons could theoretically impact each other via volume transmission of NE. We therefore set out to investigate if such interaction could be mediated through noradrenergic alpha2-receptors in a spiking neuron model of the LC. We validate our model of LC neurons through comparison with experimental patch-clamp data and identify key variables that impact alpha2-mediated inhibition of neighboring LC neurons. Our simulation confirms a reliable autoinhibition of LC neurons after episodes of high neuronal activity that continue even after neuronal activity subsided. Also, dendro-somatic synapses inhibit spontaneous spiking in the somatic compartment of connected neurons in our model. We determined the exact position of hundreds of LC neurons in the mouse brain stem via a tissue clearing approach and, based on this, further determined that 25 percent of noradrenergic neurons have a neighboring LC neuron within less than a 25-micrometer radius. By modeling NE diffusion, we estimate that more than 15 percent of alpha2-adrenergic receptors fraction can bind NE within such diffusion radius. Our spiking neuron model of LC neurons predicts that repeated or long-lasting episodes of high neuronal activity induce partitioning of the gross LC network, and reduces the spike rate in neighboring neurons at distances smaller than 25 micrometers. As these volume-mediate neighboring effects are challenging to test with the current methodology, our findings can guide future experimental approaches to test this phenomenon and its physiological consequences.

neuroscience↗

bioRxiv2018

TET-catalyzed 5-carboxylcytosine promotes CTCF binding to suboptimal sequences genome-wide

The mechanisms supporting dynamic regulation of CTCF binding sites remain poorly understood. Here we describe the TET-catalyzed 5-methylcytosine derivative, 5-carboxylcytosine (5caC) as a factor driving new CTCF binding within genomic DNA. Through a combination of in vivo and in vitro approaches, we reveal that 5caC generally strengthens CTCF association with DNA and facilitates binding to suboptimal sequences. Dramatically, profiling of CTCF binding in a cellular model that accumulates genomic 5caC identified [~]13,000 new CTCF sites. The new sites were enriched for overlapping 5caC and were marked by an overall reduction in CTCF motif strength. As CTCF has multiple roles in gene expression, these findings have wide-reaching implications and point to induced 5caC as a potential mechanism to achieve differential CTCF binding in cells.

molecular biology↗

bioRxiv2017

Independence Between Pre-mRNA Splicing And De Novo DNA Methylation In An Isogenic Minigene Resource

Actively transcribed genes adopt a unique chromatin environment with characteristic patterns of enrichment. Within gene bodies, H3K36me3 and cytosine DNA methylation are elevated at exons of spliced genes and have been implicated in the regulation of pre-mRNA splicing. H3K36me3 is further responsive to splicing, wherein splicing inhibition led to a redistribution and general reduction over gene bodies. In contrast, little is known of the mechanisms supporting elevated DNA methylation at actively spliced genic locations. Recent evidence associating the de novo DNA methyltransferase Dnmt3b with H3K36me3-rich chromatin raises the possibility that genic DNA methylation is influenced by splicing-associated H3K36me3. Here, we report the generation of an isogenic resource to test the direct impact of splicing on chromatin. A panel of minigenes of varying splicing potential were integrated into a single FRT site for inducible expression. Profiling of H3K36me3 confirmed the established relationship to splicing, wherein levels were directly correlated with splicing efficiency. In contrast, DNA methylation was equivalently detected across the minigene panel, irrespective of splicing and H3K36me3 status. In addition to revealing a degree of independence between genic H3K36me3 and DNA methylation, these findings highlight the generated minigene panel as a flexible platform for the query of splicing-dependent chromatin modifications.

molecular biology↗