Biology subjects

Kaczmarczyk, L.

Publications and source records attributed to Kaczmarczyk, L..

14 recordsLinked to original sources

bioRxiv2025

Prion shedding is reduced by chronic wasting disease vaccination

Chronic wasting disease (CWD) is a strictly fatal and highly contagious prion disease of wild and farmed cervids currently expanding in North America. Prion diseases are caused by conversion of the cellular prion protein to its pathological isoform PrPSc. Vaccination is considered a promising strategy to contain CWD, even though prion diseases do not show classical immune responses. For CWD containment, it is important that vaccines reduce shedding of prions in excreta, a major contributor to transmission. Here, we tested the effect of vaccines on prion shedding in feces and urine by vaccinating and prion infecting knock-in mice that recapitulate CWD pathogenesis as found in cervids. Vaccination reduced or even prevented CWD shedding in feces and urine collected between 30-90% of incubation time to disease. This is the first report showing that prion shedding can be blocked in a prion disease. For CWD specifically it may reduce the environmental prion burden and break the disease transmission cycle.

neuroscience↗

bioRxiv2025

Central role for fast nociceptors in mechanical nocifensive behavior and sensitization

Nociceptors, primary afferent nerve fibers that signal noxious stimuli, are broadly divided into slowly conducting unmyelinated C fibers and fast-conducting myelinated A fibers. Whereas C-nociceptors have been extensively studied, considerably less is known about the function of A-nociceptors. To address this gap, we developed an intersectional genetic approach for robust and selective interrogation and manipulation of mechanically responsive A-nociceptors (A-MNs) in mice. Optogenetic A-MN stimulation induced rapid and precise withdrawal reflexes as well as place aversion and facial expression changes consistent with pain affect, while inhibition strongly impaired mechanical nociceptive withdrawal reflexes, demonstrating that A-MNs are necessary and sufficient for rapid avoidance of noxious mechanical stimuli. Prolonged A-MN activation induced mechanical allodynia and central sensitization. In a rare individual lacking thickly myelinated A{beta} fibers, mechanical withdrawal reflexes were completely absent, and pain perception reduced. Together, these findings identify fast-conducting mechano-nociceptors as essential drivers of nocifensive behaviors in mice and humans.

neuroscience↗

bioRxiv2025

Alternative splicing generates a Ribosomal Protein S24 isoform induced by neuroinflammation and neurodegeneration

Neuroinflammation, particularly that involving reactive microglia, the brains resident immune cells, is implicated in the pathogenesis of major neurodegenerative diseases. However, early markers of this process are in high demand. Multiple studies have reported changes in ribosomal protein (RP) expression during neurodegeneration, but the significance of these changes remains unclear. Ribosomes are evolutionarily conserved protein synthesizing machines, and although commonly viewed as invariant, accumulating evidence suggest functional ribosome specialization through variation in their protein composition. By analyzing cell type-specific translating mRNAs from mouse brains, we identify distinct RP expression patterns between neurons, astrocytes, and microglia, including neuron-specific RPs, Rpl13a and Rps10. We also observed complex expression relationships between RP paralogs and their canonical counterparts, suggesting regulated mechanisms for generating heterogeneous ribosomes. Analysis across brain regions revealed that Rplp0 and Rpl13a, commonly used normalization references, show heterogeneous expression, raising important methodological considerations for gene expression studies. Importantly, we show that Rps24, an essential ribosome component that undergoes alternative splicing to produce protein variants with different C-termini, exhibits striking cell type-specific isoform expression in brain. The Rps24c isoform is predominantly expressed in microglia and is increased by neuroinflammation caused by aging, neurodegeneration, or inflammatory chemicals. We verify increased expression of S24-PKE, the protein variant encoded by Rps24c, in brains with Alzheimers disease, Parkinsons disease, and Huntingtons disease, and relevant mouse models, using isoform-specific antibodies. These findings establish heterogeneous RP expression as a feature of brain cell types and identify Rps24c/S24-PKE as a novel marker for neuroinflammation and neurodegeneration.

neuroscience↗

bioRxiv2025

Genetic targeting of myelinated primary afferent neurons using a new NefhCreERT2 knock-in mouse

Primary afferent neurons that convey somatosensory modalities comprise two large, heterogeneous populations: small-diameter neurons that give rise to slowly conducting unmyelinated axonal C fibers and medium-to-large diameter neurons with fast myelinated A fibers. Despite these two major groupings, tools to differentiate between unmyelinated and myelinated primary afferent fibers by genetic targeting have not been available; in particular, whereas numerous mouse driver lines exist to target different C fiber populations, genetic tools that target myelinated primary afferent populations are scarce. Here we describe a knock-in mouse line expressing tamoxifen-dependent CreERT2 under control of the Nefh gene, which encodes neurofilament heavy chain (NFH or NF200), a protein that is highly enriched in myelinated fibers. This mouse enables highly selective and efficient recombination of Cre-dependent reporters for functional and anatomical interrogation of myelinated fibers while excluding unmyelinated C fibers. In combination with other recombinase-expressing mouse lines, this genetic tool will be valuable for intersectional targeting of subpopulations of myelinated primary afferent fibers.

neuroscience↗

bioRxiv2025

A NaV1.8FlpO mouse enabling selective intersectional targeting of low threshold C fiber mechanoreceptors and nociceptors

Genetic targeting of select populations of cells in the mouse nervous system is often hampered by a lack of selectivity, as candidate genes for such targeting are commonly expressed by multiple cell populations, also in the same region. Intersectional targeting using two or more genes has been enabled by the development of reporter tools dependent on more than one recombinase or gene regulator. Still, widespread adoption of intersectional tools is complicated by a scarcity of driver mice expressing recombinases otherthan Cre. Here we report the generation and characterization of a new driver mouse that expresses the FlpO recombinase from the endogenous locus of the Scn10a gene encoding NaV1.8, a voltage-gated sodium channel that is almost exclusively expressed in the afferent limb of the peripheral nervous system. Moreover, among sensory neurons the channel is preferentially expressed in nociceptors and in low-threshold C-fiber mechanoreceptors (C-LTMRs). The mouse showed high recombination efficiency (97 %) and selectivity (93 %) in dorsal root ganglia. Reporter-expressing fibers were observed in a variety of peripheral tissues, including skin, skeletal muscle, genitalia, bladder and intestines. To validate the suitability of the FlpO mouse line for intersectional targeting, we crossed it with a mouse line expressing CreERT2 from the Th (tyrosine hydroxylase) locus. This approach resulted in strikingly selective and efficient targeting of C-LTMRs, showing robust visualization of nerve endings of these fibers in skin and spinal cord at the light and electron microscopic level. Thus, the NaV1.8Flpo mouse line presented here constitutes a selective and versatile tool for intersectional genetic targeting of NaV1.8 expressing primary afferent neurons.

neuroscience↗

bioRxiv2025

A fast nociceptive subsystem mediating rapid reflexive behavior but not affective pain

Spinal nociceptive withdrawal reflexes are widely believed to rely on unmyelinated and thinly myelinated nociceptive fibers that also signal affective and motivational aspects of pain. Here we discover a population of myelinated mechanoreceptive nociceptor that forms free nerve endings as well as circumferential endings around hair follicles, and exclusively terminate in the deep spinal dorsal horn. Optogenetic activation of these fibers triggers rapid withdrawal reflexes that are precise and selective for the targeted limb, while silencing increases the threshold of mechanical nociceptive withdrawal reflexes. By contrast, optogenetic stimulation of the fibers is not associated with place aversion nor with changes in facial expression. Thus, we conclude that this nerve fiber population is uniquely positioned to rapidly respond to mechanical threats via selective withdrawal of the targeted body part, whereas other fast and slow nociceptive pathways are required for affective-motivational aspects of pain.

neuroscience↗

bioRxiv2024

The Brain Interactome of a Permissive Prion Replication Substrate

Bank voles are susceptible to prion strains from many different species, yet the molecular mechanisms underlying the ability of bank vole prion protein (BVPrP) to function as a universal prion acceptor remain unclear. Potential differences in molecular environments and protein interaction networks on the cell surface of brain cells may contribute to BVPrPs unusual behavior. To test this hypothesis, we generated knock-in mice that express physiological levels of BVPrP (M109 isoform) and employed mass spectrometry to compare the interactomes of mouse (Mo) PrP and BVPrP following mild in vivo crosslinking of brain tissue. Substantial overlap was observed between the top interactors for BVPrP and MoPrP, with established PrP-interactors such as neural cell adhesion molecules, subunits of Na+/K+-ATPases, and contactin-1 being equally present in the two interactomes. We conclude that the molecular environments of BVPrP and MoPrP in the brains of mice are very similar. This suggests that the unorthodox properties of BVPrP are unlikely to be mediated by differential interactions with other proteins.

neuroscience↗

bioRxiv2023

Anti-prion drugs do not improve survival in knock-in models of inherited prion disease

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Paradoxically, the combination of Anle138b and IND24 appeared to accelerate disease by 16% and 26% in kiBVIE200K and kiBVID178N mice, respectively, and accelerated the aggregation of mutant PrP molecules in vitro. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions.

pharmacology and toxicology↗

bioRxiv2023

Convergent generation of atypical prions in knock-in mouse models of genetic prion disease

Most cases of human prion disease arise due to spontaneous misfolding of wild-type or mutant prion protein. Though recapitulating spontaneous prion conversion in animal models has proven challenging, transgenic mice expressing the misfolding-prone bank vole prion protein (BVPrP) recreate certain key aspects of sporadic and genetic prion disease. However, it remains unclear whether spontaneous prion generation can occur in the absence of protein over-expression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knock-in mice expressing physiological levels of either wild-type or mutant BVPrP with isoleucine at codon 109. While mice expressing wild-type BVPrP remained free from neurological disease, a subset of knock-in mice expressing BVPrP with mutations that cause either fatal familial insomnia (D178N) or familial Creutzfeldt-Jakob disease (E200K) developed progressive neurological illness. Brains from spontaneously ill knock-in mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant prion protein. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP knock-in mice transmitted disease to mice expressing wild-type BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical both pre- and post-transmission, suggesting that both mutations guide the formation of a highly similar atypical prion strain. These findings imply that knock-in mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. Therefore, these mice represent useful tools for studying the early stages of genetic prion diseases.

neuroscience↗

bioRxiv2023

DNA G-quadruplex is a transcriptional control device that regulates memory

The conformational state of DNA fine-tunes the transcriptional rate and abundance of RNA. Here we report that DNA G-quadruplex (G4-DNA) accumulates in neurons in an experience-dependent manner, and that this is required for the transient silencing and activation of genes that are critically involved in learning and memory. In addition, site-specific resolution of G4-DNA by dCas9-mediated deposition of the helicase DHX36 impairs fear extinction memory. Dynamic DNA structure states therefore represent a key molecular mechanism underlying memory consolidation. One-Sentence SummaryG4-DNA is a molecular switch that enables the temporal regulation of the gene expression underlying the formation of fear extinction memory.

neuroscience↗

bioRxiv2022

Cerebellar granule neurons induce Cyclin D1 in an early stage of Huntington's disease

Although Huntingtons disease (HD) is predominantly defined by selective vulnerability of striatal projection neurons, there is increasing evidence that cerebellar degeneration modulates clinical symptoms. However, little is known about cell type-specific responses of cerebellar neurons in HD. To dissect early disease mechanisms in the cerebellum and cerebrum, we analyzed translatomes of neuronal cell types from both regions in a new HD mouse model. For this, HdhQ200 knock-in mice were backcrossed with the calm, 129S4 strain, to constrain experimental noise caused by variable hyperactivity of mice in a C57BL/6 background. Behavioral and neuropathological characterization showed that these S4-HdhQ200 mice had very mild behavioral abnormalities starting around 12 months of age that remained mild up to 18 months. By 9 months, we observed abundant Huntingtin-positive neuronal intranuclear inclusions (NIIs) in the striatum and cerebellum. The translatome analysis of GABAergic cells of the cerebrum further confirmed changes typical of HD-induced striatal pathology. Surprisingly, we observed the strongest response with 626 differentially expressed genes in glutamatergic neurons of the cerebellum, a population consisting predominantly of granule cells, a cell type commonly considered disease-resistant. Our findings suggest vesicular fusion and exocytosis, as well as differentiation-related pathways are affected. Furthermore, increased expression of cyclin D1 (Ccnd1) in the granular layer and upregulated expression of polycomb group complex protein genes and cell cycle regulators Cbx2, Cbx4 and Cbx8 point to a putative role of aberrant cell cycle regulation in cerebellar granule cells in early disease.

bioinformatics↗

bioRxiv2021

Homologous recombination repair creates mutations in the non-coding genome that alter Topoisomerase-1 cleavage sites & orchestrates irinotecan resistance

Resistance to chemotherapy is a leading cause of treatment failure. Drug-resistance mechanisms involve mutations in specific proteins or changes in their expression levels. It is commonly understood that resistance mutations happen randomly prior to treatment and are selected during the treatment. However, selection of drug-resistant mutants in culture could be achieved by multiple drug exposures of cloned genetically identical cells, and thus cannot result from selection of pre-existent mutations. Accordingly, adaptation must involve generation of mutations de-novo upon drug treatment. Here we explored the origin of resistance mutations to a widely used Top1 inhibitor irinotecan, which triggers DNA breaks, causing cytotoxicity. Resistance mechanism involved gradual accumulation of recurrent mutations in non-coding regions of DNA at Top1-cleavage sites. Surprisingly, cancer cells had higher number of such sites than reference genome, which may define their increased sensitivity to irinotecan. Homologous recombination repair of DNA double strand breaks at these sites following initial drug exposures gradually reverted cleavage-sensitive "cancer" sequences back to cleavage-resistant "normal" sequences. These mutations reduced generation of DNA breaks upon subsequent exposures, thus gradually increasing the drug resistance. Together, large target size for mutations and their Top1-guided generation lead to their gradual and rapid accumulation, synergistically accelerating development of resistance. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/470089v3_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d9d93forg.highwire.dtl.DTLVardef@45b88forg.highwire.dtl.DTLVardef@157cafeorg.highwire.dtl.DTLVardef@cff4a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Reduced nuclear lamin A/C enhances cancer cell squeezing through rigid barriers, does not facilitate endothelial crossing, and impairs experimental metastasis

The mechanisms by which the nuclear lamina of tumor cells controls their migration and survival are poorly understood. Lamin A and its variant lamin C are key nuclear lamina proteins that control nucleus stiffness and chromatin conformation. Downregulation of lamin A/C levels in two metastatic lines, B16F10 melanoma and E0771 breast carcinoma, facilitated cell squeezing through rigid pores, elevated nuclear deformability and reduced heterochromatin. Unexpectedly, the transendothelial migration of both cancer cells in vitro and in vivo, through lung capillaries, was not elevated by lamin A/C knockdown. Both cancer cells with lamin A/C knockdown grew normally in primary tumors and in vitro on rigid surfaces. Strikingly, however, both lamin A/C deficient melanoma and breast cancer cells grew poorly in 3D spheroids expanded in soft agar cultures. Experimental lung metastasis of both lamin A/C knockdown cells was also markedly reduced. Taken together, our results suggest that high content of lamin A/C in multiple cancer cells promotes cancer cell survival and ability to generate lung metastasis without compromising cancer cell emigration from lung vessels.

cancer biology↗

bioRxiv2020

Insights into calcium signaling and gene expression in astrocytes uncovered with 129S4 Slc1a3-2A-CreERT2 knock-in mice

AO_SCPLOWBSTRACTC_SCPLOWGenetic variation is a primary determinant of phenotypic diversity within populations. In laboratory mice, genetic variation has often been regarded as a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains need to be made with caution. Effects of genetic background on traits need to be controlled, especially when working with complex phenotypes and disease models. Here we compared behavioral parameters of C57Bl/6 - the mouse strain most widely used for biomedical research - with those of 129S4. Our data demonstrate high within-strain and intra-litter behavioral hyperactivity in C57Bl/6. In contrast, 129S4 had relatively consistent activity levels throughout life. This consistency would be advantageous for studying neurodegeneration and aging, when mice need to be analyzed for long periods. However, the majority of mouse models and transgenic tools are on a C57Bl/6 background. We recently established six popular Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre mouse line to study astrocytes directly in 129S4, which we describe here. For functional validation, it was crossed with two Cre effector lines, each in a different genomic locus, and showed in both cases that it was functional and astrocyte-specific. Calcium currents studied with gCaMP5g-tdTomato were more heterogenous, lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes. Translatomes studied with RiboTag revealed that some genes thought to mark neurons are also expressed in astrocytes, that genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.

neuroscience↗