Biology subjects

Kaczmarek, L.

Publications and source records attributed to Kaczmarek, L..

15 recordsLinked to original sources

bioRxiv2026

CATSPERβ--δ Interaction Governs Hierarchical CatSper Holo-complex Assembly and is Essential for Male Fertility

The sperm-specific CatSper channel is macromolecular Ca2+ channel complex essential for hyperactivated motility and male fertility. The pore-forming channel (CATSPER1-4) associates with a large extracellular domain-containing canopy (CATSPER{beta}-{varepsilon}), a cytosolic Ca{superscript 2} sensing subcomplex (CATSPER{zeta}-EFCAB9-ARMH2), the putative transporter SLCO6C1, and CATSPER{theta}-, arranging into higher-order zigzag rows in the flagellar membrane. However, the molecular mechanism governing holo-complex assembly during spermatogenesis remains largely undefined. Here we demonstrate that the CATSPER{beta}-{delta} interaction represents an essential early step in CatSper biogenesis. CRISPR/Cas9 targeting of Catsperb exon 4 generated a frameshift knockout (Catsperb-/-) and an in-frame deletion mutant (Catsperb{Delta}/{Delta}) that specifically disrupts the {beta}-{delta} interaction interface. Disrupting this interface reduced CATSPER{delta} among canopy subunits, impairing canopy assembly and destabilizing the core pore-forming channel. Consequently, mature spermatozoa completely lacked the entire CatSper complex, phenocopying the knockout. A transgenic line expressing extracellular-domain-truncated CATSPER{delta} lacking the {beta}-binding region likewise phenocopied the Catsperb mutant, confirming the necessity of an intact {beta}-{delta} interface. AlphaFold-Multimer modeling and alanine-substitution mutagenesis identified key hydrogen-bonding residues at this interface that mediate canopy subunit association. Consistent with complex loss, whole-sperm patch-clamp recordings revealed complete absence of CatSper conductance in mutant spermatozoa. Both mutant lines exhibited defective sperm hyperactivation and male infertility despite normal spermatogenesis and baseline motility. Together, these findings establish that canopy formation driven by CATSPER{beta}-{delta} interaction precedes and is required for pore-forming channel assembly, defining a hierarchical assembly pathway for the CatSper holo-complex and highlighting a key structural node for male fertility and contraceptive development.

cell biology↗

bioRxiv2025

Lcn2 deficiency leads to social impairments independent of maternal immune activation.

Maternal infection during pregnancy is a well-established risk factor for neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain poorly understood. Lipocalin-2 (Lcn2), an innate immune protein that is highly upregulated during infection, also affects neuronal and glial function. This study investigates the role of Lcn2 in shaping brain development, particularly after maternal immune activation (MIA). To mimic maternal infection, pregnant mice received intraperitoneal injections of either lipopolysaccharide (LPS) or saline on embryonic days 16 to 18 to model infection during the second trimester of pregnancy in humans. We first showed that Lcn2 mRNA is expressed in the fetal brain and that MIA significantly upregulates Lcn2 mRNA and protein in the hippocampus and neocortex of both sexes. To assess functional relevance, we employed Lcn2 heterozygous females to generate wild-type and Lcn2 KO offspring from the MIA and control groups. Both female and male offspring underwent a battery of behavioral assays. Lcn2 deletion and MIA independently induced deficits in social behavior and increased repetitive behavior phenotypes relevant to NDDs in adult animals. However, their combination did not exacerbate these effects, suggesting an occlusion effect. Interestingly, no deficits were observed in the learning and memory task. To investigate potential shared molecular mechanisms, we performed RNA sequencing of the fetal forebrain 4 hours after the final LPS injection. This analysis revealed an overlapping group of differentially expressed genes in the Lcn2 KO and MIA groups, indicating convergence on similar transcriptional pathways that may underlie the observed behavioral phenotypes. These results suggest that while Lcn2 may not mediate the pathological effects of prenatal immune challenge, it plays a critical role in normal brain development.

neuroscience↗

bioRxiv2023

Matrix Metalloproteinase-9 controls structural synaptic plasticity via BDNF-dependent signaling

Synaptic plasticity underlies learning and memory processes as well as contributes, in its aberrant form, to neuropsychiatric disorders. One of its major forms is structural long-term potentiation (sLTP), an activity-dependent growth of dendritic spines that harbor excitatory synapses. The process depends on the release of brain-derived neurotrophic factor (BDNF), and activation of its receptor, TrkB. Matrix metalloproteinase-9 (MMP-9), an extracellular protease is essential for many forms of neuronal plasticity engaged in physiological as well as pathological processes. Here, we utilized two-photon microscopy and two-photon glutamate uncaging to demonstrate that MMP-9 activity is essential for sLTP and is rapidly ([~]seconds) released from dendritic spines in response to synaptic stimulation. Moreover, we show that either chemical or genetic inhibition of MMP-9 impairs TrkB activation, as measured by fluorescence lifetime imaging microscopy of FRET sensor. Furthermore, we provide evidence for a cell-free cleavage of proBDNF into mature BDNF by MMP-9. Our findings point to the autocrine mechanism of action of MMP-9 through BDNF maturation and TrkB activation during sLTP.

neuroscience↗

bioRxiv2023

Recovery from anhydrobiosis in the tardigrade Paramacrobiotus experimentalis: better to be young than old and in a group than alone

Desiccation-tolerant organisms can survive dehydration in a state of anhydrobiosis. Tardigrades can recover from anhydrobiosis at any life stage and are considered among the toughest animals on Earth. However, the factors that influence recovery from anhydrobiosis are not well understood. The study aimed to evaluate the effect of sex, age, the presence of other individuals and the combination of the number and duration of anhydrobiosis episodes on the recovery of Paramacrobiotus experimentalis. The activity of 1,200 individuals for up to 48 hours after rehydration was evaluated using ANOVA. Age was the main factor influencing return to activity, followed by the combination of number and duration of anhydrobiosis episodes, influence of the presence of other individuals, and sex. More individuals returned to activity after repeated short than repeated long anhydrobiosis episodes and older individuals were less likely to recover than younger individuals. In addition, when compared to single animals, the presence of other individuals resulted in higher number of active animals after dehydration and rehydration. The effect of sex was significant, but there was no general tendency for one sex to recover from anhydrobiosis better than the other one. The results contribute to a better understanding of the anhydrobiosis ability of Pam. experimentalis and provide background for full explanation of molecular, cellular and environmental mechanisms of anhydrobiosis.

physiology↗

bioRxiv2022

Activation of trace amine-associated receptor 1 (TAAR1) reduces alcohol drinking behaviors in mice

Alcohol dependence is characterized by the abnormal release of dopamine in the brain reward-related areas. Trace amine-associated receptor 1 (TAAR1) is a G Protein-Coupled Receptor that negatively regulates dopamine neurotransmission and thus is a promising target in the treatment of drug addiction. However, the role of TAAR1 in the regulation of alcohol abuse remains understudied. Here, we assessed the effect of TAAR1 activation on alcohol-drinking behaviors of C57Bl/6J female mice housed in IntelliCages. The animals were administered with either vehicle or TAAR1 full selective agonist, RO5256390, and tested for alcohol consumption, alcohol preference, and motivation for alcohol seeking. We found that mice with the highest preference for alcohol (high drinkers) in the RO5256390 group consumed less alcohol and had lower alcohol preference in comparison to high drinkers in the vehicle group, during 20 h of free alcohol access (FAA). We also found decreased alcohol consumption and alcohol preference comparing all animals in the RO5256390 to all animals in the vehicle group, during 20 h of FAA performed after the abstinence. These effects of RO5256390 lasted for the first 24 hours after administration that roughly corresponded to the compound level in the brain, measured by mass spectrometry. Finally, we found that administration of RO5256390 may attenuate motivation for alcohol seeking. Taken together, our findings reveal that activation of TAAR1 may transiently reduce alcohol drinking; thus, TAAR1 is a promising target for the treatment of alcohol abuse and relapse.

animal behavior and cognition↗

bioRxiv2022

Celloscope: a probabilistic model for marker-gene-driven cell type deconvolution in spatial transcriptomics data

Spatial transcriptomics maps gene expression across tissues, posing the challenge of determining the spatial arrangement of different cell types. However, spatial transcriptomics spots contain multiple cells. Therefore, the observed signal comes from mixtures of cells of different types. Here, we propose an innovative probabilistic model, Celloscope, that utilizes established prior knowledge on marker genes for cell type deconvolution from spatial transcriptomics data. Celloscope outperformed other methods on simulated data, successfully indicated known brain structures and spatially distinguished between inhibitory and excitatory neuron types based in mouse brain tissue, and dissected large heterogeneity of immune infiltrate composition in prostate gland tissue.

bioinformatics↗

bioRxiv2021

LINE-1 activation in the cerebellum drives ataxia

Previous studies have revealed that dysregulation of long interspersed nuclear element 1 (LINE-1), a dominant class of transposable elements in the human genome, correlates with neurodegeneration1-3. Yet whether LINE-1 dysregulation is causal to disease pathogenesis has not been proven directly. Here, we demonstrate that expression of evolutionarily younger LINE-1 families is elevated in the cerebella of ataxia telangiectasia (AT) patients, which was correlated with extensive downregulation of epigenetic silencers. To examine whether LINE-1 activation causes neurologic disease, we established an approach to directly target and activate the promoter of a young family of LINE-1 in mice. LINE-1 activation in the cerebellum was sufficient to lead to robust progressive ataxia. Purkinje cells in the diseased mice exhibited marked electrophysiological dysfunctions and degeneration with a significant accumulation of cytoplasmic ribonucleoprotein LINE-1Orf1p aggregates, endoplasmic reticulum (ER) stress, and DNA damage. Treatment with lamivudine, a nucleoside reverse transcriptase inhibitor, blunted the disease progression by reducing DNA damage, attenuating gliosis and interferon gene signature, and recovering the loss of key functional molecules for calcium homeostasis in Purkinje cells. This study provides direct evidence that young LINE-1 activation drives ataxia phenotype, and points to its pleiotropic effects leading to DNA damage, inflammation, and dysfunction and degeneration of neurons.

neuroscience↗

bioRxiv2021

Plasmin-mediated cleavage of EphA4 at central amygdala inhibitory synapses controls anxiety

Severe stress can trigger complex behavioural changes such as high anxiety (1). Inhibitory GABA-ergic interneurons in the lateral division of the central amygdala (CEl) control anxiety through feedforward inhibition of their target cells in the medial division (CEm) (2, 3). In particular, PKC{delta}-positive (PKC{delta}+) interneurons in CEl are critical elements of the neuronal circuitry of fear and anxiety (3-5), but the molecular mechanisms they employ are poorly understood. Here, we show that, during stress, GABA-ergic synapses of amygdala PKC{delta}+ interneurons are regulated by a serine protease plasmin. On stress, plasmin cleaves the extracellular portion of the tyrosine kinase receptor EphA4 triggering its dissociation from gephyrin, a postsynaptic GABA-receptor anchoring protein. Dynamic EphA4/gephyrin interaction leads to modification of dendritic spine morphology and synaptic GABA-receptor expression profile. Consistent with the critical role for the plasmin/EphA4/gephyrin signalling axis in anxiogenesis, viral delivery of plasmin-resistant (prEphA4) form of EphA4 into the central amygdala prevents the development of stress-induced anxiety in mice, while the delivery of plasmin-truncated EphA4 (tEphA4) dramatically enhances this effect. Thus, our studies identify a novel, critical molecular cascade regulating GABA-ergic signalling in the central amygdala synapses that allows bidirectional switching of animal behaviour from high to low anxiety states.

neuroscience↗

bioRxiv2021

Epileptiform GluN2B-driven excitation in hippocampus as a therapeutic target against temporal lobe epilepsy

NMDAR is an ionotropic glutamate receptor critically involved in excitatory synaptic transmission. The receptor properties are strongly determined by its subunit composition. One of the NMDAR subunits is GluN2B, which displays restricted and spatially different from other subunits expression in the mature brain. GluN2B-containing NMDARs are present in the hippocampus - a structure playing a major role in temporal lobe epilepsy (TLE). However, the contribution of GluN2B to pathophysiology of TLE has not been fully explored. Here, we report the functional alterations of GluN2B-containing NMDAR receptors in the hippocampus in distinct mouse models of temporal lobe epilepsy. In particular, we show the impact of GluN2B on excitatory feedback in granule cells. Based on these results, we propose a mechanism-oriented effective antiepileptic strategy that selectively antagonizes GluN2B-containing NMDARs with ifenprodil, a well-known GluN2B antagonist. Collectively, our research identifies GluN2B as one of the pivotal factors in pathogenesis of temporal lobe epilepsy and associated recurrent seizures. Furthermore, our study indicates the prospective antiepileptic properties of ifenprodil in TLE.

neuroscience↗

bioRxiv2021

A whole-brain imaging-based systems approach to understand origin of addiction in binge-like drinking model

Many fundamental questions on addiction development are still unanswered. These questions are frequently difficult to address by examining a single brain structure, but can best be addressed at the systems level. Neurons create functional networks that change over time, since brain regions may work together differently in different contexts. We offer a framework for describing the nature behind alcohol binge drinking and the transition to addiction. The present study investigated whole-brain c-Fos expression following reexposure to alcohol in a model of binge-like drinking in mice in IntelliCage. We developed a dedicated image computational workflow to identify c-Fos-positive cells in three-dimensional images obtained after optical tissue clearing and whole-brain imaging in the light-sheet microscope. We analyzed functional networks and brain modularity following reexposure to alcohol. c-Fos levels in brains from animals that were reexposed to alcohol were clearly different from binge drinking animals. Structures involved in reward processing, decision making and characteristic for addictive behaviors stood out particularly. In alcohol reexposed animals differently active structures either gained or lost correlation when compared to the control group.

neuroscience↗

bioRxiv2020

Serum response factor is essential for synaptic maturation in the hippocampus

Disturbances of gene expression patterns that occur during brain development can severely affect signal transmission, connectivity, and plasticity--key features that underlie memory formation and storage in neurons. Abnormalities at the molecular level can manifest as changes in the structural and functional plasticity of dendritic spines that harbor excitatory synapses. This can lead to such developmental neuropsychiatric conditions as Autism spectrum disorders, intellectual disabilities, and schizophrenia. The present study investigated the role of the major transcriptional regulator serum response factor (SRF) in synapse maturation and its impact on behavioral phenotypes. Using in vitro and in vivo models of early postnatal SRF deletion, we studied its influence on key morphological and physiological hallmarks of spine development. The elimination of SRF in developing neurons resulted in a phenotype of immature dendritic spines and impairments in excitatory transmission. Moreover, using a combination of molecular and imaging techniques, we showed that SRF-depleted neurons exhibited a lower level of specific glutamate receptor mRNAs and a decrease in their surface expression. Additionally, the early postnatal elimination of SRF in hippocampal CA1 excitatory neurons caused spine immaturity and a specific social deficit that is frequently observed in autism patients. Altogether, our data suggest that the regulation of structural and functional dendritic spine maturation begins at the stage of gene transcription, which underpins the crucial role of such transcription factors as SRF. Moreover, disturbances of the postnatal expression of SRF translate to behavioral changes in adult animals.

neuroscience↗

bioRxiv2020

Mitochondrial alternative oxidase contributes to successful tardigrade anhydrobiosis

Anhydrobiosis can be described as an adaptation to lack of water. This adaptation provides some organisms including tardigrades with a set of capabilities allowing them to survive extreme conditions that even do not exist on Earth. However, the underlying cellular mechanisms are still not explained. Available data assumes important contribution of mitochondrial proteins. Since mitochondrial alternative oxidase (AOX) described as a drought response element has recently been proposed for various invertebrates including tardigrades, we have decided to check if AOX is involved in successful anhydrobiosis of tardigrades. Milnesium inceptum was used as a model for the study. We confirmed functionality of M. inceptum AOX and estimated its activity contribution to anhydrobiosis of different duration. We observed that AOX activity was particularly important for M. inceptum revival after longer-term anhydrobiosis but did not affect rehydration stage. The results may contribute to explanation and then application of anhydrobiosis underlying mechanisms.

cell biology↗

bioRxiv2020

c-Fos-MMP-9 pathway in central amygdala mediates approach motivation but not reward consumption

Although impaired motivational and consummatory aspects of reward behavior are core symptoms of several psychiatric disorders, the underlying neural and molecular mechanisms remain poorly understood. c-Fos, as a component of AP-1 transcription factor, regulates the expression of matrix metalloproteinase 9 (MMP-9), an enzyme involved in synaptic remodeling and plasticity. Both proteins are expressed in the central amygdala (CeA) that orchestrates appetitive and aversive responses. We have examined the role of c-Fos and MMP-9 in CeA in reward and punishment processing. We have manipulated c-Fos and MMP-9 levels in vivo using: RNAi-based approach to block c-Fos expression, inhibitor-releasing nanoparticles to block MMP-9 activity, and lentiviral vector to increase MMP-9 expression. To assess motivation, consumption and learning reinforced by either a sweet or bitter-tasting solution, we employed automated behavioral tests in the IntelliCage cystem. We have used transgenic mouse lines to identify CeA cells expressing c-Fos and a specific fluorescently labelled substrate peptide to measure MMP-9 activity. Excitability of neurons was measured with patch-clamp. Blocking c-Fos expression has been found to disrupt both reward processing components, motivational and consummatory, while inhibition of MMP-9 activity has impaired only motivational aspects of the behavior. On the other hand, punishment processing has not been affected by those manipulations. Furthermore, we have observed that reward training induces c-Fos expression in both somatostatin (SST)+, and SST-neurons, while MMP-9 activity is increased in SST- subpopulation only. We have further linked SST+ population to consummatory reactions by showing that reward consumption increases excitability of the SST+ neurons. These findings reveal molecular mechanisms of motivational anhedonia, linking it to c-Fos-MMP-9 pathway and CeA SST- neurons, and consummatory anhedonia, linking it to c-Fos and CeA SST+ neurons.

neuroscience↗

bioRxiv2018

Deciphering defective subventricular adult neurogenesis in cyclin D2-deficient mice

Adult neurogenesis occurring in the brain of adult mammals is considered to have potential therapeutical applications. New neurons are produced constitutively from postnatal neural stem cells/precursors residing in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricles and the subgranular layer in the dentate gyrus of the hippocampus. Newly-generated neuroblasts from the SVZ migrate long distance towards the olfactory bulb and repopulate different subtypes of inhibitory interneurons modulating the olfactory processing. It was reported that cyclin D2 knockout mice (cD2-KO) present reduced generation of new hippocampal neurons, however proliferation deficiency and mechanisms responsible for dysregulation of subventricular precursors, derived progenitors, and olfactory interneurons need to be detaily investigated. In this report, proliferative activity of different subpopulations of SVZ neural precursors, cell migration, and differentiation in cD2-KO mice was characterized. For that goal, EdU, a thymidine analogue, proliferation mapping combined with multi-epitope immunohisto-chemical detection of endogenous stage-specific cell markers was carried out. Severely reduced number of newly-generated cells in the subventricular niche was demonstrated that was not accompanied by increased level of apoptotic death. Surprisingly, the number of B1 quiescent precursor subpopulation was not affected, whereas the number of B1 type active primary precursors, intermediate/transiently-amplifying progenitors (C type cells), and neuroblasts (A type cells) were reduced. The analyses suggest that cycline D2 might be critical for transition of B1 precursor quiescent cells into B1 active cells. We also demonstrate that the subpopulation of calbindin interneurons is reduced in the olfactory bulb. Deciphering processes underlying a potential modulation of intensity of adult neurogenesis at the cellular levels could lead to replacement therapies after injury, stroke, or neurodegenerative disease in the central nervous system.

neuroscience↗

bioRxiv2018

Amot regulates neuronal dendritic tree through Yap1

The Amot-Yap1 complex plays a major role in the regulation of cell contact inhibition, cellular polarity and growth. However, the function of Angiomotin (Amot) and Hippo pathway transcription co-activator Yap1 in the central nervous system remains unclear. In this study, we demonstrate that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing hippocampal neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. Conditional deletion of Amot or Yap1 in neurons leads to impaired morphogenesis of Purkinje cell dendritic trees, decreased cerebellar size, and causes defects in locomotor coordination of mutant animals. Thus, our studies identified Amot and Yap1 as novel regulators of dendritic tree morphogenesis.

neuroscience↗