Biology subjects

Janecek, M.

Publications and source records attributed to Janecek, M..

9 recordsLinked to original sources

bioRxiv2025

Late onset of striatal projection neuron hyperexcitability in Fmr1-/y mice

AbstractFragile X Syndrome (FXS), the most common genetic cause of intellectual disability and autism spectrum disorder (ASD), results from silencing of the FMR1 gene and consequent loss of Fragile X Messenger Ribonucleoprotein (FMRP). FMRP deficiency disrupts neural development, leading to behavioral and motor deficits associated with striatal dysfunction. While structural and functional abnormalities in striatal projection neurons (SPNs) have been observed in adult Fmr1 knockout (KO) mice, their developmental onset and contribution to early FXS pathophysiology remain unknown. In this study, we examined the postnatal maturation of SPN in the dorsomedial striatum (DMS) of Fmr1 KO mice, assessing glutamatergic synaptic inputs and intrinsic excitability. During postnatal development, Fmr1 deficient SPNs in DMS display normal synaptic and intrinsic properties, consistent with typical maturation. In contrast, by P60, SPNs of mice exhibit pronounced hyperexcitability, characterized by increased membrane resistance, reduced rheobase, and slower action potential kinetics. These perturbations affect both Dopamine 1 receptor-expressing (D1-SPN) and D2 receptor-expressing (D2-SPN) SPNs, though some action potential dynamics are selectively impaired in D1-SPNs. Chronic aripiprazole treatment, a widely prescribed therapy for FXS-related symptoms, fails to normalize SPN excitability, highlighting its limited efficacy in addressing core SPN dysfunction. Our findings reveal a late-onset hyperexcitability in DMS SPNs of Fmr1 KO mice, suggesting a progressive emergence of striatal neuron abnormalities over development. These results underscore the importance of developmental timing in FXS pathophysiology and emphasize the need for targeted interventions to address striatal circuit dysfunction.

neuroscience↗

bioRxiv2025

Electroporation-Based Gene Delivery and Whole-Organoid Imaging in Human Retinal Organoids

Human retinal organoids (hRetOrg) derived from human induced pluripotent stem cells (hiPSCs) have emerged as powerful in vitro systems for studying retinal development, modeling retinal diseases, and evaluating therapeutic strategies. However, current genetic manipulation approaches, such as stable hiPSC line generation and viral transduction, are laborious, costly, and inefficient, with limited spatial specificity and high variability. Here, we report a rapid, scalable, and spatially precise electroporation-based platform for efficient plasmid-based gene delivery in early-stage hRetOrg. This method enables tunable and region-specific transfection of retinal progenitor cells without viral vectors or clonal selection. Coupled with resonant-scanning two-photon microscopy, this approach allows fast live cell imaging of whole organoids with subcellular resolution. This versatile system supports high-throughput genetic manipulation and imaging in intact hRetOrg, advancing studies of human retinal development, gene function, and disease. MotivationhRetOrgs offer an unprecedented platform for functional genetic studies of human retinal development and disease. However, existing methods for gene manipulation in hRetOrg are limited by low throughput, inefficiency, and lack of scalability, hindering systematic analysis of gene function and regulatory elements. To address these limitations, we developed a streamlined, high-efficiency pipeline that enables spatially targeted electroporation of hRetOrg during early retinogenesis, combined with fast, high-resolution imaging of whole organoids using two-photon microscopy, allowing studies at both tissue and subcellular scales.

developmental biology↗

bioRxiv2024

Early Postnatal Dysfunction of mPFC PV Interneurons in Shank3B-/- Mice

Anterior cingulate cortex (ACC) dysfunction is implicated in the cognitive and social deficits associated with autism spectrum disorder (ASD), yet the developmental trajectory of ACC circuit maturation in ASD remains poorly understood. Here, we examined the postnatal development of glutamatergic synaptic connectivity and intrinsic excitability in layer 2/3 pyramidal neurons (PYR) and Parvalbumin-expressing interneurons (PVIN) in the ACC of mice harboring a deletion in SHANK3 (Shank3B-/-), a well-established genetic cause of autism. We found that ACC PVINs in Shank3B-/- mice exhibit reduced excitability and in vivo hypoactivity as early as postnatal day 15 (P15) despite receiving normal levels of glutamatergic input. This early PVIN hypoexcitability is associated with decreased feedforward inhibition from the mediodorsal thalamus and reduced hyperpolarization-activated (Ih) currents mediated by hyperpolarization-activated cyclic nucleotide gated (HCN) channels. In contrast, PYRs display normal excitability and synaptic input at this stage but already exhibit reduced Ih currents, indicating an early emergence of HCN channel dysfunction in both PYR and PVIN. By adulthood, both neuron populations undergo marked phenotypic changes, characterized by reduced glutamatergic synaptic input and divergent alterations in excitability. Together, these findings reveal a distinct sequence of early PVIN dysfunction followed by cell-type specific circuit reorganization within ACC layer 2/3 of Shank3B-/- mice and identify HCN channelopathy and impaired PVIN-mediated inhibition as early pathogenic features of SHANK3-related neurodevelopmental disorders.

neuroscience↗

bioRxiv2024

Striatal output regulates the postnatal maturation of cortical circuits

The dorsomedial prefrontal cortex (dmPFC) is interconnected with the basal ganglia (BG) through large-scale circuit loops that regulate critical motor and cognitive functions. In mice, these circuits undergo extensive postnatal maturation with marked changes in neural activity and expansion of synaptic connectivity. While cortical activity is known to regulate the development of downstream striatal circuits, the role of the basal ganglia in cortical maturation remains unknown. Here, we used mesoscale two-photon microscopy and whole-cell electrophysiology to examine whether striatal output during early postnatal development impacts the activity and maturation of upstream dmPFC circuits. We found that ablating spiny projection neurons of the direct or indirect pathways of the striatum during the first two postnatal weeks causes bidirectional changes in dmPFC neural activity, similar to observations in mature circuits. In addition, these manipulations alter the maturation of synaptic connectivity of dmPFC layer 2/3 pyramidal neurons, shifting the balance of excitation and inhibition of cortical circuits. These findings demonstrate that striatal output modulates the activity of cortical circuits during early postnatal development and suggest a regulatory role of the basal ganglia in the establishment of cortical circuits.

neuroscience↗

bioRxiv2023

Late onset and regional heterogeneity of synaptic deficits in cortical PV interneurons of Shank3B-/- mice

Epilepsy and epileptiform patterns of cortical activity are highly prevalent in autism spectrum disorders (ASDs), but the neural substrates and pathophysiological mechanisms underlying the onset of cortical dysfunction in ASD remains elusive. Reduced cortical expression of Parvalbumin (PV) has been widely observed in ASD mouse models and human postmortem studies, suggesting a crucial role of PV interneurons (PVINs) in ASD pathogenesis. Shank3B-/- mice carrying a {Delta}13-16 deletion in SHANK3 exhibit cortical hyperactivity during postnatal development and reduced sensory responses in cortical GABAergic interneurons in adulthood. However, whether these phenotypes are associated with PVIN dysfunction is unknown. Using whole-cell electrophysiology and a viral-based strategy to label PVINs during postnatal development, we performed a developmental characterization of AMPAR miniature excitatory postsynaptic currents (mEPSCs) in PVINs and pyramidal (PYR) neurons of layer (L) 2/3 mPFC in Shank3B-/- mice. Surprisingly, reduced mEPSC frequency was observed in both PYR and PVIN populations, but only in adulthood. At P15, when cortical hyperactivity is already observed, both neuron types exhibited normal mEPSC amplitude and frequency, suggesting that glutamatergic connectivity deficits in these neurons emerge as compensatory mechanisms. Additionally, we found normal mEPSCs in adult PVINs of L2/3 somatosensory cortex, revealing region-specific phenotypic differences of cortical PVINs in Shank3B-/- mice. Together, these results demonstrate that loss of Shank3 alters PVIN function but suggest that PVIN glutamatergic synapses are a suboptimal therapeutic target for normalizing early cortical imbalances in SHANK3-associated disorders. More broadly, these findings underscore the complexity of interneuron dysfunction in ASDs, prompting further exploration of region and developmental stage specific phenotypes for understanding and developing effective interventions.

neuroscience↗

bioRxiv2022

A unified in vitro to in vivo fluorescence lifetime screening platform yields amyloid β aggregation inhibitors

Inhibiting the aggregation of amyloid {beta} (1-42) is a promising strategy for the development of disease-modifying Alzheimers disease therapeutics. To date, however, no sufficiently efficacious inhibitors have been identified, despite the best efforts of >200 advanced drug development campaigns. This failure can be attributed to limitations in current compound screening and in vivo validation assays. Here, we report an in vitro to in vivo screening platform based on the use of a fluorescence lifetime aggregation sensor. The microfluidic "nanoFLIM" assay developed circumvents issues that plague conventional assays, such as lack of reproducibility, high cost and artefactual false read-outs. The fluorescence lifetime sensor can also dynamically monitor peptide aggregation in cellular and Caenorhabditis elegans disease models, providing directly comparable aggregation kinetics, which is not achievable by any other method. The power of this unified system for accelerating hit-to-lead strategies, lowering attrition rates and expediting in vivo screening, was demonstrated with a pilot screening campaign of 445 compounds, revealing a new inhibitor that can inhibit amyloid {beta} self-assembly in vitro as well as in cellular and whole organism disease models.

molecular biology↗

bioRxiv2021

Towards Convergence in Folding Simulations of RNA Tetraloops: Comparison of Enhanced Sampling Techniques and Effects of Force Field Corrections

Atomistic molecular dynamics (MD) simulations represent established technique for investigation of RNA structural dynamics. Despite continuous development, contemporary RNA simulations still suffer from suboptimal accuracy of empirical potentials (force fields, ffs) and sampling limitations. Development of efficient enhanced sampling techniques is important for two reasons. First, they allow to overcome the sampling limitations and, second, they can be used to quantify ff imbalances provided they reach a sufficient convergence. Here, we study two RNA tetraloops (TLs), namely the GAGA and UUCG motifs. We perform extensive folding simulations and calculate folding free energies ({Delta}Gfold) with the aim to compare different enhanced sampling techniques and to test several modifications of the nonbonded terms extending the AMBER OL3 RNA ff. We demonstrate that replica exchange solute tempering (REST2) simulations with 12-16 replicas do not show any sign of convergence even when extended to time scale of 120 s per replica. However, combination of REST2 with well-tempered metadynamics (ST-MetaD) achieves good convergence on a time-scale of 5-10 s per replica, improving the sampling efficiency by at least two orders of magnitude. Effects of ff modifications on {Delta}Gfold energies were initially explored by the reweighting approach and then validated by new simulations. We tested several manually-prepared variants of gHBfix potential which improve stability of the native state of both TLs by up to ~2 kcal/mol. This is sufficient to conveniently stabilize the folded GAGA TL while the UUCG TL still remains under-stabilized. Appropriate adjustment of van der Waals parameters for C-H...O5 base-phosphate interaction are also shown to be capable of further stabilizing the native states of both TLs by ~0.6 kcal/mol.

biophysics↗

bioRxiv2020

W-RESP: Well-Restrained Electrostatic Potential Derived Charges. Revisiting the Charge Derivation Model.

Representation of electrostatic interactions by a Coulombic pair-wise potential between atom-centered partial charges is a fundamental and crucial part of empirical force fields used in classical molecular dynamics simulations. The broad success of the AMBER force field family originates mainly from the restrained electrostatic potential (RESP) charge model, which derives partial charges to reproduce the electrostatic field around the molecules. However, description of the electrostatic potential around molecules by standard RESP may be biased for some types of molecules. In this study, we modified the RESP charge derivation model to improve its description of the electrostatic potential around molecules, and thus electrostatic interactions in the force field. In particular, we re-optimized the atomic radii for definition of the grid points around the molecule, redesigned the restraining scheme and included extra point charges. The RESP fitting was significantly improved for aromatic heterocyclic molecules. Thus, the suggested W-RESP(-EP) charge derivation model showed clear potential for improving the performance of the nucleic acid force fields, for which poor description of nonbonded interactions, such as underestimated base pairing, makes it difficult to describe the folding free energy landscape of small oligonucleotides.

biophysics↗

bioRxiv2019

Local-to-global signal transduction at the core of the Mn2+ sensing riboswitch

The widespread manganese-ion sensing yybP-ykoY riboswitch controls the expression of bacterial Mn2+ homeostasis genes. Here, we first determine the crystal structure of the ligand-bound yybP-ykoY riboswitch from Xanthomonas oryzae at 2.85 [A] resolution, revealing two conformations with docked four-way junction (4WJ) and incompletely coordinated metal ions. In >50 s of MD simulations, we observe that loss of divalents from the core triggers local structural perturbations in the adjacent docking interface, laying the foundation for signal transduction to the regulatory switch helix. Using single-molecule FRET, we unveil a previously unobserved extended 4WJ conformation that samples transient docked states in the presence of Mg2+. Only upon adding sub-millimolar Mn2+, however, can the 4WJ dock stably, a feature lost upon mutation of an adenosine contacting Mn2+ in the core. These observations illuminate how subtly differing ligand preferences of competing metal ions become amplified by the coupling of local with global RNA dynamics.

biophysics↗