Biology subjects

Jaworski, A.

Publications and source records attributed to Jaworski, A..

8 recordsLinked to original sources

bioRxiv2026

Evolution of NELL binding by dual-ligand-responsive axon guidance receptor Robo

Robo receptors are conserved across bilaterians and best known for their ability to mediate axonal repulsion in response to Slit family ligands. In mammals, this applies to Robo1 and Robo2, but mammalian Robo3 binds NELL proteins instead of Slits. The evolutionary origin of NELL-Robo interactions and the possible existence of dual-ligand responsiveness across species remain unknown. Here, we systematically analyzed Robo and NELL homologs across bilaterians and found that NELL-Robo binding is conserved among chordate Robos, but not in protostomes. We show that cephalochordate Robo and NELL can mediate axon repulsion in vitro, suggesting conserved functionality. We observed that conformational masking of the NELL-binding site is prevalent among chordate Robos, modulating NELL-Robo affinity. We also demonstrate that NELL-Robo complexes undergo liquid-liquid phase separation in vitro, a property preserved from cephalochordates to mammals. Our findings support a model in which an ancestral chordate Robo receptor was dual-responsive to Slit and NELL, still the case for some extant Robos, and vertebrate paralogs subfunctionalized, with full ligand specialization emerging in mammals.

biochemistry↗

bioRxiv2025

Nucleic acid strand length governs mitochondrial reprogramming and mtROS-associated antiviral responses following TLR3 engagement

Mitochondria are important rheostats that regulate innate sensing processes by producing energy, biosynthetic precursors, and bioactive molecules that affect cellular signaling. When viral nucleic acids engage endosomal and cytosolic pattern recognition receptors (PRR), antiviral immune responses are supported by mitochondrial remodeling but the role of mitochondria in fine tuning ligand-specific responses remains incompletely understood. For example, endosomal TLR3 can detect various lengths of dsRNA (0.4-8 kb) ranging from viral segmented genomes or endogenous nucleic acids have been shown to induce distinct cytokine profiles. However, it is unclear if these differences are associated with differential mitochondrial remodeling. Here, we report that TLR3 engagement with both high (HMW; 1.5-8 kb) or low molecular weight (LMW; 0.2-1 kb) Polyinosinic:polycytidylic acid (Poly(I:C)) is associated with reduced but sustained oxidative phosphorylation (OXPHOS) activity and increased mitochondrial reactive oxygen species (mtROS) production/accumulation to support antiviral responses in bone marrow-derived macrophages (BMDM). They differed in the amount of mtROS production, their spare respiratory capacity (SRC) and their mitochondrial membrane potential (MMP). Interestingly, while uncoupling protein 2 (UCP2) was found required for antiviral cytokine production, it did not contribute to ligand specific responses. Dynamic modulation of complex I of the electron transport chain (ETC), however resulted in the differential accumulation of mtROS (HMW>LMW). Further, selectively targeting the mtROS derived from Complex I leads to augmented type I IFN production. Overall, these findings highlight that targeting specific sources of mtROS without affecting electron flow may be a potential avenue for specific augmentation of antiviral responses during viral infections.

immunology↗

bioRxiv2025

Temporal control of axonal floor plate crossing through a combination of incoherent feedforward and feedback loops of gene regulatory network regulating Robo3 expression

Spinal commissural neurons play a fundamental role in motor control and sensory perception. Robo3, a receptor expressed on pre-crossing commissural axons, is required for midline crossing. Its downregulation in post-crossing axons is essential for forming synapses with contralateral CNS targets. We demonstrate that, at the transcriptional level, the dynamic expression of Robo3 in pre-crossing neurons is controlled by incoherent feed-forward loops (iFFLs) and negative feedback loops (NFLs) that precisely regulate its transience and thereby determine midline crossing. Lhx2 and Lhx9 activate Robo3 while also inducing its repressor, Barhl2. Additionally, negative feedback loops fine-tune the transience of Robo3 expression by adjusting the relative strength of the activation and repression modules. Our genomic analysis reveals that this regulatory circuitry converges on an enhancer element of the Robo3 gene. These findings imply that diverse iFFLs, together with NFLs, are essential for regulating the extent of midline crossing across different spinal commissural subtypes.

developmental biology↗

bioRxiv2023

Control of T-shaped Bifurcation by Multiple Guidance Mechanisms during Dorsal Funiculus Development in the Spinal Cord

The dorsal funiculus in the spinal cord relays somatosensory information to the brain. It is made of T-shaped bifurcation of dorsal root ganglion (DRG) sensory axons. Our previous study has shown that Slit signaling is required for proper guidance during bifurcation, but loss of Slit does not affect all DRG axons. Here, we examined the role of the extracellular molecule Netrin-1 (Ntn1). Using wholemount staining with tissue clearing, we showed that mice lacking Ntn1 have axons escaping from the dorsal funiculus at the time of bifurcation. Genetic labeling confirmed that these misprojecting axons come from DRG neurons. Single axon analysis showed that loss of Ntn1 does not affect bifurcation but rather alters turning angles. To distinguish their guidance functions, we examined mice with triple deletion of Ntn1, Slit1, and Slit2 and found a completely disorganized dorsal funiculus. Comparing mice with different genotypes using immunolabeling and single axon tracing revealed additive guidance errors, demonstrating the independent roles of Ntn1 and Slit. Moreover, the same defects were observed in embryos lacking their cognate receptors. These in vivo studies thus demonstrate the presence of multi-factorial guidance mechanisms that ensure proper formation of a common branched axonal structure during spinal cord development.

neuroscience↗

bioRxiv2023

WFIKKN2 is a bifunctional axon guidance cue that signals through divergent DCC family receptors

Axon pathfinding is controlled by attractive and repulsive molecular cues that activate receptors on the axonal growth cone, but the full repertoire of axon guidance molecules remains unknown. The vertebrate DCC receptor family contains the two closely related members DCC and Neogenin with prominent roles in axon guidance and three additional, divergent members - Punc, Nope, and Protogenin - for which functions in neural circuit formation have remained elusive. We identified a secreted Punc/Nope/Protogenin ligand, WFIKKN2, which guides mouse peripheral sensory axons through Nope-mediated repulsion. In contrast, WFIKKN2 attracts motor axons, but not via Nope. These findings identify WFIKKN2 as a bifunctional axon guidance cue that acts through divergent DCC family members, revealing a remarkable diversity of ligand interactions for this receptor family in nervous system wiring. One-Sentence SummaryWFIKKN2 is a ligand for the DCC family receptors Punc, Nope, and Prtg that repels sensory axons and attracts motor axons.

developmental biology↗

bioRxiv2023

Up-regulation of cholesterol synthesis pathways and limited neurodegeneration in a knock-in Sod1 mutant mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disorder affecting brain and spinal cord motor neurons. Mutations in the copper/zinc superoxide dismutase gene (SOD1) are associated with [~]20% of inherited and 1-2% of sporadic ALS cases. Much has been learned from mice expressing transgenic copies of mutant SOD1, which typically involve high-level transgene expression, thereby differing from ALS patients expressing one mutant gene copy. To generate a model that more closely represents patient gene expression, we created a knock-in point mutation (G85R, a human ALS-causing mutation) in the endogenous mouse Sod1 gene, leading to mutant SOD1G85R protein expression. Heterozygous Sod1G85R mutant mice resemble wild type, whereas homozygous mutants have reduced body weight and lifespan, a mild neurodegenerative phenotype, and express very low mutant SOD1 protein levels with no detectable SOD1 activity. Homozygous mutants exhibit partial neuromuscular junction denervation at 3-4 months of age. Spinal cord motor neuron transcriptome analyses of homozygous Sod1G85R mice revealed up-regulation of cholesterol synthesis pathway genes compared to wild type. Transcriptome and phenotypic features of these mice are similar to Sod1 knock-out mice, suggesting the Sod1G85R phenotype is largely driven by loss of SOD1 function. By contrast, cholesterol synthesis genes are down-regulated in severely affected human TgSOD1G93A transgenic mice at 4 months. Our analyses implicate dysregulation of cholesterol or related lipid pathway genes in ALS pathogenesis. The Sod1G85R knock-in mouse is a useful ALS model to examine the importance of SOD1 activity in control of cholesterol homeostasis and motor neuron survival. SIGNIFICANCE STATEMENTAmyotrophic lateral sclerosis is a devastating disease involving the progressive loss of motor neurons and motor function for which there is currently no cure. Understanding biological mechanisms leading to motor neuron death is critical for developing new treatments. Using a new knock-in mutant mouse model carrying a Sod1 mutation that causes ALS in patients, and in the mouse, causes a limited neurodegenerative phenotype similar to Sod1 loss-of-function, we show that cholesterol synthesis pathway genes are up-regulated in mutant motor neurons, whereas the same genes are down-regulated in transgenic SOD1 mice with a severe phenotype. Our data implicate dysregulation of cholesterol or other related lipid genes in ALS pathogenesis and provide new insights that could contribute to strategies for disease intervention.

neuroscience↗

bioRxiv2019

Differential remodeling of the electron transport chain is required to support TLR3 and TLR4 signaling and cytokine production in macrophages

Increasing evidence suggests that mitochondria play a critical role in driving innate immune responses against bacteria and viruses. However, it is unclear if differential reprogramming of mitochondrial function contributes to the fine tuning of pathogen specific immune responses. Here, we found that TLR3 and TLR4 engagement on murine bone marrow derived macrophages was associated with differential remodeling of electron transport chain complex expression. This remodeling was associated with differential accumulation of mitochondrial and cytosolic ROS, which were required to support ligand specific inflammatory and antiviral cytokine production. We also found that the magnitude of TLR3, but not TLR4, responses were modulated by glucose availability. Under conditions of low glucose conditions, TLR3 engagement was associated with increased ETC complex III expression, increased mitochondrial and cytosolic ROS and increased inflammatory and antiviral cytokine production. This amplification was selectively reversed by targeting superoxide production from the outer Q-binding site of the ETC complex III. These results suggest that ligand specific modulation of the ETC may act as a rheostat that fine-tunes innate immune responses via mitochondrial ROS production. Modulation of these processes may represent a novel mechanism to modulate the nature as well as the magnitude of antiviral versus inflammatory immune responses.

immunology↗

bioRxiv2019

Observing an antisense drug complex in intact human cells by in-cell NMR

Gaining insight into the uptake of drugs into cells, trafficking and their target engagement enhances understanding of the drugs function and efficiency. Here we study an antisense oligonucleotide drug (ASO) delivered into HEK293T and HeLa cells, by Nuclear Magnetic Resonance (NMR). Using a combination of transfection, cryoprotection and dynamic nuclear polarization (DNP), we were able to detect the drug directly in intact frozen cells. Activity of the drug was confirmed by qRT-PCR, measuring downregulation of its target mSTAT3. Applying DNP NMR to frozen cells, we overcome limitations of traditional solution-state in-cell NMR (e.g. size, stability and sensitivity) as well as of visualization techniques, where (e.g. fluorescent) tagging of the ASO decreases its activity. The possibility to study an untagged, active drug, interacting in its natural environment, will increase insights into molecular mechanisms of delivery, intracellular trafficking and target engagement in intact cells.

biophysics↗