Biology subjects

Conze, C.

Publications and source records attributed to Conze, C..

5 recordsLinked to original sources

bioRxiv2024

Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

RationaleProtein accumulation is a hallmark of many neurodegenerative and muscular diseases. Desmin-related (cardio-) myopathy (DRM), a well-studied model for cardiac muscle protein accumulation, is an autosomal dominant-inherited disease presenting with progressive muscle weakness, reduced quality of life, and shortened life span. To date, DRM patients are treated symptomatically and there is no causal treatment available. Independent of the genetic cause, most DRM patients display intracellular accumulation of desmin and its chaperone B-crystallin (CRYAB). We previously conducted an unbiased high-throughput screen to identify novel effectors that reduce cardiomyocyte aggregate levels and found that downregulation of Janus kinase 1 (JAK1) resulted in lower aggregate load in neonatal mouse cardiomyocytes. ObjectiveIn this study, we tested if the approved JAK inhibitor ruxolitinib ameliorates the disease phenotype in rodent and human CRYAB p.Arg120Gly DRM models. Methods and ResultsWe found that the mRNA levels of Jak1 and Stat3 were higher than any other JAK-signal transducer and activator of transcription (STAT) family members in neonatal rat ventricular myocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The approved JAK1/2 inhibitor ruxolitinib and the JAK1 inhibitors solcitinib, upadacitinib, and filgotinib prevented accumulation of and cleared pre-existing CRYAB p.Arg120Gly protein aggregates in NRVMs and hiPSC-CMs. Importantly, the knockdown of Jak1 and Stat3, but not Jak2 resulted in fewer aggregates. Moreover, ruxolitinib, Jak1 or Stat3 siRNA treatment enhanced the ubiquitin-proteasome system (UPS)-mediated degradation. Blocking UPS function blunted the effect of ruxolitinib or Jak1 siRNA on CRYAB p.Arg120Gly accumulation. RNAseq of NRVMs treated with Jak1 siRNA extracts revealed higher gene expression of important muscle E3 ubiquitinating enzymes. Knockdown of the E3 ligase Asb2 (Ankyrin Repeat And SOCS Box Protein 2) abolished the effect of ruxolitinib on CRYAB p.Arg120Gly aggregates. In DRM mice, phospho-STAT3 levels were markedly higher than in non-transgenic (NTG) mice with age. Ruxolitinib treatment or Jak1 knockout prevented cardiac dysfunction and reduced CRYAB p.Arg120Gly aggregate load in DRM mice. ConclusionIn this study, we uncovered the previously unknown effect of the approved drug ruxolitinib to enhance UPS-mediated degradation and prevent protein aggregates in cardiomyocytes.

pharmacology and toxicology↗

bioRxiv2024

Redox signaling by hydrogen peroxide modulates axonal microtubule organization and induces a specific phosphorylation signature of microtubule proteins distinct from distress

Many life processes are regulated by physiological redox signals, referred to as oxidative eustress. However, excessive oxidative stress can damage biomolecules and contribute to disease. The neuronal microtubule system is critically involved in axon homeostasis, regulation of axonal transport, and neurodegenerative processes. However, whether and how physiological redox signals affect axonal microtubules is largely unknown. Using live cell imaging and super- resolution microscopy, we show that subtoxic concentrations of the central redox metabolite hydrogen peroxide increase axonal microtubule dynamics, alter the structure of the axonal microtubule array, and affect the efficiency of axonal transport. We report that the mitochondria-targeting antioxidant SkQ1 and the microtubule stabilizer EpoD abolish the increase in microtubule dynamics. We found that oxidative eustress and distress specifically modulate the phosphorylation state of the microtubule system and induce a largely non- overlapping phosphorylation pattern of MAP1B as the main target. Cell-wide phosphoproteome analysis revealed that different signaling pathways are inversely activated by oxidative eustress and distress. Signaling via casein kinase (CK2) and pyruvate dehydrogenase kinases (PDK) is activated during eustress and signaling via mammalian target of rapamycin (mTOR) and serum/glucocorticoid-regulated protein kinase (SGK) is activated during distress. The results suggest that the redox metabolite and second messenger hydrogen peroxide induces rapid and local reorganization of the microtubule array in response to mitochondrial activity or as a messenger from neighboring cells by activating specific signaling cascades.

neuroscience↗

bioRxiv2024

Podocyte exopher-formation as a novel pathomechanism in membranous nephropathy

BackgroundMembranous nephropathy (MN) is caused by autoantibody binding to podocyte foot process antigens such as THSD7A and PLA2R1. The mechanisms of the glomerular antigen/autoantibody deposition and clearance are unknown. MethodsWe explore the origin and significance of glomerular accumulations in (1) diagnostic and follow-up biospecimens from THSD7A+ and PLA2R1+-MN patients compared to nephrotic non-MN patients, and (2) in experimental models of THSD7A+-MN. ResultsWe discovered podocyte exophers as correlates of histological antigen/autoantibody aggregates found in the glomerular urinary space of MN patients. Exopher vesicle formation represents a novel form of toxic protein aggregate removal in Caenorhabditis elegans neurons. In MN patients, podocytes released exophers to the urine. Enrichment of exophers from MN patient urines established them as a glomerular exit route for antigens and bound autoantibody. Exophers also carried disease-associated proteins such as complement and provided a molecular imprint of podocyte injury pathways. In experimental THSD7A+-MN, exophers were formed from podocyte processes and cell body. Their formation involved the translocation of antigen/autoantibody from the subepithelial to the urinary side of podocyte plasma membranes. Urinary exopher-release correlated with lower albuminuria and lower glomerular antigen/autoantibody burden. In MN patients the prospective monitoring of urinary exopher abundance and of exopher-bound autoantibodies was additive in the assessment of immunologic MN activity. ConclusionsExopher-formation and release is a novel pathomechanism in MN to remove antigen/autoantibody aggregates from the podocyte. Tracking exopher-release will add a non-invasive diagnostic tool with prognostic potential to clinical diagnostics and follow-up of MN patients.

cell biology↗

bioRxiv2022

Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule regulation

Tauopathies such as Alzheimers disease are characterized by the aggregation and increased phosphorylation of the microtubule-associated protein tau. The pathological changes in tau are closely linked to neurodegeneration, making tau a prime candidate for intervention. However, the multiple facets of tau function and the lack of cellular tauopathy models that could support mechanism-based drug development hampers progress. Here we report the development of a live-cell imaging approach to quantitatively monitor pathological changes of human tau as it interacts with axonal microtubules. We show that a full-length aggregation-prone tau construct exhibits reduced interaction with microtubules as it increasingly aggregates. Through chemoinformatic analyses, we identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that inhibit tau aggregation and modulate tau phosphorylation. We found that PHOX15 restores the physiological microtubule interaction of aggregation-prone tau in neurons and inhibits the first phase of tau aggregation in vitro. Furthermore, we report that PHOX15 inhibits the tau kinases GSK3{beta} and Cdk5, alters the kinome activity of model neurons, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and indicate that the binding of PHOX15 in one of the channels reduces the protofilaments ability to adopt a PHF-like conformation. The data show that our imaging approach provides a useful tool for identifying compounds that modulate tau-microtubule interaction in axons. We demonstrate that a polypharmacological approach to simultaneously treat tau aggregation and tau phosphorylation is able to restore physiological microtubule regulation, identifying PHOX15 as a promising drug candidate to counteract tau-induced neurodegeneration.

neuroscience↗

bioRxiv2022

Tau and α-synuclein shape microtubule organization and microtubule-dependent transport in neuronal dendrites

Tau and -synuclein are major players in neurodegenerative diseases, but their physiological role, particularly in dendrites, is poorly understood. Here we show that, surprisingly, lack of tau protein induces the development of a more elaborate dendritic arbor of hippocampal pyramidal cells in organotypic tissue. Using high-speed volumetric lattice light-sheet microscopy and single particle tracking, we found a more directional KIF1A-mediated transport in dendrites of Tau KO cells. Increased transport processivity correlated with longer and straighter dendritic microtubules as revealed by three-dimensional super-resolution microscopy of cultured hippocampal neurons. Unbiased mass spectrometric analysis of tissue showed highly increased expression of -synuclein in Tau KO hippocampi. Overexpression of -synuclein mimicked the transport characteristics observed in Tau KO cells. Our data indicate that tau and -synuclein shape microtubule-dependent transport in neuronal dendrites, thereby promoting dendritic arborization during maturation. Furthermore, the data demonstrate that transport efficiency and length and straightness of microtubules are correlated.

neuroscience↗