Biology subjects

Hackl, M.

Publications and source records attributed to Hackl, M..

18 recordsLinked to original sources

bioRxiv2026

Force-induced unfolding versus unbinding of a cellulosomal carbohydrate-binding module from cellulose interface

Carbohydrate-binding modules (CBMs) anchor microbial cellulosomes to cellulose surfaces, aiding biomass degradation under mechanical stress. To understand how mechanical pulling affects this process, we combine steered molecular dynamics (SMD) simulations with acoustic force spectroscopy (AFS) to investigate Clostridium thermocellum CBM3a unbinding from cellulose under different pulling configurations (C- versus N-terminus). Our SMD simulations reveal that C-terminal pulling induces substantial CBM3a unfolding prior to unbinding, whereas N-terminal pulling results in detachment with the protein largely intact. Unbinding proceeds via two distinct mechanisms across the planar binding motif: a TRP mode (initiating via TRP135) and a TYR mode (initiating via TYR84). AFS experiments corroborate the SMD findings, showing that C-terminal pulling yields lower rupture forces and a 7.5-fold higher unbinding off-rate compared to N-terminal pulling of CBM3a from cellulose. These results demonstrate that structural asymmetry and pulling orientation critically govern the mechanical stability, unfolding pathways, and cellulose-binding dynamics of cellulosomal proteins.

biophysics↗

bioRxiv2026

Castling, a novel therapeutic concept for rewiring pathological gene-expression networks, enabled by the TRIPLE technology

Pathological conditions often arise from dysregulation of complex gene networks, with microRNAs (miRNAs) acting as central modulators. Disease progression is frequently characterized by upregulation of "disease-promoting" miRNA, suppressing beneficial pathways, and concomitant downregulation of "protective/therapeutic" miRNAs, normally restraining pathological programs. Since individual miRNAs coordinately regulate multiple genes, their manipulation represents powerful therapeutic intervention, yet synthetic or ectopically overexpressed miRNA mimics or inhibitors may perturb physiological miRNA processing and/or cause off-target effects. We hypothesized that pathological gene regulatory imbalances could instead be corrected by rewiring endogenous miRNA regulation. Specifically, by placing downregulated "protective/therapeutic" miRNAs under the control of promoters activated in pathology, and driving overexpression of "disease-promoting" miRNAs, thereby disabling the pathogenic program while inducing the therapeutic one in a single editing event. We termed this concept castling, after the chess move. For effective implementation of castling, we developed TRIPLE (Targeted Replacement Induced by Persistent Locus Editing), a novel genome-editing procedure enhancing homology-directed repair through sequential cleavage. As proof of concept, we castled miRNAs inversely regulated during onset of CAR T cell dysfunction in a model of chronic antigen stimulation. Castled CAR T cells exhibited a delayed dysfunction enabled by up- and downregulation of relevant gene subsets.

molecular biology↗

bioRxiv2026

Metabolic Reprogramming of Human Macrophages Drives the Formation of Hybrid M1/M2 Pro-Regenerative Extracellular Vesicles

The coordinated activity of macrophages is essential for bone repair, with pro-inflammatory M1 macrophages driving early responses and anti-inflammatory M2 macrophages supporting later tissue remodeling. While both phenotypes are required, prolonged persistence of either subtype can impair healing, underscoring the correct transition between the two states. Macrophage polarization is closely linked to cellular metabolism, and human macrophages display distinct metabolic profiles. Macrophage-derived extracellular vesicles (EVs) carry bioactive cargo and reflect parental polarization, influencing recipient cell function. This raises critical questions about how metabolic regulation influences human macrophage function, their EVs and their effect on angiogenesis and osteogenesis. This study investigates EVs derived from polarized primary human macrophages and from macrophages exposed to DASA-58, a small molecule which activates the metabolic enzyme pyruvate kinase M2 (PKM2). Alterations in macrophage metabolism modifies the molecular cargo of their EVs, including microRNAs (miRNAs), to modulate regenerative activity. These findings demonstrate that human macrophage-derived EVs exert metabolically dependent effects on angiogenesis and osteogenesis, and that metabolic modulation enables the generation of EVs with hybrid pro-regenerative properties intermediate between M1 and M2. This establishes metabolic reprogramming within human macrophages using small molecules as a strategy to engineer novel phenotypes and EVs for bone repair.

immunology↗

bioRxiv2024

Immortalization of mesenchymal stromal cells by hTERT does not affect the functional properties of secreted extracellular vesicles

Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have emerged as promising and safe therapeutic agents, however, donor heterogeneities, limited replicative life span and changes in the cellular phenotype throughout in vitro cultivation remain major hurdles for scalable EV production. For these reasons, this study aims to investigate the use of hTERT immortalized ( telomerized) MSCs as a potential source for efficient, standardized, reliable MSC-EVs production by comparing parental primary to their telomerized MSC counterparts. We observed that hTERT expression does not affect cell morphology or cellular doubling time, while ensuring unlimited, stable in vitro propagation. In addition, telomerized WJ-MSCs maintained the canonical expression profile of surface markers and the tri-lineage differentiation potential of their primary counterparts. In terms of EV characteristics, the immortalization by hTERT expression did not affect size, number, cargo composition or biological activity regarding anti-inflammatory, anti-fibrotic and wound healing properties in vitro. In summary, the use of hTERT to immortalize MSCs leads to the creation of cell lines that continuously produce MSC-EVs without altering any key functionalities of the cells or resulting EVs. This suggests that telomerization of human cells from single donors is a promising strategy for generating cell factories that can produce EVs in standardized conditions and at scale and with standardization.

cell biology↗

bioRxiv2024

Snorkel-tag Based Affinity Chromatography for Recombinant Extracellular Vesicle Purification

Extracellular vesicles (EVs) are lipid nanoparticles and play an important role in cell-cell communications, making them potential therapeutic agents and allowing to engineer for targeted drug delivery. The expanding applications of EVs in next generation medicine are still limited by existing tools for scaling standardized EV production, single EV tracing and analytics, and thus provide only a snapshot of tissue-specific EV cargo information. Here, we present CD81, an EV surface marker protein, genetically fused to series of tags with additional transmembrane domain to be displayed on the EV surface, which we term Snorkel-tag. This system enables to affinity purify EVs from complex matrices in a non-destructive form. In future applications, this strategy will allow generating transgenic animals to enable tracing and analyzing EVs, and their cargo in physiological and pathophysiological set-ups, and facilitate the development of EV based diagnostic tools in murine models which can be translated to humans.

bioengineering↗

bioRxiv2024

Profiling microRNA expression during senescence and aging: mining for a diagnostic tool of senescent-cell burden

In the last decade cellular senescence, a hallmark of aging, has come into focus for pharmacologically targeting aging processes. Senolytics are one of these interventive strategies that have advanced into clinical trials, creating an unmet need for minimally invasive biomarkers of senescent cell load to identify patients at need for senotherapy. We created a landscape of miRNA and mRNA expression in five human cell types induced to senescence in-vitro and provide proof-of-principle evidence that miRNA expression can track senescence burden dynamically in-vivo using transgenic p21high senescent cell clearance in HFD fed mice. Finally, we profiled miRNA expression in seven different tissues, total plasma, and plasma derived EVs of young and 25 months old mice. In a systematic analysis, we identified 22 candidate senomiRs with potential to serve as circulating biomarkers of senescence not only in rodents, but also in upcoming human clinical senolytic trials.

molecular biology↗

bioRxiv2023

Characterizing the Cellulose Binding Interactions of Type-A Carbohydrate-Binding Modules using Acoustic Force Spectroscopy

A critical understanding of carbohydrate binding modules (CBMs) is vital for the manipulation of a variety of biological functions they support, including biomass deconstruction, polysaccharide biosynthesis, pathogen defence, and plant development. The unbinding characteristics of CBMs from a polysaccharide substrate surface can be studied using rupture force measurements since it enables a quantitative inference of binding properties through the application of dynamic force spectroscopy (DFS) theory. With the increase in usage of CBMs for diverse applications, it is important to engineer and characterize CBMs that have desired sets of interactions with various carbohydrate substrates. However, though the effect of mutations in the binding motif residues is known to influence CBM binding affinity, its effect on the rupture forces is still not well quantified. This is primarily due to the low experimental throughput of most single-molecule DFS techniques available to characterize the force-induced dissociation of protein-ligand interactions. Here, we have determined the rupture forces of microscopic beads functionalized with various wild-type and mutant CBMs using a highly multiplexed DFS technique called Acoustic Force Spectroscopy (AFS). We have characterized the acoustic force-induced dissociation of specifically two type A CBMs (i.e., CBM3a and CBM64) and relevant seven binding motif targeting CBM mutants unbinding from a nanocellulose surface, over a broad range of DFS loading rates (i.e., 0.1 pN/s to 100 pN/s). Our analysis of the rupture force DFS data yields apparent CBM-cellulose bond interaction parameters, which enables a quantitative comparison of the effect of corresponding mutations on cellulose-CBM binding interactions that compares favorably with results from classical bulk ensemble based binding assays. In summary, detailed insights into the rupturing mechanism of multi-CBM fused domains provides motivation for usage of specific constructs for industrial biotechnological applications.

biophysics↗

bioRxiv2023

Site-specific Effector Protein Functionalization to Create Bead-based Avidity Model Systems

The cooperative effect of multiple affinity binding interactions creating a stable bond, known as avidity, is a universal biological phenomenon seen in diverse systems. For example, avidity based biomolecular interactions are particularly important in assessing the potency of potential drugs such as monoclonal antibodies, chimeric antigen receptor (CAR) T-cell, or Natural Killer, cells to treat cancer or engineering microbes with cell surface immobilized enzyme complexes for consolidated bioprocessing (CBP) of cellulosic biomass to fuels and chemicals. However, predicting or measuring avidity based on in vitro single affinity interactions with non-complexed protein-ligand binding model systems has limitations and often fails to describe the avidity effects observed in vivo with cell surface complexed proteins interacting with multivalent ligands at solid interfaces. Acoustic force spectroscopy (AFS) based assays have recently emerged as a reliable method for direct avidity measurements, expressed as adhesion or rupture forces, which positively correlate with in vivo avidity interactions. However, to better understand and model avidity, in particular for cell-cell interactions and to correlate it with classical binding affinity, a cell mimetic model system with controlled avidity-related properties is needed. Here, we present a method for producing such a cell mimetic model system using "effector beads" that can be used in AFS-based avidity assays or any other bead-based avidity assay. The protein of interest is heterologously expressed and biotinylated in vivo in E. coli, purified, and subsequently tethered with streptavidin coated micron-sized beads to create effector beads. Our experimental results, combined with simulations of the multivalent binding phenomena, demonstrate the dependency of bead rupture force on its receptor protein surface density and force loading rate as well as the intrinsic kinetic binding parameters of the protein-ligand system of interest. These insights provide valuable information for designing future effector bead assays and cell avidity measurements for screening and characterization purposes for diverse applications.

bioengineering↗

bioRxiv2023

Oriented Display of Cello-Oligosaccharides for Pull-down Binding Assays to Distinguish Binding Preferences of Glycan Binding Proteins

The production of biofuels from lignocellulosic biomass using carbohydrate-active enzymes like cellulases is key to sustainable energy production. Understanding the adsorption mechanism of cellulases and associated binding domain proteins down to the molecular level details will help in the rational design of improved cellulases. In nature, carbohydrate-binding modules (CBMs) from families 17 and 28 often appear in tandem appended to the C-terminus of several endocellulases. Both CBMs are known to bind to the amorphous regions of cellulose non-competitively and show similar binding affinity towards soluble cello-oligosaccharides. Based on the available crystal structures, these CBMs may display a uni-directional binding preference towards cello-oligosaccharides (based on how the oligosaccharide was bound within the CBM binding cleft). However, molecular dynamics (MD) simulations have indicated no such clear preference. Considering that most soluble oligosaccharides are not always an ideal substrate surrogate to study the binding of CBMs to the native cell wall or cell surface displayed glycans, it is critical to use alternative reagents or substrates. To experimentally assess any binding directionality of CBMs towards soluble cello-oligosaccharides, we have developed a simple solid-state depletion or pull-down binding assay. Here, we specifically orient azido-labeled carbohydrates from the reducing end to alkyne-labeled micron-sized bead surfaces, using click chemistry, to mimic insoluble cell wall surface-displayed glycans. Our results reveal that both family 17 and 28 CBMs displayed a similar binding affinity towards cellohexaose-modified beads, but not cellopentaose-modified beads, which helps rationalize previously reported crystal structure and MD data. This indicates a preferred uni-directional binding of specific CBMs and could explain their co-evolution as tandem constructs appended to endocellulases to increase amorphous cellulose substrate targeting efficiency. Overall, our proposed workflow can be easily translated to measure the affinity of glycan-binding proteins to click-chemistry based immobilized surface-displayed carbohydrates or antigens.

molecular biology↗

bioRxiv2022

Multivariable Graphical User Interface for Simulation of Tethered Particle Motion

The analysis of particles bound to a surface by flexible tethers can facilitate understanding of various biophysical phenomena (e.g., molecular dynamics of DNA-protein or protein-ligand binding interactions, DNA extensibility and polymer biophysics). Being able to model such systems theoretically can aid in understanding experimentally observed motions and furthermore the limitations of such models can provide insight into modeling complex systems that basic theory sometimes cannot account for. The simulation of tethered particle motion (TPM) allows for efficient analysis of complex behaviors exhibited by such systems, however this type of experiment is rarely taught in undergraduate science classes. We have developed a MATLAB simulation package intended to be used in academic contexts to concisely model and graphically represent the behavior of different tether-particle systems. We show how analysis of the simulation results can be used in biophysical research employing single molecule force spectroscopy (SMFS). Here, our simulation package is capable of modeling any given particle-tether-substrate system and allows the user to generate a parameter space with static and dynamic model components. Our simulation was successfully able to recreate generally observed experimental trends using a recently developed SMFS technique called Acoustic Force Spectroscopy (AFS). Further, the simulation was validated through consideration of the conservation of energy of the tether-bead system, trend analyses, and comparison of particle positional data from actual TPM in silico experiments conducted to simulate data with a parameter space similar to the AFS experimental setup. Overall, our TPM simulator and graphical user interface is suitable for use in an academic context and serves as a template for researchers to set up TPM simulations to mimic their specific SMFS experimental setup.

scientific communication and education↗

bioRxiv2022

Small non-coding RNA landscape of extracellular vesicles from a post-traumatic model of equine osteoarthritis

Extracellular vesicles comprise an as yet inadequately investigated intercellular communication pathway in the field of early osteoarthritis. We hypothesised that small non-coding RNA expression pattern in synovial fluid and plasma would change during progression of experimental osteoarthritis. In this study, we used small RNA sequencing to provide a comprehensive overview of the temporal expression profiles of small non-coding transcripts carried by EVs derived from plasma and synovial fluid for the first time in a post-traumatic model of equine osteoarthritis. Additionally, we characterised synovial fluid and plasma-derived extracellular vesicles with respect to quantity, size, and surface markers. The differential expression of seven microRNAs in plasma and synovial fluid-derived extracellular vesicles; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a and four snoRNAs; U3, snord15, snord46, snord58 represent potential biomarkers for early OA. Bioinformatics analysis of the differentially expressed microRNAs in synovial fluid highlighted that in early OA these related to the inhibition of cell cycle, cell cycle progression, DNA damage and cell proliferation but increased cell viability, and differentiation of stem cells. Plasma and synovial fluid-derived extracellular vesicle small non-coding signatures have been established for the first time in a temporal model of osteoarthritis. These could serve as novel biomarkers for the evaluation of osteoarthritis progression or act as potential therapeutic targets.

molecular biology↗

bioRxiv2021

Acoustic Force Spectroscopy Reveals Subtle Differences in Cellulose Unbinding Behavior of Carbohydrate-Binding Modules

To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN). Significance StatementCellulases are multi-modular enzymes produced by numerous microbes that catalyze cellulose hydrolysis into glucose. These enzymes play an important role in global carbon cycling as well as cellulosic biofuels production. CBMs are essential components of cellulolytic enzymes involved in facilitating hydrolysis of polysaccharides by tethered catalytic domains (CD). The subtle interplay between CBM binding and CD activity is poorly understood particularly for heterogeneous reactions at solid-liquid interfaces. Here, we report a highly multiplexed single-molecule force spectroscopy method to study CBM dissociation from cellulose to infer the molecular mechanism governing substrate recognition and dissociation. This approach can be broadly applied to study multivalent protein-polysaccharide binding interactions relevant to other carbohydrates such as starch, chitin, or hyaluronan to engineer efficient biocatalysts.

biochemistry↗

bioRxiv2020

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum, frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing the substrate-bound concentration of cognate cellulase catalytic domains. However, due to the interfacial nature of cellulose hydrolysis and the structural heterogeneity of cellulose, it has been challenging to fully understand the role of CBMs on cellulase activity using classical protein-ligand binding assays. Here, we report a single-molecule CAZyme assay for an industrially relevant processive cellulase Cel7A (from T. reesei) to reveal how subtle CBM1 binding differences can drastically impact cellulase motility/velocity and commitment to initial processive motion for deconstruction of two well-studied crystalline cellulose allomorphs (namely cellulose I and III). We take a multifaceted approach to characterize the complex binding interactions of all major type-A family representative CBMs including CBM1, using an optical-tweezers based single-molecule CBM-cellulose bond rupture assay to complement several classical bulk ensemble protein-ligand binding characterization methods. While our work provides a basis for the cautious use of Langmuir-type adsorption models to characterize classical protein-ligand binding assay data, we highlight the critical limitations of using such overly simplistic models to gain a truly molecular-level understanding of interfacial protein binding interactions at heterogeneous solid-liquid interfaces. Finally, molecular dynamics simulations provided a theoretical basis for the complex binding behavior seen for CBM1 towards two distinct cellulose allomorphs reconciling experimental findings from multiscale analytical methods. Significance StatementMultimodal biomolecular binding interactions involving carbohydrate polymers (e.g., cellulose, starch, chitin, glycosaminoglycans) are fundamental molecular processes relevant to the recognition, biosynthesis, and degradation of all major terrestrial and aquatic biomass. Protein-carbohydrate binding interactions are also critical to industrial biotechnology operations such as enzymatically-catalyzed bioconversion of starch and lignocellulose into biochemicals like ethanol. However, despite the ubiquitous importance of such interfacial processes, we have a poor molecular-level understanding of protein-polysaccharide binding interactions. Here, we provide a comprehensive experimental and theoretical analysis of bulk ensemble versus single-molecule binding interactions of enzyme motors and associated non-catalytic binding domains with cellulosic polysaccharides to highlight the critical limitations of applying classical biochemical assay techniques alone to understanding protein adsorption or biological activity at solid-liquid interfaces.

biochemistry↗

bioRxiv2020

MiR-146a controls age related bone loss

Bone loss is one of the consequences of aging, leading to diseases such as osteoporosis and increased susceptibility to fragility fractures and therefore considerable morbidity and mortality in humans. Here we identify microRNA 146a as an essential epigenetic switch controlling bone loss with age. Mice deficient in miR-146a show regular development of their skeleton. However, while WT mice start to lose bone with age, animals deficient in miR-146a continue to accrue bone throughout their life span. Increased bone mass is due to increased generation and activity of osteoblasts in miR-146a deficient mice as a result of sustained activation of bone anabolic Wnt signaling during aging. Deregulation of the miR-146a target genes Wnt1 and Wnt5a parallel bone accrual and osteoblast generation, which is accompanied by reduced development of bone marrow adiposity. Furthermore, miR-146a deficient mice are protected from ovariectomy induced bone loss. In humans, levels of miR-146a are increased in patients suffering fragility fractures in comparison to those who do not. These data identify miR-146a as a crucial epigenetic temporal regulator which essentially controls bone homeostasis during aging by regulating bone anabolic Wnt signaling. Therefore, miR-146a might be a powerful therapeutic target to prevent age related bone dysfunction such as the development of bone marrow adiposity and osteoporosis.

molecular biology↗

bioRxiv2020

Cathepsin K is required for maintenance and regeneration of lymphopoiesis in vivo

In this study, we investigated the influence of the loss of Cathepsin K (Ctsk) gene on the hematopoietic system in vitro and in vivo. We found that cultures with Lineage- SCA1+ KIT+ (LSK) cells on Ctsk deficient stromal cells display reduced colony formation and proliferation, with increased differentiation, giving rise to repopulating cells with reduced ability to repopulate the donor LSK and T cell compartments in the bone marrow. Subsequent in vivo experiments showed impairment of lymphocyte numbers, but, gross effects on early hematopoiesis or myelopoiesis were not found. Most consistently in in vivo experimental settings, we found a significant reduction of (donor) T cell numbers in the bone marrow. Lymphocyte deregulation is also found in transplantation experiments, which revealed that Ctsk is required for optimal regeneration not only of T cells, but also of B cells. Interestingly, cell non-autonomous Ctsk regulates both B- and T cell numbers, but T cell numbers in the bone marrow require an additional autonomous Ctsk-dependent process. Thus, we show that Ctsk is required for the maintenance of hematopoietic stem cells in vitro, but in vivo, Ctsk deficiency most strongly affects lymphocyte homeostasis, particularly of T cells in the bone marrow.

cell biology↗

bioRxiv2019

SVF-derived extracellular vesicles carry characteristic miRNAs in lipedema

Lipedema is a chronic, progressive disease of adipose tissue with lack of consistent diagnostic criteria. The aim of this study was a thorough comparative characterization of extracellular microRNAs from the stromal vascular fraction (SVF) of healthy and lipedema adipose tissue. For this, we analyzed 187 extracellular microRNAs in concentrated conditioned media (cCM) and specifically in small extracellular vesicles (sEVs) enriched thereof by size exclusion chromatography. No significant difference in median particle size and concentration was observed between sEV fractions in healthy and lipedema. We found the majority of miRNAs located predominantly in cCM compared to sEV enriched fraction. Surprisingly, hierarchical clustering of the most variant miRNAs showed that only sEV miRNA profiles - but not cCM miRNAs - were impacted by lipedema. Seven sEV miRNAs (miR-16-5p, miR-29a-3p, miR-24-3p, miR-454-p, miR-144-5p, miR-130a-3p, let-7c-5p) were differently regulated in lipedema and healthy, whereas only one cCM miRNA (miR-188-5p) was significantly downregulated in lipedema. Comparing SVF from healthy and lipedema patients, we identified sEVs as the lipedema relevant miRNA fraction. This study contributes to identify the potential role of SVF secreted miRNAs in lipedema.

molecular biology↗

bioRxiv2019

MicroRNA levels in bone and blood change during bisphosphonate and teriparatide therapy in an animal model of postmenopausal osteoporosis

MicroRNAs control the activity of a variety of genes that are pivotal to bone metabolism. Therefore, the clinical utility of miRNAs as biomarkers and drug targets for bone diseases certainly merits further investigation. This study describes the use of an animal model of postmenopausal osteoporosis to generate a comprehensive dataset on miRNA regulation in bone tissue and peripheral blood during bone loss and specifically anti-resorptive and osteo-anabolic treatment.\n\nForty-two Sprague-Dawley rats were randomized to SHAM surgery (n=10) or ovariectomy (OVX, n=32). Eight weeks after surgery, OVX animals were further randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). After 12 weeks of treatment, bone and serum samples were used for microRNA analysis using next-generation sequencing (NGS), mRNA levels using RT-qPCR, and bone microarchitecture analysis using nanoCT.\n\nOvariectomy resulted in loss of trabecular bone, which was fully rescued using osteo-anabolic treatment, and partially rescued using anti-resorptive treatment. NGS revealed that both, anti-resorptive and anabolic treatment had a significant impact on miRNA levels in bone tissue and serum: out of 426 detected miRNAs, 46 miRNAs were regulated by teriparatide treatment an d 10 by zoledronate treatment (p-adj. < 0.1). Interestingly, teriparatide and zoledronate treatment were able to revert miRNA changes in tissue and serum of untreated OVX animals, such as the up-regulation of miR-203a-3p, a known osteo-inhibitory miRNA. We confirmed previously established mechanisms of miR-203a by analyzing its direct target Dlx5 in femoral head.\n\nOur data reveal a significant effect of ovariectomy-induced bone loss, as well as the two major types of anti-osteoporotic treatment on miRNA transcription in femoral head tissue. These changes are associated with altered activity of target genes relevant to bone formation, such as Dlx5. The observed effects of bone loss and treatment response on miRNA levels in bone are also reflected in the peripheral blood, suggesting the possibility of minimally-invasive monitoring of bone-derived miRNAs using liquid biopsies.\n\nHighlightsO_LImicroRNA expression in bone tissue is altered by osteo-anabolic and anti-resorptive therapy in OVX rats.\nC_LIO_LImicroRNA changes in untreated OVX rats are reverted by anti-osteoporotic therapy.\nC_LIO_LImiR-203a is up-regulated during bone loss and down-regulated following therapy.\nC_LIO_LIBone tissue and serum levels of miR-203a are highly correlated.\nC_LI

molecular biology↗

bioRxiv2017

Individual nephron proteomes connect morphology and function in proteinuric kidney disease

In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This \"one-segment-one-proteome-approach\" defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale

physiology↗