Beyond basic characterization and omics: Immunomodulatory roles of platelet-derived extracellular vesicles unveiled by functional testing.

Renowned for their role in haemostasis and thrombosis, platelets are also increasingly recognized for their contribution in innate immunity, immunothrombosis and inflammatory diseases. Platelets express a wide range of receptors, which allows them to reach a variety of activation endpoints and grants them immunomodulatory functions. Activated platelets release extracellular vesicles (PEVs), whose formation and molecular cargo has been shown to depend on receptor-mediated activation and environmental cues. This study compared the immunomodulatory profiles of PEVs generated via activation of platelets by different receptors, glycoprotein VI, C-type lectin-like receptor 2 and combining all thrombin-collagen receptors. Functional assays in vivo in zebrafish and in vitro in human macrophages highlighted distinct homing and secretory responses triggered by the PEVs. In contrast, omics analyses of protein and miRNA cargo combined with physicochemical particle characterization found only subtle differences between the activated PEV types, which were insufficient to predict their different immunomodulatory functions. In contrast, constitutively released PEVs, formed in the absence of an exogenous activator, displayed a distinct immunomodulatory profile from the receptor-induced PEVs. Our findings underscore that PEVs are tunable through receptor-mediated activation. To truly comprehend their role(s) in mediating platelet functions among immune cells, conducting functional assays is imperative.

HSP60 chaperone deficiency disrupts the mitochondrial matrix proteome and dysregulates cholesterol synthesis.

OBJECTIVE: Mitochondrial proteostasis is critical for cellular function. The molecular chaperone HSP60 is essential for cell function and dysregulation of HSP60 expression has been implicated in cancer and diabetes. The few reported patients carrying HSP60 gene variants show neurodevelopmental delay and brain hypomyelination. Hsp60 interacts with more than 260 mitochondrial proteins but the mitochondrial proteins and functions affected by HSP60 deficiency are poorly characterized. METHODS: We studied two model systems for HSP60 deficiency: (1) engineered HEK cells carrying an inducible dominant negative HSP60 mutant protein, (2) zebrafish HSP60 knockout larvae. Both systems were analyzed by RNASeq, proteomics, and targeted metabolomics, and several functional assays relevant for the respective model. In addition, skin fibroblasts from patients with disease-associated HSP60 variants were analyzed by proteomics. RESULTS: We show that HSP60 deficiency leads to a differentially downregulated mitochondrial matrix proteome, transcriptional activation of stress responses, and dysregulated cholesterol biosynthesis. This leads to lipid accumulation in zebrafish knockout larvae. CONCLUSIONS: Our data provide a compendium of the effects of HSP60 deficiency on the mitochondrial matrix proteome. We show that HSP60 is a master regulator and modulator of mitochondrial functions and metabolic pathways. HSP60 dysfunction also affects cellular metabolism and disrupts the integrated stress response. The effect on cholesterol synthesis explains the effect of HSP60 dysfunction on myelination observed in patients carrying genetic variants of HSP60.

The metalloproteinase PAPP-A is required for IGF-dependent chondrocyte differentiation and organization.

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel's cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel's cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

Assessing metabolic rates in zebrafish using a 3D-printed intermittent-flow respirometer and swim tunnel system.

Zebrafish have become a widely used vertebrate model in physiology and reliable measures of their metabolic rate are needed. We have developed a 3D-printed respirometer and swim tunnel system and used it for obtaining accurate measurement of standard metabolic rate (SMR) and maximal, aerobic metabolic rate (MMR) in zebrafish under rest and maximal exercise, respectively. We compared a slow (stepwise) protocol to a fast (continuous) protocol for determining MMR. The fast protocol yielded slightly (but not significantly) higher oxygen consumption rates than the slow protocol and the data, in contrast to the slow protocol, followed a normal distribution. These findings point to the fast protocol as a fast and reliable method for obtaining accurate values of MMR in zebrafish. We make the 3D drawings for printing the system available to researchers, to help streamline the field of metabolic research in zebrafish and other smaller fish species.

SorCS2 binds progranulin to regulate motor neuron development.

Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.

Generation and validation of a myoglobin knockout zebrafish model.

Previous studies using myoglobin (Mb) knockout mice and knockdown zebrafish have presented conflicting results about in vivo phenotypes resulting from the loss of this conserved and highly expressed protein, and therefore a new well-characterized knockout model is warranted. We here describe the generation of three distinct zebrafish mb knockout lines using the CRISPR/Cas system. None of the three lines exhibited any morphological phenotypes, changes in length, or lethality during embryonic and larval development. The adult homozygous knockout mb(Auzf13.2) zebrafish line were absent of Mb protein, had an almost complete degradation of mb mRNA, and showed no changes in viability, length, or heart size. Furthermore, transcriptomic analysis of adult heart tissue showed that mb knockout did not cause altered expression of other genes. Lastly, no off-targeting was observed in 36 screened loci. In conclusion, we have generated three mb knockout lines with indistinguishable phenotypes during embryonic and larval development and validated one of these lines, mb(Auzf13.2), to have no signs of genetic compensation or off-target effects in the adult heart. These findings suggests that the mb(Auzf13.2) shows promise as a candidate for investigating the biological role of Mb in zebrafish.

Dosage of the pseudoautosomal gene SLC25A6 is implicated in QTc interval duration.

The genetic architecture of the QT interval, defined as the period from onset of depolarisation to completion of repolarisation of the ventricular myocardium, is incompletely understood. Only a minor part of the QT interval variation in the general population has been linked to autosomal variant loci. Altered X chromosome dosage in humans, as seen in sex chromosome aneuploidies such as Turner syndrome (TS) and Klinefelter syndrome (KS), is associated with altered QTc interval (heart rate corrected QT), indicating that genes, located in the pseudoautosomal region 1 of the X and Y chromosomes may contribute to QT interval variation. We investigate the dosage effect of the pseudoautosomal gene SLC25A6, encoding the membrane ADP/ATP translocase 3 in the inner mitochondrial membrane, on QTc interval duration. To this end we used human participants and in vivo zebrafish models. Analyses in humans, based on 44 patients with KS, 44 patients with TS, 59 male and 22 females, revealed a significant negative correlation between SLC25A6 expression level and QTc interval duration. Similarly, downregulation of slc25a6 in zebrafish increased QTc interval duration with pharmacological inhibition of KATP channels restoring the systolic duration, whereas overexpression of SLC25A6 shortened QTc, which was normalized by pharmacological activation of KATP channels. Our study demonstrate an inverse relationship between SLC25A6 dosage and QTc interval indicating that SLC25A6 contributes to QT interval variation.

Genetic analysis identifies the SLC4A3 anion exchanger as a major gene for short QT syndrome.

BACKGROUND: A variant in the SLC4A3 anion exchanger has been identified as a novel cause of short QT syndrome (SQTS), but the clinical importance of SLC4A3 as a cause of SQTS or sudden cardiac death remains unknown. OBJECTIVE: The purpose of this study was to investigate the prevalence of potential disease-causing variants in SQTS patients using gene panels including SLC4A3. METHODS: In this multicenter study, genetic testing was performed in 34 index patients with SQTS. The pathogenicity of novel SLC4A3variants was validated in a zebrafish embryo heart model. RESULTS: Potentially disease-causing variants were identified in 9 (26%) patients and were mainly (15%) located in SLC4A3: 4 patients heterozygous for novel nonsynonymous SLC4A3 variants-p.Arg600Cys, p.Arg621Trp, p.Glu852Asp, and p.Arg952His-and 1 patient with the known p.Arg370His variant. In other SQTS genes, potentially disease-causing variants were less frequent (2× in KCNQ1, 1× in KCNJ2, and CACNA1C each). SLC4A3 variant carriers (n = 5) had a similar heart rate but shorter QT and J point to T wave peak intervals than did noncarriers (n = 29). Knockdown of slc4a3 in zebrafish resulted in shortened heart rate-corrected QT intervals (calculated using the Bazett formula) that could be rescued by overexpression of the native human SLC4A3-encoded protein (AE3), but neither by the mutated AE3 variants p.Arg600Cys, p.Arg621Trp, p.Glu852Asp nor by p.Arg952His, suggesting pathogenicity of these variants. Dysfunction in slc4a3/AE3 was associated with alkaline cytosol and shortened action potential of cardiomyocytes. CONCLUSION: In about a quarter of patients with SQTS, a potentially disease-causing variant can be identified. Nonsynonymous variants in SLC4A3 represent the most common cause of SQTS, underscoring the importance of including SLC4A3 in the genetic screening of patients with SQTS or sudden cardiac death.

Superoxide dismutase 3 is expressed in bone tissue and required for normal bone homeostasis and mineralization.

Superoxide dismutase 3 (SOD3) is an extracellular protein with the capacity to convert superoxide into hydrogen peroxide, an important secondary messenger in redox regulation. To investigate the utility of zebrafish in functional studies of SOD3 and its relevance for redox regulation, we have characterized the zebrafish orthologues; Sod3a and Sod3b. Our analyses show that both recombinant Sod3a and Sod3b express SOD activity, however, only Sod3b is able to bind heparin. Furthermore, RT-PCR analyses reveal that sod3a and sod3b are expressed in zebrafish embryos and are present primarily in separate organs in adult zebrafish, suggesting distinct functions in vivo. Surprisingly, both RT-PCR and whole mount in situ hybridization showed specific expression of sod3b in skeletal tissue. To further investigate this observation, we compared femoral bone obtained from wild-type and SOD3-/- mice to determine whether a functional difference was apparent in healthy adult mice. Here we report, that bone from SOD3-/- mice is less mineralized and characterized by significant reduction of cortical and trabecular thickness in addition to reduced mechanical strength. These analyses show that SOD3 plays a hitherto unappreciated role in bone development and homeostasis.

Tracing the In Vivo Fate of Nanoparticles with a "Non-Self" Biological Identity.

Nanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, "non-self" incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nanobioimaging, here we unravel the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona preformed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the preformed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and to inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.