Retrograde intramedullary nail fixation with oblique fixed angle screws versus locking plates in periprosthetic supracondylar fractures after total knee arthroplasty.

PURPOSE: Common surgical procedures in the treatment of periprosthetic distal femur fractures (PPFF) include osteosynthesis with fixed angle locking plates (LP) and retrograde intramedullary nails (RIN). This study aimed to compare LPs to RINs with oblique fixed angle screws in terms of complications, radiographic results and functional outcome. METHODS: 63 PPFF in 59 patients who underwent treatment in between 2009 and 2020 were included and retrospectively reviewed. The anatomic lateral and posterior distal femoral angle (aLDFA and aPDFA) were measured on post-surgery radiographs. The Fracture Mobility Score (FMS) pre- and post-surgery, information about perceived instability in the operated leg and the level of pain were obtained via a questionnaire and previous follow-up (FU) examinations in 30 patients (32 fractures). RESULTS: The collective (median age: 78 years) included 22 fractures treated with a RIN and 41 fractures fixed with a LP. There was no difference in the occurrence of complications (median FU: 21.5 months) however the rate of implant failures requiring an implant replacement was higher in fractures treated with a LP (p = 0.043). The aPDFA was greater in fractures treated with a RIN (p = 0.04). The functional outcome was comparable between both groups (median FU: 24.5 months) with a lower outcome in the post-surgery FMS (p = < 0.001). CONCLUSION: Fractures treated with RIN resulted in an increased recurvation of the femur however the rate of complications and the functional outcome were comparable between the groups. The need for implant replacements following complications was higher in the LP group.

Cytokine Profiling in Human iPSC-Derived Dopaminergic Neuronal and Microglial Cultures.

Aside from the degeneration of dopaminergic neurons, inflammation is a key component in the movement disorder Parkinson's disease (PD). Microglia activation as well as elevated cytokine levels were observed in the brains of PD patients, but the specific role of microglia in the disease process is unknown. Here, we generate human cellular models by differentiating iPSCs into dopaminergic neurons and microglia. We combine these cells in co-culture to perform cytokine profiling, representing the final functional outcome of various signaling pathways. For this, we used unstimulated conditions and treatment with inflammatory stressors. Importantly, only co-cultures but not the monocultures responded to IL-1ß treatment suggesting co-culture-related crosstalk. Moreover, we identified the main types of released cytokines and chemokines in this model system and found a preference for the activation of the chemotaxis pathway in response to all treatments, which informs future studies on the cell-type-specific reaction to inflammatory stimulation. Finally, we detected protein level changes in PD risk factor GPNMB upon stress in microglia, further confirming the link between PD-associated genes and inflammation in human-derived cellular models.

Deconstructing sarcomeric structure-function relations in titin-BioID knock-in mice.

Proximity proteomics has greatly advanced the analysis of native protein complexes and subcellular structures in culture, but has not been amenable to study development and disease in vivo. Here, we have generated a knock-in mouse with the biotin ligase (BioID) inserted at titin's Z-disc region to identify protein networks that connect the sarcomere to signal transduction and metabolism. Our census of the sarcomeric proteome from neonatal to adult heart and quadriceps reveals how perinatal signaling, protein homeostasis and the shift to adult energy metabolism shape the properties of striated muscle cells. Mapping biotinylation sites to sarcomere structures refines our understanding of myofilament dynamics and supports the hypothesis that myosin filaments penetrate Z-discs to dampen contraction. Extending this proof of concept study to BioID fusion proteins generated with Crispr/CAS9 in animal models recapitulating human pathology will facilitate the future analysis of molecular machines and signaling hubs in physiological, pharmacological, and disease context.

Resolving titin's lifecycle and the spatial organization of protein turnover in mouse cardiomyocytes.

Cardiac protein homeostasis, sarcomere assembly, and integration of titin as the sarcomeric backbone are tightly regulated to facilitate adaptation and repair. Very little is known on how the >3-MDa titin protein is synthesized, moved, inserted into sarcomeres, detached, and degraded. Here, we generated a bifluorescently labeled knockin mouse to simultaneously visualize both ends of the molecule and follow titin's life cycle in vivo. We find titin mRNA, protein synthesis and degradation compartmentalized toward the Z-disk in adult, but not embryonic cardiomyocytes. Originating at the Z-disk, titin contributes to a soluble protein pool (>15% of total titin) before it is integrated into the sarcomere lattice. Titin integration, disintegration, and reintegration are stochastic and do not proceed sequentially from Z-disk to M-band, as suggested previously. Exchange between soluble and integrated titin depends on titin protein composition and differs between individual cardiomyocytes. Thus, titin dynamics facilitate embryonic vs. adult sarcomere remodeling with implications for cardiac development and disease.

Generation and characterization of human-derived iPSC lines from three pairs of monozygotic twins discordant for Parkinson's disease.

Despite a genetic component in the development of Parkinson's disease (PD), monozygotic twin pairs often display discordance for PD. Here, we describe the generation of six human induced pluripotent stem cell (iPSC) lines from dermal fibroblasts of three pairs of monozygotic twins discordant for PD. We used non-integrating Sendai virus and the iPSC lines were comprehensively characterized. These lines provide a valuable resource for studying molecular differences between the affected and unaffected monozygotic twin and their response to genetic and non-genetic factors that might be involved in the development of PD.

Iron overload is accompanied by mitochondrial and lysosomal dysfunction in WDR45 mutant cells.

Beta-propeller protein-associated neurodegeneration is a subtype of monogenic neurodegeneration with brain iron accumulation caused by de novo mutations in WDR45. The WDR45 protein functions as a beta-propeller scaffold and plays a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins. Loss of WDR45 function due to disease-causing mutations has been linked to defects in autophagic flux in patient and animal cells. However, the role of WDR45 in iron homeostasis remains elusive. Here we studied patient-specific WDR45 mutant fibroblasts and induced pluripotent stem cell-derived midbrain neurons. Our data demonstrated that loss of WDR45 increased cellular iron levels and oxidative stress, accompanied by mitochondrial abnormalities, autophagic defects, and diminished lysosomal function. Restoring WDR45 levels partially rescued oxidative stress and the susceptibility to iron treatment, and activation of autophagy reduced the observed iron overload in WDR45 mutant cells. Our data suggest that iron-containing macromolecules and organelles cannot effectively be degraded through the lysosomal pathway due to loss of WDR45 function.

SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.

Mutations in the Parkin gene (PARK2) have been linked to a recessive form of Parkinson's disease (PD) characterized by the loss of dopaminergic neurons in the substantia nigra. Deficiencies of mitochondrial respiratory chain complex I activity have been observed in the substantia nigra of PD patients, and loss of Parkin results in the reduction of complex I activity shown in various cell and animal models. Using co-immunoprecipitation and proximity ligation assays on endogenous proteins, we demonstrate that Parkin interacts with mitochondrial Stomatin-like protein 2 (SLP-2), which also binds the mitochondrial lipid cardiolipin and functions in the assembly of respiratory chain proteins. SH-SY5Y cells with a stable knockdown of Parkin or SLP-2, as well as induced pluripotent stem cell-derived neurons from Parkin mutation carriers, showed decreased complex I activity and altered mitochondrial network morphology. Importantly, induced expression of SLP-2 corrected for these mitochondrial alterations caused by reduced Parkin function in these cells. In-vivo Drosophila studies showed a genetic interaction of Parkin and SLP-2, and further, tissue-specific or global overexpression of SLP-2 transgenes rescued parkin mutant phenotypes, in particular loss of dopaminergic neurons, mitochondrial network structure, reduced ATP production, and flight and motor dysfunction. The physical and genetic interaction between Parkin and SLP-2 and the compensatory potential of SLP-2 suggest a functional epistatic relationship to Parkin and a protective role of SLP-2 in neurons. This finding places further emphasis on the significance of Parkin for the maintenance of mitochondrial function in neurons and provides a novel target for therapeutic strategies.

Regulation of ß1 integrin-Klf2-mediated angiogenesis by CCM proteins.

Mechanotransduction pathways are activated in response to biophysical stimuli during the development or homeostasis of organs and tissues. In zebrafish, the blood-flow-sensitive transcription factor Klf2a promotes VEGF-dependent angiogenesis. However, the means by which the Klf2a mechanotransduction pathway is regulated to prevent continuous angiogenesis remain unknown. Here we report that the upregulation of klf2 mRNA causes enhanced egfl7 expression and angiogenesis signaling, which underlies cardiovascular defects associated with the loss of cerebral cavernous malformation (CCM) proteins in the zebrafish embryo. Using CCM-protein-depleted human umbilical vein endothelial cells, we show that the misexpression of KLF2 mRNA requires the extracellular matrix-binding receptor ß1 integrin and occurs in the absence of blood flow. Downregulation of ß1 integrin rescues ccm mutant cardiovascular malformations in zebrafish. Our work reveals a ß1 integrin-Klf2-Egfl7-signaling pathway that is tightly regulated by CCM proteins. This regulation prevents angiogenic overgrowth and ensures the quiescence of endothelial cells.

Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry.

Signaling by Nodal and Bmp is essential for cardiac laterality. How activities of these pathways translate into left-right asymmetric organ morphogenesis is largely unknown. We show that, in zebrafish, Nodal locally reduces Bmp activity on the left side of the cardiac field. This effect is mediated by the extracellular matrix enzyme Hyaluronan synthase 2, expression of which is induced by Nodal. Unilateral reduction of Bmp signaling results in lower expression of nonmuscle myosin II and higher cell motility on the left, driving asymmetric displacement of the entire cardiac field. In silico modeling shows that left-right differences in cell motility are sufficient to induce a robust, directional migration of cardiac tissue. Thus, the mechanism underlying the formation of cardiac left-right asymmetry involves Nodal modulating an antimotogenic Bmp activity.

Effective generation of transgenic reporter and gene trap lines of the medaka (Oryzias latipes) using the Ac/Ds transposon system.

In model teleost fishes like the medaka and the zebrafish many genes which have been identified in genome sequencing projects await their functional characterization. Techniques for the effective generation of transgenic animals are a prerequisite for this challenging task, and, due to their transparency, fish offer the possibility to combine the use of fluorescent proteins and developmental analysis in vivo. Here we describe the application of the Ac/Ds transposon system to generate transgenic medaka reporter and gene trap lines. We determined a germline transmission rate of 30% in our experiments using constructs ranging in size from 1.8 to 6 kilobase pairs. The genomic integration site of the Ds-elements can be easily identified which is an important feature for gene trap mutagenesis experiments and similar approaches. We constructed gene trap vectors with functional elements of medaka sequences that produce in frame fusions of the endogenous sequence to EGFP. These vectors mimic endogenous expression of the trapped allele in transgenic animals and are capable to interfere with the expression of the wild type allele in the homozygous individuals.

Divergent polarization mechanisms during vertebrate epithelial development mediated by the Crumbs complex protein Nagie oko.

The zebrafish MAGUK protein Nagie oko is a member of the evolutionarily conserved Crumbs protein complex and functions as a scaffolding protein involved in the stabilization of multi-protein assemblies at the tight junction. During zebrafish embryogenesis, mutations in nagie oko cause defects in both epithelial polarity and cardiac morphogenesis. We used deletion constructs of Nagie oko in functional rescue experiments to define domains essential for cell polarity, maintenance of epithelial integrity and cardiac morphogenesis. Inability of Nagie oko to interact with Crumbs proteins upon deletion of the PDZ domain recreates all aspects of the nagie oko mutant phenotype. Consistent with this observation, apical localization of Nagie oko within the myocardium and neural tube is dependent on Oko meduzy/Crumbs2a. Disruption of direct interactions with Patj or Lin-7, two other members of the Crumbs protein complex, via the bipartite L27 domains produces only partial nagie oko mutant phenotypes and does not impair correct junctional localization of the truncated Nagie oko deletion protein within myocardial cells. Similarly, loss of the evolutionarily conserved region 1 domain, which mediates binding to Par6, causes only a subset of the nagie oko mutant epithelial phenotypes. Finally, deletion of the C-terminus, including the entire guanylate kinase and the SH3 domains, renders the truncated Nagie oko protein inactive and recreates all features of the nagie oko mutant phenotype when tested in functional complementation assays. Our observations reveal a previously unknown diversity of alternative multi-protein assembly compositions of the Crumbs-Nagie-oko and Par6-aPKC protein complexes that are highly dependent on the developmental context.

Nuclear localization of the zebrafish tight junction protein nagie oko.

The tight junctions-associated MAGUK protein nagie oko is closely related to Drosophila Stardust, mouse protein associated with lin-seven 1 (Pals1), and human MAGUK p55 subfamily member 5 (Mpp5). As a component of the evolutionarily conserved Crumbs protein complex, nagie oko is essential for the maintenance of epithelial cell polarity. Here, we show that nagie oko contains a predicted nuclear export and two conserved nuclear localization signals. We find that loss of the predicted nuclear export signal results in nuclear protein accumulation. We show that nagie oko nuclear import is redundantly controlled by the two nuclear localization signals and the evolutionarily conserved region 1 (ECR1), which links nagie oko with Par6-aPKC. Finally, deletion forms of nagie oko that lack nuclear import and export signals complement several nagie oko mutant defects in cell polarity and epithelial integrity. This finding provides an entry point to potentially novel and unknown roles of this important cell polarity regulator.

Elucidation of megalin/LRP2-dependent endocytic transport processes in the larval zebrafish pronephros.

Megalin/LRP2 is an endocytic receptor in the proximal tubules of the mammalian kidney that plays a central role in the clearance of metabolites from the glomerular filtrate. To establish a genetic model system for elucidation of molecular components of this retrieval pathway, we characterized orthologous transport processes in the zebrafish. We show that expression of megalin/LRP2 and its co-receptor cubilin is conserved in the larval zebrafish pronephros and demarcates a segment of the pronephric duct that is active in clearance of tracer from the ultrafiltrate. Knock-down of megalin/LRP2 causes lack of Rab4-positive endosomes in the proximal pronephric duct epithelium and abrogates apical endocytosis. Similarly, knock-down of the megalin/LRP2 adaptor Disabled 2 also blocks renal clearance processes. These results demonstrate the conservation of the megalin/LRP2 retrieval pathway between the larval zebrafish pronephros and the mammalian kidney and set the stage for dissection of the renal endocytic machinery in a simple model organism. Using this model system, we provide first genetic evidence that renal tubular endocytosis and formation of endosomes is a ligand-induced process that crucially depends on megalin/LRP2 activity.