Ralph Witzgall

Mutations in the transcription factor LMX1B have been identified as the cause of the autosomal-dominant disease nail-patella syndrome. It manifests in small or absent patellae and dysplastic or missing toe- and fingernails, but the prognosis of the patients is determined by the development of renal symptoms due to dysfunctional podocytes. The pathogenetic mechanisms leading to podocyte damage and their association with specific mutations in the LMX1B gene are not understood, which has impeded the development of specific therapeutic strategies. Here we identify the pathogenetic mechanism affecting many patients with nail-patella syndrome, provide proof of principle for a novel therapeutic approach and suggest a domain-specific effect in gene regulation by LMX1B. Interestingly, missense mutations in the LIM domains of Lmx1b result in a recessive phenotype in two different knock-in mouse lines due to proteasomal degradation of the mutated proteins. The decreased half-life of the mutated LMX1B proteins can be attributed to ubiquitinylation and is prolonged by two proteasomal inhibitors, one of which was also tested in our mouse models and results in an alleviation of the renal symptoms. RNA sequencing of genetically altered podocytes indicates that LMX1B predominantly acts as a transcriptional repressor and furthermore suggests that the two LIM domains contribute to the distinct regulation of LMX1B target genes. We conclude that proteasomal inhibitors, drugs already approved for treatment of patients with multiple myeloma, warrant further studies to prevent renal failure in patients with nail-patella syndrome caused by mutations in the LIM domains. These studies should be designed to deliver the drug specifically to podocytes and to target specific ubiquitin ligases in order to limit side effects.

The intracellular cilia assembly pathway is a complex, multistep process that requires the continuous and coordinated incorporation of membrane material. However, how membrane remodeling occurs during early ciliogenesis is not yet understood. Moreover, the identity of the organelle(s) that supply membrane material for the nascent cilium has yet to be determined. Here, we extend the current model of primary cilia formation by showing that randomly attached distal appendage vesicles and tubules fuse laterally to generate a doughnut-shaped membrane structure. Centripetal fusion events follow to close the central hole. Our data demonstrate that both the Golgi apparatus and endocytotic pathways independently contribute to ciliogenesis. We identify the endocytotic protein GRAF1 as being essential during the early stages of ciliogenesis and for the delivery of plasma membrane-derived material to the developing ciliary membrane. Our three-dimensional ultrastructural analysis uncovers previously unrecognized intermediate stages in the intracellular cilia assembly pathway with GRAF1 as a regulator of ciliogenesis.

The intracellular cilia assembly pathway is a complex, multistep process that requires the continuous and coordinated incorporation of membrane material. However, how membrane remodeling occurs during early ciliogenesis is not yet understood. Moreover, the identity of the organelle(s) that supply membrane material for the nascent cilium has yet to be determined. Here, we extend the current model of primary cilia formation by showing that randomly attached distal appendage vesicles and tubules fuse laterally to generate a doughnut-shaped membrane structure. Centripetal fusion events follow to close the central hole. Our data demonstrate that both the Golgi apparatus and endocytotic pathways independently contribute to ciliogenesis. We identify the endocytotic protein GRAF1 as being essential during the early stages of ciliogenesis and for the delivery of plasma membrane-derived material to the developing ciliary membrane. Our three-dimensional ultrastructural analysis uncovers previously unrecognized intermediate stages in the intracellular cilia assembly pathway with GRAF1 as a novel regulator of ciliogenesis.

Achieving high target cell avidity in combination with cell selectivity are fundamental, but largely unachieved goals in the development of biomedical nanoparticle systems, which are intricately linked to the quantity of targeting functionalities on their surface. Viruses, regarded as almost ideal role models for nanoparticle design, are evolutionary optimized, so that they cope with this challenge bearing an extremely low number of spikes, and thus binding domains, on their surface. In comparison, nanoparticles are usually equipped with more than an order of magnitude more ligands. It is therefore obvious that one key factor for increasing nanoparticle efficiency in terms of avidity and selectivity lies in optimizing their ligand number. A first step along this way is to know how many ligands per nanoparticle are involved in specific binding with target cell receptors. This question is addressed experimentally for a block copolymer nanoparticle model system. The data confirm that only a fraction of the nanoparticle ligands is involved in the binding processes: with a total ligand valency of 29 ligands/100 nm² surface area a maximum 5.3 ligands/100 nm² are involved in specific receptor binding. This corresponds to an average number of 251 binding ligands per nanoparticle, a number that can be rationalized within the biological context of the model system.

The LIM homeodomain transcription factors LMX1A and LMX1B are essential mediators of midbrain dopaminergic neuronal (mDAN) differentiation and survival. Here we show that LMX1A and LMX1B are autophagy transcription factors that provide cellular stress protection. Their suppression dampens the autophagy response, lowers mitochondrial respiration, and elevates mitochondrial ROS, and their inducible overexpression protects against rotenone toxicity in human iPSC-derived mDANs in vitro. Significantly, we show that LMX1A and LMX1B stability is in part regulated by autophagy, and that these transcription factors bind to multiple ATG8 proteins. Binding is dependent on subcellular localization and nutrient status, with LMX1B interacting with LC3B in the nucleus under basal conditions and associating with both cytosolic and nuclear LC3B during nutrient starvation. Crucially, ATG8 binding stimulates LMX1B-mediated transcription for efficient autophagy and cell stress protection, thereby establishing a novel LMX1B-autophagy regulatory axis that contributes to mDAN maintenance and survival in the adult brain.

Teeth with different chemical compositions can show vastly different physical properties, so knowledge of elemental composition is required to use animal teeth as substitutes for human teeth in research. In vitro, energy dispersive X-ray spectroscopy (EDX), improved by calibration standards and Si₃N₄-window material, enables determining local elemental compositions of inorganic and organic compounds without sample destruction. Six human molars, bovine incisors, murine incisors, and murine molars were analyzed. EDX-field scans and line scans were analyzed for elements Ca, P, O, C, N, F, Na, Mg, Fe, Cl, and S. Furthermore, Ca/P- and Ca/N-Ratios were calculated. The presence of iron in murine incisor enamel was investigated using additional wavelength dispersive X-ray spectroscopy measurements (WDX) near the enamel surface. Bovine and human enamel and dentin revealed close similarities regarding elemental composition. The median (25–75% percentiles) of At%Ca was 21.1 (20.8–21.3) in human enamel, 21.0 (20.7–21.2) in bovine enamel, and in murine enamel, 18.3 (17.85–18.88) for molars and 18.35 (18.00–18.75) for incisors. In dentin, murine teeth revealed a higher At%Ca compared to human and bovine teeth. Significant differences between human and bovine teeth were found for nitrogen in dentin, with a median of 4.5 (3.3–5) At%N for human dentin and 2.7 (2.3–3.2) At%N for bovine dentin. The Ca/P-Ratio was the highest in human and bovine enamel, which did not differ significantly. Enamel from murine molars had a higher Ca/P-Ratio than murine incisors and the highest Ca/P-Ratio in dentin was observed for human teeth and murine molars (1.49). WDX revealed iron in the outer 10 µm of pre- and post-eruptive enamel of murine incisors. Pre- and post-eruptive enamel on murine incisors only differed significantly in At%Ca (p = 0.041) and At%P (p = 0.026) with both At% higher in the pre-eruptive enamel. Murine teeth differ significantly from human and bovine teeth in terms of the elemental composition of enamel and dentin.

Mutations in the PKD2 gene cause autosomal-dominant polycystic kidney disease but the physiological role of polycystin-2, the protein product of PKD2, remains elusive. Polycystin-2 belongs to the transient receptor potential (TRP) family of non-selective cation channels. To test the hypothesis that altered ion channel properties of polycystin-2 compromise its putative role in a control circuit controlling lumen formation of renal tubular structures, we generated a mouse model in which we exchanged the pore loop of polycystin-2 with that of the closely related cation channel polycystin-2L1 (encoded by PKD2L1), thereby creating the protein polycystin-2poreL1. Functional characterization of this mutant channel in Xenopus laevis oocytes demonstrated that its electrophysiological properties differed from those of polycystin-2 and instead resembled the properties of polycystin-2L1, in particular regarding its permeability for Ca2+ ions. Homology modeling of the ion translocation pathway of polycystin-2poreL1 argues for a wider pore in polycystin-2poreL1 than in polycystin-2. In Pkd2poreL1 knock-in mice in which the endogenous polycystin-2 protein was replaced by polycystin-2poreL1 the diameter of collecting ducts was increased and collecting duct cysts developed in a strain-dependent fashion.