The kidneys generate about 180 liters of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.

Background: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin,,but how it controls nephrin trafficking or other podocyte functions remains unclear. Methods: We generated a stable deletion in TBC1D8B using microhomology-mediated end joining genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulated endocytic regulators in transgenic mice provided a comprehensive functional analysis of TBC1D8B . . Results: A null allele of Drosophila TBC1D8B exhibited nephrocyte-restricted nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene.. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from TBC1D8B bearing the edited deletion predominantly localized to mature early endosomes and late endosomes and was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of TBC1D8B. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 FSGS patients and validated functional impact of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. Conclusion: Variants in TBC1D8B are not infrequent among FSGS patients. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.

Both mTOR signaling and autophagy are important modulators of podocyte homeostasis, regeneration, and aging and have been implicated in glomerular diseases. However, the mechanistic role of these pathways for the glomerular filtration barrier remains poorly understood. We used Drosophila nephrocytes as an established podocyte model and found that inhibition of mTOR signaling resulted in increased spacing between slit diaphragms. Gain-of-function of mTOR signaling did not affect spacing, suggesting that additional cues limit the maximal slit diaphragm density. Interestingly, both activation and inhibition of mTOR signaling led to decreased nephrocyte function, indicating that a fine balance of signaling activity is needed for proper function. Furthermore, mTOR positively controlled cell size, survival, and the extent of the subcortical actin network. We also showed that basal autophagy in nephrocytes is required for survival and limits the expression of the sns (nephrin) but does not directly affect slit diaphragm formation or endocytic activity. However, using a genetic rescue approach, we demonstrated that excessive, mTOR-dependent autophagy is primarily responsible for slit diaphragm misspacing. In conclusion, we established this invertebrate podocyte model for mechanistic studies on the role of mTOR signaling and autophagy, and we discovered a direct mTOR/autophagy-dependent regulation of the slit diaphragm architecture.

Neural proliferation zones mediate brain growth, and employ Delta/Notch signaling and HES/HER transcription factors to balance neural stem cell (NSC) maintenance and generation of progenitors and neurons. We investigated Notch-dependency and function of her genes in the thalamic proliferation zone of developing zebrafish larvae. Nine Notch-dependent genes, her2, her4.1-5, her12, her15.1-2, and two Notch-independent genes, her6, her9, are differentially expressed, and define distinct NSC and progenitor populations. her6 prominently executes patterning information to maintain NSCs and the zona limitans intrathalamica Shh signaling activity. her6, her9 double mutants reveal that Notch-independent her genes predominantly regulate NSC maintenance and transition into the progenitor pool. Surprisingly, combined deletion of all Notch-dependent her genes does not affect NSCs or progenitor formation. Combined genetic manipulation of up to eleven Notch-dependent and -independent her genes revealed that Notch-dependent her genes may regulate progenitor progression into neurogenesis, but not progenitor generation itself. The her gene network is partially redundant, with Notch-independent her genes better substituting for loss of Notch-dependent genes than vice versa. Together, her gene regulatory feedback loops and crossregulation contribute to the observed robustness of NSC maintenance.

Neural proliferation zones mediate brain growth and employ Delta/Notch signaling and HES/Her transcription factors to balance neural stem cell (NSC) maintenance with the generation of progenitors and neurons. We investigated Notch-dependency and function of her genes in the thalamic proliferation zone of zebrafish larvae. Nine Notch-dependent genes, her2, her4.1-4.5, her12, her15.1-15.2, and two Notch-independent genes, her6 and her9, are differentially expressed and define distinct NSC and progenitor populations. her6 prominently executes patterning information to maintain NSCs and the zona limitans intrathalamica Shh signaling activity. Surprisingly, simultaneous deletion of nine Notch-dependent her genes does not affect NSCs or progenitor formation, and her4 overexpression only caused reduction of ascl1b progenitors. Combined genetic manipulations of Notch-dependent and -independent her genes suggest that her6 in the thalamic proliferation zone prominently maintains NSCs and inhibits NSC-to-progenitor lineage transitions. The her gene network is characterized by redundant gene functions, with Notch-independent her genes better substituting for loss of Notch-dependent her genes than vice versa. Together, her gene regulatory feedback loops and cross-regulation contribute to the observed robustness of NSC maintenance.