IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma.p58 Progressive loss of functional retinal ganglion cells (RGCs) and their axons is considered the major and ultimate cause of visual impairment in glaucoma . CurrentIPK, a member of the heat shock protein (HSP) 40 family encoded by the DNAJC3 gene [IPK is believed to possess a dual topology either existing as a free luminal floating protein in the ER or residing at the ER membrane with the C-terminus facing the cytosol [IPK can bind to unfolded proteins and the major ER chaperone glucose regulated protein 78 (GRP78) promoting protein folding and homeostasis [IPK functions as an inhibitor of the eIF2α kinases, such as protein kinase double-stranded RNA-dependent (PKR), PKR-like ER kinase (PERK), and general control non-derepressible-2 (GCN2), and regulates protein translation by reducing eIF2α phosphorylation [IPK was found to inhibit PKR-dependent inflammasome activation, TNF-α and IL-6 expression, and IL-1β production, suggesting an immune regulatory function of p58IPK [IPK in the regulation of protein homeostasis and inflammatory response make it an attractive target in research concerning neurodegenerative disease of the retina. The unfolded protein response (UPR) is a highly conserved stress response mechanism that plays a critical role in maintaining cellular homeostasis and function in the central nervous system (CNS) neurons . The UPRJC3 gene ,8. Unlik cytosol . In the eostasis . When lorylation ,12. In mf p58IPK . These mIPK is highly expressed in retinal neurons including RGCs and global p58IPK knockout mice exhibit increased RGC loss during aging and in animal models of neurotoxin-induced retinal degeneration, retinal ischemia/reperfusion (I/R), and ocular hypertension (OHT) [IPK significantly reduces cell death in cultured retinal cells including primary RGCs under oxidative or ER stress conditions [IPK can protect the neural retina and RGCs from stress induced cell loss. However, it is unclear whether the protective effect of p58IPK is attributable to the intrinsic signaling in retinal neurons and/or its anti-inflammatory effect in immune cells that indirectly affects RGC survival. Herein, we investigate the cell-specific role of p58IPK in retinal neurons and in macrophages under normal and glaucomatous stress conditions taking advantage of two newly generated p58IPK conditional knockout (cKO) mouse lines. Our results suggest that the neuroprotective effect of p58IPK in OHT is mediated by its intrinsic role in retinal neurons but not its expression in macrophages. In addition, targeting RGCs directly with a neuroprotective agent will preserve vision regardless of disease pathophysiology.Because the pathogenesis of glaucoma is not fully understood and multiple pathways have been implicated in neurodegeneration, a therapeutic intervention at the critical, irreversible stage of RGC death is of interest. Previously, we demonstrated that p58on (OHT) ,15. In cnditions ,15. ThisIPK flox mouse line was generated using a targeting construct generated by inserting a loxP site upstream of the p58IPK transcription start site and a loxP-FRT-Neo-FRT cassette in the first exon of P58IPK. This construct was then used to electroporate G4 129xC57BL/6 F1 hybrid ES cells [IPK flox mice. The primers for genotyping P58IPK flox were: 5′-TAAAGGCACTGGAAGCATTT-3′ and 5′-AAGTAGGAACTATCTTCACC-3. The LysMCre line has been previously published [tm1(cre)IfoLyz2/J). The Chx10-cre line has been described elsewhere [IPK floxed (fl) mice to the LysMCre line we generated p58IPK fl/fl; LysMCre + (Lyz2-p58IPK cKO) mice, which lack p58IPK specifically in macrophages [IPK floxed mice to the Chx10-Cre line we generated p58IPK fl/fl; Chx10-Cre + (Chx10-p58IPK cKO) mice, in which p58IPK is deleted in the vast majority of retinal neurons and a subset of Müller glia [IPK flox/flox mice were backcrossed to a C57/BL6J background and crossed with LysMCre or Chx10-cre mice to generate p58IPK flox/flox; LysMCre/+ (Lyz2-p58IPK cKO) or p58IPK flox/flox; Chx10-cre (Chx10-p58IPK cKO) for experiments. Both Lyz2-p58IPK cKO and Chx10-p58IPK cKO lines were also crossed with the mTmG reporter line -Gt(ROSA)26Sortm4Luo/J), in which all cells constitutively express Tomato (red fluorescent protein) unless a cell expresses Cre recombinase, in which case Tomato production permanently ceases in those cells and constitutive green fluorescent protein (GFP) production begins [The p58ES cells . After sublished  and was lsewhere  and succlsewhere . Lysozymrophages . Similarler glia . The p58n begins . The abs7 beads/mL . Mice that developed ocular edema, hemorrhage, cataracts, or that failed to sustain OHT were excluded from the study.The microbead induced OHT model has a long history and broad acceptance as a glaucoma model across species including mice through the induction the dysfunction of the outflow facility of aqueous humor . MicrobeMouse eye cryosections were prepared for immunohistochemistry (IHC) as described previously . BrieflyAll analyses were performed blind to condition and genotype. After wholemount staining for Brn3a, retinas were flat-mounted by making 3–5 radial relief cuts and mounting in PBS with ganglion cell layer facing up. Four images per retina were taken at a position 50–70% radially from the optic disc to the periphery and absent major blood vessels. Cell counts per animal were averaged and n values represent number of mice. Images for retinal wholemount with Iba1 labeling were obtained similarly, with care taken to identify the appropriate focal planes for superficial, mid-, and deep-retinal layers. Cryosections were analyzed in regions approximately 400–900 µm from the optic nerve head and normalized per 100 µm linear distance, where appropriate. Between three and six areas across at least two sections were anlayzed per mouse. Cell counts were averaged per animal and n values represent number of mice.2 with a background of 5 cd s/m2. Electroretinogram (ERG) were performed on the Diagnosys Espion ColorDome system and manufacturer software  essentially as previously described . BrieflyDark-adapted ERGs were performed using the protocol previously described . BrieflyBMDMs were isolated and cultured as previously described ,20. AfteIPK was measured by qPCR using the SYBR green supermix (Bio-Rad) and CFX96 Touch Real-Time PCR System (Bio-Rad). The primers for qPCR were: p58IPK forward: 5′- TCCTGGTGGACCTGCAGTACG-3′ and reverse: 5′-CTGCGAGTAATTTCTTCCCC-3′; 18S ribosomal RNA forward: 5′-GTAACCCGTTGAACCCCATT-3′ and reverse: 5′-CCATCCAATCGGTAGTAGCG-3′.RNA was isolated from mouse retinas or BMDMs using Trizol following the manufacturers protocol . RNA was converted into cDNA using the iScript cDNA synthesis kit . The expression of p58TM BCA protein assay kit (ThermoFisher). Samples were loaded into a 10% SDS-PAGE gel, and transferred to nitrocellulose membrane for immunoblotting using anti-p58IPK  and anti-β-actin  antibody. After incubation with HRP-conjugated secondary antibodies, membranes were developed with Clarity and Clarity Max ECL Western Blotting Substrate  using Chemi-Doc MP Imaging System . The bands were semi-quantified by densitometry using Image J software.Mouse retinal tissue or BMDMs were lysed with radio immune precipitation assay (RIPA) buffer with a protease inhibitor mixture, PMSF, and sodium orthovanadate (Santa Cruz Biotechnology). Protein concentration was quantified using the PierceIPK-GFP [12 GC/mL were injected into the vitreous with a Hamilton syringe fitted with a 33G needle. Ophthalmic gel  was applied to the cornea immediately after injection.Mice were anesthetized with 100 mg/kg ketamine and 5 mg/kg xylazine. Both pupils were dilated with 1% atropine  followed by 2.5% Phenylephrine Hydrochloride (Bausch & Lomb). Either 1.5 µL of AAV-GFP or AAV-p58IPK-GFP  at equivp-value of less than 0.05 was considered significant.Data are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism 9 . Students’ t-test was used in the comparison of two groups and one-way ANOVA was used for comparisons between three or more groups. A result of a IPK reduces apoptosis and increases survival of cultured R28 retinal progenitor cells and primary mouse RGCs under induced ER stress and oxidative stress conditions [IPK operates in vivo, we overexpressed p58IPK in the retina by an intravitreal injection of AAVs expressing p58IPK (AAV-p58IPK) and a green fluorescent protein (GFP) into one eye in adult C57/BL6J mice. The contralateral eye of each mouse received AAVs expressing GFP (AAV-GFP) as control. Two weeks after the injections, the mice were randomly assigned to receive a microbeads injection to induce OHT  or a sham procedure (control group) in both eyes. As shown in IPK-treated and AAV-GFP-treated eyes . No gross abnormalities in growth, body weight, or blood glucose were observed in the floxed or cKO lines. Our prior work shows that global knockout of p58 and I/R ,15. We hrophages . To deliIPK cKO mice and Chx10-p58IPK cKO mice with mTmG mice, a double-fluorescent Cre reporter line which constitutively expresses a conditional tdTomato transgene in the absence of Cre recombinase but switches to expressing GFP upon exposure to Cre recombinase [IPK cKO line. In Chx10-p58IPK cKO mice the cre expression was found in the vast majority of cells throughout the retinal wholemounts (The fidelity of cre expression in each cKO line was verified by crossing Lyz2-p58ombinase . As showlemounts . IPK cKO and p58IPK fl/fl (WT) mice. RNA expression and protein level of p58IPK were determined by qRT-PCR and Western blotting, respectively. We find a reduction of 97% of p58IPK mRNA and an 86% reduction in p58IPK protein in BMDMs derived from Lyz2-p58IPK cKO mice compared to WT controls  from Lyz2-p58controls A–C. SimiT retina D–F. ThesIPK cKO mice and Chx10-p58IPK cKO mice under normal, non-diseased conditions. We find that the expression pattern and intensity of markers for multiple retinal cell types as well as synaptic markers are indistinguishable in the retinas from both p58IPK cKO lines and WT controls  on adult WT mice and p58IPK cKO mice. In dark-adapted (scotopic) ERG, we found no significant differences in a wave or b wave responses in Lyz2-p58IPK cKO mice nor in Chx10-p58IPK mice compared to WT  when compared to WT mice (28% RGC loss), whereas the RGC loss was not significantly altered in Lyz2-p58IPK cKO eyes (33% RGC loss) compared to WT -induced neurotoxicity, retinal ischemia, increased IOP, and ER stress [IPK in the retina by AAV significantly reduced RGC loss after OHT, further confirming the findings from in vitro studies that p58IPK overexpression improves retinal neuronal cell survival in cultured retinal R28 cells or primary RGCs challenged with ER stress or oxidative stress [IPK in protecting RGCs in chronic stress conditions pertinent to retinal diseases such as glaucoma.In this study, we investigated the contribution of neuron-specific and myeloid cell-specific p58R stress ,15. In ae stress ,15. CollIPK was originally identified as a cellular regulator of PKR inhibiting the PKR’s activity in phosphorylating eIF2α and reducing protein synthesis during influenza virus infection [IPK is a general inhibitor of eIF2α kinases , playing a critical role in controlling and fine-tuning the cellular stress response [IPK suppresses the PERK-UPR pathway and defects in p58IPK function lead to sustained PERK activation, reduced protein translation, and increased cell death [IPK inhibits the activity of eIF2α kinases, such as GCN2, a stress response protein induced by amino acid deprivation to modulate protein translation [IPK/eIF2α kinases pathway contributes to cell death in multiple stress conditions. Clinically, patients bearing loss-of-function mutations of DNAJC3 (p58IPK) suffer from diabetes and multisystemic neurodegeneration [IPK KO that homozygous deletion of p58IPK leads to apoptosis of pancreatic islet cells and gradual onset of glucosuria and hyperglycemia [IPK gene mutations also demonstrate impaired mitochondrial function, lipid accumulation, increased ER stress, and β cell apoptosis. These results suggest an important role of p58IPK in β cell survival and function, in part through regulation of cellular stress response [IPK on mitochondrial function and lipid metabolism in animal models.p58nfection . Subsequresponse ,12. Durill death . In contneration ,25,26. Tglycemia ,28. Cultresponse ,29,30. FIPK in the CNS neurons and neurodegenerative disease are less well defined, which is possibly due to the complex phenotype of global p58IPK KO mice with systemic metabolic abnormalities as a confounding factor [IPK that has allowed us to investigate the cell-specific role of p58IPK in the retina under normal and stressed conditions. We have demonstrated that this conditional allele of p58IPK is efficiently excised by two independent Cre lines, resulting in near complete loss of p58IPK mRNA and protein in cells expressing Cre recombinase. Further, the loss of p58IPK in Lyz2-cre-expressing myelomonocytic cells  or in Chx10-cre-expressing retinal progenitor cells does not affect retinal morphology and distribution of major retinal neuronal populations. Interestingly, we find a functional deficiency in adult Chx10-p58IPK cKO light adapted ERG b wave with no change in the a wave, indicating that p58IPK deficiency leads to dysfunction of post-synaptic retinal cells to cone photoreceptors and/or disturbance in the synapses between cone cells and bipolar cells. Recent studies have shown that ATF6, which is a major transcriptional regulator of p58IPK, is required for cone development and function [IPK in post-synaptic retinal cells in the cone signaling, such as cone bipolar cells, Müller cells, or intrinsically photosensitive retinal ganglion cells (ipRGCs), and its implication in maintaining retinal circuitry warrant future investigation.In contrast to an extensively studied role in β cells, the implications of p58g factor ,28. To afunction  and mutafunction . The exaIPK in retina reduces RGC loss in a microbead-induced OHT model, confirming a protective effect of p58IPK on RGC survival in vivo. Notably, we observed a higher degree of RGC loss induced by OHT in AAV-transduced retinas (IPK is attributed to its intrinsic signaling in retinal neurons and/or its anti-inflammatory role in macrophages. To this end, we subjected the Chx10-p58IPK cKO and Lyz2-p58IPK cKO mice, and controls, to OHT and myriad analyses. Our results indicate that knockout of p58IPK in retinal neurons significantly increased RGC loss after OHT in Chx10-p58IPK cKO compared to WT and Lyz2-p58IPK cKO mice. In contrast, deletion of p58IPK in myelomonocytic cells neither enhances RGC loss nor protects the retina from RGC loss, despite a significant increase in activated Iba1-positive cells in the superficial retina layer of Lyz2-p58IPK cKO mice after OHT. This may indicate that p58IPK deficiency does increase macrophage activation; however, the change is not sufficient to contribute to cell loss, or to their protection in disease. One caveat to this conclusion is that the time frame of analysis in our disease model is two weeks. It is feasible that the long-term effects of p58IPK-deficiency in macrophages would manifest in disease models or aging. Furthermore, macrophages and Müller cells communicate under disease conditions, and we find an increased inflammatory response in the OHT retina, as evinced by an increase in GFAP expression in Müller cells. Though we do not see a significant difference in GFAP between WT and Lyz2-p58IPK cKO mice, there is a trend for higher GFAP expression in Lyz2-p58IPK cKO. This may indicate that p58IPK in macrophages has some role in macrophage-Müller communication in relation to inflammatory response in stressed retina, which should be further investigated at additional time points after OHT and in other disease models.Extending our previous findings from in vitro studies using cultured primary RGCs or R28 cells ,15, we d retinas  compared retinas . These rIPK cKO mouse lines revealed an important role of p58IPK as an intrinsic protective factor in retinal neurons that promotes RGC survival against glaucomatous damage. Deletion of p58IPK in myelomonocytic cells increased macrophage/microglial activation in stressed neural retina but was insufficient to influence RGC survival. Overall, these findings suggest that targeting neuronal p58IPK may provide a novel approach for neuroprotection and treatment in retinal disease.In summary, the results from the present study using cell-specific p58


Little is known about the mechanistic significance of the ubiquitin proteasome system (UPS) in a kidney autoimmune environment. In membranous nephropathy (MN), autoantibodies target podocytes of the glomerular filter resulting in proteinuria. Converging biochemical, structural, mouse pathomechanistic, and clinical information we report that the deubiquitinase Ubiquitin C-terminal hydrolase L1 (UCH-L1) is induced by oxidative stress in podocytes and is directly involved in proteasome substrate accumulation. Mechanistically, this toxic gain-of-function is mediated by non-functional UCH-L1, which interacts with and thereby impairs proteasomes. In experimental MN, UCH-L1 becomes non-functional and MN patients with poor outcome exhibit autoantibodies with preferential reactivity to non-functional UCH-L1. Podocyte-specific deletion of UCH-L1 protects from experimental MN, whereas overexpression of non-functional UCH-L1 impairs podocyte proteostasis and drives injury in mice. In conclusion, the UPS is pathomechanistically linked to podocyte disease by aberrant proteasomal interactions of non-functional UCH-L1. In membranous nephropathy autoantibodies target podocytes of the kidney filter resulting in injury. Here the authors show that the ensuing proteostatic disturbances and proteinuria relate to aberrant interactions of non-functional UCH-L1 enzyme with the proteasome, curtailing its capacity. As a major quality control system responsible for degradation of proteins, the UPS comprises a cascade of E1-E2-E3 enzymes, which ubiquitinate substrate proteins and hence target them for proteasomal degradation. Deubiquitinating enzymes (DUBs) edit ubiquitin chains from substrate proteins and thereby modify their cellular fate2. The proteasome, as a complex protease, exists in multiple structural isoforms but contains two general assemblies: a proteolytic chamber (20S core particle) harboring the proteolytical active β subunits, and a regulatory particle (19S particle) which are functionally linked by a gated protein translocation channel to form the 26S/30S proteasome3. Protein degradation through the 20S core particle is achieved in an ATP and ubiquitin independent manner, whereas ATP and ubiquitin are required for degradation through the 26S/30S proteasome2.There is an unmet need for quantitative and mechanistic research into the role of the ubiquitin proteasome system (UPS) in human pathophysiology4 is one hallmark of membranous nephropathy (MN) and indicative of progressive podocyte injury4. MN is an autoimmune disease of the kidney, which targets podocytes, the key cells of the glomerular filter5. These terminally differentiated epithelial cells embrace the glomerular capillaries with their intricate net of interdigitating foot processes. Together with endothelial cells and the glomerular basement membrane, podocytes form the three-layered size and charge selective filtration barrier to blood. Nephrotic syndrome is the clinical hallmark of podocytopathies such as MN and presents with massive proteinuria, hypoalbuminemia, edema, and hyperlipidemia. Besides diabetes and focal segmental glomerulosclerosis (FSGS), MN is the most frequent cause of nephrotic range protein loss to the urine in adults7. MN is initiated by circulating autoantibodies directed to the podocyte foot process antigens such as PLA2R18 and THSD7A9. The ensuing pathomechanistic sequelae of this disease is unclear. Besides the subepithelial glomerular deposition of immune complexes and complement factors, the occurrence of oxidative stress11 within podocytes are common findings.An induction of the UPS in conjunction with an accumulation of (K48-) polyubiquitinated proteins12 and others14 described a de novo expression of Ubiquitin C-terminal hydrolase L1 (UCH-L1) in injured podocytes in human and rodents, with MN being one of the prominent glomerulonephritis forms12. The role of UCH-L1 in podocyte injury remains enigmatic. As a central neuronal DUB, with unknown direct substrates, UCH-L1 is thought to stabilize the pool of monoubiquitin in neurons15, a finding however not reproduced in a new murine model of UCH-L1 deficiency in the brain16 or kidney17. Point mutations of UCH-L1 have been identified in human neurodegenerative disorders, such as the I93M, C90S, and the E7A18 mutations, which all result in a decreased hydrolysis activity of the enzyme19. The I93M mutation additionally gives rise to a dominant toxic gain-of-function variant. While the overall crystal structure of the wildtype and the I93M variant is very similar20, comparative biophysical analysis show an apparent loss of α-helical structure in solution, hence decreasing solubility of the I93M variant22. In the brain, UCH-L1 is thought to promote anti-oxidative properties23 and to be itself modified by oxidative stress24. Oxidatively-modified UCH-L1 has been shown to acquire comparable structural and biochemical properties as the I93M-UCH-L1, including a decreased hydrolysis function and an increased tendency to insolubility and abnormal interactions25 for example with Lamp226 and tubulin22.In 2009 weThis report unravels the role of UCH-L1 in podocyte injury by integrating experimental evidence from 1) biopsies and sera from well-defined MN patient cohorts, 2) podocyte culture and mouse models of experimental MN, 3) genetically modified mice with podocyte-specific UCH-L1-deletion compared to overexpression of functional versus non-functional (I93M) UCH-L1, and 4) mechanistic studies on proteasome function. Our data demonstrate that UCH-L1 is not only induced by oxidative stress in podocytes, but also presents as an enzymatic non-functional protein in experimental MN. Non-functional  UCH-L1 drives podocyte injury and ubiquitin accumulation in experimental MN, mechanistically by binding to the proteasome and reducing constitutive proteasomal subunit activities. MN patients exhibit autoantibodies to UCH-L1 with a differential reactivity to non-functional UCH-L1, an indirect indication that UCH-L1 modification might occur in humans.We established whether oxidative stress, a prominent feature of MN, induces UCH-L1 in podocytes. In biopsy samples of patients diagnosed with ischemic glomerular collapse and MN, podocyte UCH-L1 immunoreactivity correlated with an enhanced expression of superoxide dismutase 2 (SOD2), a mitochondrial scavenger of reactive oxygen species (ROS) Fig. . Mouse p4 -UCH-L1 and modified UCH-L1 proteins. However, both, the I93M mutant and the ROS-modified UCH-L1, are less positively charged around the entrance to the active site compared to the (WT)-UCH-L1 based on identified oxidative-modified amino acids of human UCH-L124. Further, the dorsal side of the oxidative-modified variant shows positive surface charge which is missing in the wildtype . This probe covalently binds to the catalytic center of only active DUBs, including UCH-L1 , mice with overexpression of functional wildtype UCH-L1 , and mice with non-functional I93M-UCH-L1 . Glomerular UCH-L1 protein abundance was comparable in both overexpressing transgenic mouse lines . Consistent with podocyte culture data  polyubiquitinated proteins. The factors triggering—and—the nature of proteasome impairment in MN are unclear but could range from clogging of the proteasome through free ubiquitin chains33, to posttranslational34 or ROS-mediated modification of proteasomes35, as described in non-renal disorders. In the autoimmunologic environment of MN we now demonstrate proteasomal impairment and aggravated disease to arise from a toxic gain-of-function UCH-L1, as one of the strongest upregulated UPS proteins in injured podocytes14.In this study we converge biochemical, structural, mouse pathomechanistic, and clinical information to unravel the significance of the ubiquitin proteasome system in human MN. Our work is centered on the hypothesis that protein degradation through the UPS is a common modifier of MN progression, severity, and outcome. The factors leading to and indicating progressive podocyte injury are much sought for, especially for the guidance of clinical decisions if disease outcome varies as strongly as in MN . Independent of the clinical outcome, podocytes respond uniformly to injury with structural damage such as foot process effacement. An upregulation of important UPS players, however, occurs in progressive but not in transient podocyte injury17, and the glomerular filtration rate36. In neurons, UCH-L1 is not only part of an antioxidant response23 but is itself modified by ROS, thus reducing its enzymatic activity, and making it prone to a toxic gain-of-function24. In line with this proposed neuronal function of UCH-L1, we can show that podocytes upregulate UCH-L1 in response to oxidative stress, which is prominent in experimental MN11. Upregulated UCH-L1 in experimental MN exhibited reduced enzymatic activity, strongly suggesting oxidative damage to occur during podocyte injury. We could now demonstrate that UCH-L1 promotes podocyte injury, as UCH-L1Δpod mice were protected from experimental MN37. In line, earlier investigations in rat experimental MN had shown that chemical modulation of UCH-L1 enzymatic activity enhances glomerular proteasomal activity and ameliorates the clinical course of disease38. Our efforts to correlate UCH-L1 protein expression to the clinical course of MN patients were inconclusive, even though in a previous study UCH-L1 expression differentiated patients with remitting minimal change disease from those that progressed to focal segmental glomerulosclerosis (FSGS)4. Besides technical issues, the distinct pathogenesis of podocyte injury  could explain the difference between the studies, as podocytes exhibit no UPS alterations in minimal change disease, whereas podocytes in FSGS and MN patients do12. Nonetheless, these findings add to the idea, that the presence of non-functional UCH-L1 rather than of functional UCH-L1 might be pivotal in the promotion of podocyte injury.In the mouse kidney, a conditional deficiency of UCH-L1 influences ubiquitin and protein homeostasis, proteasome composition19); (2) the lack of knowledge regarding specific UCH-L1 targets in podocytes and (3) multiple UCH-L1 protein modifications, which alter UCH-L1 function and subcellular localization19. To unravel the disease-promoting effect of UCH-L1 in podocytes we therefore generated transgenic mice which expressed the enzymatic functional UCH-L1 wildtype protein or non-functional I93M-UCH-L1 in podocytes. We expected that expression of functional UCH-L1 would negatively affect podocyte function through the initiation of dedifferentiation12, through the induction of necroptosis39, or through the altered degradation of podocyte proteins40. However, UCH-L1WT mice showed no major difference in their naive phenotype or in the clinical course of experimental MN compared to controls. This discrepancy might be related to the fact that most mechanistic findings to UCH-L1 are based on cell culture observations40, and relate on the analyses of whole glomeruli, where constitutive UCH-L1 expression in endothelial cells41 could mask de novo injury-induced UCH-L1 effects in podocytes. A further explanation could be related to technical issues, as potentially the overall expression level of UCH-L1WT might have been too low . To avoid too high UCH-L1 levels, most experiments were performed in heterozygous mice resulting in a UCH-L1 expression in 30-80% of podocytes within individual glomeruli.The mechanisms by which UCH-L1 perpetuates podocyte injury are not understood. This is partly due to (1) the complex biochemical functions of UCH-L1  medical conditions such as for example tumors45, which are a frequent co-occurrence in patients with MN. However, besides the primary autoantibodies to PLA2R1 and THSD7A, the additional presence of autoantibodies to intracellular proteins is thought to enhance the autoimmune burden of podocytes in MN47. Why MN patients exhibit autoantibodies to UCH-L1 is at this state not only an enigma for UCH-L1, but also for the main MN antigens PLA2R1 and THSD7A. One could speculate that UCH-L1 autoantibody development is the result of podocyte-expressed  UCH-L1 being presented to the immune system by an unidentified glomerular exit route such as extracellular vesicles, in which UCH-L1 is present49. However, UCH-L1 autoantibody development could also represent a by-standing result from another underlying medical condition. Nevertheless, as non-functional UCH-L1 drives podocyte injury in experimental MN, further investigations in larger cohorts are warranted to establish whether there is a translational significance for autoantibodies to non-functional UCH-L1 in patients with podocytopathies.Autoantibodies directed to intracellular proteins such as UCH-L1 have been identified in MN patient seraIn summary, we provide evidence, that non-functional UCH-L1 is present in podocytopathies such as MN. In addition, we show that non-functional UCH-L1 drives podocyte injury through binding to and inhibiting proteasomal activity, which lastly results in severe proteotoxic stress and in the aggravation of MN.All uncropped and unprocessed scans of the most important blots are presented in the Source Data file or as a supplementary figure in the 39); mouse anti-UCH-L1 ; rabbit anti-UCH-L1 ; rabbit anti-SOD2 ; mouse anti-Flag ; rabbit anti-α2 ; rabbit anti-α4 ; mouse anti-α6 ; rabbit anti-β1c ; rabbit anti-β2c ; rabbit anti-β5c ; rabbit anti-β5c ; rabbit anti-β5i ; rabbit anti-ubiquitin ; mouse anti-ubiquitin ; rabbit anti-K48-polyubiquitin ; rabbit anti-K48-polyubiquitin ; guinea-pig anti-nephrin ; rabbit anti-α-actinin 4 ; rabbit anti-p57 ; rhodamine-wheat germ agglutinin ; rat anti-HA ; rabbit pan-proteasomal antibody ; mouse anti-β-actin ; for the detection of aggresomes PROTEOSTAT® Protein aggregation assay . All secondary antibodies used were either biotinylated, HRP- or fluorescent dye-conjugated affinity purified donkey antibodies (Jackson ImmunoResearch).Primary antibodies used for the study were: rat anti-UCH-L1 (immunofluorescence microscopy mouse (IF) 1:50, immunoblot (WB) 1:250, Sosna et al.AA AAA AGG CGC GCC GCG AAG ATG CAG CTG AAG CCG AT ’3 and -reverse (Rev) 5’ AA AAA AGG CGC GCC TTA AGC TGC TTT GCA GAG AGC CAC GG ’3 primers, respectively, including an AA overhang (cursive) and AscI sites (underlined) at their 5´ ends according to the mUCH-L1 sequence (NM_011670). PCR was performed for 35 cycles  using 5 U Phusion-DNA polymerase (Thermo Fisher Scientific) and subsequently cut for two hours at 37 °C with 10 U AscI . For cloning, 5 µg of the Rosa26/CAG/Stop/eGFP knock-in target vector  was also digested over night at 37 °C with 25 U AscI (NEB)50. The DNA-fragment and the vector were purified by phenol/chloroform extraction and ethanol-precipitation for one hour at −80 °C. Ligation was performed with 100 ng vector-DNA and different molar ratios of the PCR-fragment  using 1 U T4-DNA ligase (NEB) at 12 °C overnight and subsequently transformed in competent XL10 E. coli  for 30 seconds at 42 °C. The cloning was verified by PCR screening and DNA-sequencing of the complete mUCH-L1 cDNA at Seqlab  according to their recommendations. Finally, 70 µg of one positive mUCH-L1 full-length clone was linearized in 300 µl 10 mM Tris/HCl pH 8.0 with 200 U AsiSI (NEB) over night at 37 °C, purified by sequential phenol and chloroform extractions and ethanol precipitated. The dried DNA-pellet was reconstituted in H2O and integrity was verified by agarose gel electrophoresis.To generate the Rosa26 based knock-in, target vector construct of the complete mouse wildtype or I93M UCH-L1 protein, 1 µg of total RNA preparation was reverse transcribed using M-MLV-reverse transcriptase  and 100 ng Oligo dT-primer. For PCR, 2 µl cDNA was specifically amplified with 10 mM mUCH-L1-forward (Fw) 5’ Uchl1fl/fl mice were generated by Genoway17 by insertion of loxP sites flanking the putative promoter region and exon 3 of the murine Uchl1 gene. To obtain a podocyte-specific deletion of Uchl1, Uchl1fl/fl mice were crossed to Nphs2-Cre mice51 and backcrossed to the C57BL/6 background. For experiments, Uchl1d/d;cre+ 17 were used and compared to Uchl1+/+;cre+ or Uchl1d/d;cre- littermates.Uchl1WT and Uchl1I93M overexpressing mice were generated as follows. Briefly, R1-embryonic stem (ES)-cells  were obtained at passage 11 and expanded to passage 14 and 15. R1-ES-cells were grown on gelatinated cell culture plates supplemented with a confluent layer of inactivated mouse embryo fibroblast (MEF) feeder cells. R1-ES-cells were passaged every second day. In order to obtain mice that could overexpress mouse WT or I93M-UCH-L1, ES-cells containing a Rosa26/CAG/Stop/mWT-UCH-L1 or mI93M-UCH-L1 knock-in target vector50 were generated as detailed above and 1 × 107 R1-ES-cells were electroporated with 50 µg of the linearized target vector in a 0.4 cm gap-electroporation-cuvette (Life Technologies) and subsequently selected in a medium containing 200 µg geneticin  for one week. Correct targeting was verified by Southern blotting of EcoRI digested genomic DNA derived from isolated clones using a 5’ external ROSA probe. Positive R1-ES-cell clones were further expanded before injection into blastocysts. For blastocyst injection, 15 trypsinized R1-ES-cells with clearly visible, small, round nuclei without vacuoles were collected and injected into one blastocyst. Eight of these blastocysts were implanted into the uterus of a female mouse mated with vasectomized males at day 2.5 post coitum. The resulting chimeric males were mated with wild-type C57BL/6 females. Littermates of the F1 generation were genotyped by PCR to obtain offsprings that pass on the mWT or mI93M-UCH-L1 knock-in. These mice carried the transgenic information of mWT- or mI93M-UCH-L1 under the control of a floxed stop-cassette to prevent transactivation by the CAG-enhancer. These animals are referred to as mWT-or mI93M-UCH-L1-negative precursor mice and were further used for breeding with Nphs2-Cre mice51 to obtain a podocyte-specific mWT- or mI93M-UCH-L1 expression. Due to the podocyte-specific action of the Nphs2 (podocin) promoter-triggered Cre-recombinase, the stop cassette was removed and mWT- or mI93M-UCH-L1 overexpression on glomerular podocytes was initiated. For experiments Uchl1WT+/fl;cre+  and Uchl1I93M+fl/;cre+ were backcrossed to the C75BL/6 background and compared to Uchl1WT+/fl;cre- or Uchl1I93M+/fl;cre- littermates, respectively. Figure Podocyte-specific Uchl1fl/fl mice flox-Fw: 5’-GCA CCA GTG ATT CAG CTT TCT AAA AGG AAC-3’ and flox Rev: 5’-TCT CAC CTG ACT GTA AAC TGA TGA GGG C-3’; for the R26-knockin locus Fw: 5’-GTT ACT ATG GGA ACA TAC GTC ATT-3’ and Uchl1 Rev: 5’-GTT GCA ATA CCT TTC TGG GAG TT-3’; and for Uchl1 wildtype Fw: 5’-CGA AAA TCT GTG GGA AGT CTT GT-3’ combined with Uchl1 Rev primer. Gel electrophoresis was performed with a 1,5% agarose gel.The different genotypes were verified by PCR. Briefly, for PCR reaction, tail biopsies were lysed in DirectPCR (Tail) (Viagen 102-T) with Proteinase K  overnight at 55 °C. Primers for the Cre-locus were: cre-Fw: 5’-GCA TAA CCA GTG AAA CAG CAT TGC TG-3’ and cre-Rev: 5’-GGA CAT GTT CAG GGA TCG CCA GGC G-3’; for the flox-locus of 27 by intravenous injection of 225 µl of sheep anti-podocyte antibodies or equal amounts of unspecific pre-immune sheep IgG. Urine was collected on day −1 prior to induction of experimental MN and on days 3, 6, 9 and 13 after induction of experimental MN. Kidneys were removed and serum collected on day 14 after disease induction. For induction of THSD7A-associated MN I93M-UCH-L1 mice were backcrossed for 3 generations with BALB/c to enhance susceptibility to the rabbit anti-THSD7A antibodies30. THSD7A-MN was induced by intravenous injection of purified anti-THSD7A rabbit IgG or unspecific control IgG as described30. Kidneys were removed and serum collected on day 12 after disease induction. Animal euthanasia was performed following buprenorphine  administration for analgesia 30 minutes prior to cervical neck dissection under 3.5% isoflurane inhalation narcosis.Mice were housed in a pathogen-free animal facility at the University Medical Center Hamburg-Eppendorf. Mice used in the study were older than 8 weeks of age and were predominantly analyzed at 10–38 weeks of age. Mice were kept at ambient temperature and humidity, had free access to water and standard animal chow (Altromin 1328 P) and were synchronized to a 12 h light/12 h dark cycle. All experimental procedures were performed according to the institutional guidelines. Anti-podocyte nephritis was induced as published53 through the renal arteries. The kidney capsule was removed, the kidney shred into small pieces and digested with 1,2 mg/ml collagenase (Sigma), 100 U/ml DNase (Roche) in HBSS (Thermo Fisher Scientific) for 15 min at 37 °C under constant agitation at 1300 rpm. The suspension was homogenized through a 100 µm filter and pelleted in a magnetic field. Glomeruli were washed multiple times with HBSS supplemented with 0.05% BSA (Sigma) and preparation quality and glomerular count was assessed microscopically.To isolate glomeruli, mice were euthanized. Kidneys were dissected, perfused with HBSS containing magnetic beads Urinary albumin to creatinine ratio: Mouse urine was collected in metabolic cages. Urine albumin content was quantified using a commercially available ELISA system (Bethyl) according to the manufacturer’s instructions, using an ELISA plate reader (BioTek), as described37. Urinary albumin values were standardized against urine creatinine values of the same individuals determined by Jaffe  and plotted.Stainfree SDS-PAGE: Stainfree protein profile analyses of creatinine-adapted urines on day 14 after disease induction were performed by loading creatinine-adapted urines . Similarly to Coomassie or silver stain approaches, this method allows for the “unspecific” visualization of protein by utilizing a polyacrylamide containing trihalo compound (BioRad), which is covalently bound to tryptophan residues. Upon photoactivation with UV light, proteins fluoresce directly in-gel.54 were cultured in RPMI supplied with 10% FCS, 1% penicillin/streptomycin, 15 mM HEPES, 1 mM sodium pyruvate and 10 U/ml Interferon γ at 32 °C and 5% CO2. For differentiation, interferon γ was removed from the medium and podocytes were cultured at 37 °C and 5% CO2 for at least 14 days. For inducible overexpression of UCH-L1, the Retro-XTM Tet-On Advanced Inducible expression system (Clontech) was used as described39. To generate the WT-UCH-L1 and I93M-UCH-L1 tet-on cells, podocytes were transduced with the pRetroX-Tet-On Advanced vector and with the pRetroX-Tight-Pur-WT-UCH-L1 or pRetroX-Tight-Pur-I93M-UCH-L1 vector. For the generation of negative control cells , podocytes were transduced with the pRetroX-Tet-On Advanced vector and the pRetroX-Tight-Pur empty vector. For induction of UCH-L1 overexpression, UCH-L1 tet-on or tet- podocytes were cultured with 5 ng/ml doxycycline40. For stable knockdown experiments, shRNA to murine UCH-L1 (shRNA627 and shRNA817) or scrambled shRNA for control was overexpressed in podocytes38. To analyze podocytes in the experimental MN cell culture model, podocytes were treated for 1, 2, 4 h with 1% sheep anti-podocyte antibody or pre-immune sheep IgG in standard medium and then prepared for further mRNA and protein analysis.Undifferentiated murine podocytesFor the induction of oxidative stress, podocytes were exposed to 150 µM xanthine  and 200 mU xanthine oxidase  in standard medium for 1, 2, 4, 6 h. Controls were treated instead with equal volumes of solvents sodium hydroxide (xanthine) or potassium phosphate buffer (xanthine oxidase). Harvested cells were processed for further mRNA and protein analysis. Reactive oxygen species were scavenged with 10 mM sodium pyruvate (Gibco), 5000 U/ml pancreatic catalase (Sigma), 100 mM mannitol (Sigma), 100 µM carboxy-PTIO (Sigma), beginning 30 min after initiation of X/XO exposure.2. Transient transfection of HEK293T cells with hu-WT-UCH-L1, hu-I93M-UCH-L1 and hu-THSD7A constructs was performed by the calcium phosphate precipitation method. For that purpose, 10 µg of respective plasmid DNA was thoroughly mixed with 2 M CaCl2 and 2x HBS  and after incubation for 30 min at room temperature added to <50% confluent HEK293T cells followed by overnight incubation at 37 °C and 5% CO2 and medium change on day 2. Harvested cells were prepared for subsequent proteasomal activity assays and screening of patient sera.HEK293T cells  were cultured in DMEM supplied with 10% FCS and 1% penicillin/streptomycin at 37 °C and 5% CO2O. 200 µg of extracted RNA were reverse transcribed with random hexamer primer (Invitrogen) and RevertAid reverse transcriptase (Thermo Scientific) in a thermocycler (Biometra) at 25 °C for 10 min, 42 °C for 1 h, 70 °C for 10 min, 4 °C forever. mRNA expression was quantified with the QuantStudio 3 (Applied Biosystems) qPCR cycler using SYBR green. Exon spanning primer pairs to murine cDNA were used: Uchl1 , Sod2 , Gpx4 , Trx . 18 S  was used as housekeeping control to normalize mRNA levels. Relative gene expression was calculated appropriating the ΔΔCT method.Total messenger RNA was extracted from murine podocytes using phenol/chloroform extraction. Cell pellets were lysed with TRIzol (Ambion) and incubated with 1/6 volume chloroform to separate RNA. For RNA purification isopropanol was added to the aqueous phase for 30 min at 4 °C. RNA pellet was washed twice with 80% ethanol and afterwards solved in purified H2O) in a TransBlot Turbo System (BioRad). After the transfer, all proteins were visualized by ponceau staining. PVDF membranes (Millipore) were blocked (3% non-fat milk) prior to incubation with primary antibodies diluted in Superblock reagent (Thermo Fisher Scientific) or non-fat milk. Binding was detected by incubation with HRP-coupled secondary antibodies . Protein expression was visualized with ECL SuperSignal (Thermo Fisher Scientific) according to manufacturer’s instructions on an Amersham Imager 600 . Immunoblots were analyzed using software from ImageJ55. β-actin stainings of the same membrane are shown and were used as loading control and for densitometric normalization. Bands of the same membrane are shown, fine dashed black lines indicate, where bands were not adjacent to another on the membrane.All immunoblots were performed with isolated glomeruli or cultured podocytes. Samples were lysed in T-Per (Thermo Fisher Scientific) containing complete EDTA-free (Roche) and denatured for 10 min at 95 °C in SDS solubilization buffer. Samples were separated on a 4-15% MiniProtean TGX gel (BioRad) in a Tris-glycine migration buffer . Protein transfer was performed in transfer buffer  of most DUBs, including UCH-L1, which is only accessible in active enzymes. The N-terminal epitope Cy5-tag allows detecting the modified DUBs after the separation of a protein mixture by SDS-PAGE, followed by fluorescent detection. Briefly, glomeruli-adapted lysates (TPer lysis buffer (Pierce) with 250 U/ml benzonase) in an end volume of 20 µl labeling buffer  were labeled with 2.24 µM ubiquitin derived activity-based ubiquitin-vinylmethylester probe (Cy5-Ub-VME) and incubated for 1 h at 37 °C. The reaction was stopped while adding DTT containing solubilizer and heating at 70 °C for 10 min. Proteins were resolved by SDS-PAGE on 12.5% tris-glycine gels, fluorescent detection was performed applying a Capsule 640 nm and F-710 interference filter and using a FUSION FX07 (Vilber).Levels of enzymatic-active UCH-L1 or DUBs in glomerular lysates were assessed, using the ubiquitin derived activity-based ubiquitin-vinylmethylester probe (Ub-VME), which is an active-site directed probe with broad reactivity towards DUBs with the exception of the few metalloproteasesOverexpressed WT- or I93M-UCH-L1-flag constructs from HEK293T cells were purified using anti-Flag M2 Affinity Gel (Sigma) according to the manufacturer’s instructions. Therefore approximately 10 µg total HEK293T lysates (TPer lysis buffer supplemented with complete EDTA-free) were used and flag protein could be eluted by IgG elution buffer pH 2.8 (ThermoScientific) with subsequent rebuffering into PBS using Zebra Spin Desalting Columns, 7 K MWCO (ThermoScientific). The outcome and purity was examined by SDS-PAGE on 4-15% MiniProtean TGX gels (BioRad).In vitro chymotrypsin-like activity assay was performed using 10 µg purified WT- or I93M-UCH-L1-flag constructs incubated with 1 nM purified human (hu) 20S and 15 nM purified hu26S proteasome (BostonBiochem), respectively as well as hu20S activator (0.035%) SDS in incubation buffer  to a final volume of 50 µl. After preincubation for 2 hours at 4 °C, 60 µM Suc-LLVY-AMC substrate  in incubation buffer was mixed with the samples. Proteasomal activity was measured at 355 nm and 460 nm using a fluorescent spectrophotometer  after incubation at 37 °C for 30, 60, 90, 120 and 180 min in the dark.2, 2 mM ATP). The chymotrypsin-like activity of proteasomal complexes could be visualized by measuring fluorescence applying a Capsule 365 nm and F-450 interference filter using a FUSION FX07 (Vilber).For further evaluation of chymotrypsin-like activity 20 µg HEK293T cells overexpressing WT- or I93M-UCH-L1 mixed with 5x clear native loading buffer  excluding heating were resolved using a native PAGE. After electrophoresis gels were incubated with 100 µM Suc-LLVY-AMC substrate (see above) in activity buffer  using an ethanol/dry ice mixture following centrifugation for 10 min with 16000 g at 4 °C. Protein concentration of the supernatant was determined by BCA assay. 20 µg total protein was mixed with 0.5 µM MVB003 and filled up to a total volume of 20 µl with TSDG buffer and incubated for 1 h at 37 °C. As a control, 20 µg of total protein from kidney tissue of BALB/c mice were incubated with either 2 µM epoxomicin (negative control) or equal volumes DMSO (positive control) for 1 h at 37 °C, followed by an incubation with 0.5 µM MVB003. After solubilization with DTT containing loading buffer for 10 min at 70 °C samples were resolved by SDS-PAGE on 12.5% tris-glycine gels with 3.3% crosslinker (acrylamide:bis 29:1). Fluorescent detection was performed applying a Capsule 530 nm and F-595 interference filter using a FUSION FX07 (Vilber) followed by subsequent blotting of the gel to determine total expression of β subunits for activity normalization.Proteasomal subunit activities were investigated using a pan-reactive β subunits activity-based probe MVB003. To this end HEK293T cells were freeze-thawed (7 cycles) in TSDG buffer  over night at 4 °C under gentle rotation with 2 µg antibody as indicated. The antibody protein complex was incubated for 2 hours with protein G sepharose (Roche) and pulled-down by centrifugation. After 2 washing steps with PBS the proteins were eluted by SDS-PAGE sample buffer and employed for immunoblotting.Proteins or protein complexes were precipitated from 150 µg total cell lysate in 1 ml TSDG buffer  and 20 µl were analyzed by immunoblotting.Protein complexes of 3 mg total protein in TSDG buffer were separated by glycerol gradient ultracentrifugation using a gradient from 10–40% glycerol and ultracentrifugation at 283.000 Kidney cortex was embedded in paraffin for light or high-resolution confocal microscopy. For light microscopic evaluation, 1.5 µm thick sections were cut on a rotation microtome and stained with periodic-acid-Schiff reagent (Merck) according to the manufacturer’s instructions. For immunofluorescent stainings, 3 µm paraffin sections were deparaffinized and antigen retrieval was performed by steamer boiling  or by protease XXIV  digestion. Unspecific binding was blocked in 5% horse serum and 0.05% TritonX-100 for 30 min. Primary antibody incubations  were followed by incubation with biotinylated or AF488-, AF647-, AF586-, Cy3-, or Cy5-coupled secondary antibodies . Nuclei were visualized using Hoechst . Stainings were evaluated with an LSM510 meta microscope for conventional microscopy or with an LSM800 with airyscan for high-resolution confocal microscopy using LSM or ZENblue software .For quantification of podocyte loss, 3 µM paraffin sections were stained for the nuclear marker p57 using the ZytoChem-Plus AP Polymer Kit (Zytomed Systems) and Neufuchsin (Merck) according to the manufacturer’s instructions for color development. 50 glomeruli per mouse per condition were analyzed for glomerular tuft area and respective number of p57-positive podocytes in a double blinded fashion at a 400fold magnification.58. In brief, 3 μm paraffin sections mounted on high-precision coverslips coated with Poly-L-lysine (Sigma) were stained for nephrin. Stained sections were mounted with ProLong Gold (Thermo Fisher) for imaging. For visualization super-resolution STED microscopy was performed using a 4 channel STED microscope (Abberior). For quantification, 3D-SIM z-stacks of nephrin-stained kidney sections were acquired with a Zeiss Elyra PS.1 SIM microscope using the ZEN software  with five horizontal shifts and five rotations of the illumination pattern with a slice-to-slice distance of 0.3 µm. Reconstruction of 3D-SIM images was performed using the Zen Black software. PEMP analysis was performed using the PEMP macro for FIJI58. For this, the capillary area was encircled, and the slit diaphragm length was determined. Filtration slit density (FSD) values were calculated from the ratio of slit diaphragm length and capillary area. Per animal, 12 glomeruli with 31-39 ROIs were analyzed by PEMP.Visualization of foot process morphology, and analysis of filtration slit density was performedFor electron-microscopical analyses, small cortical samples fixed in 4% buffered paraformaldehyde were used. Tissue was post-fixed with 1% osmium in 0.1 M phosphate buffer (1 h at RT), stained with 1% uranylacetate (1 h at RT in 70% ethanol), dehydrated and embedded in epoxy-resin . Ultrathin sections were cut  and contrasted with lead citrate. Micrographs were generated with a transmission-electron microscope .t-test and Fisher’s exact test.For the correlative analysis of clinical outcome to podocyte UCH-L1 expression in membranous nephropathy, 28 patient biopsies from the Hamburger GN register were stained for UCH-L1 using the mouse mAB anti-UCH-L1 antibody , nuclei were counterstained using hematoxylin. Biopsies with no glomeruli or with an absent UCH-L1 signal in distal tubuli were excluded from the analyses, due to technical issues in antigen-preservation and detectability. UCH-L1 immunosignal was classified into “low-medium-high” in relation to the distal tubular staining intensity within the 27  included biopsies in a double-blinded manner. Correlation analyses to clinical outcome were performed using Student The determination of sero-positivity for anti-UCH-L1 autoantibodies was performed in a double-blinded manner. Briefly, for the detection of anti-UCH-L1 autoantibodies in 78 MN patient sera and 9 healthy control sera, human WT or mutant I93M UCH-L1 protein fused to both an N-terminal flag and a myc tag were expressed in Freestyle293-F cells  and purified by affinity chromatography using anti-Flag M2 Affinity Gel (Sigma Aldrich). UCH-L1 protein was eluted by pH shift. The purity of the elution fraction and protein concentration was checked by SDS PAGE and the stainfree technology (Biorad). Additionally, protein concentration was confirmed by photometrics, and proteins were separated by SDS PAGE  and blotted onto PVDF membrane (Merck Millipore) using a Transblot Turbo device (Biorad). After blotting, the membrane was blocked in 3% non-fat milk in TBST  for 1 h at RT. Membrane snippets were cut in a way that each snippet contained one lane molecular weight marker (HighQu), one lane WT protein and one lane mutant protein. Each snippet was incubated in a 2 mL tube with 20 µL of patient serum in 1 mL of 3% non-fat milk in TBST on a roller shaker (Cole-Parmer) over night at 4 °C. Snippets were washed 3 times for 10 minutes with TBST followed by an incubation with affinity purified HRP-coupled human anti-IgG+M . For sera analyses, 700 ng of each WT and I93M protein were mixed with reducing sample buffer  and boiled at 95 °C for 5 min. Samples Immunoresearch Laboratories) for 1 h at RT. After another 3 washes with TBST and an incubation of 5 min in SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher) the snippets were imaged using a Vilber Fusion FX07 system. Maximum detection time was set at 3 min. To confirm that protein was loaded on each snippet, the snippets were stripped for 15 minutes in 200 mM Glycin, 0.1% SDS, 1% Tween 20 pH 2.2 followed by a reprobe with an anti-myc antibody .To quantify the intensity of the lanes, densitometry was done by using the rolling ball method for background substraction with the EvolutionCapt Software (Vilber). The Software was allowed to determine the optimal rolling ball size automatically. Also, the UCH-L1 signals that were detected by using the patient sera as primary antibodies were normalized to the intensity of the signals detected with the myc antibody.WT, PDB entry 2ETL, and its I93M variant UCH-L1I93M, PDB entry 3IRT) were used. The respective wildtype structure was used to calculate the electrostatic surface potential of an oxidative-modified variant (UCH-L1WT_ox_mod). The oxidative modifications were defined as determined by Choi et al.24. Using the PyMOL plugin PyTMs (https://github.com/Pymol-Scripts/Pymol-script-repo/blob/master/plugins/pytms.py) the wildtype residues Met1, Met6, Met12, Met124 and Met179 were in silico mutated to their oxidated counterparts methionine sulfoxide. Additionally, the wildtype residue Cys220 was mutated in silico to its corresponding cysteine sulfonic acid. The published PDB files 2ETL and 3IRT as well as the calculated PDB file for the oxidative-modified variant were used to calculate the electrostatic surface potential applying the APBS-PDB2PQR software suite using the corresponding server (https://server.poissonboltzmann.org/). The software package PyMOL (https://pymol.org/2/) was used for visualization.For the calculation of the electrostatic surface potential the published crystal structures of Ubiquitin carboxy-terminal hydrolase L1 (UCH-L1WT (PDB entry 2ETL) or its I93M variant UCH-L1I93M (PDB entry 3IRT) the respective published crystal structures were applied to Cluspro (https://cluspro.bu.edu) to calculate representative sets of lowest energy configurations for the docking of UCH-L1 variants to the human 20S proteasome. The software package PyMOL (https://pymol.org/2/) was used for visualization.For the prediction of putative interactions between the human 20S proteasome (PDB entry 5LE5) and UCH-L1www.graphpad.com. Effects of genotype were analyzed with t-test (for two genotypes), or One-way ANOVA (for three genotypes) followed by Bonferroni post-hoc tests when appropriate. If data did not reach the criteria for parametric statistics, non-parametric statistics using Mann-Whitney U test or Kruskal-Wallis test followed by Dunn’s multiple comparisons if two or three genotypes, respectively, were tested. In time course analyses a mixed two-way ANOVA with Bonferroni post-hoc test was used. Non-parametric statistic was selected to enable robust conclusions on effects significance in case of departures from normality associated with small sample sizes. Replicates used were biological replicates, which were measured using samples derived from distinct mice. All animals were littermates and were blindly assigned to the experimental groups. Patient data were analyzed using ordinary One-way ANOVA with Tukey’s multiple comparisons test, Wilcoxon test, and Spearman correlation. Unless reported otherwise, all tests were two-tailed and significance was set at p ≤ 0.05.All values are expressed as mean ± SEM and analyzed using Prism 8.2.1 for Mac OS X, GraphPad Software, San Diego, California USA, Further information on research design is available in the Supplementary InformationReporting Summary


Rehabilitation after rotator cuff repair is crucial for functional recovery and for minimizing the risk of retear. There are two rehabilitation protocols  and the debate about which is the best is open. This umbrella review aimed to compare the effect of these rehabilitation protocols in terms of reduction in pain, functional recovery, and retear risk. We selected systematic reviews and meta-analyses published between 2012 and 2022 dealing with the aim. Nineteen systematic reviews were included. No significant differences were found between early and traditional protocols in terms of pain reduction. Early rehabilitation provided better short-term results regarding Range of Motion improvement, but long-term functional outcomes were similar. Retear risk remains a significant concern for the early protocol. We found major differences between the analyzed protocols. This review suggests that both protocols are useful to recover global shoulder function, but the standard protocol has a greater safety profile for larger tears. On the other hand, the early protocol may be preferable for smaller lesions, allowing a faster recovery and having less impact on medical costs. Further research is needed to identify optimal rehabilitation strategies tailored to the individual patient’s needs and characteristics. Rotator cuff tear is a common cause of shoulder pain, decreased range of motion, and weakness of the upper limb, limiting people’s daily activities such as brushing hair or putting on clothes, caused by repetitive overhead lifting or shoulder injuries . MechaniThe evidence suggests that the early protocol may prevent postoperative stiffness, fatty infiltration, and muscle atrophy but it can compromise the tendon healing and increase cuff retears ,26. The We reported this umbrella review according to the Preferred Reporting Items for Overviews of Reviews (PRIOR) .The Population, Intervention, Comparison, and Outcome (PICO) method was selected to arrange this review .Population: patients with rotator cuff tear undergoing surgical repair, over 18 years old;Intervention: early rehabilitation protocol;Comparison: standard/delayed rehabilitation protocol;Primary outcome: pain;Secondary outcome: function  and risk of retear.We included systematic reviews, with or without meta-analysis, comparing the efficacy of early rehabilitation protocol with the traditional one after rotator cuff surgical repair, published between 2012 and 2022, in English language and with available full text, reporting outcomes for at least one parameter among pain, shoulder functional and retear rates, with a clinically relevant follow-up time ranging from 3 to 24 months. The definitions of early rehabilitation and traditional rehabilitation were used as described in each study.We excluded studies with different aims, published before 2012 and studies that considered tears caused by traumatic events.Search strategy was independently applied by three independent reviewers. The main MeSH terms and keywords used were: rotator cuff, arthroscopy, shoulder, shoulder joint, rehabilitation, physiotherapy, and physical therapy. The search was conducted in these databases: PubMed, EMBASE, Cochrane Library, PEDro, SCOPUS, and Web of Science (WoS).The data extracted and summarized by three independent reviewers were: name of the authors and year of publication, design of the primary studies included, inclusion criteria of the primary studies, intervention group and comparison with the primary study, tools used to evaluate the results for variables of interest , and primary study references. The data collection process was performed through the reading of full texts and their relevant data were inserted in tables.Three reviewers independently completed assessments of the methodological quality of the included systematic reviews via the AMSTAR-2  and any A total of 19 systematic reviews were included , compariThe systematic review of Bandara et al. includes six Randomized Controlled Trial (RCT) for a total of 531 patients undergoing either early or delayed rehabilitation protocol after rotator cuff repair. A total of 42 patients with stage 2 or 3 full thickness tear underwent arthroscopic side to side surgery, 124 patients with full thickness tear < 30 mm in width underwent arthroscopic double row surgery, 130 patients with small to large tear size underwent arthroscopic single or double row surgery, 103 patients with full thickness tear underwent arthroscopic single row surgery, the data about the remaining 132 patients were not reported. The results suggest a major functional outcome in the EP, durable for the first six months after surgery, but in the long-term this superiority is not so evident. No statistical significance was found for the recurrence retear risk after EP . The metHuang et al. analyzed 100 patients with partial thickness tear, undergoing arthroscopic repair, 29 patients with partial thickness tear, undergoing side to side repair, 68 patients with full thickness tear undergoing arthroscopic suture bridge, 95 patients with small to medium tears undergoing single or double row or suture bridge repair, 64 patients with medium to large tear undergoing arthroscopic single row repair, and 92 patients with partial or full thickness tear undergoing single or double row repair. They found in the EP group a better achievement in ROM and shoulder function, but the early rehabilitation may increase the risk of retear and bad tendon healing. In the EP, pain outcome was better in the first weeks of treatment, but no differences were found at six or twelve months follow up . The fivSince the aim of this umbrella review was to examine the effectiveness of post-surgical rotator cuff repair rehabilitation protocol , we decided to divide the discussion into three main points: pain, functional recovery, and risk of retear.Most of the included systematic reviews showed that there was no significant difference in pain relief between the early rehabilitation and traditional rehabilitation protocols ,40,42. HFunctional recovery, concerning range of motion, strength, and quality of life, was one of the key aspects examined in this review. The findings showed that early rehabilitation protocols provide quicker improvements in range of motion ,40,42,43Risk of retear represents a crucial disadvantage in early rehabilitation protocols, as stated in some of the analyzed systematic reviews ,38,39,43More comparisons based on other patient characteristics, such as age, smoking habit, gender, and occupation would be useful to define increasingly personalized and effective rehabilitation protocols. Further research is needed to establish the most effective rehabilitation strategies for different patients and injury characteristics.This study is primarily limited by the fact that we did not report all the surgical techniques used for the repair, often missing in the articles included in the review. In particular, large, retracted tears of the cuff often exclude the double row technique which has proven to have superior biomechanical properties especially in the initial phase of healing. For this reason, in fact, this represents the major limitation of our article. Secondly, the size of the lesions was also not always reported by all the articles we included, as was the adherence to different rehabilitation protocols. This information, not reported by the authors, represents a gap that requires future studies. Thirdly, we decided to focus this work only on chronic tears because collecting the data of acute and chronic conditions together would have created too much dispersion since they have different outcomes ,51.This umbrella review showed that both early and delayed rehabilitation protocols after arthroscopic rotator cuff repair surgery can provide adequate pain relief and functional recovery. Early rehabilitation protocols generally lead to better short-term ROM outcomes and strength improvement, potentially. However, these advantages may not persist in the long term. The fastest recovery provided by the EP may bring a reduction in the costs of medical assistance for both patient and medical system. Risk of recurrence remains a concern for early rehabilitation, particularly for large injuries; clinicians should carefully consider the patient’s individual characteristics, injury severity and specific therapy modalities when determining the most appropriate rehabilitation protocol after rotator cuff repair. Based on the information collected, the only patient-related characteristic that can be useful to guide the choice between the two protocols is the size of the lesion. Future studies are needed to better quantify the possible differences and characteristics that can influence the choice of one protocol over another.


However, an alternative paradigm has emerged which indicates that FOXOs operate as mediators of cellular homeostasis and/or resistance in both ‘normal’ and pathophysiological scenarios. Distinct FOXO family members fulfil discrete roles during normal B cell maturation and function, and it is now clear that FOXOs are aberrantly expressed and mutated in discrete B-cell malignancies. While active FOXO function is generally associated with disease suppression in chronic lymphocytic leukemia for example, FOXO expression is associated with disease progression in diffuse large B cell lymphoma, an observation also seen in other cancers. The opposing functions of the FOXO family drives the debate about the circumstances in which FOXOs favour or hinder disease progression, and whether targeting FOXO-mediated processes would be effective in the treatment of B-cell malignancies. Here, we discuss the disparate roles of FOXO family members in B lineage cells, the regulatory events that influence FOXO function focusing mainly on post-translational modifications, and consider the potential for future development of therapies that target FOXO activity.Forkhead box (FOX) class O (FOXO) proteins are a dynamic family of transcription factors composed of four family members: FOXO1, FOXO3, FOXO4 and FOXO6. As context-dependent transcriptional activators and repressors, the FOXO family regulates diverse cellular processes including cell cycle arrest, apoptosis, metabolism, longevity and cell fate determination. A central pathway responsible for negative regulation of FOXO activity is the phosphatidylinositol-3-kinase (PI3K)-AKT signalling pathway, enabling cell survival and proliferation. FOXO family members can be further regulated by distinct kinases, both positively  and negatively , with additional post-translational modifications further impacting on FOXO activity. Evidence has suggested that FOXOs behave as ‘ Mammalian FOXO family members were initially reported as part of pro-oncogenic fusion proteins of paired box protein 3/7 (PAX)-FOXO1 in alveolar rhabdomyosarcoma, whereby FOXO1 trans-activation and the FOXO-dependent TGF-β response was inhibited, thus promoting tumorigenesis . The FOigenesis , 3. Simiigenesis –6. FOXO igenesis –10. Withigenesis . Partnerigenesis . Therefoigenesis –14, withigenesis . In addiigenesis ). As FOX22.12 to PIP3 and recruitment of PDK1 and AKT to the plasma membrane. PDK1 phosphorylates and activates AktT308, enabling further phosphorylation/activation of AKTS473 by mechanistic target of rapamycin complex 2  upregulation, which in turn activates tuberous sclerosis 1 and 2 (TSC1 & TSC2) via AMPK  ligation is a key signalling component responsible for inducing cell growth and survival , 20. Upo mTORC2 , 21). AK. AK2 to ion site , 23). AKP motifs , 28. DowP motifs . FOXOs h2.2383 and Thr649 (GPX1 (oxidative damage) and PGC1α (metabolism)  is negatively regulated by MAPK/ERK, CDK2 and casein kinase 1-mediated phosphorylation –36. Convd Thr649 , 38. In abolism) . FOXOs atination , 40. Sevtination –44, whiltination , 45. miRof FOXO1 .33.1FOXO1 enables the differentiation of common lymphoid progenitor (CLP) cells towards the pro-B cell stage. This is initiated by E2A and HeLa E-box binding (HEB) proteins, with the ablation of these proteins diminishing FOXO1 expression and inducing a block at the CLP stage  and lowered BCL2L1 (BCL-XL) expression  proteins RAG1 and RAG2, which are responsible for initiating Ig gene rearrangement –52. In aectively . An abseCCND3 or BCL6 respectively. CCND3 (Cyclin D3) is a FOXO target and crucial for pre-B cell proliferation  and peripheral blood  reduced B-cell lymph node populations, presenting FOXO1 expression as being crucial for correct B cell migration  resulting in the generation of IgG, IgA and IgE antibodies. However, evidence indicates that CSR mainly occurs earlier in pre-GC cells (Myc and Prdm1 (encoding Blimp1) expression which, together with FOXO1 activity, assist in normal GC function  upregulation, while inactivation of FOXO1 through AKT-mediated signals led to IRF4-driven PC differentiation  are specialised structures within secondary lymphoid organs (SLO) in which B-cells undergo clonal expansion and somatic hypermutation (SHM), leading to the generation of antibodies that possess a higher affinity for antigen (affinity maturation) and the export of long-lived plasma cells (PC) and memory B cells , 65. FolGC cells . Bcl-6 afunction –73. Indefunction . This ha program , 73, 74.ntiation . In addiferation , demonstsurvival . Furtherxpansion . CollectFOXO3-deficient mice exhibit lower levels of IL-21, anti-ovalbumin antibodies when challenged with ovalbumin, and decreased Tfh and B-cell populations. Additionally, there is a marked reduction in the occurrence of T cell co-stimulator (ICOS)-induced Tfh differentiation . Indeedmagiomas . Howevermagiomas , more remagiomas –82. The in vitro studies, which showed escape from PI3K/AKT regulation through loss of AKT phosphorylation; increased nuclear sequestration and increased DNA binding compared to wildtype . Mutations of the other FOXO family members are less frequent within B cell malignancies  and BL (FOXO3 is frequently deleted in DLBCL (with deletions of 6q21-q22 occurring in 40% of ABC-DLBCL cases and 22% of GCB-DLBCL cases) , follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL), with a frequency of ~11%, 6% and ~5% respectively –18, 83).wildtype , or; loswildtype , 88. Add) and BL Figure 1) and BL . It shouL cases)  and is aL cases) . DeletioL cases) , 92.4.1FOXO1 expression is not altered in BCP-ALL  is the most common childhood malignancy originating from pro-B and pre-B cells , 93. BCP BCP-ALL , 96. In ll death . Bhansalll death . In contgulation , 100. FO BCP-ALL . In mousgression . Interespression . This su4.2+ B-lymphocytes in the blood, BM and SLOs (FOXO family genes occurred in 4% (22/551) of CLL patients, of which 2% (11/551) occurred in FOXO1, with a functionally “high impact” missense mutation of FOXO1 encoded in just one patient   . Interesrylation . These srylation , 108. Inrylation . While t4.3in vivo   translocation resulting in the overexpression of BCL-2 . Somaticagenesis . While Fagenesis . Downstr7-FLIPI) , 115. Mo7-FLIPI) .4.5MYC oncogene is the focal point driving BL pathogenesis . CRer of BL . Gehringhibition . Thus, t4.6de novo DLBCL is well characterised, with common genomic alterations, including chromosomal translocations and mutations found between subtypes, contributing to promotion of pro-survival pathways  , while FOXO1 ablation prevented R406-induced apoptosis and cell cycle inhibition  or activated B-cells (ABC-DLBCL), with ABC-DLBCL associated with an inferior outcome . These spathways . Within 9% cases . Howevermutation . Furthers. 8.6%) . FOXO famutation Figure 1mutation . This comutation . Within unctions . A furth is rare . The FOXon motif , 128), shibition .FOXO4 knockdown reduced colony forming ability of DLBCL cell lines   , but alsormation . Supportormation , 132, orormation , 139. Hoormation , 111 andrs (CRC) . Furtherrs (CRC) , 114, imsistance . This stsistance –82, and In the broader context, studies investigating FOXO expression as a prognostic biomarker have contrasting results. In CRC, low expression of FOXO3 is associated with cancer progression in specimens with normal tissue , while hJL, JH and MM wrote the manuscript and JL and JH prepared figures. AM reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.


Left ventricular assist devices (LVADs) provide a clear benefit to patients with end-stage heart failure. Despite this, device-related infections such as driveline infections, device pocket infections, and LVAD-related bloodstream infections occur in up to 39% of implanted patients. Pseudomonas aeruginosa is a common causative organism in LVAD-associated infections and frequently develops resistance, limiting treatment options. This study aims to analyze the relationships between the frequency of acquired resistance (AR) and single-agent antibiotic courses, as well as the frequency of AR and duration of antibiotic courses; and compare the likelihood of AR between different antibiotic classes.This was a single-center retrospective cohort study involving all patients with LVAD-associated P. aeruginosa infections between 2011-2023. Patients treated with piperacillin-tazobactam, cefepime, ciprofloxacin, or meropenem, were included (n = 37). Each antibiotic course (n = 117) was associated with a pre- and post-antibiotic sensitivity. AR was defined as culture-proven, newly developed resistance to the utilized antibiotic after starting therapy.The incidence and cumulative proportion of AR were estimated using Poisson regression and Kaplan-Meier curve analysis, respectively. A marginal proportional hazards model was used to analyze time to AR between different antibiotic groups.There was a significantly lower incidence of AR with cefepime [0.8 (95% CI 0.2-3.3) per 100 person-weeks] compared to piperacillin-tazobactam [3.6 ], meropenem [7.6 ], and ciprofloxacin [6.4 ] (p=0.04). Similarly, the cumulative proportion of AR was significantly lower with cefepime as compared to other antibiotic classes . The hazard rate of AR was approximately 3.5 to 6.7 times higher with each antibiotic in comparison to cefepime (p = 0.02) (Table 1).Resistance occurs early and more frequently with piperacillin-tazobactam, meropenem, and ciprofloxacin, but less frequently and late with cefepime. The cumulative proportion of acquired resistance over time differed significantly across antibiotic types (p = 0.02).Cefepime had a statistically significantly lower hazard of AR when compared to the rest of the antibiotic groups as a whole, and when compared individually, had a significantly lower hazard than ciprofloxacin and meropenem.This study suggests a lower incidence of AR for LVAD-associated P. aeruginosa infection treated with cefepime. AR also seems to develop later in patients treated with cefepime. There is a 3.5-6.7-fold increase in the risk of acquiring resistance with other antibiotics compared to cefepime.Jeffrey Alexis, MD, Abbott: Grant/Research Support