Newly synthesized polycystin-1 takes different trafficking pathways to the apical and ciliary membranes.

Mutations in the genes encoding polycystin-1 (PC1) and polycystin 2 (PC2) cause autosomal dominant polycystic kidney disease. These transmembrane proteins colocalize in the primary cilia of renal epithelial cells, where they may participate in sensory processes. PC1 is also found in the apical membrane when expressed in cultured epithelial cells. PC1 undergoes autocatalytic cleavage, producing an extracellular N-terminal fragment that remains noncovalently attached to the transmembrane C-terminus. Exposing cells to alkaline solutions elutes the N-terminal fragment while the C-terminal fragment is retained in the cell membrane. Utilizing this observation, we developed a "strip-recovery" synchronization protocol to study PC1 trafficking in polarized LLC-PK1 renal epithelial cells. Following alkaline strip, a new cohort of PC1 repopulates the cilia within 30 minutes, while apical delivery of PC1 was not detectable until 3 hours. Brefeldin A (BFA) blocked apical PC1 delivery, while ciliary delivery of PC1 was BFA insensitive. Incubating cells at 20°C to block trafficking out of the trans-Golgi network also inhibits apical but not ciliary delivery. These results suggest that newly synthesized PC1 takes distinct pathways to the ciliary and apical membranes. Ciliary PC1 appears to by-pass BFA sensitive Golgi compartments, while apical delivery of PC1 traverses these compartments.

LIN7 regulates the filopodium- and neurite-promoting activity of IRSp53.

The insulin receptor substrate protein of 53 kDa (IRSp53) is crucially involved in the formation of filopodia and neurites through mechanisms that have only partially been clarified. We have investigated the role of the small scaffold protein LIN7, which interacts with IRSp53. We found that formation of actin-filled protrusions in neuronal NSC34 cells and neurites in neuroblastoma N2A cells depends on motifs mediating the LIN7:IRSp53 association, as both the coexpression of LIN7 with IRSp53 or the expression of the L27-IRSp53 chimera (a fusion protein between IRSp53 and the LIN7L27 domain for plasma membrane protein complexes association) prevented actin-deficient protrusions induced by overexpressed IRSp53, and enhanced the formation of actin-filled protrusions. The regulatory role of LIN7 in IRSp53-mediated extension of filopodia in neuronal N2A cells was demonstrated by live-cell imaging experiments. Moreover, LIN7 silencing prevented the extension of filopodia and neurites, induced by ectopic expression of IRSp53 or serum starvation, respectively, in undifferentiated and differentiated N2A cells. The expression of full-length IRSp53 or the LIN7ΔPDZ mutant lacking the domain for association with IRSp53 was unable to restore neuritogenesis in LIN7-silenced cells. Conversely, defective neuritogenesis could be rescued by the expression of RNAi-resistant full-length LIN7 or chimeric L27-IRSp53. Finally, LIN7 silencing prevented the recruitment of IRSp53 in Triton X-100-insoluble complexes, otherwise occurring in differentiated cells. Collectively these data indicate that LIN7 is a novel regulator of IRSp53, and that the association of these proteins is required to promote the formation of actin-dependent filopodia and neurites.

The POF1B candidate gene for premature ovarian failure regulates epithelial polarity.

POF1B is a candidate gene for premature ovarian failure (POF); it is mainly expressed in polarised epithelial tissues, but its function in these tissues and the relationship with the disorder are unknown. Here we show colocalisation of POF1B with markers of both adherens and tight junctions in human jejunum. The tight junction localisation was maintained by the human POF1B stably expressed in the MDCK polarised epithelial cell line, whereas it was lost by the POF1B R329Q variant associated with POF. Localisation of apico-basal polarity markers and ultrastructure of the tight junctions were maintained in cells expressing the mutant. However, tight junction assembly was altered, cells were dysmorphic and the monolayer organisation was also altered in three-dimensional culture systems. Moreover, cells expressing the POF1B R329Q variant showed defects in ciliogenesis and cystogenesis as a result of misorientation of primary cilia and mitotic division. All of these defects were explained by interference of the mutant with the content and organisation of F-actin at the junctions. A role for POF1B in the regulation of the actin cytoskeleton was further verified by shRNA silencing of the endogenous protein in human intestinal Caco-2 cells. Taken together, these data indicate that localisation of POF1B to tight junctions has a key role in the organisation of epithelial monolayers by regulating the actin cytoskeleton.

The C-terminal tail of polycystin-1 suppresses cystic disease in a mitochondrial enzyme-dependent fashion.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent potentially lethal monogenic disorder. Mutations in the PKD1 gene, which encodes polycystin-1 (PC1), account for approximately 78% of cases. PC1 is a large 462-kDa protein that undergoes cleavage in its N and C-terminal domains. C-terminal cleavage produces fragments that translocate to mitochondria. We show that transgenic expression of a protein corresponding to the final 200 amino acid (aa) residues of PC1 in two Pkd1-KO orthologous murine models of ADPKD suppresses cystic phenotype and preserves renal function. This suppression depends upon an interaction between the C-terminal tail of PC1 and the mitochondrial enzyme Nicotinamide Nucleotide Transhydrogenase (NNT). This interaction modulates tubular/cyst cell proliferation, the metabolic profile, mitochondrial function, and the redox state. Together, these results suggest that a short fragment of PC1 is sufficient to suppress cystic phenotype and open the door to the exploration of gene therapy strategies for ADPKD.

LIN7 mediates the recruitment of IRSp53 to tight junctions.

In this study, we examined the role of the L27 [(LIN2-LIN7) domain] and PDZ domain (domain previously found in PSD95-DlgA-ZO-1) for protein-protein interaction of the scaffold protein LIN7 in tight junction (TJ) assembly in Madin-Darby canine kidney (MDCK) cells and found that the stable expression of a LIN7 mutant lacking the L27 domain (DeltaL27 mutant) acts as a dominant interfering protein by inhibiting TJ localization of endogenous LIN7. The loss of LIN7 did not alter the localization of the PALS1 (protein associated with LIN7) partner of the L27 domain but prevented TJ localization of the insulin receptor substrate p53 (IRSp53), a partner of the PDZ domain of LIN7. The function of both L27 and PDZ domains of LIN7 in IRSp53 localization to TJs has been further demonstrated by reducing the expression of LIN7 (LIN7 small hairpin RNA experiments) and by expression of IRSp53 deleted of its motif for PDZ interaction (IRSp53Delta5) or fused to the L27 domain of LIN7 (L27-IRSp53Delta5). Cell lines with decreased localization of LIN7 and IRSp53 to TJs showed defects during assembly of TJs and cyst polarization and failed to activate Rac1, a member of the Rho guanosine triphosphatases family crucially involved in actin organization and orientation of apicobasal polarity. These data therefore indicate that LIN7-IRSp53 association plays a role during assembly of functional TJs and surface polarization in epithelial cells.

The proinflammatory action of microglial P2 receptors is enhanced in SOD1 models for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of lower and upper motoneurons. The pathology is imputable in approximately 2% of cases to mutations in the ubiquitous enzyme Cu, Zn superoxide dismutase (SOD1). Common theories to explain the pathogenic mechanisms of ALS include activation of microglia, responsible for the release of proinflammatory factors. However, how mutant SOD1 affects microglial activation and subsequently injures neurons is still unclear. Considering that extracellular ATP, through purinergic P2 receptors, constitutes a well recognized neuron-to-microglia alarm signal, the aim of this study was to investigate how the expression of mutant SOD1 affects P2 receptor-mediated proinflammatory microglial properties. We used primary and immortalized microglial cells from mutant SOD1 mice to explore several aspects of activation by purinergic ligands and to analyze the overall effect of such stimulation on the viability of NSC-34 and SH-SY5Y neuronal cell lines. We observed up-regulation of P2X(4), P2X(7), and P2Y(6) receptors and down-regulation of ATP-hydrolyzing activities in mutant SOD1 microglia. This potentiation of the purinergic machinery reflected into enhanced sensitivity mainly to 2'-3'-O-(benzoyl-benzoyl) ATP, a P2X(7) receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-alpha and cyclooxygenase-2 content, and finally into toxic effects exerted on neuronal cell lines by microglia expressing mutant SOD1. All these parameters were prevented by the antagonist Brilliant Blue G. The purinergic activation of microglia may thus constitute a new route involved in the progression of ALS to be exploited to potentially halt the disease.

A cut above (and below): Protein cleavage in the regulation of polycystin trafficking and signaling.

The polycystin-1 and 2 proteins, encoded by the genes mutated in Autosomal Dominant Polycystic Kidney Disease, are connected to a large number of biological pathways. While the nature of these connections and their relevance to the primary functions of the polycystin proteins have yet to be fully elucidated, it is clear that many of them are mediated by or depend upon cleavage of the polycystin-1 protein. Cleavage of polycystin-1 at its G protein coupled receptor proteolytic site is an obligate step in the protein's maturation and in aspects of its trafficking. This cleavage may also serve to prime polycystin-1 to play a role as a non-canonical G protein coupled receptor. Cleavage of the cytoplasmic polycystin-1C terminal tail releases fragments that are able to enter the nucleus and the mitochondria and to influence their activities. Understanding the nature of these cleavages, their regulation and their consequences is likely to provide valuable insights into both the physiological functions served by the polycystin proteins and the pathological consequences of their absence.

Chloride channels regulate differentiation and barrier functions of the mammalian airway.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

A High Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury during the COVID-19 pandemic.

Drug repurposing is the only method capable of delivering treatments on the shortened time-scale required for patients afflicted with lung disease arising from SARS-CoV-2 infection. Mucin-1 (MUC1), a membrane-bound molecule expressed on the apical surfaces of most mucosal epithelial cells, is a biochemical marker whose elevated levels predict the development of acute lung injury (ALI) and respiratory distress syndrome (ARDS), and correlate with poor clinical outcomes. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce MUC1 protein abundance. Our screen identified Fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo , Fostamatinib reduced MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro , SYK inhibition by Fostamatinib promoted MUC1 removal from the cell surface. Our work reveals Fostamatinib as a repurposing drug candidate for ALI and provides the rationale for rapidly standing up clinical trials to test Fostamatinib efficacy in patients with COVID-19 lung injury.

A High-Content Screen for Mucin-1-Reducing Compounds Identifies Fostamatinib as a Candidate for Rapid Repurposing for Acute Lung Injury.

Drug repurposing has the advantage of identifying potential treatments on a shortened timescale. In response to the pandemic spread of SARS-CoV-2, we took advantage of a high-content screen of 3,713 compounds at different stages of clinical development to identify FDA-approved compounds that reduce mucin-1 (MUC1) protein abundance. Elevated MUC1 levels predict the development of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and correlate with poor clinical outcomes. Our screen identifies fostamatinib (R788), an inhibitor of spleen tyrosine kinase (SYK) approved for the treatment of chronic immune thrombocytopenia, as a repurposing candidate for the treatment of ALI. In vivo, fostamatinib reduces MUC1 abundance in lung epithelial cells in a mouse model of ALI. In vitro, SYK inhibition by the active metabolite R406 promotes MUC1 removal from the cell surface. Our work suggests fostamatinib as a repurposing drug candidate for ALI.

PKC induces internalization and retention of the EAAC1 glutamate transporter in recycling endosomes of MDCK cells.

Here we show that stimulation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) treatment induces a time-dependent decrease in glutamate transport activity due to relocalization of the excitatory amino acid carrier 1 (EAAC1) glutamate transporter from the apical surface of polarized epithelial Madin-Darby canine kidney (MDCK) cells to intracellular compartments. The PKC-induced internalization of EAAC1 is negatively regulated by the calcineurin inhibitor cyclosporine A and by the expression of a dominant-negative mutant of the endocytic protein dynamin 1, a well-known target of the phosphatase activity of calcineurin. Using 32P-metabolic labeling experiments, we found unchanged levels of phosphorylated EAAC1, indicating that EAAC1 relocalization does not depend on PKC and calcineurin modification of the transporter, while we found that a target of these modifications was the serine778 residue of dynamin, a calcineurin substrate that in its dephosphorylated form activates the endocytic functions of dynamin. These data suggest that PMA stimulates endogenous dynamin and that this activation is required to mediate internalization of EAAC1 in MDCK cells. By immunofluorescence experiments with endosomal markers we demonstrated that internalized EAAC1 accumulates in endosomes also containing the basolateral betaine-GABA transporter BGT1 and activated PKCalpha. The sustained activation of PKC was required to maintain the transporters in the endosomal compartment, while a posttreatment with a PKC-specific inhibitor induced the recycling of the transporters to their appropriate surfaces. Taken together, our data indicate that PKC activity regulates EAAC1 surface density in MDCK cells by inducing its internalization and retention in PKCalpha-labeled recycling endosomes common to apical and basolateral proteins.

Metabolism and mitochondria in polycystic kidney disease research and therapy.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common, potentially lethal, monogenic diseases and is caused predominantly by mutations in polycystic kidney disease 1 (PKD1) and PKD2, which encode polycystin 1 (PC1) and PC2, respectively. Over the decades-long course of the disease, patients develop large fluid-filled renal cysts that impair kidney function, leading to end-stage renal disease in ~50% of patients. Despite the identification of numerous dysregulated pathways in ADPKD, the molecular mechanisms underlying the renal dysfunction from mutations in PKD genes and the physiological functions of the polycystin proteins are still unclear. Alterations in cell metabolism have emerged in the past decade as a hallmark of ADPKD. ADPKD cells shift their mode of energy production from oxidative phosphorylation to alternative pathways, such as glycolysis. In addition, the polycystins seem to play regulatory roles in modulating mechanisms and machinery related to energy production and utilization, including AMPK, PPARα, PGC1α, calcium signalling at mitochondria-associated membranes, mTORC1, cAMP and CFTR-mediated ion transport as well as the expression of crucial components of the mitochondrial energy production apparatus. In this Review, we explore these metabolic changes and discuss in detail the relationship between energy metabolism and ADPKD pathogenesis and identify potential therapeutic targets.

The polycystins are modulated by cellular oxygen-sensing pathways and regulate mitochondrial function.

Autosomal dominant polycystic kidney disease is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), which form an ion channel complex that may mediate ciliary sensory processes and regulate endoplasmic reticulum (ER) Ca2+ release. Loss of PC1 expression profoundly alters cellular energy metabolism. The mechanisms that control the trafficking of PC1 and PC2, as well as their broader physiological roles, are poorly understood. We found that O2 levels regulate the subcellular localization and channel activity of the polycystin complex through its interaction with the O2-sensing prolyl hydroxylase domain containing protein EGLN3 (or PHD3), which hydroxylates PC1. Moreover, cells lacking PC1 expression use less O2 and show less mitochondrial Ca2+ uptake in response to bradykinin-induced ER Ca2+ release, indicating that PC1 can modulate mitochondrial function. These data suggest a novel role for the polycystins in sensing and responding to cellular O2 levels.

POF1B localizes to desmosomes and regulates cell adhesion in human intestinal and keratinocyte cell lines.

By means of morphological and biochemical criteria, we here provide evidence for the localization and function of premature ovarian failure, 1B (POF1B) in desmosomes. In monolayers of Caco-2 intestinal cells and in stratified HaCaT keratinocytes, endogenous POF1B colocalized with desmoplakin at desmosome plaques and in cytoplasmic particles aligned along intermediate filaments (IFs). POF1B predominantly co-fractionated with desmosomes and IF components and exhibited properties characteristic of desmosomes (i.e., detergent insolubility and calcium independence). The role of NH2 and COOH domains in the association of POF1B with desmosomes and IFs was revealed by transient expression of the truncated protein in Caco-2 cells and in cells lacking desmosomes. The function of POF1B in desmosomes was investigated in HaCaT keratinocytes stably downregulated for POF1B expression. Transmission electron microscopy analysis revealed a decrease in desmosome number and size, and desmosomes of the downregulated keratinocytes displayed weak electron-dense plaques. Desmosome alterations were associated with defects in cell adhesion, as revealed by the reduced resistance to mechanical stress in the dispase fragmentation assay. Moreover, desmosome localization of POF1B was restricted to granular layers in human healthy epidermis, whereas it largely increased in hyperproliferative human skin diseases, thus demonstrating the localization of POF1B also in desmosomes of multistratified epithelia.