Disease causing mutations in inverted formin 2 regulate its binding to G-actin, F-actin capping protein (CapZ α-1) and profilin 2

Focal segmental glomerulosclerosis (FSGS) is a devastating form of nephrotic syndrome which ultimately leads to end stage renal failure (ESRF). Mutations in inverted formin 2 (INF2), a member of the formin family of actin-regulating proteins, have recently been associated with a familial cause of nephrotic syndrome characterized by FSGS. INF2 is a unique formin that can both polymerize and depolymerize actin filaments. How mutations in INF2 lead to disease is unknown. In the present study, we show that three mutations associated with FSGS, E184K, S186P and R218Q, reduce INF2 auto-inhibition and increase association with monomeric actin. Furthermore using a combination of GFP–INF2 expression in human podocytes and GFP-Trap purification coupled with MS we demonstrate that INF2 interacts with profilin 2 and the F-actin capping protein, CapZ α-1. These interactions are increased by the presence of the disease causing mutations. Since both these proteins are involved in the dynamic turnover and restructuring of the actin cytoskeleton these changes strengthen the evidence that aberrant regulation of actin dynamics underlies the pathogenesis of disease

Expression of HIV-1 Vpu Leads to Loss of the Viral Restriction Factor CD317/Tetherin from Lipid Rafts and Its Enhanced Lysosomal Degradation

CD317/tetherin (aka BST2 or HM1.24 antigen) is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts). It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i) no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii) the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii) internalised tetherin is present in non-raft fractions, iv) expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v) internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi) lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells

Adeno-associated virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome

Gene therapy for kidney diseases has proven challenging. Adeno-associated virus (AAV) is used as a vector for gene therapy targeting other organs, with particular success demonstrated in monogenic diseases. We aimed to establish gene therapy for the kidney by targeting a monogenic disease of the kidney podocyte. The most common cause of childhood genetic nephrotic syndrome is mutations in the podocyte gene NPHS2, encoding podocin. We used AAV-based gene therapy to rescue this genetic defect in human and mouse models of disease. In vitro transduction studies identified the AAV-LK03 serotype as a highly efficient transducer of human podocytes. AAV-LK03–mediated transduction of podocin in mutant human podocytes resulted in functional rescue in vitro, and AAV 2/9–mediated gene transfer in both the inducible podocin knockout and knock-in mouse models resulted in successful amelioration of kidney disease. A prophylactic approach of AAV 2/9 gene transfer before induction of disease in conditional knockout mice demonstrated improvements in albuminuria, plasma creatinine, plasma urea, plasma cholesterol, histological changes, and long-term survival. A therapeutic approach of AAV 2/9 gene transfer 2 weeks after disease induction in proteinuric conditional knock-in mice demonstrated improvement in urinary albuminuria at days 42 and 56 after disease induction, with corresponding improvements in plasma albumin. Therefore, we have demonstrated successful AAV-mediated gene rescue in a monogenic renal disease and established the podocyte as a tractable target for gene therapy approaches

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor

Aims/hypothesis: Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. Methods: Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. Results: Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. Conclusions/interpretation: Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.</p

A longitudinal study of men with male genital schistosomiasis (MGS) in Southern Malawi associated with human, zoonotic and hybrid schistosomes

In sub-Saharan Africa s endemic areas for urogenital schistosomiasis, Male Genital Schistosomiasis (MGS) can cause significant morbidity. As part of the Hybridization in UroGenital Schistosomiasis (HUGS) investigation, a MGS sub-study examined a cohort of adult men over a calendar year to better ascertain general infection dynamics and putative zoonotic schistosome transmission. During follow-up, demographic, health and socioeconomic data were collected through individual questionnaire interviews. Collected urine and semen were analysed using urine filtration, urine and semen microscopy and molecular DNA analyses of semen. Ten participants with reported MGS-associated symptoms had Schistosoma eggs in their urine and semen at 6 months follow up, with seven at 12 months. Ten out of eleven participants with S. haematobium eggs on semen microscopy at baselinehad persistent infection at 6 months follow-up, together with 6 new participants, giving an MGS prevalence of 84.2% (n=19). Two also had S. mattheei eggs co-infection. Four of the 13 participants at 12 months follow-up had S. haematobium eggs in their semen which were persistent at all the time-points. Using semen PCR, 14 participants (73.7%) had Schistosoma infection at 6 months, with only 2 participants being infected for first time. Upon DNA analysis, three participants also had hybrid co-infection at this time-point. At 12 months, only six participants had Schistosoma infection with no hybrids detected. In summary, like S. haematobium and despite praziquantel treatment, both zoonotic and hybrid schistosomes can continue to cause MGS, which pose a further tangible challenge in future management and control measures.</p

An investigation of female genital schistosomiasis and associated genital infections in southern Malawi

Urogenital schistosomiasis caused by zoonotic or hybrid schistosome infection(s) is an emerging public health concern in Malawi and we describe a 1-year clinical sub-study with three inspection time-points for female genital schistosomiasis upon selecting 86 women with proven urogenital schistosomiasis. This sub-study was set within a broader 2-year longitudinal 'Hybridization in UroGenital Schistosomiasis (HUGS)' investigation. A detailed cervicovaginal examination with a portable colposcope was conducted, examining cervicovaginal lavage (CVL), cervical swabs, cervical biopsy and urine with traditional parasitological and molecular diagnostic methods. At baseline, overt FGS by colposcopy was 72.1%, 64.3% by CVL real-time PCR and 51.2% by both colposcopy and CVL-PCR, noting urine-microscopy could often be negative. Human papilloma virus was detected in 31.0%, with 8.3% also FGS positive by colposcopy and real-time PCR. Over the year, FGS prevalence by colposcopy increased by 32.7% during the study to 84.6%, homogenous yellow and grainy sandy patches being very common in the youngest 18-25 age group, where 51.9% were positive. FGS appears widespread locally and we discuss difficulties in its detection without invasive sampling. In addition to the presence of S. haematobium, S. mattheei was noted alongside key concurrent sexually transmitted infections. From our findings, we point out that improved prevention and management of FGS is required, foremost, better availability and regular accessibility to praziquantel treatment is needed. Furthermore, targeted health education, raised community awareness and dovetailing synergistic public health activities within Sexual and Reproductive Health services and local HIV/AIDS programmes could develop an appropriate holistic health intervention package.</p

Three annual cross-sectional community-based knowledge, attitudes and practices (KAP) and prevalence surveys for urogenital schistosomiasis infection in two rural communities within Mangochi and Nsanje Districts, southern Malawi

In 2022 the World Health Organization (WHO) issued guidelines with key interventions to control and eliminate schistosomiasis in endemic countries. In Malawi, whilst praziquantel Mass Drug Administration (MDA) campaigns have been ongoing for over a decade, implementation of other interventions have not been formally assessed. To help formulation of an integrated country-specific control strategy, we assessed the Knowledge, Attitudes and Practices (KAP) and infection prevalences in two representative rural communities in Mangochi and Nsanje Districts. Longitudinal cross-sectional community-based questionnaire surveys were undertaken with participants aged from 6 to 45 years in 2022 and later repeated in 2023 and in 2024. Participants (including children aged 2 to 5 years) provided urine samples for parasitological tests. Comparative analysis involved calculation of percentages, tabulations, frequencies, and a logistic regression (logit) model to assess the effect of education level, gender, age, and study area on general and correct knowledge of schistosomiasis. A total of 1964 participants took part in the KAP surveys in 2022, and 1789 and 1908 participants were followed up in 2023 and 2024 respectively, while for the parasitological surveys, 2,319 participants took part in 2022, and 2,006 and 2,014 participants were followed up in 2023 and 2024 surveys respectively. In total, 53.2 % were from Mangochi, 55.5 % were females, 62.1 % were School-Aged Children (SAC) and 37.9 % were adults with their mean ages at 11 and 28 years, respectively. Overall, 65.5 % of respondents demonstrated satisfactory (≥50.0 % – ≤70.0 %) knowledge of schistosomiasis while only 5.1 % correctly mentioned freshwater snails as intermediate hosts. In 2022, prevalence of urogenital schistosomiasis by urine microscopy was 43.6 %, which despite annual MDA increased to 44.1 % in 2023, then after biannual MDA decreased to 27.0 % in 2024. In 2022, 10.5 % of all participants had heavy-intensity infections which increased to 11.4 % in 2023 before decreasing to 7.7 % in 2024. The majority (91.3 %) used a borehole or piped source of drinking water and used a latrine to urinate or defecate (93.8 %) although many (59.6 %) reported to have visited a freshwater body more than once in a day. Since MDA has taken place over several years in these areas and only had insufficient local impact, we strongly encourage addition of complementary methods to bolster its impact. It is therefore essential to engage individuals and communities, improving their understanding of disease and behaviour change to more effectively control and potentially eliminate schistosomiasis

Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2

Tetherin, also known as BST2, CD317 or HM1.24, was recently identified as an interferon-inducible host–cell factor that interferes with the detachment of virus particles from infected cells. HIV-1 overcomes this restriction by expressing an accessory protein, Vpu, which counteracts tetherin. Since lentiviruses of the SIVsmm/mac/HIV-2 lineage do not have a vpu gene, this activity has likely been assumed by other viral gene products. We found that deletion of the SIVmac239 nef gene significantly impaired virus release in cells expressing rhesus macaque tetherin. Virus release could be restored by expressing Nef in trans. However, Nef was unable to facilitate virus release in the presence of human tetherin. Conversely, Vpu enhanced virus release in the presence of human tetherin, but not in the presence of rhesus tetherin. In accordance with the species-specificity of Nef in mediating virus release, SIV Nef downregulated cell-surface expression of rhesus tetherin, but did not downregulate human tetherin. The specificity of SIV Nef for rhesus tetherin mapped to four amino acids in the cytoplasmic domain of the molecule that are missing from human tetherin, whereas the specificity of Vpu for human tetherin mapped to amino acid differences in the transmembrane domain. Nef alleles of SIVsmm, HIV-2 and HIV-1 were also able to rescue virus release in the presence of both rhesus macaque and sooty mangabey tetherin, but were generally ineffective against human tetherin. Thus, the ability of Nef to antagonize tetherin from these Old World primates appears to be conserved among the primate lentiviruses. These results identify Nef as the viral gene product of SIV that opposes restriction by tetherin in rhesus macaques and sooty mangabeys, and reveal species-specificity in the activities of both Nef and Vpu in overcoming tetherin in their respective hosts

CD317/tetherin is an organiser of membrane microdomains

The integral membrane protein tetherin has been associated with an eclectic mix of cellular processes, including restricting the release of a range of enveloped viruses from infected cells. The unusual topology of tetherin (it possesses both a conventional transmembrane domain and a glycosylphosphatidylinositol anchor), its localisation to membrane microdomains (lipid rafts) and the fact that its cytosolic domain can be linked (indirectly) to the actin cytoskeleton, led us to speculate that tetherin might form a 'tethered picket fence' and thereby play a role in the organisation of lipid rafts. We now show that knocking down expression of tetherin leads to changes in the distribution of lipid raft-localised proteins and changes in the organisation of lipids in the plasma membrane. These changes can be reversed by re-expression of wild-type tetherin, but not by any of a range of tetherin-based constructs, indicating that no individual feature of the tetherin sequence is dispensable in the context of its lipid raft organising function