Search

Lowe Syndrome (LS) is a lethal genetic disorder caused by mutations in theOCRL1gene which encodes the lipid 5’ phosphatase Ocrl1. Patients exhibit a characteristic triad of symptoms including eyes, brain and kidneys abnormalities with renal failure as the most common cause of premature death. Over 200OCRL1mutations have been identified in LS, but their specific impact on cellular processes is unknown. Despite observations of heterogeneity in patient symptom severity, there is little understanding of the correlation between genotype and its impact on phenotype.Here, we show that different mutations had diverse effects on protein localization and on triggering LS cellular phenotypes. In addition, some mutations affecting specific domains imparted unique characteristics to the resulting mutated protein. We also propose that certain mutations conformationally affect the 5’-phosphatase domain of the protein, resulting in loss of enzymatic activity and causing common and specific phenotypes.This study is the first to show the differential effect of patient 5’-phosphatase mutations on cellular phenotypes and introduces a conformational disease component in LS. This work provides a framework that can help stratify patients as well as to produce a more accurate prognosis depending on the nature and location of the mutation within theOCRL1gene.

Lowe syndrome (LS) is an X-linked developmental disease characterized by cognitive deficiencies, bilateral congenital cataracts and renal dysfunction. Unfortunately, this disease leads to the early death of affected children often due to kidney failure. Although this condition was first described in the early 1950s and the affected gene (OCRL1) was identified in the early 1990s, its pathophysiological mechanism is not fully understood and there is no LS-specific cure available to patients. Here we report two important signaling pathways affected in LS patient cells. While RhoGTPase signaling abnormalities led to adhesion and spreading defects as compared to normal controls, PI3K/mTOR hyperactivation interfered with primary cilia assembly (scenario also observed in other ciliopathies with compromised kidney function). Importantly, we identified two FDA-approved drugs able to ameliorate these phenotypes. Specifically, statins mitigated adhesion and spreading abnormalities while rapamycin facilitated ciliogenesis in LS patient cells. However, no single drug was able to alleviate both phenotypes. Based on these and other observations, we speculate that Ocrl1 has dual, independent functions supporting proper RhoGTPase and PI3K/mTOR signaling. Therefore, this study suggest that Ocrl1-deficiency leads to signaling defects likely to require combinatorial drug treatment to suppress patient phenotypes and symptoms.

Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid (CO) model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband COs exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control COs. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-β-Catenin signaling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of COs to uncover molecular mechanisms underlying AUTS2 syndrome.

Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband cerebral organoids exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control cerebral organoids. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-β-catenin signalling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of cerebral organoids to investigate molecular mechanisms underlying AUTS2 syndrome.

Lowe Syndrome (LS) is a lethal developmental disease characterized by mental retardation, cataracts at birth and kidney dysfunction. LS children unfortunately die by adolescence from renal failure. The gene responsible for the disease (OCRL1) encodes an inositol 5’ phosphatase Ocrl1. In addition to its 5’ phosphatase domain, this protein has other domains that allow protein-protein interactions, facilitating diverse sub-cellular distribution and functions. LS patient cells lacking Ocrl1 display defects in cell spreading, ciliogenesis and vesicle trafficking. Currently the mechanisms underlying these cellular defects are not known, and hence no LS-specific therapies exist. We have uncovered the mechanisms underlying two LS-specific cellular phenotypes- namely cell spreading and ciliogenesis and identified 2 FDA-approved candidates- statins and rapamycin that could revert these abnormalities. We found that Ocrl1-deficient cells exhibit hyperactivation in mTOR signaling, resulting in ciliogenesis as well as autophagy defects, which were rescued by administering rapamycin. We also identified a novel RhoGTPase signaling-dependent cell adhesion defect in LS patient cells which resulted in focal adhesion abnormalities and sensitivity to fluid shear stress (critical for kidney function). Both RhoGTPase signaling dependent cell spreading and adhesion defects were corrected by treatment with statins. Importantly, over 200 unique mutations in OCRL1 cause LS and patients demonstrate heterogeneity in symptoms. However, the correlation between genotype and cellular phenotypes is unknown. We have determined that different OCRL1 patient mutations have a differential impact on the two cellular phenotypes described above. Mutants exhibit behavior, sub-cellular distribution and cellular phenotypes unique to the domain and relevant to LS pathogenesis. We also propose that a subset of non-catalytic phosphatase domain mutations are conformationally affecting the protein, suggesting that LS has a conformational disease component. Importantly, we tested an FDA-approved drug, 4-phenyl butyric acid (4-PBA), used as a therapeutic in conformational diseases and found that it could revert phenotypes and restore the catalytic activity of these mutants. These findings collectively contribute to provide the cellular basis for LS patient heterogeneity as well as to propose a conformational disease component for LS (allowing the use of chemical chaperones as a therapeutic strategy for a subset of LS patients). Together, we hope that these studies will help lay the foundation of better prognosis and tailoring personalized therapeutic strategies for LS patients.

Noncoding and synonymous coding variants that exert their effects via alternative splicing are increasingly recognized as an important category of disease-causing variants. In this report, we describe two siblings who presented with hypotonia, profound developmental delays, and seizures. Brain MRI in the proband at 5 years showed diffuse cerebral and cerebellar white matter volume loss. Both siblings later developed ventilator-dependent respiratory insufficiency, scoliosis and are currently nonverbal and non-ambulatory. Extensive molecular testing including oligo array and clinical exome sequencing was non-diagnostic. Research genome sequencing under an IRB-approved study protocol revealed that both affected children were compound-heterozygous for variants in the SEPSECS gene. One variant was an initiator codon change (c.1A>T) that disrupted protein translation, consistent with the observation that most disease-causing variants are loss-of-function changes. The other variant was a coding change (c.846G>A) that was predicted to be synonymous but had been demonstrated to disrupt mRNA splicing in a minigene assay. SEPSECS gene encodes O-phosphoseryl-tRNA(Sec) selenium transferase; an enzyme that participates in the biosynthesis and transport of selenoproteins in the body. Variations in SEPSECS cause autosomal recessive pontocerebellar hypoplasia type 2D (PCHT 2D; OMIM #613811), a neurodegenerative condition characterized by progressive cerebrocerebellar atrophy, microcephaly, and epileptic encephalopathy. The identification of biallelic pathogenic variants in this family- one of which was a synonymous change not identified by prior clinical testing- not only ended the diagnostic odyssey for this family, but also highlights the contribution of occult pathogenic variants that may not be recognized by standard genetic testing methodologies.

Bladder cancer is the sixth most common cancer in the United States, and it exhibits an alarming 70% recurrence rate. Thus, the development of more efficient antibladder cancer approaches is a high priority. Accordingly, this work provides the basis for a transformative anticancer strategy that takes advantage of the unique characteristics of the bladder. Unlike mucin-shielded normal bladder cells, cancer cells are exposed to the bladder lumen and overexpress EGFR. Therefore, we used an EGF-conjugated anthrax toxin that after targeting EGFR was internalized and triggered apoptosis in exposed bladder cancer cells. This unique agent presented advantages over other EGF-based technologies and other toxin-derivatives. In contrast to known agents, this EGF-toxin conjugate promoted its own uptake via receptor microclustering even in the presence of Her2 and induced cell death with a LC(50) < 1 nM. Furthermore, our data showed that exposures as short as ≈3 min were enough to commit human (T24), mouse (MB49) and canine (primary) bladder cancer cells to apoptosis. Exposure of tumor-free mice and dogs with the agent resulted in no toxicity. In addition, the EGF-toxin was able to eliminate cells from human patient tumor samples. Importantly, the administration of EGF-toxin to dogs with spontaneous bladder cancer, who had failed or were not eligible for other therapies, resulted in ~30% average tumor reduction after one treatment cycle. Because of its in vitro and in vivo high efficiency, fast action (reducing treatment time from hours to minutes) and safety, we propose that this EGF–anthrax toxin conjugate provides the basis for new, transformative approaches against bladder cancer.

Lowe syndrome is an X-linked condition characterized by congenital cataracts, neurological abnormalities and kidney malfunction. This lethal disease is caused by mutations in the OCRL1 gene, which encodes for the phosphatidylinositol 5-phosphatase Ocrl1. While in the past decade we witnessed substantial progress in the identification and characterization of LS patient cellular phenotypes, many of these studies have been performed in knocked-down cell lines or patient's cells from accessible cell types such as skin fibroblasts, and not from the organs affected. This is partially due to the limited accessibility of patient cells from eyes, brain and kidneys. Here we report the preparation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts and their reprogramming into kidney cells. These reprogrammed kidney cells displayed primary cilia assembly defects similar to those described previously in cell lines. Additionally, the transcription factor and cap mesenchyme marker Six2 was substantially retained in the Golgi complex and the functional nuclear-localized fraction was reduced. These results were confirmed using different batches of differentiated cells from different iPSC colonies and by the use of the human proximal tubule kidney cell line HK2. Indeed, OCRL1 KO led to both ciliogenesis defects and Six2 retention in the Golgi complex. In agreement with Six2's role in the suppression of ductal kidney lineages, cells from this pedigree were over-represented among patient kidney-reprogrammed cells. We speculate that this diminished efficacy to produce cap mesenchyme cells would cause LS patients to have difficulties in replenishing senescent or damaged cells derived from this lineage, particularly proximal tubule cells, leading to pathological scenarios such as tubular atrophy.

Lowe syndrome is a lethal X-linked genetic disorder characterized by congenital cataracts, mental retardation, and kidney dysfunction. It is caused by mutations in the OCRL1 (oculocerebrorenal syndrome of Lowe) gene that encodes a phosphatidylinositol 5-phosphatase (EC 3.1.3.36). The gene product Ocrl1 has been linked to a multitude of functions due to the central role played by phosphoinositides in signaling. Moreover, this protein also has the ability to bind Rho GTPases, the master regulators of the actin cytoskeleton, and to interact with elements of the vesicle trafficking machinery. It is currently under investigation how deficiencies in Ocrl1 affect these different processes and contribute to patient symptoms. This chapter outlines the known physiological roles of Ocrl1 which might be relevant to the mechanism underlying Lowe syndrome.

Lowe syndrome is a lethal X-linked genetic disorder characterized by congenital cataracts, mental retardation, and kidney dysfunction. It is caused by mutations in the OCRL1 (oculocerebrorenal syndrome of Lowe) gene that encodes a phosphatidylinositol 5-phosphatase (EC 3.1.3.36). The gene product Ocrl1 has been linked to a multitude of functions due to the central role played by phosphoinositides in signaling. Moreover, this protein also has the ability to bind Rho GTPases, the master regulators of the actin cytoskeleton, and to interact with elements of the vesicle trafficking machinery. It is currently under investigation how deficiencies in Ocrl1 affect these different processes and contribute to patient symptoms. This chapter outlines the known physiological roles of Ocrl1 which might be relevant to the mechanism underlying Lowe syndrome.

Background. Renal cell carcinoma (RCC) is characterized by lack of early warning signs, diverse clinical manifestations, absence of a reliable diagnostic and predictive biomarker, and resistance to targeted therapy. Thus, novel approaches for diagnosis, management and treatment of RCC are urgently needed. Kidney Injury Molecule-1 (KIM-1) is not expressed in normal kidney tissues but markedly up-regulated in dedifferentiated proximal tubular epithelial cells following renal injury. We demonstrated that KIM-1 is expressed ubiquitoulsy by both primary and metastatic lesions of clear cell and papillary renal cell carcinoma, while barely detectable in other subtypes of RCC. Additionally, the ectodomain of KIM-1 is cleaved by matrix metalloproteinases and sheds into the surrounding milieu. Methods. KIM-1 was stably transfected in immortalized renal tubular epithelial cells (LLC-PK1, gain-of-function) and KIM-1 was downmodulated in RCC cell lines (loss-of-function) using a siRNA lentiviral approach. KIM-1 function was assessed using these two systems. Results. Stable expression of KIM-1 in non-malignant renal epithelial cells increased cell proliferation, migration and invasion potential, induced anchorage independent growth, and increased the secretion of tumor promoting and angiogenic factors including VEGF, Ang2, TGF beta and IL-6 (PK1-KIM-1); while depletion of KIM-1 in RCC cells led to G1 cell cycle arrest phase and senescence. Recently, we and others have demonstrated that KIM-1 is a phosphatildyl serine receptor and the expression of KIM-1 converts a normal epithelial cell into a “semi professional phagocyte” and facilitates the removal of apoptotic and necrotic cells by recognizing phosphotidyl serine (PS) on their cell surface. Our data suggests that during this process, tumor epithelial cells undergo reprogramming and secrete and activate cytokines involved in cell survival and angiogenesis programs. Conclusions. The present study shows that KIM-1-expression promotes tumorigenesis primarily by two distinct mechanisms: i) by directly conferring oncogenic properties to renal epithelial cells; ii) by resulting in production of a variety of chemokines and cytokines that support tumor progression and angiogenesis. We believe that our findings can significantly contribute to the development of novel therapeutic apporaches and in the advancement of management strategies in RCC and help reduce associated mortality and morbidity. Citation Format: Venkata Sabbisetti, Rupal Bhatt, Swetha Ramadesikan, Joseph Bonventre. Kidney injury molecule-1: a novel therapeutic target in renal cell carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2013-5195 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.