Modulating native GABAA receptors in medulloblastoma with positive allosteric benzodiazepine-derivatives induces cell death.

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.

Rapid discrimination of pediatric brain tumors by mass spectrometry imaging.

PURPOSE: Medulloblastoma, the most common primary pediatric malignant brain tumor, originates in the posterior fossa of the brain. Pineoblastoma, which originates within the pineal gland, is a rarer malignancy that also presents in the pediatric population. Medulloblastoma and pineoblastoma exhibit overlapping clinical features and have similar histopathological characteristics. Histopathological similarities confound rapid diagnoses of these two tumor types. We have conducted a pilot feasibility study analyzing the molecular profile of archived frozen human tumor specimens using mass spectrometry imaging (MSI) to identify potential biomarkers capable of classifying and distinguishing between medulloblastoma and pineoblastoma. METHODS: We performed matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry imaging on eight medulloblastoma biopsy specimens and three pineoblastoma biopsy specimens. Multivariate statistical analyses were performed on the MSI dataset to generate classifiers that distinguish the two tumor types. Lastly, the molecules that were discriminative of tumor type were queried against the Lipid Maps database and identified. RESULTS: In this pilot study we show that medulloblastoma and pineoblastoma can be discriminated using molecular profiles determined by MSI. The highest-ranking discriminating classifiers of medulloblastoma and pineoblastoma were glycerophosphoglycerols and sphingolipids, respectively. CONCLUSION: We demonstrate proof-of-concept that medulloblastoma and pineoblastoma can be rapidly distinguished by using MSI lipid profiles. We identified biomarker candidates capable of distinguishing these two histopathologically similar tumor types. This work expands the current molecular knowledge of medulloblastoma and pineoblastoma by characterizing their lipidomic profiles, which may be useful for developing novel diagnostic, prognostic and therapeutic strategies.

Transcript profile of cellular senescence-related genes in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a genetically heterogeneous disease. Hypothesizing that cellular senescence may be relevant in FECD pathogenesis, genetically undifferentiated late-onset FECD endothelial samples were analyzed to identify common changes of specific senescence-related transcripts. Total RNA was extracted from 21 FECD endothelial samples retrieved from patients undergoing lamellar keratoplasty due to clinically diagnosed end-stage FECD and from 12 endothelial samples retrieved from normal autopsy eyes. Taqman low density array (TLDA) cards were used to analyze differential expression of 89 cellular senescence-related transcripts. Result validation was performed using individual real-time PCR assays. TLDA-analysis demonstrated differential expression of 31 transcripts (fold-change >1.5; p < 0.05). Thereof, 27 showed significant up-regulation and 4 significant down-regulation. Markedly elevated mRNA-levels of the constitutively active and reactive oxygen species-generating enzyme NOX4 were found in all evaluable FECD samples. In addition, increased expression of CDKN2A and its transcriptional activators ETS1 and ARHGAP18 (SENEX) along with decreased expression of CDKN2A inhibitor ID1 were detected in FECD samples. Consistent over-expression of NOX4 in FECD endothelial samples suggests a role as pathogenic factor and as a potential new treatment target in FECD. Transcriptional up-regulation of the CDKN2A-pathway provides further evidence for increased cellular senescence in FECD endothelium.

GABA(A) Receptor Activation Drives GABARAP-Nix Mediated Autophagy to Radiation-Sensitize Primary and Brain-Metastatic Lung Adenocarcinoma Tumors.

In non-small cell lung cancer (NSCLC) treatment, radiotherapy responses are not durable and toxicity limits therapy. We find that AM-101, a synthetic benzodiazepine activator of GABA(A) receptor, impairs the viability and clonogenicity of both primary and brain-metastatic NSCLC cells. Employing a human-relevant ex vivo 'chip', AM-101 is as efficacious as docetaxel, a chemotherapeutic used with radiotherapy for advanced-stage NSCLC. In vivo, AM-101 potentiates radiation, including conferring a significant survival benefit to mice bearing NSCLC intracranial tumors generated using a patient-derived metastatic line. GABA(A) receptor activation stimulates a selective-autophagic response via the multimerization of GABA(A) receptor-associated protein, GABARAP, the stabilization of mitochondrial receptor Nix, and the utilization of ubiquitin-binding protein p62. A high-affinity peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis that triggers autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity.

Unconventional chemiosmotic coupling of NHA2, a mammalian Na+/H+ antiporter, to a plasma membrane H+ gradient.

Human NHA2, a newly discovered cation proton antiporter, is implicated in essential hypertension by gene linkage analysis. We show that NHA2 mediates phloretin-sensitive Na(+)-Li(+) counter-transport (SLC) activity, an established marker for hypertension. In contrast to bacteria and fungi where H(+) gradients drive uptake of metabolites, secondary transport at the plasma membrane of mammalian cells is coupled to the Na(+) electrochemical gradient. Our findings challenge this paradigm by showing coupling of NHA2 and V-type H(+)-ATPase at the plasma membrane of kidney-derived MDCK cells, resulting in a virtual Na(+) efflux pump. Thus, NHA2 functionally recapitulates an ancient shared evolutionary origin with bacterial NhaA. Although plasma membrane H(+) gradients have been observed in some specialized mammalian cells, the ubiquitous tissue distribution of NHA2 suggests that H(+)-coupled transport is more widespread. The coexistence of Na(+) and H(+)-driven chemiosmotic circuits has implications for salt and pH regulation in the kidney.

Screening Ion Channels in Cancer Cells.

Ion channels are critical for cell development and maintaining cell homeostasis. The perturbation of ion channel function contributes to the development of a broad range of disorders or channelopathies. Cancer cells utilize ion channels to drive their own development, as well as to improve as a tumor and to assimilate in a microenvironment that includes various non-cancerous cells. Furthermore, increases in levels of growth factors and hormones within the tumor microenvironment can result in enhanced ion channel expression, which contributes to cancer cell proliferation and survival. Thus, the pharmacological targeting of ion channels is potentially a promising approach to treating solid malignancies, including primary and metastatic brain cancers. Herein, protocols to characterize the function of ion channels in cancerous cells and approaches to analyze modulators of ion channels to determine their impact on cancer viability are described. These include staining a cell(s) for an ion channel(s), testing the polarized state of mitochondria, establishing ion channel function using electrophysiology, and performing viability assays to assess drug potency.

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity. Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation.

The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.

Therapeutically leveraging GABAA receptors in cancer.

γ-aminobutyric acid or GABA is an amino acid that functionally acts as a neurotransmitter and is critical to neurotransmission. GABA is also a metabolite in the Krebs cycle. It is therefore unsurprising that GABA and its receptors are also present outside of the central nervous system, including in immune cells. This observation suggests that GABAergic signaling impacts events beyond brain function and possibly human health beyond neurological disorders. Indeed, GABA receptor subunits are expressed in pathological disease states, including in disparate cancers. The role that GABA and its receptors may play in cancer development and progression remains unclear. If, however, those cancers have functional GABA receptors that participate in GABAergic signaling, it raises an important question whether these signaling pathways might be targetable for therapeutic benefit. Herein we summarize the effects of modulating Type-A GABA receptor signaling in various cancers and highlight how Type-A GABA receptors could emerge as a novel therapeutic target in cancer.

Reuse of Molecules for Glioblastoma Therapy.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. The current standard of care for GBM is the Stupp protocol which includes surgical resection, followed by radiotherapy concomitant with the DNA alkylator temozolomide; however, survival under this treatment regimen is an abysmal 12-18 months. New and emerging treatments include the application of a physical device, non-invasive 'tumor treating fields' (TTFs), including its concomitant use with standard of care; and varied vaccines and immunotherapeutics being trialed. Some of these approaches have extended life by a few months over standard of care, but in some cases are only available for a minority of GBM patients. Extensive activity is also underway to repurpose and reposition therapeutics for GBM, either alone or in combination with the standard of care. In this review, we present select molecules that target different pathways and are at various stages of clinical translation as case studies to illustrate the rationale for their repurposing-repositioning and potential clinical use.

Melanoma Cell Intrinsic GABAA Receptor Enhancement Potentiates Radiation and Immune Checkpoint Inhibitor Response by Promoting Direct and T Cell-Mediated Antitumor Activity.

PURPOSE: Most patients with metastatic melanoma show variable responses to radiation therapy and do not benefit from immune checkpoint inhibitors. Improved strategies for combination therapy that leverage potential benefits from radiation therapy and immune checkpoint inhibitors are critical. METHODS AND MATERIALS: We analyzed metastatic melanoma tumors in the TCGA cohort for expression of genes coding for subunits of type A γ-aminobutyric acid (GABA) receptor (GABAAR), a chloride ion channel and major inhibitory neurotransmitter receptor. Electrophysiology was used to determine whether melanoma cells possess intrinsic GABAAR activity. Melanoma cell viability studies were conducted to test whether enhancing GABAAR mediated chloride transport using benzodiazepine-impaired viability. A syngeneic melanoma mouse model was used to assay the effect of benzodiazepine on tumor volume and its ability to potentiate radiation therapy or immunotherapy. Treated tumors were analyzed for changes in gene expression by RNA sequencing and presence of tumor-infiltrating lymphocytes by flow cytometry. RESULTS: Genes coding for subunits of GABAARs express functional GABAARs in melanoma cells. By enhancing GABAAR-mediated anion transport, benzodiazepines depolarize melanoma cells and impair their viability. In vivo, benzodiazepine alone reduces tumor growth and potentiates radiation therapy and α-PD-L1 antitumor activity. The combination of benzodiazepine, radiation therapy, and α-PD-L1 results in near complete regression of treated tumors and a potent abscopal effect, mediated by increased infiltration of polyfunctional CD8+ T cells. Treated tumors show expression of cytokine-cytokine receptor interactions and overrepresentation of p53 signaling. CONCLUSIONS: This study identifies an antitumor strategy combining radiation and/or an immune checkpoint inhibitor with modulation of GABAARs in melanoma using benzodiazepine.

Impact of Sequencing Radiation Therapy and Immune Checkpoint Inhibitors in the Treatment of Melanoma Brain Metastases.

PURPOSE: Melanoma brain metastases (MBM) occur in ∼50% of melanoma patients. Although both radiation therapy (RT) and immune checkpoint inhibitor (ICI) are used alone or in combination for MBM treatment, the role of this combination and how these treatments could best be sequenced remains unclear. METHODS AND MATERIALS: We conducted a retrospective analysis of patients with resected MBM who underwent treatment with RT, ICI, or a combination of RT and ICI. Among the latter, we specifically investigated the differential gene expression via RNA-sequencing between patients who received RT first then ICI (RT → ICI) versus ICI first then RT (ICI → RT). We used a glycoprotein-transduced syngeneic melanoma mouse model for validation experiments. RESULTS: We found that for patients with resected MBM, a combination of RT and ICI confers superior survival compared with RT alone. Specifically, we found that RT → ICI was superior compared with ICI → RT. Transcriptome analysis of resected MBM revealed that the RT → ICI cohort demonstrated deregulation of genes involved in apoptotic signaling and key modulators of inflammation that are most implicated in nuclear factor kappa-light-chain-enhancer of activated B cells signaling. In a preclinical model, we showed that RT followed by anti-programmed death-ligand 1 therapy was superior to the reverse sequence of therapy, supporting the observations we made in patients with MBM. CONCLUSIONS: Our study provides initial insights into the optimal sequence of RT and ICI in the treatment of MBM after surgical resection. Prospective studies examining the best sequence of RT and ICI are necessary, and our study contributes to the rationale to pursue these.

Vestibular hair bundles control pH with (Na+, K+)/H+ exchangers NHE6 and NHE9.

In hair cells of the inner ear, robust Ca2+/H+ exchange mediated by plasma-membrane Ca2+-ATPase would rapidly acidify mechanically sensitive hair bundles without efficient removal of H+. We found that, whereas the basolateral membrane of vestibular hair cells from the frog saccule extrudes H+ via an Na+-dependent mechanism, bundles rapidly remove H+ in the absence of Na+ and HCO3(-), even when the soma is acidified. K+ was fully effective and sufficient for H+ removal; in contrast, Rb+ failed to support pH recovery. Na+/H+-exchanger isoform 1 (NHE1) was present on hair-cell soma membranes and was likely responsible for Na+-dependent H+ extrusion. NHE6 and NHE9 are organellar isoforms that can appear transiently on plasma membranes and have been proposed to mediate K+/H+ exchange. We identified NHE6 in a subset of hair bundles; NHE9 was present in all bundles. Heterologous expression of these isoforms in yeast strains lacking endogenous exchangers conferred pH-dependent tolerance to high levels of KCl and NaCl. NHE9 preferred cations in the order K+, Na+ >> Rb+, consistent with the relative efficacies of these ions in promoting pH recovery in hair bundles. Electroneutral K+/H+ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K+ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the +80 mV endocochlear potential, and uses mechanisms already present in the ear for K+ recycling. This mechanism allows the hair cell to remove H+ generated by Ca2+ pumping without ATP hydrolysis in the cell.

Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking.

Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

MicroRNA-29b Overexpression Decreases Extracellular Matrix mRNA and Protein Production in Human Corneal Endothelial Cells.

PURPOSE: MicroRNAs are small noncoding RNAs that regulate gene expression at the posttranscriptional level. We reported that levels of microRNA (miR)-29 family are decreased in corneas of patients with Fuchs endothelial corneal dystrophy (FECD). The miR-29 family regulates the production of extracellular matrix (ECM) proteins. Accumulation of ECM proteins in Descemet membrane is an important pathologic change in FECD. In this study, we transfected miR-29b into human corneal endothelial cells and tissues and evaluated ECM protein expression levels. METHODS: An immortalized Fuchs human corneal endothelial cell line (iFECD) was established by infection of corneal endothelial cells from patients with FECD with hTERT lentivirus. MiR-29b was transfected into iFECD, and the expression levels of ECMs collagen type 1 alpha 1 (COL1A1), collagen type 4 alpha 1 (COL4A1), and laminin gamma 1 (LAMC1) were evaluated with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot. Expression level of LAMC1 protein in miR-29b-transfected donor corneal endothelium was also evaluated by Western blot. RESULTS: Compared with control, miR-29b expression level after transfection of iFECD was increased to 335.6% (±91.0%), and ECM expression levels were significantly decreased. Compared with control, qRT-PCR demonstrated reduction of ECM to the following levels: COL1A1: 1.9% (±0.4%); COL4A1: 7.1% (±1.7%); and LAMC1: 21.5% (±2.7%). Western blot showed reduced protein expression: COL1A1: 4.8% (±3.2%); COL4A1: 42.5% (±25.0%); and LAMC1: 44.8% (±3.1%). In miR-29b-transfected corneal tissue, LAMC1 protein expression level was decreased to 14.4% (±20.5%). CONCLUSIONS: Overexpression of miR-29b decreased ECM protein production in human corneal endothelial cells. Thus, miR-29 replacement therapy might be a new treatment strategy for FECD aimed at reducing pathologic production of ECM proteins in Descemet membrane.

Use of human serum for human corneal endothelial cell culture.

BACKGROUND/AIMS: To study human corneal endothelial cells (HCECs) cultured in vitro with human serum (HS) supplemented media (HS-SM) compared with HCEC cultured in fetal bovine serum (FBS) supplemented media (FBS-SM). METHODS: One cornea from a donor aged 21 years and a pair of corneas from a 16 year-old donor were obtained from the eye bank and used to create two different cell populations. At the first passage, the cell populations were equally divided and seeded in two different wells containing FBS-SM or HS-SM. In subsequent passages, HS-SM was compared with FBS-SM by morphology, growth curves, immunohistochemistry and real-time reverse-transcriptase PCR for endothelial cell markers. RESULTS: No difference in morphology could be seen in P2, P5 or in any other passages for cells grown in the two media. By growth curves, cell counts were similar in FBS-SM and HS-SM from days 1 to 5, with a trend towards higher cell counts in HS-SM at day 7. Cells grown in FBS-SM and HS-SM media showed similar expression of endothelial cell markers when assessed by immunohistochemistry and real-time reverse-transcriptase PCR. CONCLUSIONS: HS-SM was similar to FBS-SM for HCEC culture when assessed by cell morphology, proliferation and protein/gene expression.

Endothelial cell microRNA expression in human late-onset Fuchs' dystrophy.

PURPOSE: MicroRNAs (miRNAs) are a class of endogenous noncoding RNA and post transcriptionally modulate gene expression during development and disease. Our study investigated the differential miRNA expression in human Fuchs' endothelial corneal dystrophy (FECD) compared with normal endothelium to identify miRNA sequences that are involved in the pathogenesis of FECD. METHODS: Comparative miRNA expression profiles of endothelial samples obtained from FECD patients during lamellar corneal transplant surgery and from normal donor globes were generated using OpenArray plate technology. Differential expression of individual miRNAs was validated in the original and in independent samples using stem-loop RT qPCR assays. Expression of miRNA target genes was assessed using qPCR and tissue microarray (TMA) immunolabeling. RESULTS: Our results demonstrate downregulation of 87 miRNAs in FECD compared with normal endothelium (>3-fold change; P < 0.01). Correspondingly, DICER1, (encoding an endoribonuclease critical to miRNA biogenesis) showed a moderate but significant decrease in FECD samples (P < 0.05). Significant repression of three miR-29 family members (miR-29a-3p, miR-29b-2-5p, and miR-29c-5p) was paralleled by upregulation of their extracellular matrix associated mRNA targets collagen I and collagen IV. Tissue microarray immunolabeling showed histologically verifiable subendothelial collagen I and collagen IV deposition and increased endothelial laminin protein expression in FECD samples. CONCLUSIONS: The present study provides the first miRNA profile in FECD and normal endothelial cells and demonstrates widespread miRNA downregulation in FECD. Decreased endothelial expression of miR-29 family members may be associated with increased subendothelial extracellular matrix accumulation in FECD.

Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v)) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v) (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pH(v) under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v) dysregulation in a neo1(ts) mutant restored viability whereas cholesterol accumulation in human NPC1(-/-) fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

Scribble associates with two polarity proteins, Lgl2 and Vangl2, via distinct molecular domains.

Scribble (Scrib) is a large multi-domain cytoplasmic protein that was first identified through its requirement for the establishment of epithelial polarity. We tested the hypotheses that Scrib asssociates with the basolateral membrane via multiple domains, binds specific protein partners, and is part of a multimeric complex. We generated a series of EGFP-tagged Scrib fusion proteins and examined their membrane localizations in two types of polarized mammalian epithelial cells using biochemical and morphological approaches. We found that Scrib's Leucine-rich-repeat (LRR) and PDS-95/Discs Large/ZO-1 (PDZ) domains independently associate with the plasma membrane in both cell types. We identified multiple large Scrib complexes, demonstrated that Scrib and the cytoplasmic protein Lethal giant larvae2 (Lgl2) co-IP and that this association occurs via Scrib's LRR domain. Further, this report demonstrates that the membrane protein Vangl2 binds selectively to specific PDZ domains in Scrib. Our identification of Scrib's associations highlights its function in multiple biologic pathways and sets the stage for future identification of more proteins that must interact with Scrib's remaining domains. J. Cell. Biochem. 99: 647-664, 2006. (c) 2006 Wiley-Liss, Inc.