Matthew F. Starost

Mutation in the key enzyme of sialic acid biosynthesis causes severe glomerular proteinuria and is rescued by N -acetylmannosamine

Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

Deafness and Stria Vascularis Defects in S1P2 Receptor-null Mice

The S1P2 receptor is a member of a family of G protein-coupled receptors that bind the extracellular sphingolipid metabolite sphingosine 1-phosphate with high affinity. The receptor is widely expressed and linked to multiple G protein signaling pathways, but its physiological function has remained elusive. Here we have demonstrated that S1P2 receptor expression is essential for proper functioning of the auditory and vestibular systems. Auditory brainstem response analysis revealed that S1P2 receptor-null mice were deaf by one month of age. These null mice exhibited multiple inner ear pathologies. However, some of the earliest cellular lesions in the cochlea were found within the stria vascularis, a barrier epithelium containing the primary vasculature of the inner ear. Between 2 and 4 weeks after birth, the basal and marginal epithelial cell barriers and the capillary bed within the stria vascularis of the S1P2 receptor-null mice showed markedly disturbed structures. JTE013, an S1P2 receptor-specific antagonist, blocked the S1P-induced vasoconstriction of the spiral modiolar artery, which supplies blood directly to the stria vascularis and protects its capillary bed from high perfusion pressure. Vascular disturbance within the stria vascularis is a potential mechanism that leads to deafness in the S1P2 receptor-null mice.

TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is a clonal disease that evolves through the accrual of genetic rearrangements and/or mutations within the dominant clone. The TEL-AML1 (ETV6-RUNX1) fusion in precursor-B (pre-B) ALL is the most common genetic rearrangement in childhood cancer; however, the cellular origin and the molecular pathogenesis of TEL-AML1-induced leukemia have not been identified. To study the origin of TEL-AML1-induced ALL, we generated transgenic zebrafish expressing TEL-AML1 either ubiquitously or in lymphoid progenitors. TEL-AML1 expression in all lineages, but not lymphoid-restricted expression, led to progenitor cell expansion that evolved into oligoclonal B-lineage ALL in 3% of the transgenic zebrafish. This leukemia was transplantable to conditioned wild-type recipients. We demonstrate that TEL-AML1 induces a B cell differentiation arrest, and that leukemia development is associated with loss of TEL expression and elevated Bcl2/Bax ratio. The TEL-AML1 transgenic zebrafish models human pre-B ALL, identifies the molecular pathways associated with leukemia development, and serves as the foundation for subsequent genetic screens to identify modifiers and leukemia therapeutic targets.

Inflammatory cardiac valvulitis in TAX1BP1‐deficient mice through selective NF‐κB activation

Nuclear factor kappa B (NF‐κB) is a key mediator of inflammation. Unchecked NF‐κB signalling can engender autoimmune pathologies and cancers. Here, we show that Tax1‐binding protein 1 (TAX1BP1) is a negative regulator of TNF‐α‐ and IL‐1β‐induced NF‐κB activation and that binding to mono‐ and polyubiquitin by a ubiquitin‐binding Zn finger domain in TAX1BP1 is needed for TRAF6 association and NF‐κB inhibition. Mice genetically knocked out for TAX1BP1 are born normal, but develop age‐dependent inflammatory cardiac valvulitis, die prematurely, and are hypersensitive to low doses of TNF‐α and IL‐1β. TAX1BP1−/− cells are more highly activated for NF‐κB than control cells when stimulated with TNF‐α or IL‐1β. Mechanistically, TAX1BP1 acts in NF‐κB signalling as an essential adaptor between A20 and its targets.

Accumulation of the inner nuclear envelope protein Sun1 is pathogenic in progeric and dystrophic laminopathies.

Human LMNA gene mutations result in laminopathies that include Emery-Dreifuss muscular dystrophy (AD-EDMD) and Hutchinson-Gilford progeria, the premature aging syndrome (HGPS). The Lmna null (Lmna(-/-)) and progeroid LmnaΔ9 mutant mice are models for AD-EDMD and HGPS, respectively. Both animals develop severe tissue pathologies with abbreviated life spans. Like HGPS cells, Lmna(-/-) and LmnaΔ9 fibroblasts have typically misshapen nuclei. Unexpectedly, Lmna(-/-) or LmnaΔ9 mice that are also deficient for the inner nuclear membrane protein Sun1 show markedly reduced tissue pathologies and enhanced longevity. Concordantly, reduction of SUN1 overaccumulation in LMNA mutant fibroblasts and in cells derived from HGPS patients corrected nuclear defects and cellular senescence. Collectively, these findings implicate Sun1 protein accumulation as a common pathogenic event in Lmna(-/-), LmnaΔ9, and HGPS disorders.

IDEC‐131 (Anti‐CD154), Sirolimus and Donor‐Specific Transfusion Facilitate Operational Tolerance in Non‐Human Primates

CD154‐specific antibody therapy prevents allograft rejection in many experimental transplant models. However, initial clinical transplant trials with anti‐CD154 have been disappointing suggesting the need for as of yet undetermined adjuvant therapy. In rodents, donor antigen (e.g., a donor blood transfusion), or mTOR inhibition (e.g., sirolimus), enhances anti‐CD154s efficacy. We performed renal transplants in major histocompatibility complex‐(MHC) mismatched rhesus monkeys and treated recipients with combinations of the CD154‐specific antibody IDEC‐131, and/or sirolimus, and/or a pre‐transplant donor‐specific transfusion (DST). Therapy was withdrawn after 3 months. Triple therapy prevented rejection during therapy in all animals and led to operational tolerance in three of five animals including donor‐specific skin graft acceptance in the two animals tested. IDEC‐131, sirolimus and DST are highly effective in preventing renal allograft rejection in primates. This apparently clinically applicable regimen is promising for human renal transplant trials.

Identification of Small Molecules That Suppress MicroRNA Function and Reverse Tumorigenesis

MicroRNAs (miRNAs) act in post-transcriptional gene silencing and are proposed to function in a wide spectrum of pathologies, including cancers and viral diseases. Currently, to our knowledge, no detailed mechanistic characterization of small molecules that interrupt miRNA pathways have been reported. In screening a small chemical library, we identified compounds that suppress RNA interference activity in cultured cells. Two compounds were characterized; one impaired Dicer activity while the other blocked small RNA-loading into an Argonaute 2 (AGO2) complex. We developed a cell-based model of miRNA-dependent tumorigenesis, and using this model, we observed that treatment of cells with either of the two compounds effectively neutralized tumor growth. These findings indicate that miRNA pathway-suppressing small molecules could potentially reverse tumorigenesis.

Identification and Characterization of SSTK, a Serine/Threonine Protein Kinase Essential for Male Fertility

ABSTRACT Here we describe and characterize a small serine/threonine kinase (SSTK) which consists solely of the N- and C-lobes of a protein kinase catalytic domain. SSTK protein is highly conserved among mammals, and no close homologues were found in the genomes of nonmammalian organisms. SSTK specifically interacts with HSP90-1β, HSC70, and HSP70 proteins, and this association appears to be required for SSTK kinase activity. The SSTK transcript was most abundant in human and mouse testes but was also detected in all human tissues tested. In the mouse testis, SSTK protein was localized to the heads of elongating spermatids. Targeted deletion of the SSTK gene in mice resulted in male sterility due to profound impairment in motility and morphology of spermatozoa. A defect in DNA condensation in SSTK null mutants occurred in elongating spermatids at a step in spermiogenesis coincident with chromatin displacement of histones by transition proteins. SSTK phosphorylated histones H1, H2A, H2AX, and H3 but not H2B or H4 or transition protein 1 in vitro. These results demonstrate that SSTK is required for proper postmeiotic chromatin remodeling and male fertility. Abnormal sperm chromatin condensation is common in sterile men, and our results may provide insight into the molecular mechanisms underlying certain human infertility disorders.

Xanthine Oxidoreductase Is an Endogenous Regulator of Cyclooxygenase-2

Xanthine oxidoreductase (XOR) is the enzyme responsible for the final step in purine degradation resulting in the generation of uric acid. Here we have generated mice deficient in XOR. As expected, these animals lack tissue XOR activity and have low to undetectable serum levels of uric acid. Although normal at birth, XOR−/− mice fail to thrive after 10 to 14 days, and most die within the first month. The cause of death appears to be a form of severe renal dysplasia, a phenotype that closely resembles what has been observed previously in cyclooxygenase-2 (COX-2)–deficient mice. We further demonstrate that in the first month of life, a period in which the mouse kidney is undergoing rapid maturation and remodeling, wild-type mice exhibit an ≈30-fold increase in renal XOR activity, with a corresponding induction of COX-2 expression. In contrast, during this same period, XOR−/− animals fail to augment renal COX-2 expression. Finally, we show that in vitro and in vivo, uric acid can stimulate basal COX-2 expression. These results demonstrate that XOR activity is an endogenous physiological regulator of COX-2 expression and thereby provide insight into previous epidemiological evidence linking elevated serum uric levels with systemic hypertension and increased mortality from cardiovascular diseases. In addition, these results suggest a novel molecular link between cellular injury and the inflammatory response.

Ring Expanded Nucleoside Analogues Inhibit RNA Helicase and Intracellular Human Immunodeficiency virus type 1 Replication

A series of ring expanded nucleoside (REN) analogues were synthesized and screened for inhibition of cellular RNA helicase activity and human immunodeficiency virus type 1 (HIV-1) replication. We identified two compounds, 1 and 2, that inhibited the ATP dependent activity of human RNA helicase DDX3. Compounds 1 and 2 also suppressed HIV-1 replication in T cells and monocyte-derived macrophages. Neither compound at therapeutic doses was significantly toxic in ex vivo cell culture or in vivo in mice. Our findings provide proof-of-concept that a cellular factor, an RNA helicase, could be targeted for inhibiting HIV-1 replication.