Faculty Bibliography

Mechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved alpha-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration

We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), encoded by nonstructural protein 5B (NS5B), is absolutely essential for the viral replication. Here we describe the development, characterization, and functional properties of the panel of monoclonal antibodies (mAbs) and specifically describe the mechanism of action of two mAbs inhibiting the NS5B RdRp activity. These mAbs recognize and bind to distinct linear epitopes in the fingers subdomain of NS5B. The mAb 8B2 binds the N-terminal epitope of the NS5B and inhibits both primer-dependent and de novo RNA synthesis. mAb 8B2 selectively inhibits elongation of RNA chains and enhances the RNA template binding by NS5B. In contrast, mAb 7G8 binds the epitope that contains motif G conserved in viral RdRps and inhibits only primer-dependent RNA synthesis by specifically targeting the initiation of RNA synthesis, while not interfering with the binding of template RNA by NS5B. To reveal the importance of the residues of mAb 7G8 epitope for the initiation of RNA synthesis, we performed site-directed mutagenesis and extensively characterized the functionality of the HCV RdRp motif G. Comparison of the mutation effects in both in vitro primer-dependent RdRp assay and cellular transient replication assay suggested that mAb 7G8 epitope amino acid residues are involved in the interaction of template-primer or template with HCV RdRp. The data presented here allowed us to describe the functionality of the epitopes of mAbs 8B2 and 7G8 in the HCV RdRp activity and suggest that the epitopes recognized by these mAbs may be useful targets for antiviral drugs.

Gap junctions provide a low-resistance pathway for cardiac electric propagation. The role of GJ regulation in arrhythmia is unclear, partly because of limited availability of pharmacological tools. Recently, we showed that a peptide called 'RXP-E' binds to the carboxyl terminal of connexin43 and prevents chemically induced uncoupling in connexin43-expressing N2a cells. Here, pull-down experiments show RXP-E binding to adult cardiac connexin43. Patch-clamp studies revealed that RXP-E prevented heptanol-induced and acidification-induced uncoupling in pairs of neonatal rat ventricular myocytes. Separately, RXP-E was concatenated to a cytoplasmic transduction peptide (CTP) for cytoplasmic translocation (CTP-RXP-E). The effect of RXP-E on action potential propagation was assessed by high-resolution optical mapping in monolayers of neonatal rat ventricular myocytes, containing approximately 20% of randomly distributed myofibroblasts. In contrast to control experiments, when heptanol (2 mmol/L) was added to the superfusate of monolayers loaded with CTP-RXP-E, action potential propagation was maintained, albeit at a slower velocity. Similarly, intracellular acidification (pH(i) 6.2) caused a loss of action potential propagation in control monolayers; however, propagation was maintained in CTP-RXP-E-treated cells, although at a slower rate. Patch-clamp experiments revealed that RXP-E did not prevent heptanol-induced block of sodium currents, nor did it alter voltage dependence or amplitude of Kir2.1/Kir2.3 currents. RXP-E is the first synthetic molecule known to: (1) bind cardiac connexin43; (2) prevent heptanol and acidification-induced uncoupling of cardiac gap junctions; and (3) preserve action potential propagation among cardiac myocytes. RXP-E can be used to characterize the role of gap junctions in the function of multicellular systems, including the heart

GroEL is an Escherichia coli chaperonin that is composed of two heptameric rings stacked back-to-back. GroEL assists protein folding with its cochaperonin GroES in an ATP-dependent manner in vitro and in vivo. However, it is still unclear whether GroES binds to both rings of GroEL simultaneously under physiological conditions. In this study, we monitored the GroEL-GroES interaction in the reaction cycle using fluorescence resonance energy transfer. We found that nearly equivalent amounts of symmetric GroEL-(GroES)(2) (football-shaped) complex and asymmetric GroEL-GroES (bullet-shaped) complex coexist during the functional reaction cycle. We also found that D398A, an ATP hydrolysis defective mutant of GroEL, forms a football-shaped complex with ATP bound to the two rings. Furthermore, we showed that ADP prevents the association of ATP to the trans-ring of GroEL, and as a consequence, the second GroES cannot bind to GroEL. Considering the concentrations of ADP and ATP in E. coli, ADP is expected to have a small effect on the inhibition of GroES binding to the trans-ring of GroEL in vivo. These results suggest that we should reconsider the chaperonin-mediated protein-folding mechanism that involves the football-shaped complex.

Induction of apoptosis by tensile forces is an important determinant of connective tissue destruction in osteoarthritis and periodontal diseases. We examined the role of molecular components of the unfolded protein response in force-induced apoptosis. Magnetic fields were used to apply tensile force through integrins to cultured fibroblasts bound with collagen-coated magnetite beads. Tensile force induced caspase 3 cleavage, DNA fragmentation, depolarization of mitochondria, and induction of CHOP10, all indicative of activation of apoptosis. Immunoblotting, immunocytochemistry, and release of Ca(2+) from the endoplasmic reticulum showed evidence for both physical and functional associations between bound beads and the endoplasmic reticulum. Force-induced apoptosis was not detected in PERK null cells, but reconstitution of wild-type PERK in PERK null cells restored the apoptotic response. Force-induced apoptosis did not require PKR, GCN2, eIF2alpha, or CHOP10. Furthermore, force more than 24 h did not activate other initiators of the unfolded protein response including IRE-1 and ATF6. However, force-induced activation of caspase 3 was dependent on caspase 9 but was independent of mitochondria. We conclude that force-induced apoptosis depends on a novel function of PERK that occurs in addition to its canonical role in the unfolded protein response

BACKGROUND: Inflammatory changes are a prominent feature of brains affected by Alzheimer's disease (AD). Activated glial cells release inflammatory cytokines which modulate the neurodegenerative process. These cytokines are encoded by genes representing several interleukins and TNFA, which are associated with AD. The gene coding for HLA-B associated transcript 1 (BAT1) lies adjacent to TNFA in the central major histocompatibility complex (MHC). BAT1, a member of the DEAD-box family of RNA helicases, appears to regulate the production of inflammatory cytokines associated with AD pathology. In the current study TNFA and BAT1 promoter polymorphisms were analysed in AD and control cases and BAT1 mRNA levels were investigated in brain tissue from AD and control cases. METHODS: Genotyping was performed for polymorphisms at positions -850 and -308 in the proximal promoter of TNFA and position -22 in the promoter of BAT1. These were investigated singly or in haplotypic association in a cohort of Australian AD patients with AD stratified on the basis of their APOE epsilon4 genotype. Semi-quantitative RT-PCR was also performed for BAT1 from RNA isolated from brain tissue from AD and control cases. RESULTS: APOE epsilon4 was associated with an independent increase in risk for AD in individuals with TNFA -850*2, while carriage of BAT1 -22*2 reduced the risk for AD, independent of APOE epsilon4 genotype. Semi-quantitative mRNA analysis in human brain tissue showed elevated levels of BAT1 mRNA in frontal cortex of AD cases. CONCLUSION: These findings lend support to the application of TNFA and BAT1 polymorphisms in early diagnosis or risk assessment strategies for AD and suggest a potential role for BAT1 in the regulation of inflammatory reactions in AD pathology.

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.