Faculty Bibliography

Autophagy is a lysosomal degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and protein aggregates. These substrates reach lysosomes by several distinct mechanisms, including delivery within endosomes as well as autophagosomes. Completion of digestion involves dynamic interactions among compartments of the autophagic and endocytic pathways. Neurons are particularly vulnerable to disruptions of these interactions, especially as the brain ages. Not surprisingly, mutations of genes regulating autophagy cause neurodegenerative diseases across the age spectrum with exceptional frequency. In late-onset disorders such as Alzheimer's disease, amyotrophic lateral sclerosis and familial Parkinson's disease, defects arise at different stages of the autophagy pathway and have different implications for pathogenesis and therapy. This Review provides an overview of the role of autophagy in neurodegenerative disease, focusing particularly on less frequently considered lysosomal clearance mechanisms and their considerable impact on disease. Various therapeutic strategies for modulating specific stages of autophagy and the current state of drug development for this purpose are also evaluated.

Mechanical factors such as strain, pressure, and shear stress are key regulators of cell function, but the molecular mechanisms underlying the detection and responses to such stimuli are poorly understood. Whether the angiotensin II (AngII) AT1 receptor (AT1R) transduces shear stress in endothelial cells (ECs) is unknown. We exposed human umbilical cord endothelial cells (HUVECs) to a shear stress of 0 (control) or 15 dyn/cm(2) for 5 or 10 min. The colocalization of AT1R with caveolin-1 (Cav1), endosomal markers Rab5, EEA1, and Rab7, and lysosomal marker Lamp-1 increased in shear stimulated cells, detected by immunocytochemistry. Shear stress reduced labeling of wild-type mouse ECs (18+/-3% of unsheared control, P<0.01) but not Cav1(-/-) ECs (90+/-10%) with fluorescent AngII, confirming that internalization of AT1R requires Cav1. Shear stress activated ERK1/2 2-fold (P<0.01), which was prevented by the AT1R blocker losartan. NADPH oxidase inhibition with apocynin prevented both the colocalization of AT1R with Cav1 and the induction of ERK1/2 by shear stress. Moreover, shear-dependent ERK1/2 activation was minimal in CHO cells expressing an AT1Ra mutant that does not internalize, compared with cells expressing wild-type AT1Ra (P<0.05). Hence, AT1R may be an important transducer of shear stress-dependent activation of ERK1/2.

TGFbeta is secreted in a latent state and must be "activated" by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFbeta mobilisation in vivo. Here we review the process of TGFbeta secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFbeta is secreted, sequestered and released. This allows the separation of more ancient TGFbeta activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.

Stem cells embody the tremendous potential of the human body to develop, grow, and repair throughout life. Understanding the biologic mechanisms that underlie stem cell-mediated tissue regeneration is key to harnessing this potential. Recent advances in molecular biology, genetic engineering, and material science have broadened our understanding of stem cells and helped bring them closer to widespread clinical application. Specifically, innovative approaches to optimize how stem cells are identified, isolated, grown, and utilized will help translate these advances into effective clinical therapies. Although there is growing interest in stem cells worldwide, this enthusiasm must be tempered by the fact that these treatments remain for the most part clinically unproven. Future challenges include refining the therapeutic manipulation of stem cells, validating these technologies in randomized clinical trials, and regulating the global expansion of regenerative stem cell therapies.

Background: Retinoic acid (RA) signaling plays a critical role in vertebrate development. Transcriptional reporters of RA signaling in zebrafish, thus far, have not reflected the broader availability of embryonic RA, necessitating additional tools to enhance our understanding of the spatial and temporal activity of RA signaling in vivo. Results: We have generated novel transgenic RA sensors in which a RA receptor (RAR) ligand-binding domain (RLBD) is fused to the Gal4 DNA binding domain (GDBD) or a VP16-GDBD (VPBD) construct. Stable transgenic lines expressing these proteins when crossed with UAS reporter lines are responsive to RA. Interestingly, the VPBD RA sensor is significantly more sensitive than the GDBD sensor and demonstrates there may be almost ubiquitous availability of RA within the early embryo. Using confocal microscopy to compare the expression of the GDBD RA sensor to our previously established RA signaling transcriptional reporter line, Tg(12XRARE:EGFP), illustrates these reporters have significant overlap, but that expression from the RA sensor is much broader. We also identify previously unreported domains of expression for the Tg(12XRARE:EGFP) line. Conclusions: Our novel RA sensor lines will be useful and complementary tools for studying RA signaling during development and anatomical structures independent of RA signaling. Developmental Dynamics, 2013. (c) 2013 Wiley Periodicals,Inc.

We sought to identify fibroblast growth factor receptor 2 (FGFR2) kinase domain mutations that confer resistance to the pan-FGFR inhibitor, dovitinib, and explore the mechanism of action of the drug-resistant mutations. We cultured BaF3 cells overexpressing FGFR2 in high concentrations of dovitinib and identified 14 dovitinib-resistant mutations, including the N550K mutation observed in 25% of FGFR2(mutant) endometrial cancers (ECs). Structural and biochemical in vitro kinase analyses, together with BaF3 proliferation assays, showed that the resistance mutations elevate the intrinsic kinase activity of FGFR2. BaF3 lines were used to assess the ability of each mutation to confer cross-resistance to PD173074 and ponatinib. Unlike PD173074, ponatinib effectively inhibited all the dovitinib-resistant FGFR2 mutants except the V565I gatekeeper mutation, suggesting ponatinib but not dovitinib targets the active conformation of FGFR2 kinase. EC cell lines expressing wild-type FGFR2 were relatively resistant to all inhibitors, whereas EC cell lines expressing mutated FGFR2 showed differential sensitivity. Within the FGFR2(mutant) cell lines, three of seven showed marked resistance to PD173074 and relative resistance to dovitinib and ponatinib. This suggests that alternative mechanisms distinct from kinase domain mutations are responsible for intrinsic resistance in these three EC lines. Finally, overexpression of FGFR2(N550K) in JHUEM-2 cells (FGFR2(C383R)) conferred resistance (about five-fold) to PD173074, providing independent data that FGFR2(N550K) can be associated with drug resistance. Biochemical in vitro kinase analyses also show that ponatinib is more effective than dovitinib at inhibiting FGFR2(N550K). We propose that tumors harboring mutationally activated FGFRs should be treated with FGFR inhibitors that specifically bind the active kinase.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Abeta42 and gamma-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including alpha-synuclein (alpha-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on alpha-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with alpha-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of alpha-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate alpha-syn accumulation in transgenic mouse models of alpha-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Metabolic diseases are characterized by the failure of regulatory genes or proteins to effectively orchestrate specific pathways involved in the control of many biological processes. In addition to the classical regulators, recent discoveries have shown the remarkable role of small non-coding RNAs (microRNAs) in the post-transcriptional regulation of gene expression. In this regard, we have recently demonstrated that miR-33a/b, intronic microRNAs (miRNA) located within the sterol regulatory element-binding protein (SREBP) genes, regulate lipid metabolism in concert with their host genes. Here, we show that miR-33b also cooperates with SREBP1 in regulating glucose metabolism by targeting phosphoenolpyruvate carboxykynase (PCK1) and glucose-6-phosphatase (G6PC), key regulatory enzymes of hepatic gluconeogenesis. Overexpression of miR-33b in human hepatic cells inhibits PCK1 and G6PC expression leading to a significant reduction of glucose production. Importantly, hepatic SREBP1c/miR-33b levels correlate inversely with the expression PCK1 and G6PC upon glucose infusion in rhesus monkeys. Taken together, these results suggest that miR-33b works in concert with its host gene to ensure a fine-tuned regulation of lipid and glucose homeostasis, highlighting the clinical potential of miR-33a/b as novel therapeutic targets for a range of metabolic diseases.