Faculty Bibliography

Dendrites are the primary sites on neurons for receiving and integrating inputs from their presynaptic partners. Defects in dendrite development perturb the formation of neural circuitry and impair information processing in the brain. Extracellular cues are important for shaping the dendritic morphogenesis, but the underlying molecular mechanisms are not well understood. In this study, we examined the role of ARMS (ankyrin repeat-rich membrane spanning protein), also known as Kidins220 (kinase D-interacting substrate of 220 kDa), previously identified as a downstream target of neurotrophin and ephrin receptors, in dendrite development. We report here that knockdown of ARMS/Kidins220 by in utero electroporation impairs dendritic branching in mouse cerebral cortex, and silencing of ARMS/Kidins220 in primary rat hippocampal neurons results in a significant decrease in the length, number, and complexity of the dendritic arbors. Overexpression of cell surface receptor tyrosine kinases, including TrkB and EphB2, in ARMS/Kidins220-deficient neurons can partially rescue the defective dendritic phenotype. More importantly, we show that PI3K (phosphoinositide-3-kinase)- and Akt-mediated signaling pathway is crucial for ARMS/Kidins220-dependent dendrite development. Furthermore, loss of ARMS/Kidins220 significantly reduced the clustering of EphB2 receptor signaling complex in neurons. Our results collectively suggest that ARMS/Kidins220 is a key player in organizing the signaling complex to transduce the extracellular stimuli to cellular responses during dendrite development.

Stromal responses elicited by early stage neoplastic lesions can promote tumor growth. However, the molecular mechanisms that underlie the early recruitment of stromal cells to sites of neoplasia remain poorly understood. Here, we demonstrate an oncogenic Kras(G12D)-dependent upregulation of GM-CSF in mouse pancreatic ductal epithelial cells (PDECs). An enhanced GM-CSF production is also observed in human PanIN lesions. Kras(G12D)-dependent production of GM-CSF in vivo is required for the recruitment of Gr1(+)CD11b(+) myeloid cells. The suppression of GM-CSF production inhibits the in vivo growth of Kras(G12D)-PDECs, and, consistent with the role of GM-CSF in Gr1(+)CD11b(+) mobilization, this effect is mediated by CD8(+) T cells. These results identify a pathway that links oncogenic activation to the evasion of antitumor immunity.

The housekeeping sarco(endo)plasmic reticulum Ca(2+) ATPase SERCA2b transports Ca(2+) across the endoplasmic reticulum membrane maintaining a vital Ca(2+) gradient. Compared with the muscle-specific isoforms SERCA2a and SERCA1a, SERCA2b houses an 11th transmembrane segment (TM11) and a short luminal extension (LE) at its C terminus (2b-tail). The 2b-tail imposes a 2-fold higher apparent Ca(2+) affinity and lower V(max). Previously, we assumed that LE is the sole functional region of the 2b-tail and that TM11 is a passive element providing an additional membrane passage. However, here we show that peptides corresponding to the TM11 region specifically modulate the activity of the homologous SERCA1a in co-reconstituted proteoliposomes and mimic the 2b-tail effect (i.e. lower V(max) and higher Ca(2+) affinity). Using truncated 2b-tail variants we document that TM11 regulates SERCA1a independently from LE, confirming that TM11 is a second, previously unrecognized functional region of the 2b-tail. A phylogenetic analysis further indicates that TM11 is the oldest and most conserved feature of the 2b-tail, found in the SERCA pump of all Bilateria, whereas LE is only present in Nematoda and vertebrates. Considering remarkable similarities with the Na(+),K(+)-ATPase alpha-beta interaction, we now propose a model for interaction of TM11 with TM7 and TM10 in the anchoring subdomain of the Ca(2+) pump. This model involves a TM11-induced helix bending of TM7. In conclusion, more than just a passive structural feature, TM11 acts as a genuine regulator of Ca(2+) transport through interaction with the pump.

RATIONALE: High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well-characterized. OBJECTIVE: This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol-requiring enzyme 1beta (Ire1beta) would affect the development of hyperlipidemia and atherosclerosis in Apoe(-/-) mice. METHODS AND RESULTS: We used Ire1beta-deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here, we show that Ire1b(-/-)/Apoe(-/-) mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, exhibit hyperlipidemia, and have higher levels of atherosclerotic plaques compared with Apoe(-/-) mice when fed chow and western diets. CONCLUSIONS: These studies indicate that Ire1beta regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the alpha-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organization. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayer. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in 3D culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker Scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, while Par-3 displayed cytoplasmic localization, suggesting incomplete apico-basal polarity. Despite membrane localization of E-cadherin and beta-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex, interdigitating membrane protrusions. Single cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces, but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes reformed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active, individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression may reflect partial recapitulation of a normal developmental program.

Homeostasis of the protein-folding environment in the endoplasmic reticulum (ER) is maintained by signal transduction pathways that collectively constitute an unfolded protein response (UPR). These affect bulk protein synthesis and thereby the levels of ER stress, but also culminate in regulated expression of specific mRNAs, such as that encoding the transcription factor ATF4. Mechanisms linking eukaryotic initiation factor 2 (eIF2) phosphorylation to control of unfolded protein load in the ER were elucidated more than 10 years ago, but recent work has highlighted the diversity of processes that impinge on eIF2 activity and revealed that there are multiple mechanisms by which changes in eIF2 activity can modulate the translation of individual mRNAs. In addition, the potential for affecting this step of translation initiation pharmacologically is becoming clearer. Furthermore, it is now clear that another strand of the UPR, controlled by the endoribonuclease inositol-requiring enzyme 1 (IRE1), also affects rates of protein synthesis in stressed cells and that its effector function, mediated by the transcription factor X-box-binding protein 1 (XBP1), is subject to important mRNA-specific translational regulation. These new insights into the convergence of translational control and the UPR will be reviewed here.

Genomewide-association studies have revealed that SNPs (single nucleotide polymorphisms) in FTO (fat mass and obesity-associated) are robustly associated with BMI (body mass index) and obesity. FTO is an Fe(II) 2-OG (2-oxoglutarate)-dependent dioxygenase that can demethylate 3-meT (3-methylthymine) in single-stranded DNA, as well as 3-meU (3-methyluracil) and N6-methyl adenosine in RNA. In the present paper we describe the development of an RNase-cleavage assay measuring the demethylation activity of FTO on 3-meU. RNase A cleaves at the 3'-end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, an RNA loop containing a single 3-meU as the only RNase A-cleavage site, a fluorescent reporter on one end and a quencher at the other end. FTO demethylation of the unique 3-meU enables RNase A cleavage, releasing the quencher and enabling a fluorescent signal. In the presence of excess RNase A, FTO activity is limiting to the development of fluorescent signal, which can be read continuously and is able to discriminate between wild-type and the catalytically dead R316Q FTO. 2-OG is a co-substrate of FTO and, as a metabolite in the citric acid cycle, is a marker of intracellular nutritional status. The assay described in the present paper was used to measure, for the first time, the K(m) of FTO for 2-OG. The K(m) of 2.88 muM is up to 10-fold lower than the estimated intracellular concentrations of 2-OG, rendering it unlikely that FTO functions as a sensor for 2-OG levels.

Aerobic metabolism is fundamental for almost all animal life. Cellular consumption of oxygen (O(2)) and production of carbon dioxide (CO(2)) signal metabolic states and physiological stresses. These respiratory gases are also detected as environmental cues that can signal external food quality and the presence of prey, predators and mates. In both contexts, animal nervous systems are endowed with mechanisms for sensing O(2)/CO(2) to trigger appropriate behaviors and maintain homeostasis of internal O(2)/CO(2). Although different animal species show different behavioral responses to O(2)/CO(2), some underlying molecular mechanisms and pathways that function in the detection of respiratory gases are fundamentally similar and evolutionarily conserved. Studies of Caenorhabditis elegans and Drosophila melanogaster have identified roles for cyclic nucleotide signaling and the hypoxia inducible factor (HIF) transcriptional pathway in mediating behavioral responses to respiratory gases. Understanding how simple invertebrate nervous systems detect respiratory gases to control behavior might reveal general principles common to nematodes, insects and vertebrates that function in the molecular sensing of respiratory gases and the neural control of animal behaviors.

ATP-binding cassette transporter A1 (ABCA1) is a cholesterol transporter that transfers excess cellular cholesterol onto lipid-poor apolipoproteins. Given its critical role in cholesterol homeostasis, ABCA1 has been studied as a therapeutic target for Alzheimer's disease. Transcriptional regulation of ABCA1 by liver X receptor has been well characterized. However, whether ABCA1 expression is regulated at the posttranscriptional level is largely unknown. Identification of a novel pathway that regulates ABCA1 expression may provide new strategy for regulating cholesterol metabolism and amyloid beta (Abeta) levels. Since ABCA1 has an unusually long 3' untranslated region, we investigated whether microRNAs could regulate ABCA1 expression. We identified miR-106b as a novel regulator of ABCA1 expression and Abeta metabolism. miR-106b significantly decreased ABCA1 levels and impaired cellular cholesterol efflux in neuronal cells. Furthermore, miR-106b dramatically increased levels of secreted Abeta by increasing Abeta production and preventing Abeta clearance. Alterations in Abeta production and clearance were rescued by expression of miR-106b-resistant ABCA1. Taken together, our data suggest that miR-106b affects Abeta metabolism by suppressing ABCA1 expression.