Nighttime restlessness and daytime drowsiness are common and early symptoms of Alzheimer's Disease (AD). This symptomology implicates dysfunctional biological timing, yet the role of the circadian system in AD pathogenesis is unknown. To evaluate the role of the circadian clock in amyloid-β (Aβ) dynamics and pathology, we used a mouse model of β-amyloidosis and disrupted circadian clock function either globally or locally in the brain via targeted deletion of the core clock gene Bmal1 Our results demonstrate that loss of central circadian rhythms leads to disruption of daily hippocampal interstitial fluid Aβ oscillations and accelerates amyloid plaque accumulation, whereas loss of peripheral Bmal1 in the brain parenchyma increases expression of Apoe and promotes fibrillar plaque deposition. These results provide evidence that both central circadian rhythms and local clock function influence Aβ dynamics and plaque formation and demonstrate mechanisms by which poor circadian hygiene may directly influence AD pathogenesis.

Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults and plays a critical role in brain health and disease. We report that the core circadian clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism and a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression in vitro and in vivo, mediated in part by suppression of glutathione-Stransferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a mechanism by which the circadian clock could influence many aspects of brain function and neurological disease.

Abstract Antibodies vs CD47 (CD47mAbs) that block the CD47-SIRPalpha interaction promote the phagocytosis of cancer cells and have efficacy in several tumor models. A select few CD47mAbs also directly kill cancer cells by lowering cellular cAMP levels leading to mitochondrial damage and cell death. These CD47mAbs are thus referred to as “dual-function” mAbs. Activation of protein kinase A prevents CD47mAb-mediated death suggesting that phosphorylation of one or more target proteins in the cancer cell can block the death mechanism. BNIP3, a member of the BH3-only family, is induced by hypoxia and oncogenes and is necessary for induction of cell death by dual-function CD47mAbs. BNIP3 can activate autophagy, a pro-survival function, and can also induce cell death by damaging mitochondria. The role of BNIP3 in cancer is controversial and context-dependent with some cancers over-expressing BNIP3 compared to low levels of expression in normal tissue, while other cancers cannot tolerate BNIP3 expression and silence the gene, often by methylation of the BNIP3 promoter. This suggests that cancers that tolerate BNIP3 expression employ an as yet unknown mechanism to protect themselves from its toxic effects. The C-terminal transmembrane (TM) domain of BNIP3 (residues 164-184) penetrates the outer mitochondrial membrane allowing the extreme C-terminal ten residue tail of BNIP3 (RRLTTSTSTF, residues 185-194) to extend across the intermembrane space to bind OPA1 on the inner mitochondrial membrane, a key step in the death mechanism. We used a phosphosite-specific antibody to detect phosphorylation of BNIP3 immunoprecipitated from Jurkat leukemia cells at residue T188 which resides in a canonical protein kinase A site (RRLT, amino acids 185-188). Using mass spectrometry of 6His-tagged BNIP3 isolated from HEK293 cells treated with 8BrcAMP, we identified as many as 4 additional phosphorylated sites in the C-terminal tail sequence (residues 189-194, TSTSTF). We generated phosphomimetic (S/T to D) and unphosphorylated (S/T to A or N) mutations at these residues and expressed the mutant BNIP3 proteins in 293 cells. All of the BNIP3 mutants associated with mitochondria but only the phosphomimetic mutants prevented BNIP3-induced mitochondrial damage and cell death. In contrast, mutation of the phosphorylated S/T residues to unphosphorylated residues resulted in rapid and extensive cell death. Importantly, phosphomimetic C-terminal BNIP3 residues blocked cell death without preventing autophagy, providing evidence that the two roles of BNIP3 can be regulated independently. We replicated these results in one lung cancer and three breast cancer cell lines. These findings suggest that phosphorylation at the C-terminus of BNIP3 is a switch that determines the pro-survival vs pro-death effects of BNIP3. Dual-function CD47mAbs may act by dephosphorylating BNIP3 expressed in tumor cells thus unleashing its killing potential. Citation Format: William Frazier, Katherine Liu, Julie Dimitry, Benjamin Capoccia, Pamela Manning, Robert Karr. Phosphorylation of BNIP3 is a switch between life and death in cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2015-2

CD47 functions as a marker of "self" by inhibiting phagocytosis of autologous cells. CD47 has been shown to be overexpressed by various tumor types as a means of escaping the antitumor immune response. The goal of this research was to investigate the utility of CD47 imaging using positron emission tomography (PET) in both human xenograft and murine allograft tumor models. Anti-CD47 antibodies were conjugated with p-isothiocyanatobenzyldesferrioxamine (Df-Bz-NCS) and labeled with 89Zr. We employed xenograft and allograft small-animal models of cancer in biodistribution and PET imaging studies to investigate the specificity and PET imaging robustness of CD47. Ab-Df-Bz-NCS conjugates were labeled with 89Zr with specific activity of 0.9 to 1.6 μCi/μg. Biodistribution studies in the xenograft and allograft model showed similar specific tumor uptake of the antihuman and antimouse CD47 antibodies. However, the tracer retention in the liver, spleen, and kidneys was significantly higher in the allograft-bearing animals, suggesting uptake mediated by the CD47 normally expressed throughout the reticular endothelial system. CD47, a marker of "self," was evaluated as a diagnostic PET biomarker in xenograft and allograft cancer animal models. CD47 imaging is feasible, warranting further studies and immunoPET tracer development.

CD47, a receptor for thrombospondin-1, limits two important regulatory axes: nitric oxide-cGMP signaling and cAMP signaling, both of which can promote mitochondrial biogenesis. Electron microscopy revealed increased mitochondrial densities in skeletal muscle from both CD47 null and thrombospondin-1 null mice. We further assessed the mitochondria status of CD47-null vs WT mice. Quantitative RT-PCR of RNA extracted from tissues of 3month old mice revealed dramatically elevated expression of mRNAs encoding mitochondrial proteins and PGC-1α in both fast and slow-twitch skeletal muscle from CD47-null mice, but modest to no elevation in other tissues. These observations were confirmed by Western blotting of mitochondrial proteins. Relative amounts of electron transport enzymes and ATP/O2 ratios of isolated mitochondria were not different between mitochondria from CD47-null and WT cells. Young CD47-null mice displayed enhanced treadmill endurance relative to WTs and CD47-null gastrocnemius had undergone fiber type switching to a slow-twitch pattern of myoglobin and myosin heavy chain expression. In 12month old mice, both skeletal muscle mitochondrial volume density and endurance had decreased to wild type levels. Expression of myosin heavy chain isoforms and myoglobin also reverted to a fast twitch pattern in gastrocnemius. Both CD47 and TSP1 null mice are leaner than WTs, use less oxygen and produce less heat than WT mice. CD47-null cells produce substantially less reactive oxygen species than WT cells. These data indicate that loss of signaling from the TSP1–CD47 system promotes accumulation of normally functioning mitochondria in a tissue-specific and age-dependent fashion leading to enhanced physical performance, lower reactive oxygen species production and more efficient metabolism.

Control of alphaIIb beta3 and alphav beta3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin alpha and beta subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or "off" conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of alphav beta3. In this bent structure, a segment of alphav (301-308) and beta3 (560-567) are juxtaposed. Here we provide evidence that these regions of alphav/alphaIIb and beta3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the alphaIIb and beta3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three beta3 LIBS antibodies. Mutation of the clasp region of alphav or beta3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the alphav beta3 structure yield a refined model for the alphav beta3 clasp and provide plausible explanations for the effects of the activating mutations.

Control of RIIb!3 and Rv!3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin R and ! subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or “off” conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of Rv!3. In this bent structure, a segment of Rv (301-308) and !3 (560-567) are juxtaposed. Here we provide evidence that these regions of Rv/RIIb and !3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the RIIb and !3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three !3 LIBS antibodies. Mutation of the clasp region of Rv or !3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the Rv!3 structure yield a refined model for the Rv!3 clasp and provide plausible explanations for the effects of the activating mutations. Control of integrin activity is of crucial importance in platelet aggregation (1), leukocyte adherence and trafficking (2) (3), angiogenesis (4), and apoptosis (5). Crystal structures of integrin R chain domains (2) and the structure of the entire extracellular domain of Rv!3 (6, 7) have provided new hypotheses for integrin regulation. Multiple mechanisms contribute to regulation of integrin activation. This is not unanticipated given the critical role of integrins in so many important biological scenarios and the disastrous consequences of inappropriate activation of platelet or leukocyte integrins. Two general mechanisms regulate the functional state of integrins. Conformational changes of the R! dimer are clearly involved in transitions from lowto high-affinity states (8), usually judged by the binding of soluble ligands or the exposure of binding sites for mAbs that recognize an activated conformation stabilized by ligands or a “ligandinduced binding site” (anti-LIBS mAbs) (9-11). Integrin dimers competent for ligand binding may also be clustered resulting in a high “avidity” state that increases binding to multivalent, usually immobilized ligands (12-14). Early studies of RIIb!3 activation suggested that association of juxtamembrane cytoplasmic ion pairs opposite each other in the R and ! subunit tails could restrain activation (15) (16). This notion of a cytoplasmic “clasp” of the R and ! subunits was strengthened by the effects of additional mutations in the juxtamembrane regions of RIIb and !3 that resulted in constitutive activation (17). Further, the addition of non-native, coiled-coil dimerizing peptides to the cytoplasmic tailsofRand! subunitsconstrainedactivation (18,19). A role for the TM domains of R and ! subunits in integrin activation has also been proposed based on mutations that constitutively activate RIIb!3 (17, 20). These data suggest a model in which a specific R-! TM helix interface contributes to stabilizing the off state, likely acting in concert with the juxtamembrane clasp in the cytoplasmic tails. The publication of the crystal structure of free and RGDbound extracellular domains of Rv!3 (6, 7) gave rise to an entirely new model for activation. The bent or genuflected integrin seen in the crystal structure suggested that massive conformational changes of the entire Rv!3 extracellular domain must accompany integrin activation, if indeed the fully active integrin were to assume the extended, upright conformation expected from earlier EM studies (21-24). A growing body of data supports the idea that the bent structure seen in the crystals of Rv!3 is likely the physiologically relevant “off” or low-affinity state and an extended, erect integrin represents the fully active state (8, 13, 14, 25, 26). Particularly revealing was an experiment in which non-native Cys residues were inserted in the R and ! subunits of Rv!3 (or the homologous position of RIIb) at locations that were within disulfide bond distance in the bent structure but were predicted to be very far apart in an extended structure (RvG307 > C, and !3-R563 > C). When the mutant R and ! subunits were expressed in cells, they formed R! dimers that were inactive, even in Mn2+, until mild reducing agent was applied, or either one of the mutant R or ! subunits was replaced with the wild-type (non-Cys) construct (8). This † Supported by NIH Grant HL54390 (W.A.F.) and by the NSF sponsored UAB ADVANCE program (Y.S.). * To whom inquires should be addressed. Phone: 314-362-3348. Fax: 314-362-7183. E-mail: frazier@biochem.wustl.edu. ‡ Washington University School of Medicine. § The University of Alabama at Birmingham. 1 Abbreviations: 4N1K, thrombospondin-1 peptide with sequence KRFYVVMWKK; LIBS, ligand-induced binding site; RGD peptide, peptide with sequence GRGDSP; TM, transmembrane; Vn, vitronectin; WT, wild type. Biochemistry 2008, 47, 11616–11624 11616 10.1021/bi8015108 CCC: $40.75  2008 American Chemical Society Published on Web 10/09/2008 experiment demonstrates that the bent structure can exist on the cell surface and is inactive. Not addressed is whether these juxtaposed regions of the R (G307) and ! (R563) subunits normally have a role in regulating integrin activation by forming an extracellular clasp. Here we test this hypothesis. EXPERIMENTAL PROCEDURES Reagents, Cell Lines, and Peptides. Human K562 erythroleukemic cells (ATCC: CCL-243), stably expressing Rv!3 integrin (27), HEK 293 cells (ATCC: CRL1573), and C32 human melanoma cells (ATCC: CRL-1585) were grown as described (28). Ligand-induced binding site (LIBS) antibodies, LIBS1 and LIBS6, were generously provided by Dr. Mark Ginsberg (Scripps Research Institute) (9), and the LIBS antibody D3 was a gift from Dr. Lisa Jennings (The University of Tennessee, Memphis, TN) (11). Peptides were synthesized, purified, and verified by mass spectrometry as previously described (29). All other reagents were purchased from Sigma-Aldrich unless otherwise stated. Adhesion and spreading assays of C32 cells on Vn were performed as described (30). Collection of human blood was performed under an approved protocol of the Washington University School of Medicine Human Studies Committee. Washed platelets were prepared as described (31) and stored on ice for 1-48 h or treated as described below. Binding of anti!3 mAb AP3 and anti-LIBS mAbs LIBS1, LIBS6, and D3 to cells and platelets under indicated conditions was quantified using a Coulter EPICS flow cytometer. Data were analyzed using WinMDI software. Preparation and Expression of Mutants of RV and !3. Fulllength cDNA clones of human Rv and !3 integrin subunits were provided by Dr. Scott Blystone. Restriction fragments containing the mutation sites were subcloned into Bluescript BSKS+ for PCR mutagenesis using overlapping primers containing the mutant bases. Details are provided in Supporting Information. After confirmation by DNA sequencing, the restriction fragment containing the desired mutation was reassembled in pCDNA3 for Rv (G418 selection) and either pREP10 or pBLY100 for !3 (hygromycin selection). Initial tests of the !3 mutants were performed by transfecting them into human ovarian carcinoma clone OV10 which expresses WT Rv (largely as Rv!5) but no !3 (32). For cotransfections of both subunits, HEK 293 cells were used (33) (34). Expression was determined by flow cytometry with mAbs L230 (Rv) and AP3 (!3). LIBS binding was determined as above and normalized to AP3 binding or the binding of the LIBS mAb in the presence of excess RGDS peptide and Mn2+ to yield an activation index. Molecular Dynamics Simulations. The reported crystal structure of Rv!3 (PDB ID: 1JV2) (6) was subjected to energy minimization and equilibration using GROMACS version 3.3 (35). Details of the method are provided in Supporting Information.

CD36 is necessary for inhibition of some angiogenic responses by the matricellular glycoprotein thrombospondin-1 and is therefore assumed to be the receptor that mediates its anti-angiogenic activities. Although ligation of CD36 by antibodies, recombinant type 1 repeats of thrombospondin-1, or CD36-binding peptides was sufficient to inhibit nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells, picomolar concentrations of native thrombospondin-1 similarly inhibited NO signaling in vascular cells from wild-type and CD36-null mice. Ligation of the thrombospondin-1 receptor CD47 by recombinant C-terminal regions of thrombospondin-1, thrombospondin-1 peptides, or CD47 antibodies was also sufficient to inhibit NO-stimulated phenotypic responses and cGMP signaling in vascular cells. Thrombospondin-1 did not inhibit NO signaling in CD47-null vascular cells or NO-stimulated vascular outgrowth from CD47-null muscle explants in three-dimensional cultures. Furthermore, the CD36-binding domain of thrombospondin-1 and anti-angiogenic peptides derived from this domain failed to inhibit NO signaling in CD47-null cells. Therefore, ligation of either CD36 or CD47 is sufficient to inhibit NO-stimulated vascular cell responses and cGMP signaling, but only CD47 is necessary for this activity of thrombospondin-1 at physiological concentrations.

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.

Integrin-associated protein (IAP; CD47) is a thrombospondin receptor that forms a signaling complex with beta3 integrins resulting in enhanced alphavbeta3-dependent cell spreading and chemotaxis and, in platelets, alphaIIbbeta3-dependent spreading and aggregation. These actions of CD47 are all specifically abrogated by pertussis toxin treatment of cells. Here we report that CD47, its beta3 integrin partner, and Gi proteins form a stable, detergent-soluble complex that can be recovered by immunoprecipitation and affinity chromatography. Gialpha is released from this complex by treatment with GTP or AlF4. GTP and AlF4 also reduce the binding of CD47 to its agonist peptide (4N1K) derived from thrombospondin, indicating a direct association of CD47 with Gi. 4N1K peptide causes a rapid decrease in intraplatelet cyclic AMP levels, a Gi-dependent event necessary for aggregation. Finally, 4N1K stimulates the binding of GTPgamma35S to membranes from cells expressing IAP and alphavbeta3. This functional coupling of CD47 to heterotrimeric G proteins provides a mechanistic explanation for the biological effects of CD47 in a wide variety of systems.