Yoshiko Maeda

Pyramidal Neurons Are Generated from Oligodendroglial Progenitor Cells in Adult Piriform Cortex

Previous studies have shown that oligodendroglial progenitor cells (OPCs) can give rise to neurons in vitro and in perinatal cerebral cortex in vivo. We now report that OPCs in adult murine piriform cortex express low levels of doublecortin, a marker for migratory and immature neurons. Additionally, these OPCs express Sox2, a neural stem cell marker, and Pax6, a transcription factor characteristic of progenitors for cortical glutamatergic neurons. Genetic fate-mapping by means of an inducible Cre–LoxP recombination system proved that these OPCs differentiate into pyramidal glutamatergic neurons in piriform cortex. Several lines of evidence indicated that these newly formed neurons became functionally integrated into the cortical neuronal network. Our data suggest that NG2+/PDGFRα+ proteolipid protein promoter-expressing progenitors generate pyramidal glutamatergic neurons within normal adult piriform cortex.

Adenomatous polyposis coli regulates oligodendroglial development

The expression of the gut tumor suppressor gene adenomatous polyposis coli (Apc) and its role in the oligodendroglial lineage are poorly understood. We found that immunoreactive APC is transiently induced in the oligodendroglial lineage during both normal myelination and remyelination following toxin-induced, genetic, or autoimmune demyelination murine models. Using the Cre/loxP system to conditionally ablate APC from the oligodendroglial lineage, we determined that APC enhances proliferation of oligodendroglial progenitor cells (OPCs) and is essential for oligodendrocyte differentiation in a cell-autonomous manner. Biallelic Apc disruption caused translocation of β-catenin into the nucleus and upregulated β-catenin-mediated Wnt signaling in early postnatal but not adult oligodendroglial lineage cells. The results of conditional ablation of Apc or Ctnnb1 (the gene encoding β-catenin) and of simultaneous conditional ablation of Apc and Ctnnb1 revealed that β-catenin is dispensable for postnatal oligodendroglial differentiation, that Apc one-allele deficiency is not sufficient to dysregulate β-catenin-mediated Wnt signaling in oligodendroglial lineage cells, and that APC regulates oligodendrocyte differentiation through β-catenin-independent, as well as β-catenin-dependent, mechanisms. Gene ontology analysis of microarray data suggested that the β-catenin-independent mechanism involves APC regulation of the cytoskeleton, a result compatible with established APC functions in neural precursors and with our observation that Apc-deleted OPCs develop fewer, shorter processes in vivo. Together, our data support the hypothesis that APC regulates oligodendrocyte differentiation through both β-catenin-dependent and additional β-catenin-independent mechanisms.

Disruption of NMDA Receptors in Oligodendroglial Lineage Cells Does Not Alter Their Susceptibility to Experimental Autoimmune Encephalomyelitis or Their Normal Development

Pharmacological studies have suggested that oligodendroglial NMDA glutamate receptors (NMDARs) mediate white matter injury in a variety of CNS diseases, including multiple sclerosis (MS). We tested this hypothesis in experimental autoimmune encephalomyelitis (EAE), a model of human MS, by timed conditional disruption of oligodendroglial NR1, an essential subunit of functional NMDARs, using an inducible proteolipid protein (Plp) promoter-driven Cre-loxP recombination system. We found that selective ablation of oligodendroglial NR1 did not alter the clinical severity of EAE elicited in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35–55 (MOG-peptide), nor were there significant differences between the oligodendroglial NR1 KO and non-KO mice in numbers of axons lost in spinal cord dorsal funiculi or severity of spinal cord demyelination. Similarly, constitutive deletion of NR3A, a modulatory subunit of oligodendroglial NMDARs, did not alter the course of MOG-peptide EAE. Furthermore, conditional and constitutive ablation of NR1 in neonatal oligodendrocyte progenitor cells did not interrupt their normal maturation and differentiation. Collectively, our data suggest that oligodendroglial lineage NMDARs are neither required for timely postnatal development of the oligodendroglial lineage, nor significant participants in the pathophysiology of MOG-peptide EAE.

Macroglial Plasticity and the Origins of Reactive Astroglia in Experimental Autoimmune Encephalomyelitis

Accumulations of hypertrophic, intensely glial fibrillary acidic protein-positive (GFAP+) astroglia, which also express immunoreactive nestin and vimentin, are prominent features of multiple sclerosis lesions. The issues of the cellular origin of hypertrophic GFAP+/vimentin+/nestin+ “reactive” astroglia and also the plasticities and lineage relationships among three macroglial progenitor populations—oligodendrocyte progenitor cells (OPCs), astrocytes and ependymal cells—during multiple sclerosis and other CNS diseases remain controversial. We used genetic fate-mappings with a battery of inducible Cre drivers (Olig2-Cre-ERT2, GFAP-Cre-ERT2, FoxJ1-Cre-ERT2 and Nestin-Cre-ERT2) to explore these issues in adult mice with myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis (EAE). The proliferative rate of spinal cord OPCs rose fivefold above control levels during EAE, and numbers of oligodendroglia increased as well, but astrogenesis from OPCs was rare. Spinal cord ependymal cells, previously reported to be multipotent, did not augment their low proliferative rate, nor give rise to astroglia or OPCs. Instead, the hypertrophic, vimentin+/nestin+, reactive astroglia that accumulated in spinal cord in this multiple sclerosis model were derived by proliferation and phenotypic transformation of fibrous astroglia in white matter, and solely by phenotypic transformation of protoplasmic astroglia in gray matter. This comprehensive analysis of macroglial plasticity in EAE helps to clarify the origins of astrogliosis in CNS inflammatory demyelinative disorders.

The Use of One-Bead One-Compound Combinatorial Library Technology to Discover High-Affinity αvβ3 Integrin and Cancer Targeting Arginine-Glycine-Aspartic Acid Ligands with a Built-in Handle

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68 ± 0.08 μmol/L) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. Mol Cancer Ther; 9(10); 2714–23. ©2010 AACR.

Rapid discovery of death ligands with one-bead-two-compound combinatorial library methods

The one-bead-one-compound (OBOC) technology enables one to generate thousands to millions of chemical molecules on resin beads (90 μm diameter) such that each bead displays 10(13) copies of the same chemical entity. Whole-cell binding assays have been developed to screen OBOC combinatorial libraries for ligands that bind to specific cell surface receptors. While very powerful, this screening method does not address the downstream cell signaling properties of the binding ligand. We have modified the OBOC technology by introducing a fixed known cell adhesion ligand to the outer layer of each bead. This one-bead-two-compound (OB2C) library configuration allows the bound cells to interact with the random immobilized chemical molecules on each bead. The bound cells can then be probed for specific cellular responses such as apoptosis and activation or inhibition of a specific cell signaling pathway. To validate this concept, an OB2C combinatorial library was created such that a random hexapeptide plus a high affinity lymphoma targeting ligand LLP2A were displayed on each bead. This LLP2A-X(6) OB2C library was then screened with human T-cell leukemia cells (Molt-4) for cell death responses. After 5 days of incubation, propidium iodide was added to the bead library to stain dead cells. Beads coated by red fluorescent cells were isolated for sequence analysis. Two ligands identified by this method, when added to the lymphoid cancer cells, were able to induce cell death.

Canine malignant melanoma alpha-3 integrin binding peptides

There is a need to develop novel targeted imaging and therapeutic agents that can aid in early diagnosis, detection of metastasis and treatment of melanoma. Alpha-3 integrin is overexpressed in 82% of metastatic melanomas in humans and may be a potential target for peptide ligands carrying therapeutic agents. Five melanoma cell lines were generated from canine primary oral and metastatic canine tumors, grown in mice, and validated with melanoma markers Melan A, S-100, Micropthalmia transcription factor (MITF), Tyrosinase, and MART-1. The melanoma cell lines were tested for binding affinity to previously published alpha-3 integrin-binding peptides containing the cdGXGXXc motif. Fluorescent conjugates of the alpha-3 integrin binding OA02 peptide were used to quantify receptor affinity in the cell lines, a specimen of canine primary oral melanoma, and melanoma xenografts. Alpha-3 integrin was expressed by all 5 canine melanoma cell lines. Four of the 5 lines as well as the primary canine tumor showed affinity to alpha-3 integrin binding peptides with the cdGXGXXc motif. Optical imaging of canine melanoma xenografts in nude mice indicates rapid, strong uptake of the optical tracer in the tumor with an average persistence of approximately 48 h. Ex vivo images showed high tumor-to-background ratio, with tumor signals more than twice that of the kidney and other vital organs. We propose that integrin alpha-3 integrin binding ligands could potentially become useful probes for imaging and delivery of cytotoxic agents for the treatment of melanoma.

Abstract 366: Novel self-folding tricyclic cholic acid-peptide hybrids as nanotherapeutic ligands targeting prostate and breast cancers and human embryonic stem cells

Prostate cancer is the second leading cause of death in American men accounting for 10% of all cancer-related mortalities. According to NCI statistics from 2005-2007, 1 in 6 men will be diagnosed with prostate cancer during their lifetime. Despite the use of chemotherapy for the treatment of advanced or metastatic disease, cellular resistance to anticancer drugs and adverse side effects to healthy organs limit therapeutic efficacy. A targeted pro-apoptotic drug delivery system has been developed for the selective destruction of malignant tumors and tumorigenic stem cells. A novel self-folding tricyclic branched library was synthesized. Fixed hydrophobic amino acids near the N-terminus (position 5) of twin branched peptide arms fold back onto the hydrophobic face of the planar cholic acid molecule to form a compact tricyclic molecule. The novel bracelet library was screened using a One Bead Two Compounds (OB2C) combinatorial chemistry approach. Receptors expressed on cancerous cells serve as the targets for the tricyclic molecules to induce apoptotic signaling. Receptors for LDO 18, found on prostate cancer and stem cells, serve as the carrier vector, enabling the targeted delivery of the anticancer drug to the tumor mass. This approach spares normal tissues from toxic side effects of the peptidomimetic compounds. Preliminary studies suggest that tumors of ovarian and breast origin can also be targeted using LDO18. Lead therapeutic compounds will be conjugated to LDO18 nanocarriers along with LHRH analogues to evaluate the efficacy and combined potency to prostate, ovarian, and breast cancer xenografts in nude mice and orthotropic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2011-366

Abstract 2316: Therapeutic targeting of novel human stem cell and cancer associated glycans

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Host and tumor surface glycans are important in regulating pivotal pathophysiological events during the development of cancer and tumor progression. Changes in glycosylation allow tumorigenic cells to seize control over developmental processes which allow cells to proliferate, invade, promote angiogenesis, and metastasize. Malignant transformation is accompanied by the expression of oncofetal antigens which are expressed on embryonic cells, tumor cells, and a few adult cells. Glycans common to human embryonic stem cells and cancer cells can be targeted using combinatorial chemistry. Several peptidomimetic ligands have been discovered using One Bead One Compound (OBOC) and One Bead Two Compound (OB2C) methods. These cell adhering ligands possess the potential to target tumorigenic cells and mediate intracellular signaling events by binding to high mannose glycans expressed on the surface of Jurkat, TK6, Her2 expressing MCF7, chemoresistant MCF7, HeLa, and SiHa cell lines. Three lead linear peptides with alternating natural and unnatural amino acids have been investigated for their in vivo targeting potential in the lymphoid leukemia, breast, and cervical cancer mouse xenograph models. In vitro and in vivo stem and cancer cell experiments, which demonstrate genetically altered glycan function, are currently being investigated for their anticancer potential. These novel agents may be used alone or in combination with chemoradiation and anti-angiogenesis strategies for treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2011-2316

Abstract 2593: A novel small molecule drug (LWKS) for pancreatic cancer therapy

Pancreatic ductal adenocarcinoma (PDAC) currently stands at approximately 29,000 new cases per year in North America and lack of effective therapies leads to an extremely poor prognosis. Since pancreatic cancers respond poorly to chemotherapy, more effective and less toxic treatment is a crucial need. We have employed a biological assay (MTS assay) to identify small molecular compounds that induce cytotoxicity specifically in pancreatic cancer cells and not in normal cells by screening large number of compounds from various combinatorial chemical library. Commercially available PDAC cells derived from various stages of pancreatic cancer were used for screening. For normal cells, immortalized epithelial cells isolated from pancreatic duct (h-tert HPNE) were used. One of the compounds, LWKS (MW: 547.94) kills pancreatic cancer cells and not the normal HPNE cells. LWKS kills pancreatic cancer cells isolated from all the four stages and the EC50 is in the range of 2 – 10 µM, whereas for normal cells is 70 µM. Maximum tolerated dose is 80 mg/kg of body weight. At 20 mg/Kg body weight, it showed significant tumor shrinkage in human PDAC xenografts mice model when compared to PBS controls. The tumor reduction was monitored by measuring the tumor volume for two times per week for 8 weeks. All the animal safety procedures were followed as described by AALAC. At this dosage level no toxicity was observed in the mice model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2593. doi:10.1158/1538-7445.AM2011-2593