In the human brain, microRNAs (miRNAs) from the microRNA-376 (miR-376) cluster undergo programmed "seed" sequence modifications by adenosine-to-inosine (A-to-I) editing. Emerging evidence suggests a link between impaired A-to-I editing and cancer, particularly in high-grade gliomas. We hypothesized that disruption of A-to-I editing alters expression of genes regulating glioma tumor phenotypes. By sequencing the miR-376 cluster, we show that the overall miRNA editing frequencies were reduced in human gliomas. Specifically in high-grade gliomas, miR-376a* accumulated entirely in an unedited form. Clinically, a significant correlation was found between accumulation of unedited miR-376a* and the extent of invasive tumor spread as measured by magnetic resonance imaging of patient brains. Using both in vitro and orthotopic xenograft mouse models, we demonstrated that the unedited miR-376a* promoted glioma cell migration and invasion, while the edited miR-376a* suppressed these features. The effects of the unedited miR-376a* were mediated by its sequence-dependent ability to target RAP2A and concomitant inability to target AMFR. Thus, the tumordependent introduction of a single base difference in the miR-376a* sequence dramatically alters the selection of its target genes and redirects its function from inhibiting to promoting glioma cell invasion. These findings uncover a new mechanism of miRNA deregulation and identify unedited miR-376a* as a potential therapeutic target in glioblastoma cells. IntroductionMicroRNAs (miRNAs) are short, noncoding RNAs that mediate post-transcriptional silencing of a set of target genes. The target gene specificity of each miRNA is dictated by sequence-dependent interaction between approximately 22-nt-long mature miRNAs, especially their 6-or 7-nucleotide "seed" sequences at the 5′ end, and the 3′ untranslated regions of mRNAs (1). It has been shown that the epigenetic process of adenosineto-inosine (A-to-I) editing of certain miRNAs can lead to a single base substitution in their seed sequence and generate variant "edited" mature miRNA species with target gene specificity drastically different from that of the unedited, genomically encoded miRNA (2). This was demonstrated for miR-376 cluster transcripts collected from normal human and mouse tissues. The miR-376 cluster encodes 4 primary miRNAs (primiRs), including pri-miR-376a1, -376a2, -376b, and -376c, that are processed to 5 distinct mature miRNAs, miR-376a, -376a*, -376a2-5p, -376b, and -376c. In the human brain, 9 adenosines within this miRNA cluster are subject to specific and high-level A-to-I RNA editing (2).In gliomas, the most frequent primary brain tumors, Alu repeats and several protein-coding substrates of adenosine deaminases acting on RNA (ADARs; a family of enzymes that mediate A-to-I editing of RNAs) have been found to be edited to lower than normal frequencies (3-5). Particularly in high-grade gliomas, glioblastoma multiforme (GBMs), emerging lines of evidence suggest a

Neurons and glia in the vertebrate central nervous system arise in temporally distinct, albeit overlapping, phases. Neurons are generated first followed by astrocytes and oligodendrocytes from common progenitor cells. Increasing evidence indicates that axon-derived signals spatiotemporally modulate oligodendrocyte maturation and myelin formation. Our previous observations demonstrate that F3/contactin is a functional ligand of Notch during oligodendrocyte maturation, revealing the existence of another group of Notch ligands. Here, we establish that NB-3, a member of the F3/contactin family, acts as a novel Notch ligand to participate in oligodendrocyte generation. NB-3 triggers nuclear translocation of the Notch intracellular domain and promotes oligodendrogliogenesis from progenitor cells and differentiation of oligodendrocyte precursor cells via Deltex1. In primary oligodendrocytes, NB-3 increases myelin-associated glycoprotein transcripts. Thus, the NB-3/Notch signaling pathway may prove to be a molecular handle to treat demyelinating diseases. Neural progenitor cells (NPCs)1 are self-renewing multipotent cells that can give rise to all types of neural cells, namely neurons, oligodendrocytes (OLs), and astrocytes. Increasing evidence suggests that this fate commitment of NPCs requires molecular cues provided by extracellular molecules and intrinsic signaling involving various transcription factors (1, 2). Our recent study (3) has demonstrated that the F3/Notch signaling pathway via Deltex1 (DTX1) promotes oligodendrocyte precursor cell (OPC) differentiation into oligodendrocytes (OLs) and up-regulates myelin-associated glycoprotein (MAG) expression in both primary OLs and OLN-93 cells, an OL cell line.

Mutations in the parkin gene, which encodes a ubiquitin ligase, are a major genetic cause of parkinsonism. Interestingly, parkin also plays a role in cancer as a putative tumor suppressor, and the gene is frequently targeted by deletion and inactivation in human malignant tumors. Here, we investigated a potential tumor suppressor role for parkin in gliomas. We found that parkin expression was dramatically reduced in glioma cells. Restoration of parkin expression promoted G 1 phase cell-cycle arrest and mitigated the proliferation rate of glioma cells in vitro and in vivo. Notably, parkin-expressing glioma cells showed a reduction in levels of cyclin D1, but not cyclin E, and a selective downregulation of Akt serine-473 phosphorylation and VEGF receptor levels. In accordance, cells derived from a parkin-null mouse model exhibited increased levels of cyclin D1, VEGF receptor, and Akt phosphorylation, and divided significantly faster when compared with wild-type cells, with suppression of these changes following parkin reintroduction. Clinically, analysis of parkin pathway activation was predictive for the survival outcome of patients with glioma. Taken together, our study provides mechanistic insight into the tumor suppressor function of parkin in brain tumors and suggests that measurement of parkin pathway activation may be used clinically as a prognostic tool in patients with brain tumor. Cancer Res; 72(10); 2543-53. Ó2012 AACR.

Cancer stem cells have been shown to initiate and sustain tumor growth. In many instances, clinical material is limited, compounded by a lack of methods to preserve such cells at convenient time points. Although brain tumor-initiating cells grown in a spheroid manner have been shown to maintain their integrity through serial transplantation in immune-compromised animals, practically, it is not always possible to have access to animals of suitable ages to continuously maintain these cells. We therefore explored vitrification as a cryopreservation technique for brain tumor-initiating cells. Tumor neurospheres were derived from five patients with glioblastoma multiforme (GBM). Cryopreservation in 90% serum and 10% dimethyl sulfoxide yielded greatest viability and could be explored in future studies. Vitrification yielded cells that maintained self-renewal and multipotentiality properties. Karyotypic analyses confirmed the presence of GBM hallmarks. Upon implantation into NOD/SCID mice, our vitrified cells reformed glioma masses that could be serially transplanted. Transcriptome analysis showed that the vitrified and nonvitrified samples in either the stem-like or differentiated states clustered together, providing evidence that vitrification does not change the genotype of frozen cells. Upon induction of differentiation, the transcriptomes of vitrified cells associated with the original primary tumors, indicating that tumor stem-like cells are a genetically distinct population from the differentiated mass, underscoring the importance of working with the relevant tumor-initiating population. Our results demonstrate that vitrification of brain tumor-initiating cells preserves the biological phenotype and genetic profiles of the cells. This should facilitate the establishment of a repository of tumor-initiating cells for subsequent experimental designs.

Knockdown of ABCG2 transporters did not abrogate the SP cell response to temozolomide. Upregulation of several other ABC drug transporter genes is proposed to account for this chemoresistance.

We report Nogo-A as an oligodendroglial component congregating and interacting with the Caspr±F3 complex at paranodes. However, its receptor Nogo-66 receptor (NgR) does not segregate to speci®c axonal domains. CHO cells cotransfected with Caspr and F3, but not with F3 alone, bound speci®cally to substrates coated with Nogo-66 peptide and GST±Nogo-66. Binding persisted even after phosphatidylinositolspeci®c phospholipase C (PI-PLC) removal of GPIlinked F3 from the cell surface, suggesting a direct interaction between Nogo-66 and Caspr. Both Nogo-A and Caspr co-immunoprecipitated with Kv1.1 and Kv1.2, and the developmental expression pattern of both paralleled compared with Kv1.1, implicating a transient interaction between Nogo-A±Caspr and K + channels at early stages of myelination. In pathological models that display paranodal junctional defects (EAE rats, and Shiverer and CGT ±/± mice), distances between the paired labeling of K + channels were shortened signi®cantly and their localization shifted toward paranodes, while paranodal Nogo-A congregation was markedly reduced. Our results demonstrate that Nogo-A interacts in trans with axonal Caspr at CNS paranodes, an interaction that may have a role in modulating axon±glial junction architecture and possibly K + -channel localization during development.

Autografts have been extensively studied to facilitate optic nerve (ON) regeneration in animal experiments, but the clinical application of this approach to aid autoregeneration has not yet been attempted. This study aims to explore the guided regeneration by an artificial polyglycolic acid-chitosan conduit coated with recombinant L1-Fc. Consistent with previous studies; in vitro assay showed that both chitosan, a natural biomaterial, and the neural cell adhesion molecule L1-Fc enhanced neurite outgrowth. Rat optic nerve transection was used as an in vivo model. The implanted PGA-chitosan conduit was progressively degraded and absorbed, accompanied by significant axonal regeneration as revealed by immunohistochemistry, anterograde and retrograde tracing. The polyglycolic acid-chitosan conduit coated with L1-Fc showed more effective to promote axonal regeneration and remyelination. Taken together, our observations demonstrated that the L1-Fc coated PGA-chitosan conduits provided a compatible and supportive canal to guild the injured nerve regeneration and remyelination.

No beneficial effect on survival after ICH of prior statin use could be demonstrated in our large multi-ethnic Asian patient cohort.