Research on human glioma stem cells in China.

Research on human glioma stem cells began early in the 21st century and since then has become a rapidly growing research field with the number of publications increasing year by year. The research conducted by our diverse group of investigators focused primarily on cell culture techniques, molecular regulation, signaling pathways, cancer treatment, the stem cell microenvironment and the cellular origin and function of glioma stem cells. In particular, we put forward our view that there are inverse or forward transformations among neural stem cells, glial cells and glioma stem cells in glioma tissues under certain conditions. Based on the background of the progress of international research on human glioma stem cells, we aim to share our progress and current findings of human glioma stem cell research in China with colleagues around the world.

Rab27b is Involved in Lysosomal Exocytosis and Proteolipid Protein Trafficking in Oligodendrocytes.

Myelination by oligodendrocytes in the central nervous system requires coordinated exocytosis and endocytosis of the major myelin protein, proteolipid protein (PLP). Here, we demonstrated that a small GTPase, Rab27b, is involved in PLP trafficking in oligodendrocytes. We showed that PLP co-localized with Rab27b in late endosomes/lysosomes in oligodendrocytes. Short hairpin-mediated knockdown of Rab27b not only reduced lysosomal exocytosis but also greatly diminished the surface expression of PLP in oligodendrocytes. In addition, knockdown of Rab27b reduced the myelin-like membranes induced by co-culture of oligodendrocytes and neurons. Our data suggest that Rab27b is involved in myelin biogenesis by regulating PLP transport from late endosomes/lysosomes to the cell membrane in oligodendrocytes.

Rab27a/Slp2-a complex is involved in Schwann cell myelination.

Myelination of Schwann cells in the peripheral nervous system is an intricate process involving myelin protein trafficking. Recently, the role and mechanism of the endosomal/lysosomal system in myelin formation were emphasized. Our previous results demonstrated that a small GTPase Rab27a regulates lysosomal exocytosis and myelin protein trafficking in Schwann cells. In this present study, we established a dorsal root ganglion (DRG) neuron and Schwann cell co-culture model to identify the signals associated with Rab27a during myelination. First, Slp2-a, as the Rab27a effector, was endogenously expressed in Schwann cells. Second, Rab27a expression significantly increased during Schwann cell myelination. Finally, Rab27a and Slp2-a silencing in Schwann cells not only reduced myelin protein expression, but also impaired formation of myelin-like membranes in DRG neuron and Schwann cell co-cultures. Our findings suggest that the Rab27a/Slp2-a complex affects Schwann cell myelination in vitro.

HMGB1 Promotes Mitochondrial Dysfunction-Triggered Striatal Neurodegeneration via Autophagy and Apoptosis Activation.

Impairments in mitochondrial energy metabolism are thought to be involved in many neurodegenerative diseases. The mitochondrial inhibitor 3-nitropropionic acid (3-NP) induces striatal pathology mimicking neurodegeneration in vivo. Previous studies showed that 3-NP also triggered autophagy activation and apoptosis. In this study, we focused on the high-mobility group box 1 (HMGB1) protein, which is important in oxidative stress signaling as well as in autophagy and apoptosis, to explore whether the mechanisms of autophagy and apoptosis in neurodegenerative diseases are associated with metabolic impairment. To elucidate the role of HMGB1 in striatal degeneration, we investigated the impact of HMGB1 on autophagy activation and cell death induced by 3-NP. We intoxicated rat striata with 3-NP by stereotaxic injection and analyzed changes in expression HMGB1, proapoptotic proteins caspase-3 and phospho-c-Jun amino-terminal kinases (p-JNK). 3-NP-induced elevations in p-JNK, cleaved caspase-3, and autophagic marker LC3-II as well as reduction in SQSTM1 (p62), were significantly reduced by the HMGB1 inhibitor glycyrrhizin. Glycyrrhizin also significantly inhibited 3-NP-induced striatal damage. Neuronal death was replicated by exposing primary striatal neurons in culture to 3-NP. It was clear that HMGB1 was important for basal autophagy which was shown by rescue of cells through HMGB1 targeting shRNA approach.3-NP also induced the expression of HMGB1, p-JNK, and LC3-II in striatal neurons, and p-JNK expression was significantly reduced by shRNA knockdown of HMGB1, an effect that was reversed by exogenously increased expression of HMGB1. These results suggest that HMGB1 plays important roles in signaling for both autophagy and apoptosis in neurodegeneration induced by mitochondrial dysfunction.

Glioma stem cells are more aggressive in recurrent tumors with malignant progression than in the primary tumor, and both can be maintained long-term in vitro.

BACKGROUND: Despite the advances made during decades of research, the mechanisms by which glioma is initiated and established remain elusive. The discovery of glioma stem cells (GSCs) may help to elucidate the processes of gliomagenesis with respect to their phenotype, differentiation and tumorigenic capacity during initiation and progression. Research on GSCs is still in its infancy, so no definitive conclusions about their role can yet be drawn. To understand the biology of GSCs fully, it is highly desirable to establish permanent and biologically stable GSC lines. METHODS: In the current study, GSCs were isolated from surgical specimens of primary and recurrent glioma in a patient whose malignancy had progressed during the previous six months. The GSCs were cryopreserved and resuscitated periodically during long-term maintenance to establish glioma stem/progenitor cell (GSPC) lines, which were characterized by immunofluorescence, flow cytometry and transmission electronic microscopy. The primary and recurrent GSPC lines were also compared in terms of in vivo tumorigenicity and invasiveness. Molecular genetic differences between the two lines were identified by array-based comparative genomic hybridization and further validated by real-time PCR. RESULTS: Two GSPC lines, SU-1 (primary) and SU-2 (recurrent), were maintained in vitro for more than 44 months and 38 months respectively. Generally, the potentials for proliferation, self-renewal and multi-differentiation remained relatively stable even after a prolonged series of alternating episodes of cryopreservation and resuscitation. Intracranial transplantation of SU-1 cells produced relatively less invasive tumor mass in athymic nude mice, while SU-2 cells led to much more diffuse and aggressive lesions strikingly recapitulated their original tumors. Neither SU-1 nor SU-2 cells reached the terminal differentiation stage under conditions that would induce terminal differentiation in neural stem cells. The differentiation of most of the tumor cells seemed to be blocked at the progenitor cell phase: most of them expressed nestin but only a few co-expressed differentiation markers. Transmission electron microscopy showed that GSCs were at a primitive stage of differentiation with low autophagic activity. Array-based comparative genomic hybridization revealed genetic alterations common to both SU-1 and SU-2, including amplification of the oncogene EGFR and deletion of the tumor suppressor PTEN, while some genetic alterations such as amplification of MTA1 (metastasis associated gene 1) only occurred in SU-2. CONCLUSION: The GSPC lines SU-1 and SU-2 faithfully retained the characteristics of their original tumors and provide a reliable resource for investigating the mechanisms of formation and recurrence of human gliomas with progressive malignancy. Such investigations may eventually have major impacts on the understanding and treatment of gliomas.