Joerg Dietrich

CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo

Background Chemotherapy in cancer patients can be associated with serious short- and long-term adverse neurological effects, such as leukoencephalopathy and cognitive impairment, even when therapy is delivered systemically. The underlying cellular basis for these adverse effects is poorly understood. Results We found that three mainstream chemotherapeutic agents – carmustine (BCNU), cisplatin, and cytosine arabinoside (cytarabine), representing two DNA cross-linking agents and an antimetabolite, respectively – applied at clinically relevant exposure levels to cultured cells are more toxic for the progenitor cells of the CNS and for nondividing oligodendrocytes than they are for multiple cancer cell lines. Enhancement of cell death and suppression of cell division were seen in vitro and in vivo. When administered systemically in mice, these chemotherapeutic agents were associated with increased cell death and decreased cell division in the subventricular zone, in the dentate gyrus of the hippocampus and in the corpus callosum of the CNS. In some cases, cell division was reduced, and cell death increased, for weeks after drug administration ended. Conclusion Identifying neural populations at risk during any cancer treatment is of great importance in developing means of reducing neurotoxicity and preserving quality of life in long-term survivors. Thus, as well as providing possible explanations for the adverse neurological effects of systemic chemotherapy, the strong correlations between our in vitro and in vivo analyses indicate that the same approaches we used to identify the reported toxicities can also provide rapid in vitro screens for analyzing new therapies and discovering means of achieving selective protection or targeted killing.

Characterization of A2B5+ glial precursor cells from cryopreserved human fetal brain progenitor cells.

The identification and characterization of human neural precursor cells are critical in extending our understanding of central nervous system development from model animal systems to our own species. Moreover, availability of well‐characterized populations of human cells is of potential value in endeavors ranging from cell transplantation to drug screening. We have isolated a population of continuously dividing glial‐restricted precursor cells from commercially available cryopreserved 18–20 weeks old fetal brain neural progenitor cells. These human glial‐restricted precursor cells are A2B5+ and do not express polysialylated E‐NCAM (PSA‐NCAM). They can be grown as purified populations in serum‐free medium supplemented with basic fibroblast growth factor (bFGF) and can be induced to generate cells with the antigenic characteristics of oligodendrocytes and distinct astrocytic populations. GLIA 40:65–77, 2002.

Glutamate transporter expression and function in human glial progenitors

Glutamate is the major neurotransmitter of the brain, whose extracellular levels are tightly controlled by glutamate transporters. Five glutamate transporters in the human brain (EAAT1–5) are present on both astroglia and neurons. We characterize the profile of three different human astroglial progenitors in vitro: human glial restricted precursors (HGRP), human astrocyte precursors (HAPC), and early‐differentiated astrocytes. EAAT 1, EAAT3, and EAAT4 are all expressed in GRPs with a subsequent upregulation of EAAT1 following differentiation of GRPs into GRP‐derived astrocytes in the presence of bone morphogenic protein (BMP‐4). This corresponds to a significant increase in the glutamate transport capacity of these cells. EAAT2, the transporter responsible for the bulk of glutamate transport in the adult brain, is not expressed as a full‐length protein, nor does it appear to have functional significance (as determined by the EAAT2 inhibitor dihydrokainate) in these precursors. A splice variant of EAAT2, termed EAAT2b, does appear to be present in low levels, however. EAAT3 and EAAT4 expression is reduced as glial maturation progresses both in astrocyte precursors and early‐differentiated astrocytes and is consistent with their role in adult tissues as primarily neuronal glutamate transporters. These human glial precursors offer several advantages as tools for understanding glial biology because they can be passaged extensively in the presence of mitogens, afford the potential to study the temporal changes in glutamate transporter expression in a tightly controlled fashion, and are cultured in the absence of neuronal coculture, allowing for the independent study of astroglial biology.

Infection with an endemic human herpesvirus disrupts critical glial precursor cell properties.

Human herpesvirus 6 (HHV-6), a common resident virus of the human CNS, has been implicated in both acute and chronic inflammatory–demyelinating diseases. Although HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes, nothing is known about the susceptibility of glial precursors, the ancestors of myelin-producing oligodendrocytes, to viral infection. We show that HHV-6 infects human glial precursor cells in vitro. Active infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins, with subsequent formation of cell syncytia. Infection leads to alterations in cell morphology and impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by bromodeoxyuridine uptake, which was confirmed by blunting of the cell growth rate of infected cells compared with uninfected controls over time. The detailed analysis using novel, fluorescent-labeled HHV-6A or HHV-6B reagents demonstrated strong G1/S phase inhibition in infected precursor cells. Cell cycle arrest in HHV-6-infected cells was associated with a profound decrease in the expression of the glial progenitor cell marker A2B5 and a corresponding increase in the oligodendrocyte differentiation marker GalC. These data demonstrate for the first time that infection of primary human glial precursor cells with a neurologically relevant human herpesvirus causes profound alterations of critical precursor cell properties. In light of recent observations that repair of CNS demyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in demyelinating diseases and the disruption of normal glial maturation.

Intersections between neurobiology and oncology: tumor origin, treatment and repair of treatment-associated damage

The number of potentially intimate relationships between brain tumors and the precursor cells that contribute to normal central nervous system (CNS) development and repair now appears to be somewhat larger than would have been anticipated only a few years ago. It also appears that understanding the vulnerabilities of CNS precursor cells, and of the specific cells that they generate, might help us to reveal the biological basis for the cognitive impairment that is increasingly recognized as an adverse effect of systemic cancer therapies. Using neural stem cells as therapeutic vehicles in the treatment of brain tumors could be modified to allow repair of the damage caused by brain tumors themselves and of the neurological impairment that is frequently associated with traditional cancer treatment approaches.

The complex identity of brain tumors: emerging concerns regarding origin, diversity and plasticity

Elucidation of genetic and epigenetic mechanisms underlying neoplasia is one of the great success stories of modern science, but this success has not been associated with parallel improvements in the treatment of malignant tumors. One possible explanation for this failure is that the most important variables that support growth of malignancies are not yet identified. Another possible explanation, however, is that multiple variables important in neoplastic progression combine to create a level of disease complexity not taken into account by current therapeutic approaches. The study of development and neoplasia in the CNS provides some of the strongest support for the latter view--a view that, if correct, would suggest that a radical rethinking of the biology of malignancy is required if we are to make progress in the treatment of this important medical condition.

Redox biology in normal cells and cancer: restoring function of the redox/Fyn/c-Cbl pathway in cancer cells offers new approaches to cancer treatment.

This review discusses a unique discovery path starting with novel findings on redox regulation of precursor cell and signaling pathway function and identification of a new mechanism by which relatively small changes in redox status can control entire signaling networks that regulate self-renewal, differentiation, and survival. The pathway central to this work, the redox/Fyn/c-Cbl (RFC) pathway, converts small increases in oxidative status to pan-activation of the c-Cbl ubiquitin ligase, which controls multiple receptors and other proteins of central importance in precursor cell and cancer cell function. Integration of work on the RFC pathway with attempts to understand how treatment with systemic chemotherapy causes neurological problems led to the discovery that glioblastomas (GBMs) and basal-like breast cancers (BLBCs) inhibit c-Cbl function through altered utilization of the cytoskeletal regulators Cool-1/βpix and Cdc42, respectively. Inhibition of these proteins to restore normal c-Cbl function suppresses cancer cell division, increases sensitivity to chemotherapy, disrupts tumor-initiating cell (TIC) activity in GBMs and BLBCs, controls multiple critical TIC regulators, and also allows targeting of non-TICs. Moreover, these manipulations do not increase chemosensitivity or suppress division of nontransformed cells. Restoration of normal c-Cbl function also allows more effective harnessing of estrogen receptor-α (ERα)-independent activities of tamoxifen to activate the RFC pathway and target ERα-negative cancer cells. Our work thus provides a discovery strategy that reveals mechanisms and therapeutic targets that cannot be deduced by standard genetics analyses, which fail to reveal the metabolic information, isoform shifts, protein activation, protein complexes, and protein degradation critical to our discoveries.

Developing concepts in neural stem cells

Stem Cells in the Mammalian Brain: the 4th Brain Research Interactive Symposium, at the 2001 Annual Conference of the Society for Neuroscience, San Diego, CA, USA from November 8-10 2001.

Characterization of specific HHV-6 and cell cycle genes implicated in virus-mediated G1/S cell-cycle arrest of glial precursors

Human herpesvirus 6 (HHV-6), a common resident virus of the human central nervous system (CNS), has been implicated in both acute and chronic inflammatory-demyelinating diseases. HHV-6 persists within the human CNS and has been described to infect mature oligodendrocytes and astrocytes. We recently demonstrated that HHV-6 infects human glial precursor cells, the ancestors of myelin producing oligodendrocytes, in vitro. Productive infection was demonstrated by both electron microscopy and expression of viral gene transcripts and proteins. Infection led to impairment of cell replication but not increased cell death. Infected cells showed decreased proliferation as measured by BrdU uptake and demonstrated strong G1/S-phase inhibition by FACS. We now present evidence detailing both specific viral and cell cycle genes inducing cellular G1/S cell cycle arrest. In parallel with human cells, the well characterized rodent oligodendroglial progenitor cell (OPC) system was used. Restricted infection of the murine cells was demonstrated by quantitative PCR, EM, and RT-PCR, and IFA for viral late proteins but, intriguingly, reproduced the G1/S arrest seen after productive virus infection of human progenitors. Restricted infection of murine OPCs was also found to be sufficient to induce the activation of DNA damage pathways previously reported by our group in human glial progenitors (ISNV 2004) and after productive infection in human lymphocytes (Oster et al. 2005). IFA and immunoblot again demonstrated expression of phospho-ATM, CHK-2, and p53 in infected murine precursors and two viral IE proteins were sufficient to induce G1/S arrest. Virus-induced cell-cycle arrest was accompanied by GalC+ differentiation with corresponding loss of the OPC pool as had been previously seen in the human precursors. Enhanced GFP-expressing murine OPCs have been created that can be readily infected with ALEXA-632-labeled HHV-6, allowing both sorting for infected cells and their identification in evaluating repair in demyelinated mice. In light of recent observations that repair of CNS remyelination is dependent on the generation of mature oligodendrocytes from the glial precursor cell pool, these findings may have broad implications for both the ineffective repair seen in human demyelinating diseases and the disruption of normal glial differentiation.

Analysis of Neural Precursor Cells in the Context of Origin of Brain Tumors, Their Treatment, and Repair of Treatment-Associated Damage

The treatment of any disease always begins with a correct diagnosis. It is here, before any treatment has been initiated, that enormous problems are faced by those involved in investigating brain tumors. Despite longstanding attempts to generate unambiguous diagnostic criteria for tumors of the central nervous system (CNS), the extent of disagreement between neuropathologists diagnosing the same tumor is often considerable (17,47,53,91). The importance of this disagreement cannot be understated. If pathologists are going to place identical tumors in different categories, then further analyses of outcomes as they pertain to diagnosis are severely compromised.