Margot Mayer-Pröschel

Isolation of Lineage-Restricted Neuronal Precursors from Multipotent Neuroepithelial Stem Cells

We have identified a neuronal-restricted precursor (NRP) cell that expresses E-NCAM (high polysialic-acid NCAM) and is morphologically distinct from multipotent neuroepithelial (NEP) cells (Kalyani et al., 1997) and spinal glial progenitors (Rao and Mayer-Proschel, 1997). NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin-3 (NT-3) and differentiate in the presence of retinoic acid and the absence of FGF into postmitotic neurons. NRP cells can also be generated from multipotent E10.5 NEP cells. Clonal analysis shows that NRP cells arise from a NEP progenitor that generates other restricted CNS precursors. The NEP-derived NRPs undergo self renewal and can differentiate into multiple neuronal phenotypes. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at E13.5 in the spinal cord.

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.

Systemic 5-fluorouracil treatment causes a syndrome of delayed myelin destruction in the central nervous system

Background Cancer treatment with a variety of chemotherapeutic agents often is associated with delayed adverse neurological consequences. Despite their clinical importance, almost nothing is known about the basis for such effects. It is not even known whether the occurrence of delayed adverse effects requires exposure to multiple chemotherapeutic agents, the presence of both chemotherapeutic agents and the bodys own response to cancer, prolonged damage to the blood-brain barrier, inflammation or other such changes. Nor are there any animal models that could enable the study of this important problem. Results We found that clinically relevant concentrations of 5-fluorouracil (5-FU; a widely used chemotherapeutic agent) were toxic for both central nervous system (CNS) progenitor cells and non-dividing oligodendrocytes in vitro and in vivo. Short-term systemic administration of 5-FU caused both acute CNS damage and a syndrome of progressively worsening delayed damage to myelinated tracts of the CNS associated with altered transcriptional regulation in oligodendrocytes and extensive myelin pathology. Functional analysis also provided the first demonstration of delayed effects of chemotherapy on the latency of impulse conduction in the auditory system, offering the possibility of non-invasive analysis of myelin damage associated with cancer treatment. Conclusions Our studies demonstrate that systemic treatment with a single chemotherapeutic agent, 5-FU, is sufficient to cause a syndrome of delayed CNS damage and provide the first animal model of delayed damage to white-matter tracts of individuals treated with systemic chemotherapy. Unlike that caused by local irradiation, the degeneration caused by 5-FU treatment did not correlate with either chronic inflammation or extensive vascular damage and appears to represent a new class of delayed degenerative damage in the CNS.

Gliogenesis in the central nervous system

Multipotential neuroepithelial stem cells are thought to give rise to all the differentiated cells of the central nervous system (CNS). The developmental potential of these multipotent stem cells becomes more restricted as they differentiate into progressively more committed cells and ultimately into mature neurons and glia. In studying gliogenesis, the optic nerve and spinal cord have become invaluable models and the progressive stages of differentiation are being clarified. Multiple classes of glial precursors termed glial restricted precursors (GRP), oligospheres, oligodendrocyte‐type2 astrocyte (O‐2A) and astrocyte precursor cells (APC) have been identified. Similar classes of precursor cells can be isolated from human neural stem cell cultures and from embryonic stem (ES) cell cultures providing a non‐fetal source of such cells. In this review, we discuss gliogenesis, glial stem cells, putative relationships of these cells to each other, factors implicated in gliogenesis, and therapeutic applications of glial precursors. GLIA 30:105–121, 2000.

Astrocytes Secrete Exosomes Enriched with Proapoptotic Ceramide and Prostate Apoptosis Response 4 (PAR-4): POTENTIAL MECHANISM OF APOPTOSIS INDUCTION IN ALZHEIMER DISEASE (AD)*

Background: In AD, amyloid protein is associated with neurodegeneration, which may involve amyloid effects on astrocytes. Results: In astrocytes, amyloid peptide triggers secretion of proapoptotic exosomes (“apoxosomes”) that are associated with ceramide and PAR-4. Conclusion: Activation of nSMase2 and expression of PAR-4 is critical for the secretion of apoxosomes and glial apoptosis. Significance: Apoxosomes may contribute to glial apoptosis, and therefore, neurodegeneration in AD. Amyloid protein is well known to induce neuronal cell death, whereas only little is known about its effect on astrocytes. We found that amyloid peptides activated caspase 3 and induced apoptosis in primary cultured astrocytes, which was prevented by caspase 3 inhibition. Apoptosis was also prevented by shRNA-mediated down-regulation of PAR-4, a protein sensitizing cells to the sphingolipid ceramide. Consistent with a potentially proapoptotic effect of PAR-4 and ceramide, astrocytes surrounding amyloid plaques in brain sections of the 5xFAD mouse (and Alzheimer disease patient brain) showed caspase 3 activation and were apoptotic when co-expressing PAR-4 and ceramide. Apoptosis was not observed in astrocytes with deficient neutral sphingomyelinase 2 (nSMase2), indicating that ceramide generated by nSMase2 is critical for amyloid-induced apoptosis. Antibodies against PAR-4 and ceramide prevented amyloid-induced apoptosis in vitro and in vivo, suggesting that apoptosis was mediated by exogenous PAR-4 and ceramide, potentially associated with secreted lipid vesicles. This was confirmed by the analysis of lipid vesicles from conditioned medium showing that amyloid peptide induced the secretion of PAR-4 and C18 ceramide-enriched exosomes. Exosomes were not secreted by nSMase2-deficient astrocytes, indicating that ceramide generated by nSMase2 is critical for exosome secretion. Consistent with the ceramide composition in amyloid-induced exosomes, exogenously added C18 ceramide restored PAR-4-containing exosome secretion in nSMase2-deficient astrocytes. Moreover, isolated PAR-4/ceramide-enriched exosomes were taken up by astrocytes and induced apoptosis in the absence of amyloid peptide. Taken together, we report a novel mechanism of apoptosis induction by PAR-4/ceramide-enriched exosomes, which may critically contribute to Alzheimer disease.

Acute transplantation of glial-restricted precursor cells into spinal cord contusion injuries: survival, differentiation, and effects on lesion environment and axonal regeneration

Transplantation of stem cells and immature cells has been reported to ameliorate tissue damage, induce axonal regeneration, and improve locomotion following spinal cord injury. However, unless these cells are pushed down a neuronal lineage, the majority of cells become glia, suggesting that the alterations observed may be potentially glially mediated. Transplantation of glial-restricted precursor (GRP) cells--a precursor cell population restricted to oligodendrocyte and astrocyte lineages--offers a novel way to examine the effects of glial cells on injury processes and repair. This study examines the survival and differentiation of GRP cells, and their ability to modulate the development of the lesion when transplanted immediately after a moderate contusion injury of the rat spinal cord. GRP cells isolated from a transgenic rat that ubiquitously expresses heat-stable human placental alkaline phosphatase (PLAP) were used to unambiguously detect transplanted GRP cells. Following transplantation, some GRP cells differentiated into oligodendrocytes and astrocytes, retaining their differentiation potential after injury. Transplanted GRP cells altered the lesion environment, reducing astrocytic scarring and the expression of inhibitory proteoglycans. Transplanted GRP cells did not induce long-distance regeneration from corticospinal tract (CST) and raphe-spinal axons when compared to control animals. However, GRP cell transplants did alter the morphology of CST axons toward that of growth cones, and CST fibers were found within GRP cell transplants, suggesting that GRP cells may be able to support axonal growth in vivo after injury.

Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury

Background Two critical challenges in developing cell-transplantation therapies for injured or diseased tissues are to identify optimal cells and harmful side effects. This is of particular concern in the case of spinal cord injury, where recent studies have shown that transplanted neuroepithelial stem cells can generate pain syndromes. Results We have previously shown that astrocytes derived from glial-restricted precursor cells (GRPs) treated with bone morphogenetic protein-4 (BMP-4) can promote robust axon regeneration and functional recovery when transplanted into rat spinal cord injuries. In contrast, we now show that transplantation of GRP-derived astrocytes (GDAs) generated by exposure to the gp130 agonist ciliary neurotrophic factor (GDAsCNTF), the other major signaling pathway involved in astrogenesis, results in failure of axon regeneration and functional recovery. Moreover, transplantation of GDACNTF cells promoted the onset of mechanical allodynia and thermal hyperalgesia at 2 weeks after injury, an effect that persisted through 5 weeks post-injury. Delayed onset of similar neuropathic pain was also caused by transplantation of undifferentiated GRPs. In contrast, rats transplanted with GDAsBMP did not exhibit pain syndromes. Conclusion Our results show that not all astrocytes derived from embryonic precursors are equally beneficial for spinal cord repair and they provide the first identification of a differentiated neural cell type that can cause pain syndromes on transplantation into the damaged spinal cord, emphasizing the importance of evaluating the capacity of candidate cells to cause allodynia before initiating clinical trials. They also confirm the particular promise of GDAs treated with bone morphogenetic protein for spinal cord injury repair.

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system.

Redox State as a Central Modulator of Precursor Cell Function

Abstract: In our attempts to understand how the balance between self‐renewal and differentiation is regulated in dividing precursor cells, we have discovered that intracellular redox state appears to be a critical modulator of this balance in oligodendrocyte‐type‐2 astrocyte (O‐2A) progenitor cells. The intracellular redox state of freshly isolated progenitor cells allows prospective isolation of cells with different self‐renewal characteristics, which can be further modulated in opposite directions by prooxidants and antioxidants. Redox state is itself modulated by cell‐extrinsic signaling molecules that alter the balance between self‐renewal and differentiation: growth factors that promote self‐renewal cause progenitors to become more reduced, while exposure to signaling molecules that promote differentiation causes progenitors to become more oxidized. Moreover, pharmacological antagonists of the redox effects of these cell‐extrinsic signaling molecules antagonize their effects on self‐renewal and differentiation, further suggesting that cell‐extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism. A further example of the potential relevance of intracellular redox state to development processes emerges from our attempts to understand why different central nervous system (CNS) regions exhibit different temporal patterns of oligodendrocyte generation and myelinogenesis. Characterization of O‐2A progenitor cells (O‐2A/OPCs) isolated from different regions indicates that these developmental patterns are consistent with properties of the specific O‐2A/OPCs resident in each region. Marked differences were seen in self‐renewal and differentiation characteristics of O‐2A/OPCs isolated from cortex, optic nerve, and optic chiasm. In conditions where optic nerve‐derived O‐2A/OPCs generated oligodendrocytes within 2 days, oligodendrocytes arose from chiasm‐derived cells after 5 days and from cortical O‐2A/OPCs after only 7–10 days. These differences, which appear to be cell intrinsic, were manifested both in reduced percentages of clones producing oligodendrocytes and in a lesser representation of oligodendrocytes in individual clones. In addition, responsiveness of optic nerve‐, chiasm‐, and cortex‐derived O‐2A/OPCs to thyroid hormone (TH) and ciliary neurotrophic factor (CNTF), well‐characterized inducers of oligodendrocyte generation, was inversely related to the extent of self‐renewal observed in basal division conditions. These results demonstrate hitherto unrecognized complexities among the precursor cells thought to be the immediate ancestors of oligodendrocytes and suggest that the properties of these different populations may contribute to the diverse time courses of myelination in different CNS regions. Strikingly, O‐2A/OPCs isolated from cortex and analyzed immediately upon isolation were more reduced in their redox state than were optic nerve‐derived cells, precisely as would be predicted from our analysis of the role of redox state in modulating the balance between self‐renewal and differentiation. Chiasm‐derived cells, which exhibited self‐renewal properties intermediate between cortex‐ and optic nerve‐derived cells, were more reduced than optic nerve cells but more oxidized that cortical O‐2A/OPCs.

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.