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1.  Iron deficiency disrupts axon maturation of the developing auditory nerve 
The Journal of Neuroscience  2012;32(14):5010-5015.
Iron is critical in multiple aspects of CNS development but its role in neurodevelopment the ability of iron deficiency to alter normal development is difficult to dissociate from the effects of anemia. We developed a novel dietary restriction model that allows us to study the effects of iron deficiency in the absence of severe anemia. Using a combination of auditory brain stem response analyses (ABR) and electron microscopy, we identified an unexpected impact of non-anemic iron deficiency on axonal diameter and neurofilament regulation in the auditory nerve. These changes are associated with altered ABR latency during development. In contrast to models of severe iron deficiency with anemia, we did not find consistent and prolonged defects in myelination. Our data demonstrate that iron deficiency in the absence of anemia disrupts normal development of the auditory nerve and results in altered conduction velocity.
doi:10.1523/JNEUROSCI.0526-12.2012
PMCID: PMC3327472  PMID: 22492056
2.  Neural Precursor Cell Proliferation Is Disrupted Through Activation of the Aryl Hydrocarbon Receptor by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin 
Stem Cells and Development  2010;20(2):313-326.
Neurogenesis involves the proliferation of multipotent neuroepithelial stem cells followed by differentiation into lineage-restricted neural precursor cells (NPCs) during the embryonic period. Interestingly, these progenitor cells express robust levels of the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that regulates expression of genes important for growth regulation, and xenobiotic metabolism. Upon binding 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a pervasive environmental contaminant and potent AhR ligand, AhR, is activated and disrupts gene expression patterns to produce cellular toxicity. Because of its widespread distribution in the brain during critical proliferative phases of neurogenesis, it is conceivable that AhR participates in NPC expansion. Therefore, this study tested the hypothesis that AhR activation by TCDD disrupts signaling events that regulate NPC proliferation. The C17.2 NPC line served as a model system to (1) assess whether NPCs are targets for TCDD-induced neurotoxicity and (2) characterize the effects of TCDD on NPC proliferation. We demonstrated that C17.2 NPCs express an intact AhR signaling pathway that becomes transcriptionally active after TCDD exposure. 3H-thymidine and alamar blue reduction assays indicated that TCDD suppresses NPC proliferation in a concentration-dependent manner without the loss of cell viability. Cell cycle distribution analysis by flow cytometry revealed that TCDD-induced growth arrest results from an impaired G1 to S cell cycle transition. Moreover, TCDD exposure altered p27 kip1 and cyclin D1 cell cycle regulatory protein expression levels consistent with a G1 phase arrest. Initial studies in primary NPCs isolated from the ventral forebrain of embryonic mice demonstrated that TCDD reduced cell proliferation through a G1 phase arrest, corroborating our findings in the C17.2 cell line. Together, these observations suggest that the inappropriate or sustained activation of AhR by TCDD during neurogenesis can interfere with signaling pathways that regulate neuroepithelial stem cell/NPC proliferation, which could adversely impact final cell number in the brain and lead to functional impairments.
doi:10.1089/scd.2009.0529
PMCID: PMC3128757  PMID: 20486776
3.  A composite likelihood approach to the analysis of longitudinal clonal data on multitype cellular systems under an age-dependent branching process 
Biostatistics (Oxford, England)  2010;12(1):173-191.
A recurrent statistical problem in cell biology is to draw inference about cell kinetics from observations collected at discrete time points. We investigate this problem when multiple cell clones are observed longitudinally over time. The theory of age-dependent branching processes provides an appealing framework for the quantitative analysis of such data. Likelihood inference being difficult in this context, we propose an alternative composite likelihood approach, where the estimation function is defined from the marginal or conditional distributions of the number of cells of each observable cell type. These distributions have generally no closed-form expressions but they can be approximated using simulations. We construct a bias-corrected version of the estimating function, which also offers computational advantages. Two algorithms are discussed to compute parameter estimates. Large sample properties of the estimator are presented. The performance of the proposed method in finite samples is investigated in simulation studies. An application to the analysis of the generation of oligodendrocytes from oligodendrocyte type-2 astrocyte progenitor cells cultured in vitro reveals the effect of neurothrophin-3 on these cells. Our work demonstrates also that the proposed approach outperforms the existing ones.
doi:10.1093/biostatistics/kxq050
PMCID: PMC3006127  PMID: 20732974
Bias correction; Cell differentiation; Composite likelihood; Discrete data; Monte Carlo; Neurotrophin-3; Oligodendrocytes; Precursor cell; Stochastic model
4.  Glial Restricted Precursor Cell Transplant with Cyclic Adenosine Monophosphate Improved Some Autonomic Functions but Resulted in a Reduced Graft Size after Spinal Cord Contusion Injury in Rats 
Experimental neurology  2010;227(1):159-171.
Transplantation of glial restricted precursor (GRP) cells has been shown to reduce glial scarring after spinal cord injury (SCI) and, in combination with neuronal restricted precursor (NRP) cells or enhanced expression of neurotrophins, to improve recovery of function after SCI. We hypothesized that combining GRP transplants with rolipram and cAMP would improve functional recovery, similar to that seen after combining Schwann cell transplants with increasing cAMP. A short term study, 1)uninjured control, 2)SCI+vehicle, and 3)SCI+cAMP, showed that spinal cord [cAMP] were increased 14 days after SCI. We used 51 male rats subjected to a thoracic SCI for a 12-week survival study: 1)SCI+vehicle, 2)SCI+GRP, 3)SCI+cAMP, 4)SCI+GRP+cAMP, and 5)uninjured endpoint age-matched control (AM). Rolipram was administered for 2 weeks after SCI. At 9 days after SCI, GRP transplantation and injection of dibutyryl-cAMP into the spinal cord were performed. GRP cells survived, differentiated, and formed extensive transplants that were well integrated with host tissue. Presence of GRP cells increased the amount of tissue in the lesion; however, cAMP reduced the graft size. White matter sparing at the lesion epicenter was not affected. Serotonergic input to the lumbosacral spinal cord was not affected by treatment, but the amount of serotonin immediately caudal to the lesion was reduced in the cAMP groups. Using telemetric monitoring of corpus spongiosum penis pressure we show that the cAMP groups regained the same number of micturitions per 24 hrs when compared to the AM group, however, the frequency of peak pressures was increased in these groups compared to the AM group. In contrast, the GRP groups had similar frequency of peak pressures compared to baseline and the AM group. Animals that received GRP cells regained the same number of erectile events per 24 hrs compared to baseline and the AM group. Since cAMP reduced the GRP transplant graft, and some modest positive effects were seen that could be attributable to both GRP or cAMP, future research is required to determine how cAMP affects survival, proliferation, and / or function of progenitor cells and how this is related to function. cAMP may not always be a desirable addition to a progenitor cell transplantation strategy after SCI.
doi:10.1016/j.expneurol.2010.10.011
PMCID: PMC3018678  PMID: 21040723
rolipram; micturition; sexual function; chronic; telemetry; hPLAP transgenic rat; serotonin; therapeutic strategy; astrocytes; oligodendrocytes
5.  Oligodendrocyte progenitors reversibly exit the cell cycle and give rise to astrocytes in response to interferon-γ 
Oligodendrocyte – type-2 astrocyte progenitor cells (O-2A/OPCs) populate the CNS and generate oligodendrocytes and astrocytes in vitro and in vivo. Understanding how O-2A/OPCs respond to their environment is crucial to understanding how these cells function in the CNS and how to best promote their therapeutic proliferation and differentiation. We show that IFN-γ was not toxic to highly purified perinatal or adult rat O-2A/OPCs. IFN-γ treatment led to downregulation of PDGFR-α and Ki-67 and decreased self-renewal in clonal populations. IFN-γ also significantly increased the proportion of cells in the G0/G1 phase of the cell cycle, decreased BrdU incorporation, and led to increased expression of the cell cycle inhibitors Rb and p27kip1. While p27kip1 expression was not necessary for IFN-γ – mediated quiescence, its upstream regulator IRF-1 was required. The quiescent state of O-2A/OPCs caused by INF-γ was reversible as the withdrawal of IFN-γ allowed O-2A/OPCs to appropriately respond to both proliferation and differentiation signals. Differentiation into oligodendrocytes induced by either thyroid hormone or CNTF was also abrogated by IFN-γ. This inhibition was specific to the oligodendrocyte pathway, as O-2A/OPC differentiation into astrocytes was not inhibited. IFN-γ alone also led to the generation of GFAP positive astrocytes in a subset of O-2A/OPCs. Taken together these results demonstrate a reversible inhibitory effect of IFN-γ on O-2A/OPC proliferation with a concomitant generation of astrocytes. We propose that neuroinflammation involving increased IFN-γ can reduce progenitor numbers and inhibit differentiation, which has significant clinical relevance for injury repair, but may also contribute to the generation of astrocytes.
doi:10.1523/JNEUROSCI.5905-10.2011
PMCID: PMC3104669  PMID: 21508246
Oligodendrocyte precursor cell; oligodendrocyte; astrocyte; interferon; multiple sclerosis; cell cycle
6.  Precursor Cell Biology and the Development of Astrocyte Transplantation Therapies: Lessons from Spinal Cord Injury 
Neurotherapeutics  2011;8(4):677-693.
This review summarizes current progress on development of astrocyte transplantation therapies for repair of the damaged central nervous system. Replacement of neurons in the injured or diseased central nervous system is currently one of the most popular therapeutic goals, but if neuronal replacement is attempted in the absence of appropriate supporting cells (astrocytes and oligodendrocytes), then the chances of restoring neurological functional are greatly reduced. Although the past 20 years have offered great progress on oligodendrocyte replacement therapies, astrocyte transplantation therapies have been both less explored and comparatively less successful. We have now developed successful astrocyte transplantation therapies by pre-differentiating glial restricted precursor (GRP) cells into a specific population of GRP cell-derived astrocytes (GDAs) by exposing the GRP cells to bone morphogenetic protein-4 (BMP) prior to transplantation. When transplanted into transected rat spinal cord, rat and human GDAsBMP promote extensive axonal regeneration, rescue neuronal cell survival, realign tissue structure, and restore behavior to pre-injury levels on a grid-walk analysis of volitional foot placement. Such benefits are not provided by GRP cells themselves, demonstrating that the lesion environment does not direct differentiation in a manner optimally beneficial for the restoration of function. Such benefits also are not provided by transplantation of a different population of astrocytes generated from GRP cells exposed to ciliary neurotrophic factor (GDAsCNTF), thus providing the first transplantation-based evidence of functional heterogeneity in astrocyte populations. Moreover, lessons learned from the study of rat cells are strongly predictive of outcomes using human cells. Thus, these studies provide successful strategies for the use of astrocyte transplantation therapies for restoration of function following spinal cord injury.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0071-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-011-0071-z
PMCID: PMC3210359  PMID: 21918888
Glial-restricted precursor cells; Glial precursor cell-derived astrocyte; Spinal cord injury; Regeneration; Astrocyte transplantation therapy; Astrocyte heterogeneity
7.  Precursor Cell Biology and the Development of Astrocyte Transplantation Therapies: Lessons from Spinal Cord Injury 
Neurotherapeutics  2011;8(4):677-693.
This review summarizes current progress on development of astrocyte transplantation therapies for repair of the damaged central nervous system. Replacement of neurons in the injured or diseased central nervous system is currently one of the most popular therapeutic goals, but if neuronal replacement is attempted in the absence of appropriate supporting cells (astrocytes and oligodendrocytes), then the chances of restoring neurological functional are greatly reduced. Although the past 20 years have offered great progress on oligodendrocyte replacement therapies, astrocyte transplantation therapies have been both less explored and comparatively less successful. We have now developed successful astrocyte transplantation therapies by pre-differentiating glial restricted precursor (GRP) cells into a specific population of GRP cell-derived astrocytes (GDAs) by exposing the GRP cells to bone morphogenetic protein-4 (BMP) prior to transplantation. When transplanted into transected rat spinal cord, rat and human GDAsBMP promote extensive axonal regeneration, rescue neuronal cell survival, realign tissue structure, and restore behavior to pre-injury levels on a grid-walk analysis of volitional foot placement. Such benefits are not provided by GRP cells themselves, demonstrating that the lesion environment does not direct differentiation in a manner optimally beneficial for the restoration of function. Such benefits also are not provided by transplantation of a different population of astrocytes generated from GRP cells exposed to ciliary neurotrophic factor (GDAsCNTF), thus providing the first transplantation-based evidence of functional heterogeneity in astrocyte populations. Moreover, lessons learned from the study of rat cells are strongly predictive of outcomes using human cells. Thus, these studies provide successful strategies for the use of astrocyte transplantation therapies for restoration of function following spinal cord injury.
Electronic supplementary material
The online version of this article (doi:10.1007/s13311-011-0071-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s13311-011-0071-z
PMCID: PMC3210359  PMID: 21918888
Glial-restricted precursor cells; Glial precursor cell-derived astrocyte; Spinal cord injury; Regeneration; Astrocyte transplantation therapy; Astrocyte heterogeneity
8.  Identifying a Window of Vulnerability during Fetal Development in a Maternal Iron Restriction Model 
PLoS ONE  2011;6(3):e17483.
It is well acknowledged from observations in humans that iron deficiency during pregnancy can be associated with a number of developmental problems in the newborn and developing child. Due to the obvious limitations of human studies, the stage during gestation at which maternal iron deficiency causes an apparent impairment in the offspring remains elusive. In order to begin to understand the time window(s) during pregnancy that is/are especially susceptible to suboptimal iron levels, which may result in negative effects on the development of the fetus, we developed a rat model in which we were able to manipulate and monitor the dietary iron intake during specific stages of pregnancy and analyzed the developing fetuses. We established four different dietary-feeding protocols that were designed to render the fetuses iron deficient at different gestational stages. Based on a functional analysis that employed Auditory Brainstem Response measurements, we found that maternal iron restriction initiated prior to conception and during the first trimester were associated with profound changes in the developing fetus compared to iron restriction initiated later in pregnancy. We also showed that the presence of iron deficiency anemia, low body weight, and changes in core body temperature were not defining factors in the establishment of neural impairment in the rodent offspring.
Our data may have significant relevance for understanding the impact of suboptimal iron levels during pregnancy not only on the mother but also on the developing fetus and hence might lead to a more informed timing of iron supplementation during pregnancy.
doi:10.1371/journal.pone.0017483
PMCID: PMC3057971  PMID: 21423661
9.  Transplanted astrocytes derived from BMP- or CNTF-treated glial-restricted precursors have opposite effects on recovery and allodynia after spinal cord injury 
Journal of Biology  2008;7(7):24.
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.
doi:10.1186/jbiol85
PMCID: PMC2776404  PMID: 18803859
10.  Systemic 5-fluorouracil treatment causes a syndrome of delayed myelin destruction in the central nervous system 
Journal of Biology  2008;7(4):12.
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 body's 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.
doi:10.1186/jbiol69
PMCID: PMC2397490  PMID: 18430259
11.  Identification of two novel glial-restricted cell populations in the embryonic telencephalon arising from unique origins 
Background
Considerably less attention has been given to understanding the cellular components of gliogenesis in the telencephalon when compared to neuronogenesis, despite the necessity of normal glial cell formation for neurological function. Early proposals of exclusive ventral oligodendrocyte precursor cell (OPC) generation have been challenged recently with studies revealing the potential of the dorsal telencephalon to also generate oligodendrocytes. The identification of OPCs generated from multiple regions of the developing telencephalon, together with the need of the embryonic telencephalon to provide precursor cells for oligodendrocytes as well as astrocytes in ventral and dorsal areas, raises questions concerning the identity of the precursor cell populations capable of generating macroglial subtypes during multiple developmental windows and in differing locations.
Results
We have identified progenitor populations in the ventral and dorsal telencephalon restricted to the generation of astrocytes and oligodendrocytes. We further demonstrate that the dorsal glial progenitor cells can be generated de novo from the dorsal telencephalon and we demonstrate their capacity for in vivo production of both myelin-forming oligodendrocytes and astrocytes upon transplantation.
Conclusion
Based on our results we offer a unifying model of telencephalic gliogenesis, with the generation of both oligodendrocytes and astrocytes from spatially separate, but functionally similar, glial restricted populations at different developmental times in the dorsal and ventral CNS.
doi:10.1186/1471-213X-7-33
PMCID: PMC1858687  PMID: 17439658
13.  CNS progenitor cells and oligodendrocytes are targets of chemotherapeutic agents in vitro and in vivo 
Journal of Biology  2006;5(7):22.
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.
doi:10.1186/jbiol50
PMCID: PMC2000477  PMID: 17125495

Results 1-13 (13)