Developmental neurotoxicity of inhaled ambient ultrafine particle air pollution: Parallels with neuropathological and behavioral features of autism and other neurodevelopmental disorders.

Accumulating evidence from both human and animal studies show that brain is a target of air pollution. Multiple epidemiological studies have now linked components of air pollution to diagnosis of autism spectrum disorder (ASD), a linkage with plausibility based on the shared mechanisms of inflammation. Additional plausibility appears to be provided by findings from our studies in mice of exposures from postnatal day (PND) 4-7 and 10-13 (human 3rd trimester equivalent), to concentrated ambient ultrafine (UFP) particles, considered the most reactive component of air pollution, at levels consistent with high traffic areas of major U.S. cities and thus highly relevant to human exposures. These exposures, occurring during a period of marked neuro- and gliogenesis, unexpectedly produced a pattern of developmental neurotoxicity notably similar to multiple hypothesized mechanistic underpinnings of ASD, including its greater impact in males. UFP exposures induced inflammation/microglial activation, reductions in size of the corpus callosum (CC) and associated hypomyelination, aberrant white matter development and/or structural integrity with ventriculomegaly (VM), elevated glutamate and excitatory/inhibitory imbalance, increased amygdala astrocytic activation, and repetitive and impulsive behaviors. Collectively, these findings suggest the human 3rd trimester equivalent as a period of potential vulnerability to neurodevelopmental toxicity to UFP, particularly in males, and point to the possibility that UFP air pollution exposure during periods of rapid neuro- and gliogenesis may be a risk factor not only for ASD, but also for other neurodevelopmental disorders that share features with ASD, such as schizophrenia, attention deficit disorder, and periventricular leukomalacia.

cFLIP is critical for oligodendrocyte protection from inflammation.

Neuroinflammation associated with degenerative central nervous system disease and injury frequently results in oligodendrocyte death. While promoting oligodendrocyte viability is a major therapeutic goal, little is known about protective signaling strategies. We report that in highly purified rat oligodendrocytes, interferon gamma (IFNγ) activates a signaling pathway that protects these cells from tumor necrosis factor alpha (TNFα)-induced cytotoxicity. IFNγ protection requires Jak (Janus kinase) activation, components of the integrated stress response and NF-κB activation. Although NF-κB activation also occurred transiently in the absence of IFNγ and presence of TNFα, this activation was not sufficient to prevent induction of the TNFα-responsive cell death pathway. Genetic inhibition of NF-κB translocation to the nucleus abrogated IFNγ-mediated protection and did not change the cell death induced by TNFα, suggesting that NF-κB activation via IFNγ induces a different set of responses than activation of NF-κB via TNFα. A promising candidate is the NF-κB target cFLIP (cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein), which is protease-deficient caspase homolog that inhibits caspase-3 activation. We show that IFNγ-mediated protection led to upregulation of cFLIP. Overexpression of cFLIP was sufficient for oligodendrocyte protection from TNFα and short hairpin RNA knockdown of cFLIP-abrogated IFNγ -mediated protection. To determine the relevance of our in vitro finding to the more complex in vivo situation, we determined the impact on oligodendrocyte death of regional cFLIP loss of function in a murine model of neuroinflammation. Our data show that downregulation of cFLIP during inflammation leads to death of oligodendrocytes and decrease of myelin in vivo. Taken together, we show that IFNγ-mediated induction of cFLIP expression provides a new mechanism by which this cytokine can protect oligodendrocytes from TNFα-induced cell death.

Saddlepoint approximations to the moments of multitype age-dependent branching processes, with applications.

This article proposes saddlepoint approximations to the expectation and variance-covariance function of multitype age-dependent branching processes. The proposed approximations are found accurate, easy to implement, and much faster to compute than by simulating the process. Multiple applications are presented, including the analyses of clonal data on the generation of oligodendrocytes from their immediate progenitor cells, and on the proliferation of Hela cells. New estimators are also constructed to analyze clonal data. The proposed methods are finally used to approximate the distribution of the generation, which has recently found several applications in cell biology.

The cortical ancestry of oligodendrocytes: common principles and novel features.

Studies on the development of cortical oligodendrocytes indicate that although general principles that apply to other parts of the CNS are applicable, there are important differences that appear to be critical to the analysis of this lineage in the cortex. Herein, we review previous studies demonstrating that oligodendrocyte-type-2 astrocyte progenitor cells (or oligodendrocyte precursor cells; aka O-2A/OPCs) of the developing postnatal cortex exhibit a striking cell-intrinsic bias towards undergoing prolonged self-renewal in the relative absence of oligodendrocyte generation [Power et al., Dev Biol 2002;245:362-375]. This phenotype is quite distinct from that observed in comparable cells isolated from the optic tract. This predilection for self-renewal is associated with a lessened response to inducers of oligodendrocyte generation and of possible mechanistic importance in regards to these other properties. We also review studies on stem/progenitor cells isolated from the embryonic cortex that are able to generate oligodendrocytes. As for the studies on O-2A/OPCs, important differences also distinguish these early cells from those studied in other CNS regions in their response to signaling molecules and expression of the Dlx family of transcriptional regulators [He et al., J Neurosci 2001;21:8854-8862; Yung et al., Proc Natl Acad Sci USA 2002;99:16273-16278]. We also present new data on clonal analysis of A2B5+ precursor cells isolated from the E13.5 cortex, demonstrating that this tissue appears to contain a cell similar in properties to the tripotential glial-restricted precursor cell that has been isolated from embryonic spinal cord [Rao et al., Proc Natl Acad Sci USA 1998;95:3996-4001]. Moreover, the A2B5+ precursor cells isolated from embryonic cortex are much more heterogeneous than is seen in the spinal cord at this age, even to the point of including an A2B5/PSA-NCAM double-positive cell that can generate neurons.

Iron modulates the differentiation of a distinct population of glial precursor cells into oligodendrocytes.

Iron deficiency in children is associated with a number of neural defects including hypomyelination. It has been hypothesized by others that this hypomyelination is due to a failure in myelin production. Other possibilities include failure in the generation of oligodendrocytes from their precursor cells or an interruption in oligodendrocyte maturation. These hypotheses are based on the observations that there is a peak in brain iron uptake in vivo that coincides with the period of greatest myelination and that a shortage of iron leads to myelination deficiency. We now demonstrate that iron availability modulates the generation of oligodendrocytes from tripotential-glial restricted precursor (GRP) cells isolated from the embryonic day 13.5 rat spinal cord. In contrast, we found no effects of iron on oligodendrocyte maturation or survival in vitro, nor did we find that increasing iron availability above basal levels increases oligodendrocyte generation from bipotential oligodendrocyte-type-2 astrocyte/oligodendrocyte precursor cells (O-2A/OPCs). Our results raise the possibility that iron may affect oligodendrocyte development at stages during early embryogenesis rather than during later development.

Embryonic-derived glial-restricted precursor cells (GRP cells) can differentiate into astrocytes and oligodendrocytes in vivo.

We have isolated and characterized a unique glial-restricted precursor cell (GRP) from the embryonic spinal cord. Clonal analysis demonstrated that these cells are able to generate oligodendrocytes and two distinct type of astrocytes (type 1 and type 2) when exposed to appropriate signals in vitro. We now show that many aspects of these cells are retained in vivo. GRP cells are restricted to the glial lineage in vivo as they seem to be unable to generate neuronal phenotypes in an in vivo neurogenic environment. GRP cells survive and migrate in the neonatal and adult brain. Transplanted GRP cells differentiate into myelin-forming oligodendrocytes in a myelin-deficient background and also generate immature oligodendrocytes in the normal neonatal brain. In addition, GRP cells also consistently generated glial fibrillary protein-expressing cells in the neonatal and adult brain, a property not consistently expressed by other glial precursor cells like the O-2A/OPC cells. We suggest that the lineage restriction of GRP cells and their ability to generate both oligodendrocytes and astrocytes in vivo together with their embryonic character that allows for extensive in vitro expansion of the population makes the cell useful for clinical application.

Isolation and generation of oligodendrocytes by immunopanning.

This unit presents a procedure for the purification of oligodendrocyte progenitor cells, their expansion in vitro, and differentiation of these cells to yield oligodendrocyte cultures. A variation is also presented, detailing the direct isolation of differentiated oligodendrocytes from postnatal brain. The purification of the target cell population is achieved by exploiting the differential binding of cells to tissue culture dishes coated with an antibody directed against a specific cell-surface antigen. Cells expressing this surface antigen are retained on the dish and are thereby separated from the remaining cell population.

An alternative stochastic model of generation of oligodendrocytes in cell culture.

According to our previous model, oligodendrocyte--type 2 (O-2A) astrocyte progenitor cells become competent for differentiation in vitro after they complete a certain number of critical mitotic cycles. After attaining the competency to differentiate, progenitor cells divide with fixed probability p in subsequent cycles. The number of critical cycles is random; analysis of data suggests that it varies from zero to two. The present paper presents an alternative model in which there are no critical cycles, and the probability that a progenitor cell will divide again decreases gradually to a plateau value as the number of completed mitotic cycles increases. In particular all progenitor cells have the ability to differentiate from the time of plating. The Kiefer-Wolfowitz procedure is used to fit the new model to experimental data on the clonal growth of purified O-2A progenitor cells obtained from the optic nerves of 7 day old rats. The new model is shown to fit the experimental data well, indicating that it is not possible to determine whether critical cycles exist on the basis of these experimental data. In contrast to the fit of the previous model, which suggested that the addition of thyroid hormone increased the limiting probability of differentiation as the number of mitotic cycles increases, the fit of the new model suggests that the addition of thyroid hormone has almost no effect on the limiting probability of differentiation.

Are hypothyroidism and iron deficiency precursor cell diseases?

It is neither known why hormonal and nutritional deficiencies only cause neurological abnormalities at particular periods of development, nor is it known why repair only occurs if normal metabolic status is restored within a particular temporal window. We propose that these maladies are precursor cell diseases, in which the normal balance between self-renewal and differentiation is compromised in dividing precursor cells. According to this hypothesis, the windows of vulnerability in these disorders correspond to the timing of particular transitions in CNS precursor cells, as seen in our studies on the effects of thyroid hormone and iron on the generation of oligodendrocytes and their immediate ancestor, the O-2A progenitor cell.

Estimation problems associated with stochastic modeling of proliferation and differentiation of O-2A progenitor cells in vitro.

Our previous research effort has resulted in a stochastic model that provides an excellent fit to our experimental data on proliferation and differentiation of oligodendrocyte type-2 astrocyte progenitor cells at the clonal level. However, methods for estimation of model parameters and their statistical properties still remain far away from complete exploration. The main technical difficulty is that no explicit analytic expression for the joint distribution of the number of progenitor cells and oligodendrocytes, and consequently for the corresponding likelihood function, is available. In the present paper, we overcome this difficulty by using computer-intensive simulation techniques for estimation of the likelihood function. Since the output of our simulation model is essentially random, stochastic optimization methods are necessary to maximize the estimated likelihood function. We use the Kiefer-Wolfowitz procedure for this purpose. Given sufficient computing resources, the proposed estimation techniques significantly extend the spectrum of problems that become approachable. In particular, these techniques can be applied to more complex branching models of multi-type cell systems with dependent evolutions of different types of cells.

Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell.

We have discovered that intracellular redox state appears to be a necessary and sufficient modulator of the balance between self-renewal and differentiation in dividing oligodendrocyte-type-2 astrocyte progenitor cells. The intracellular redox state of freshly isolated progenitors allows prospective isolation of cells with different self-renewal characteristics. 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 signaling molecules that promote differentiation cause 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, indicating that cell-extrinsic signaling molecules that modulate this balance converge on redox modulation as a critical component of their effector mechanism.

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.

Heparin is a unique marker of progenitors in the glial cell lineage.

The oligodendrocyte-type-2 astrocyte progenitor cells (precursors of oligodendrocytes and type-2 astrocytes) are an excellent system in which to study differentiation as they can be manipulated in vitro. Maintenance of oligodendrocyte-type-2 astrocyte progenitor cells requires basic fibroblast growth factor, a growth factor whose action normally depends on a heparan sulfate coreceptor. Biochemical analysis revealed a most surprising result: that the oligodendrocyte-type-2 astrocyte progenitors did not synthesize heparan sulfate, the near ubiquitous N-sulfated cell surface polysaccharide, but the chemically related heparin in a form that was almost completely N- and O-sulfated. The heparin was detected in the pericellular fraction of the cells and the culture medium. In contrast the differentiated glial subpopulations (oligodendrocytes and type-2 astrocytes) synthesized typical heparan sulfate but with distinctive fine structural features for each cell type. Thus heparin is a unique differentiation marker in the glial lineage. Previously heparin has been found only in a subset of mature mast cells called the connective tissue mast cells. Its presence within the developing nervous system on a precise population of progenitors may confer specific and essential recognition properties on those cells in relation to binding soluble growth and/or differentiation factors and the extracellular matrix.

A random walk model of oligodendrocyte generation in vitro and associated estimation problems.

A branching stochastic process proposed earlier to model oligodendrocyte generation by O-2A progenitor cells under in vitro conditions does not allow invoking the maximum likelihood techniques for estimation purposes. To overcome this difficulty, we propose a partial likelihood function based on an embedded random walk model of clonal growth and differentiation of O-2A progenitor cells. Under certain conditions, the partial likelihood function yields consistent estimates of model parameters. The usefulness of this approach is illustrated with computer simulations and data analyses.

A stochastic model of temporally regulated generation of oligodendrocytes in cell culture.

The results of our previous analyses suggest that O-2A progenitor cells become competent for differentiation in vitro after they complete a certain number of critical mitotic cycles. The number of critical cycles varies from clone to clone and should be thought of as a random variable. We propose an approach to the analysis of oligodendrocyte generation in vitro based on a stochastic model allowing for an arbitrary distribution of this random variable with a finite support. When applied to experimental data on clonal growth and differentiation of purified O-2A progenitor cells obtained from optic nerves of 1 and 7 day-old rats, the model provides a good quantitative description not only of the first two moments (mean and variance) of the number of O-2A progenitor cells and oligodendrocytes at different times after the start of experiment, but of the corresponding distributions as well. As our estimates show, there are scarcely any O-2A progenitor cells that divide in vitro more than twice before they acquire the competence for differentiation. Those O-2A cells that have undergone the critical divisions differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability. We give estimates of this probability for O-2A cells under different growth conditions. Our analysis suggests that the effect of thyroid hormone is twofold: it reduces the mean duration of the mitotic cycle for progenitor cells, and it increases the probability of their transformation into oligodendrocytes.

Cell differentiation in the embryonic mammalian spinal cord.

The acquisition of cell type specific properties in the spinal cord is a process of a sequential restriction in developmental potential. Multipotent neuroepithelial stem cells (NEP cells) can give rise to all the major cell types in the central nervous system. The generation of these multiple cell types occurs via the generation of intermediate precursor cells, which are restricted in their differentiation potential, but are still able to give rise to more than one cell type. These intermediate precursor cells are different from NEP cells and are different from each other. We have identified neuronal restricted precursor cells (NRP's) which can only generate neurons but no longer glial cells and glial restricted precursor cells (GRP's), which give rise to glial cells but not to neurons. These intermediate precursor cells can be purified and expanded in vitro and might offer a new tool for gene discovery, drug screening and transplantation approaches.

Quantitative insight into proliferation and differentiation of oligodendrocyte type 2 astrocyte progenitor cells in vitro.

As part of our attempts at understanding fundamental principles that underlie the generation of nondividing terminally differentiated progeny from dividing precursor cells, we have developed approaches to a quantitative analysis of proliferation and differentiation of oligodendrocyte type 2 astrocyte (O-2A) progenitor cells at the clonal level. Owing to extensive previous studies of clonal differentiation in this lineage, O-2A progenitor cells represent an excellent system for such an analysis. Previous studies have resulted in two competing hypotheses; one of them suggests that progenitor cell differentiation is symmetric, the other hypothesis introduces an asymmetric process of differentiation. We propose a general model that incorporates both such extreme hypotheses as special cases. Our analysis of experimental data has shown, however, that neither of these extreme cases completely explains the observed kinetics of O-2A progenitor cell proliferation and oligodendrocyte generation in vitro. Instead, our results indicate that O-2A progenitor cells become competent for differentiation after they complete a certain number of critical mitotic cycles that represent a period of symmetric development. This number varies from clone to clone and may be thought of as a random variable; its probability distribution was estimated from experimental data. Those O-2A cells that have undergone the critical divisions then may differentiate into an oligodendrocyte in each of the subsequent mitotic cycles with a certain probability, thereby exhibiting the asymmetric type of differentiation.

A stochastic model of brain cell differentiation in tissue culture.

The timing of cell differentiation can be controlled both by cellintrinsic mechanisms and by cell-extrinsic signals. Oligodendrocyte type-2 astrocyte progenitor cells are known to be the precursor cells that give rise to oligodendrocytes. When stimulated to divide by purified cortical astrocytes or by platelet-derived growth factor, these progenitor cells generate oligodendrocytes in vitro with a timing like that observed in vivo. The most widely accepted model of this process assumes a cell-intrinsic biological clock that resides in the progenitor cell. The intrinsic clock model originally proposed in 1986 remains as the dominant theoretical concept for the analysis of timed differentiation in this cell lineage. However, the results of a recent experimental study (Ibarrola et al., Developmental Biology, vol. 180, 1-21, 1996) are most consistent with the hypothesis that the propensity of a clone of dividing O-2A progenitor cells initially to generate at least one oligodendrocyte may be regulated by cell-intrinsic mechanisms, but that environmental signals regulate the extent of further oligodendrocyte generation. We propose a stochastic model of cell differentiation in culture to accommodate the most recent experimental findings. Our model is an age-dependent branching stochastic process with two types of cells. The model makes it possible to derive analytical expressions for the expected number of progenitor cells and of oligodendrocytes as functions of time. The model parameters were estimated by fitting these functions through data on the average (sample mean) number of both types of cells per colony at different time intervals from start of experiment. Using this method we provide a biologically meaningful interpretation of the observed pattern of oligodendrocyte generation in vitro and its modification in the presence of thyroid hormone.

A common neural progenitor for the CNS and PNS.

Cultured spinal cord neuroepithelial (NEP) cells can differentiate into neurons, oligodendrocytes and astrocytes and are morphologically and antigenically distinct from neural crest stem cells (NCSCs) that generate the PNS. NEP cells, however, can generate p75/nestin-immunoreactive cells that are morphologically and antigenically similar to previously characterized NCSCs. NEP-derived p75-immunoreactive cells differentiate into peripheral neurons, smooth muscle, and Schwann cells in mass and clonal culture. Clonal analysis of NEP cells demonstrates that a common NEP progenitor cell generated both CNS and PNS phenotypes. Differentiation into NCSCs was promoted by BMP-2/4 and differentiation did not require cells to divide, indicating that BMP played an instructive role in the differentiation process. Thus, individual NEP cells are multipotent and can differentiate into most major types of cell in the CNS and PNS and that PNS differentiation involves a transition from a NEP stem to another more limited, p75-immunoreactive, neural crest stem cell.