Induction of neuron-specific tropomyosin mRNAs by nerve growth factor is dependent on morphological differentiation

We have examined the expression of brain-specific tropomyosins during neuronal differentiation. Both TmBr-1 and TmBr-3 were shown to be neuron specific. TmBr-1 and TmBr-3 mRNA levels increased during the most active phase of neurite outgrowth in the developing rat cerebellum. In PC12 cells stimulated by nerve growth factor (NGF) to differentiate to the neuronal phenotype, TmBr-1 and TmBr-3 levels increased with an increasing degree of morphological differentiation. Induction of TmBr-1 and TmBr-3 expression only occurred under conditions where PC12 cells were permitted to extend neurites. NGF was unable to maintain levels of TmBr-1 and TmBr-3 with the loss of neuronal phenotype by resuspension of differentiated PC12 cells. The unique cellular expression and regulation in vivo and in vitro of TmBr- 1 and TmBr-3 strongly suggests a critical role of these tropomyosins in neuronal microfilament function. The findings reveal that the induction and maintenance of the neuronal tropomyosins is dependent on morphological differentiation and the maintenance of the neuronal phenotype.     

Glutathione transferase P silencing promotes neuronal differentiation of retinal R28 cells

Glutathione transferases (GSTs) play an important role in retinal pathophysiology. Within this family, the GSTP isoform is known as an endogenous regulator of cell survival and proliferation pathways and of cellular responses to oxidative stress. In the present study we silenced GSTP in R28 cells, a retinal precursor cell line with markers of both glial and neuronal origin, and obtained stable clones which were viable and, unexpectedly, characterized by a more neuronal phenotype. The degree of neuronal differentiation was inversely correlated with GSTP residual expression levels. The clone with the lowest expression of GSTP showed metabolic reprogramming, a more favorable redox status and, despite its neuronal phenotype, a sensitivity to glutamate and 4-hydroxynonenal toxicity comparable to that of control cells. Altogether, our evidence shows that near full depletion of GSTP in retinal precursor cells, triggers neuronal differentiation and prosurvival metabolic changes.     

Nitrotyrosine as a marker for peroxynitrite‐induced neurotoxicity: the beginning or the end of the end of dopamine neurons?

This review examines the involvement of nitrotyrosine as a marker for peroxynitrite-mediated damage in the dopamine neuronal system. We propose that the dopamine neuronal phenotype can influence the cytotoxic signature of peroxynitrite. Dopamine and tetrahydrobiopterin are concentrated in dopamine neurons, and both are essential for their proper neurochemical function. It is not well appreciated that dopamine and tetrahydrobiopterin are also powerful blockers of peroxynitrite-induced tyrosine nitration. What is more, the reaction of peroxynitrite with either dopamine or tetrahydrobiopterin forms chemical species (i.e. o-quinones and pterin radicals, respectively) whose cytotoxic effects may be manifested far earlier than nitrotyrosine formation in the course of dopamine neuronal damage. A better understanding of how the dopamine neuronal phenotype modulates the effects of reactive nitrogen species could reveal early steps in drug- and disease-induced damage to the dopamine neuron and form the basis for rational, protective therapies.     

APC protein is required for initiation of neuronal differentiation in rat pheochromocytoma PC12 cells

The adenomatous polyposis (APC) gene product is highly expressed in the central nervous system. To elucidate the contribution of the APC protein to neuronal differentiation, we used an inducible antisense mRNA vector to suppress APC protein expression and examined neuronal differentiation of PC12 cells induced by nerve growth factor (NGF). When antisense mRNA was induced, APC protein expression was suppressed to 20% of the noninduced level. In those cells, neurite extension induced by NGF and expression of microtubule-associated protein 2 (MAP2) was completely inhibited. However, once cells had differentiated, antisense APC mRNA expression and subsequent suppression of APC protein expression had no effect on either cell morphology or MAP2 protein expression. These results suggest that the wild type APC is critically involved only in the initiation of neuronal differentiation, but not in the maintenance of the differentiated phenotype, or that the neuronal phenotype could be maintained at lower level of APC protein.     

Epidermal growth factor promotes a neural phenotype in thymic epithelial cells and enhances neuropoietic cytokine expression

Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus-dependent immune functions by promoting neuronal gene expression in neural crest- derived cells.     

Switching from bone marrow-derived neurons to epithelial cells through dedifferentiation and translineage redifferentiation

While the ability of stem cells to switch lineages has been suggested, the route(s) through which this may happen is unclear. To date, the best characterized adult stem cell population considered to possess transdifferentiation capacity is BM-MSCs (bone marrow mesenchymal stem cells). We investigated whether BM-MSCs that had terminally differentiated into the neural or epithelial lineage could be induced to transdifferentiate into the other phenotype in vitro. Our results reveal that neuronal phenotypic cells derived from adult rat bone marrow cells can be switched to epithelial phenotypic cells, or vice versa, by culture manipulation allowing the differentiated cells to go through, first, dedifferentiation and then redifferentiation to another phenotype. Direct transdifferentiation from differentiated neuronal or epithelial phenotype to the other differentiated phenotype cannot be observed even when appropriate culture conditions are provided. Thus, dedifferentiation appears to be a prerequisite for changing fate and differentiating into a different lineage from a differentiated cell population.     

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.     

Dissection of the faint little ball (flb) phenotype: determination of the development of the Drosophila central nervous system by early interactions in the ectoderm.

The complex embryonic phenotype of mutations in the faint little ball (flb) locus, encoding the Drosophila EGF receptor homolog (DER), was dissected by temperature shifts of a temperature-sensitive allele. We show that the phenotype can be resolved into at least five components, which are temporally and spatially distinct. Most notably, the central nervous system (CNS) phenotype is determined at two separate phases. A severe collapse results from early defects in the DER-expressing ectodermal cells from which neuroblasts and midline glial cells deaminate. We thus suggest that DER activity is crucial for interactions that occur in the ectoderm at an early stage, and determine the fate of neuronal and glial cell lineages. This finding explains how a severe CNS phenotype is generated in flb embryos, in spite of the absence of expression of the protein in neuronal cells. In a second phase, during germ band retraction, the flb function is required specifically in the three pairs of midline glial cells (MG). In the absence of a functional DER protein, these cells die or fail to differentiate correctly, resulting in a fused commissure phenotype.     

Immortalization of neuronal progenitors using SV40 large T antigen and differentiation towards dopaminergic neurons

Transplantation is common in clinical practice where there is availability of the tissue and organ. In the case of neurodegenerative disease such as Parkinson's disease (PD), transplantation is not possible as a result of the non‐availability of tissue or organ and therefore, cell therapy is an innovation in clinical practice. However, the availability of neuronal cells for transplantation is very limited. Alternatively, immortalized neuronal progenitors could be used in treating PD. The neuronal progenitor cells can be differentiated into dopaminergic phenotype. Here in this article, the current understanding of the molecular mechanisms involved in the differentiation of dopaminergic phenotype from the neuronal progenitors immortalized with SV40 LT antigen is discussed. In addition, the methods of generating dopaminergic neurons from progenitor cells and the factors that govern their differentiation are elaborated. Recent advances in cell‐therapy based transplantation in PD patients and future prospects are discussed.     

A rapid exocytosis mode in chromaffin cells with a neuronal phenotype

We have used astrocyte-conditioned medium (ACM) to promote the transdifferentiation of bovine chromaffin cells and study modifications in the exocytotic process when these cells acquire a neuronal phenotype. In the ACM-promoted neuronal phenotype, secretory vesicles and intracellular Ca2+ rise were preferentially distributed in the neurite terminals. Using amperometry, we observed that the exocytotic events also occurred mainly in the neurite terminals, wherein the individual exocytotic events had smaller quantal size than in undifferentiated cells. Additionally, duration of pre-spike current was significantly shorter, suggesting that ACM also modifies the fusion pore stability. After long exposure (7-9 days) to ACM, the kinetics of catecholamine release from individual vesicles was markedly accelerated. The morphometric analysis of vesicle diameters suggests that the rapid exocytotic events observed in neurites of ACM-treated cells correspond to the exocytosis of large dense-core vesicles (LDCV). On the other hand, experiments performed in EGTA-loaded cells suggest that ACM treatment promotes a better coupling between voltage-gated calcium channels (VGCC) and LDCV. Thus, our findings reveal that ACM promotes a neuronal phenotype in chromaffin cells, wherein the exocytotic kinetics is accelerated. Such rapid exocytosis mode could be caused at least in part by a better coupling between secretory vesicles and VGCC.     

Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes

Hox genes are instrumental in assigning segmental identity in the developing hindbrain. Auto-, cross- and para-regulatory interactions help establish and maintain their expression. To understand to what extent such regulatory interactions shape neuronal patterning in the hindbrain, we analysed neurogenesis, neuronal differentiation and motoneuron migration in Hoxa1, Hoxb1 and Hoxb2 mutant mice. This comparison revealed that neurogenesis and differentiation of specific neuronal subpopulations in r4 was impaired in a similar fashion in all three mutants, but with different degrees of severity. In the Hoxb1 mutants, neurons derived from the presumptive r4 territory were re-specified towards an r2-like identity. Motoneurons derived from that territory resembled trigeminal motoneurons in both their migration patterns and the expression of molecular markers. Both migrating motoneurons and the resident territory underwent changes consistent with a switch from an r4 to r2 identity. Abnormally migrating motoneurons initially formed ectopic nuclei that were subsequently cleared. Their survival could be prolonged through the introduction of a block in the apoptotic pathway. The Hoxa1 mutant phenotype is consistent with a partial misspecification of the presumptive r4 territory that results from partial Hoxb1 activation. The Hoxb2 mutant phenotype is a hypomorph of the Hoxb1 mutant phenotype, consistent with the overlapping roles of these genes in facial motoneuron specification. Therefore, we have delineated the functional requirements in hindbrain neuronal patterning that follow the establishment of the genetic regulatory hierarchy between Hoxa1, Hoxb1 and Hoxb2.     

Area-based analyzing technique at cell array experiment using neuronal cell line

We propose a simple procedure for the identification and quantitative analysis of neurite outgrowth in neuronal cell lines that were uniformly differentiated. Upon stimulation most neuronal cell lines extend neurites in the differentiation process, resulting, according to our observation, in the increase of cell surface area. This increase is dependent on the length and the number of extended neurites. Furthermore, we use this method for the phenotype analysis of cell array experiments to perform large-scale functional evaluation of genes involved in the neurite outgrowth during neuronal differentiation.     

The neurological phenotype of ataxia-telangiectasia: solving a persistent puzzle

Human genomic instability syndromes affect the nervous system to different degrees of severity, attesting to the vulnerability of the CNS to perturbations of genomic integrity and the DNA damage response (DDR). Ataxia-telangiectasia (A-T) is a typical genomic instability syndrome whose major characteristic is progressive neuronal degeneration but is also associated with immunodeficiency, cancer predisposition and acute sensitivity to ionizing radiation and radiomimetic chemicals. A-T is caused by loss or inactivation of the ATM protein kinase, which mobilizes the complex, multi-branched cellular response to double strand breaks in the DNA by phosphorylating numerous DDR players. The link between ATM's function in the DDR and the neuronal demise in A-T has been questioned in the past. However, recent studies of the ATM-mediated DDR in neurons suggest that the neurological phenotype in A-T is indeed caused by deficiency in this function, similar to other features of the disease. Still, major issues concerning this phenotype remain open, including the presumed differences between the DDR in post-mitotic neurons and proliferating cells, the nature of the damage that accumulates in the DNA of ATM-deficient neurons under normal life conditions, the mode of death of ATM-deficient neurons, and the lack of a major neuronal phenotype in the mouse model of A-T. A-T remains a prototype disease for the study of the DDR's role in CNS development and maintenance.     

oko meduzy mutations affect neuronal patterning in the zebrafish retina and reveal cell-cell interactions of the retinal neuroepithelial sheet

Mutations of the oko meduzy (ome) locus cause drastic neuronal patterning defect in the zebrafish retina. The precise, stratified appearance of the wild-type retina is absent in the mutants. Despite the lack of lamination, at least seven retinal cell types differentiate in oko meduzy. The ome phenotype is already expressed in the retinal neuroepithelium affecting morphology of the neuroepithelial cells. Our experiments indicate that previously unknown cell-cell interactions are involved in development of the retinal neuroepithelial sheet. In genetically mosaic animals, cell-cell interactions are sufficient to rescue the phenotype of oko meduzy retinal neuroepithelial cells. These cell-cell interactions may play a critical role in the patterning events that lead to differentiation of distinct neuronal laminae in the vertebrate retina.     

Echinoid mutants exhibit neurogenic phenotypes and show synergistic interactions with the Notch signaling pathway

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has previously been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, we show that Ed has a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed we show, furthermore, that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. Our work establishes a role of Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway.