Tiam1 as a Signaling Mediator of Nerve Growth Factor-Dependent Neurite Outgrowth

Nerve Growth Factor (NGF)-induced neuronal differentiation requires the activation of members of the Rho family of small GTPases. However, the molecular mechanisms through which NGF regulates cytoskeletal changes and neurite outgrowth are not totally understood. In this work, we identify the Rac1-specific guanine exchange factor (GEF) Tiam1 as a novel mediator of NGF/TrkA-dependent neurite elongation. In particular, we report that knockdown of Tiam1 causes a significant reduction in Rac1 activity and neurite outgrowth induced by NGF. Physical interaction between Tiam1 and active Ras (Ras-GTP), but not tyrosine phosphorylation of Tiam1, plays a central role in Rac1 activation by NGF. In addition, our findings indicate that Ras is required to associate Tiam1 with Rac1 and promote Rac1 activation upon NGF stimulation. Taken together, these findings define a novel molecular mechanism through which Tiam1 mediates TrkA signaling and neurite outgrowth induced by NGF

The orphan G protein-coupled receptor gene GPR178 is evolutionary conserved and altered in response to acute changes in food intake

G protein-coupled receptors (GPCRs) are a class of integral membrane proteins mediating physiological functions fundamental for survival, including energy homeostasis. A few years ago, an amino acid sequence of a novel GPCR gene was identified and named GPR178. In this study, we provide new insights regarding the biological significance of Gpr178 protein, investigating its evolutionary history and tissue distribution as well as examining the relationship between its expression level and feeding status. Our phylogenetic analysis indicated that GPR178 is highly conserved among all animal species investigated, and that GPR178 is not a member of a protein family. Real-time PCR and in situ hybridization revealed wide expression of Gpr178 mRNA in both the brain and periphery, with high expression density in the hypothalamus and brainstem, areas involved in the regulation of food intake. Hence, changes in receptor expression were assessed following several feeding paradigms including starvation and overfeeding. Short-term starvation (12-48h) or food restriction resulted in upregulation of Gpr178 mRNA expression in the brainstem, hypothalamus and prefrontal cortex. Conversely, short-term (48h) exposure to sucrose or Intralipid solutions downregulated Gpr178 mRNA in the brainstem; long-term exposure (10 days) to a palatable high-fat and high-sugar diet resulted in a downregulation of Gpr178 in the amygdala but not in the hypothalamus. Our results indicate that hypothalamic Gpr178 gene expression is altered during acute exposure to starvation or acute exposure to palatable food. Changes in gene expression following palatable diet consumption suggest a possible involvement of Gpr178 in the complex mechanisms of feeding reward

Inactivation of Pmel Alters Melanosome Shape But Has Only a Subtle Effect on Visible Pigmentation

PMEL is an amyloidogenic protein that appears to be exclusively expressed in pigment cells and forms intralumenal fibrils within early stage melanosomes upon which eumelanins deposit in later stages. PMEL is well conserved among vertebrates, and allelic variants in several species are associated with reduced levels of eumelanin in epidermal tissues. However, in most of these cases it is not clear whether the allelic variants reflect gain-of-function or loss-of-function, and no complete PMEL loss-of-function has been reported in a mammal. Here, we have created a mouse line in which the Pmel gene has been inactivated (Pmel−/−). These mice are fully viable, fertile, and display no obvious developmental defects. Melanosomes within Pmel−/− melanocytes are spherical in contrast to the oblong shape present in wild-type animals. This feature was documented in primary cultures of skin-derived melanocytes as well as in retinal pigment epithelium cells and in uveal melanocytes. Inactivation of Pmel has only a mild effect on the coat color phenotype in four different genetic backgrounds, with the clearest effect in mice also carrying the brown/Tyrp1 mutation. This phenotype, which is similar to that observed with the spontaneous silver mutation in mice, strongly suggests that other previously described alleles in vertebrates with more striking effects on pigmentation are dominant-negative mutations. Despite a mild effect on visible pigmentation, inactivation of Pmel led to a substantial reduction in eumelanin content in hair, which demonstrates that PMEL has a critical role for maintaining efficient epidermal pigmentation

GABA Maintains the Proliferation of Progenitors in the Developing Chick Ciliary Marginal Zone and Non-Pigmented Ciliary Epithelium

GABA is more than the main inhibitory neurotransmitter found in the adult CNS. Several studies have shown that GABA regulates the proliferation of progenitor and stem cells. This work examined the effects of the GABAA receptor system on the proliferation of retinal progenitors and non-pigmented ciliary epithelial (NPE) cells. qRT-PCR and whole-cell patch-clamp electrophysiology were used to characterize the GABAA receptor system. To quantify the effects on proliferation by GABAA receptor agonists and antagonists, incorporation of thymidine analogues was used. The results showed that the NPE cells express functional extrasynaptic GABAA receptors with tonic properties and that low concentration of GABA is required for a baseline level of proliferation. Antagonists of the GABAA receptors decreased the proliferation of dissociated E12 NPE cells. Bicuculline also had effects on progenitor cell proliferation in intact E8 and E12 developing retina. The NPE cells had low levels of the Cl–transporter KCC2 compared to the mature retina, suggesting a depolarising role for the GABAA receptors. Treatment with KCl, which is known to depolarise membranes, prevented some of the decreased proliferation caused by inhibition of the GABAA receptors. This supported the depolarising role for the GABAA receptors. Inhibition of L-type voltage-gated Ca2+ channels (VGCCs) reduced the proliferation in the same way as inhibition of the GABAA receptors. Inhibition of the channels increased the expression of the cyclin-dependent kinase inhibitor p27KIP1, along with the reduced proliferation. These results are consistent with that when the membrane potential indirectly regulates cell proliferation with hyperpolarisation of the membrane potential resulting in decreased cell division. The increased expression of p27KIP1 after inhibition of either the GABAA receptors or the L-type VGCCs suggests a link between the GABAA receptors, membrane potential, and intracellular Ca2+ in regulating the cell cycle

The Heterogenic Final Cell Cycle of Retinal Horizontal Cells

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

The Heterogenic Final Cell Cycle of Retinal Horizontal Cells

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

The Heterogenic Final Cell Cycle of Retinal Horizontal Cells

The cell cycle is a highly complex process that is under the control of several pathways.  Failure to regulate and/or complete the cell cycle often leads to cell cycle arrest, which may be followed by programmed cell death (apoptosis). One cell type that has a variety of unique cell cycle properties is the horizontal cell of the chicken retina. In this thesis we aimed to characterize the final cell cycle of retinal horizontal cells. In addition, the regulation of the cell cycle and the resistance to apoptosis of retinal horizontal cells are investigated. Our results show that the final cell cycle of Lim1-expressing horizontal progenitor cells is heterogenic and three different cell cycle behaviors can be distinguished. The horizontal cells are generated by: (i) an interkinetic nuclear migration with an apical mitosis; (ii) a final cell cycle with an S-phase that is not followed by mitosis, such cells remain with a fully or partially replicated genome; or (iii) non-apical (basal) mitoses. Furthermore, we show that the DNA damage response pathway is not triggered during the heterogenic final cell cycle of horizontal progenitor cells. However, chemically induced DNA damage activated the DNA damage response pathway without leading to cell cycle arrest, and the horizontal progenitor cells entered mitosis in the presence of DNA damage. This was not followed by apoptosis, despite the horizontal cells being able to functionally activate p53, p21CIP1/waf1, and caspase-3. Finally, we show that FoxN4 is expressed in horizontal progenitor cells and is required for their generation. Over-expression of FoxN4 causes cell death in several neuronal retinal cell types, except horizontal cells, where it results in an overproduction. In conclusion, in this thesis, a novel cell cycle behavior, which includes endoreplication not caused by DNA damage and a basal mitosis that can proceed in the presence of DNA damage, is described. The cell cycle and cell survival processes are of particular interest since retinal horizontal cells are suggested to be the cell-of-origin for retinoblastoma.

The terminal basal mitosis of chicken retinal Lim1 horizontal cells is not sensitive to cisplatin-induced cell cycle arrest

For proper development, cells need to coordinate proliferation and cell cycle-exit. This is mediated by a cascade of proteins making sure that each phase of the cell cycle is controlled before the initiation of the next. Retinal progenitor cells divide during the process of interkinetic nuclear migration, where they undergo S-phase on the basal side, followed by mitoses on the apical side of the neuroepithelium. The final cell cycle of chicken retinal horizontal cells (HCs) is an exception to this general cell cycle behavior. Lim1 expressing (+) horizontal progenitor cells (HPCs) have a heterogenic final cell cycle, with some cells undergoing a terminal mitosis on the basal side of the retina. The results in this study show that this terminal basal mitosis of Lim1+ HPCs is not dependent on Chk1/2 for its regulation compared to retinal cells undergoing interkinetic nuclear migration. Neither activating nor blocking Chk1 had an effect on the basal mitosis of Lim1+ HPCs. Furthermore, the Lim1+ HPCs were not sensitive to cisplatin-induced DNA damage and were able to continue into mitosis in the presence of γ-H2AX without activation of caspase-3. However, Nutlin3a-induced expression of p21 did reduce the mitoses, suggesting the presence of a functional p53/p21 response in HPCs. In contrast, the apical mitoses were blocked upon activation of either Chk1/2 or p21, indicating the importance of these proteins during the process of interkinetic nuclear migration. Inhibiting Cdk1 blocked M-phase transition both for apical and basal mitoses. This confirmed that the cyclin B1-Cdk1 complex was active and functional during the basal mitosis of Lim1+ HPCs. The regulation of the final cell cycle of Lim1+ HPCs is of particular interest since it has been shown that the HCs are able to sustain persistent DNA damage, remain in the cell cycle for an extended period of time and, consequently, survive for months

Horizontal Cells, the Odd Ones Out in the Retina, Give Insights into Development and Disease

Thorough investigation of a neuronal population can help reveal key aspects regarding the nervous system and its development. The retinal horizontal cells have several extraordinary features making them particularly interesting for addressing questions regarding fate assignment and subtype specification. In this review we discuss and summarize data concerning the formation and diversity of horizontal cells, how morphology is correlated to molecular markers, and how fate assignment separates the horizontal lineage from the lineages of other retinal cell types. We discuss the novel and unique features of the final cell cycle of horizontal cell progenitors and how they may relate to retinoblastoma carcinogenesis