Necdin is required for terminal differentiation and survival of primary dorsal root ganglion neurons

Necdin is expressed predominantly in postmitotic neurons and serves as a growth suppressor that is functionally similar to the retinoblastoma tumor suppressor protein. Using primary cultures of dorsal root ganglion (DRG) of mouse embryos, we investigated the involvement of necdin in the terminal differentiation of neurons. DRG cells were prepared from mouse embryos at 12.5 days of gestation and cultured in the presence of nerve growth factor (NGF). Immunocytochemistry revealed that necdin accumulated in the nucleus of differentiated neurons that showed neurite extension and expressed the neuronal markers microtubule-associated protein 2 and synaptophysin. Suppression of necdin expression in DRG cultures treated with antisense oligonucleotides led to a marked reduction in the number of terminally differentiated neurons. The antisense oligonucleotide-treated cells did not attempt to reenter the cell cycle, but underwent death with characteristics of apoptosis such as caspase-3 activation, nuclear condensation, and chromosomal DNA fragmentation. Furthermore, a caspase-3 inhibitor rescued antisense oligonucleotide-treated cells from apoptosis and significantly increased the population of terminally differentiated neurons. These results suggest that necdin mediates the terminal differentiation and survival of NGF-dependent DRG neurons and that necdin-deficient nascent neurons are destined to caspase-3-dependent apoptosis.     

Depolarization promotes survival of ciliary ganglion neurons by BDNF-dependent and -independent mechanisms

Membrane activity upregulates brain derived neurotrophic factor (BDNF) expression to coordinately support neuronal survival in many systems. In parasympathetic ciliary ganglion (CG) neurons, activity mimicked by KCl depolarization provides nearly full trophic support. While BDNF has been considered unable to influence CG neuronal survival, we now document its expression during CG development and show that low concentrations do support survival via high-affinity TrkB receptors. Furthermore, a contribution of BDNF to activity-induced trophic support was demonstrated by showing that KCl depolarization increased BDNF mRNA and protein in, and release of BDNF from, CG neuron cultures. Application of anti-BDNF blocking antibody or mitogen activated protein kinase (MAPK) kinase inhibitor, attenuated depolarization-supported survival, implicating canonical BDNF/TrkB signaling. Ca2+-Calmodulin kinase II (CaMKII) was also required since its inhibition combined with anti-BDNF or MAPK kinase inhibitor abolished or greatly reduced the trophic effects of depolarization. Membrane activity may thus support CG neuronal survival both by stimulating release of BDNF that binds high-affinity TrkB receptors to activate MAPK and by recruiting CaMKII. This mechanism could have relevance late in development in vivo as ganglionic transmission and the effectiveness of BDNF over other growth factors both increase.     

BDNF is essentially required for the early postnatal survival of nociceptors

Neurotrophins promote the survival of specific types of neurons during development and ensure proper maintenance and function of mature responsive neurons. Significant effects of BDNF (Brain-Derived Neurotrophic Factor) on pain physiology have been reported but the contribution of this neurotrophin to the development of nociceptors has not been investigated. We present evidence that BDNF is required for the survival of a significant fraction of peptidergic and non-peptidergic nociceptors in dorsal root ganglia (DRG) postnatally. Bdnf homozygous mutant mice lose approximately half of all nociceptive neurons during the first 2 weeks of life and adult heterozygotes exhibit hypoalgesia and a loss of 25% of all nociceptive neurons. Our in vitro analyses indicate that BDNF-dependent nociceptive neurons also respond to NGF and GDNF. Expression analyses at perinatal times indicate that BDNF is predominantly produced within sensory ganglia and is more abundant than skin-derived NGF or GDNF. Function-blocking studies with BDNF specific antibodies in vitro or cultures of BDNF-deficient sensory neurons suggest that BDNF acts in an autocrine/paracrine way to promote the early postnatal survival of nociceptors that are also responsive to NGF and GDNF. Altogether, the data demonstrate an essential requirement for BDNF in the early postnatal survival of nociceptive neurons.     

p18INK4c and p27KIP1 are required for cell cycle arrest of differentiated myotubes

Myogenic differentiation is characterized by permanent and irreversible cell cycle withdrawal and increased resistance to apoptosis. These functions correlate with changes in expression and activity of several cyclin-dependent kinase inhibitors, including p18, p21, and p27. In this study, we examined the requirements for p18, p21, and p27 in initiating growth arrest in multinucleated myotubes under differentiation conditions and in maintaining terminal arrest upon restimulation of differentiated myotubes with mitogenic signals. Under differentiation conditions, only p27(-/-) or p18(-/-)p27(-/-) myotubes are capable of reentering the cell cycle and synthesizing DNA at a very low frequency. Escape from cell cycle arrest was significantly greater in p18(-/-)p27(-/-) myotubes than in p27(-/-) myotubes. Stimulation of differentiated cultures with a mitogen-rich growth medium enhances p18(-/-)p27(-/-) myotube proliferation to encompass approximately half of the nuclei. p18(-/-)p21(-/-) and p21(-/-)p27(-/-) myotubes remain terminally arrested. Nuclei within individual restimulated p18(-/-)p27(-/-) myotubes can be found in all phases of the cell cycle, and a myotube can be multiphasic without any obvious deleterious effects. Increasing the time of differentiation or serum stimulation of p18(-/-)p27(-/-) myotubes neither increases the proliferation index of the myotube nuclei, nor does it alter the percentage of nuclei in each of the cell cycle phases. During the first 24 h of serum stimulation, the p18(-/-)p27(-/-) myotube nuclei that escape G0 arrest will rearrest in either S or G2 phase, without either mitosis or endoreplication. Apoptosis is increased in restimulated p18(-/-)p27(-/-) myotube nuclei, but is not specific for any cell cycle phase. These results suggest a collaborative role for p18 and p27 in initiating and maintaining G0 arrest during myogenic differentiation. While p18 and p27 appear to be essential in initiating G0 arrest in a proportion of postmitotic myotube nuclei, there must be another cell cycle inhibitor protein that functions with p18 and p27 in maintaining terminal arrest. We propose that the combined rate-limiting expressions of p18, p27, and this other inhibitor determine whether the myotube nuclei will remain postmitotic, or reenter the cell cycle, and if the nuclei escape G0 arrest, in which phase of the cell cycle the nuclei will ultimately rearrest.     

The orphan G protein-coupled receptor 161 is required for left-right patterning

Gpr161 (also known as RE2) is an orphan G protein-coupled receptor (GPCR) that is expressed during embryonic development in zebrafish. Determining its biological function has proven difficult due to lack of knowledge regarding its natural or synthetic ligands. Here, we show that targeted knockdown of gpr161 disrupts asymmetric gene expression in the lateral plate mesoderm, resulting in aberrant looping of the heart tube. This is associated with elevated Ca²⁺ levels in cells lining the Kupffer's vesicle and normalization of Ca²⁺ levels, by over-expression of ncx1 or pmca-RNA, is able to partially rescue the cardiac looping defect in gpr161 knockdown embryos. Taken together, these data support a model in which gpr161 plays an essential role in left-right (L-R) patterning by modulating Ca²⁺ levels in the cells surrounding the Kupffer's vesicle.     

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53^wt) or being p(HCT-116 p53^−/−), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53^−/− xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53^wt cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53^wt cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75^NTR, p53 and Bax.     

Gustatory papillae and taste bud development and maintenance in the absence of TrkB ligands BDNF and NT-4

Taste buds and the peripheral nerves innervating them are two important components of the peripheral gustatory system. They require appropriate connections for the taste system to function. Neurotrophic factors play crucial roles in the innervation of peripheral sensory organs and tissues. Both brain-derived neurotrophic factor (BDNF) null-mutated and neurotrophin-4 (NT-4) null-mutated mice exhibit peripheral gustatory deficits. BDNF and NT-4 bind to a common high affinity tyrosine kinase receptor, TrkB (NTRK-2), and a common p75 neurotrophin receptor (NGFR). We are currently using a transgenic mouse model to study peripheral taste system development and innervation in the absence of both TrkB ligands. We show that taste cell progenitors express taste cell markers during early stages of taste bud development in both BDNF^−/−xNT-4^−/− and wild-type mice. At early embryonic stages, taste bud progenitors express Troma-1, Shh, and Sox2 in all mice. At later stages, lack of innervation becomes a prominent feature in BDNF^−/−xNT-4^−/− mice leading to a decreasing number of fungiform papillae and morphologically degenerating taste cells. A total loss of vallate taste cells also occurs in postnatal transgenic mice. Our data indicate an initial independence but a later permissive and essential role for innervation in taste bud development and maintenance. This work was supported by NIH-NIDCD R01-RDC007628.     

Ginsenoside Rg3 antagonizes NMDA receptors through a glycine modulatory site in rat cultured hippocampal neurons

We previously reported that ginseng, a well-known herbal medicine, inhibited NMDA receptors in cultured hippocampal neurons. Here, we further examined the detailed mechanism of ginseng-mediated inhibition using its main active ingredient, ginsenoside Rg3. Co-application of ginsenoside Rg3 with increasing concentrations of NMDA did not change the EC50 of NMDA to the receptor, suggesting that ginsenoside Rg3 inhibits NMDA receptors without competing with the NMDA-binding site. Ginsenoside Rg3-mediated inhibition also occurred in a distinctive manner from the well-characterized NMDA receptor open channel blocker, MK-801. However, ginsenoside Rg3 produced its effect in a glycine concentration-dependent manner and shifted the glycine concentration-response curve to the right without changing the maximal response, suggesting the role of ginsenoside Rg3 as a competitive NMDA receptor antagonist. We also demonstrated that ginsenoside Rg3 significantly protected neurons against NMDA insults. Therefore, these results suggest that ginsenoside Rg3 protects NMDA-induced neuronal death via a competitive interaction with the glycine-binding site of NMDA receptors in cultured hippocampal neurons.     

Role of PI 3-kinase, Akt and Bcl-2-related proteins in sustaining the survival of neurotrophic factor-independent adult sympathetic neurons

By adulthood, sympathetic neurons have lost dependence on NGF and NT-3 and are able to survive in culture without added neurotrophic factors. To understand the molecular mechanisms that sustain adult neurons, we established low density, glial cell-free cultures of 12-wk rat superior cervical ganglion neurons and manipulated the function and/or expression of key proteins implicated in regulating cell survival. Pharmacological inhibition of PI 3-kinase with LY294002 or Wortmannin killed these neurons, as did dominant-negative Class IA PI 3-kinase, overexpression of Rukl (a natural inhibitor of Class IA PI 3-kinase), and dominant-negative Akt/PKB (a downstream effector of PI 3-kinase). Phospho-Akt was detectable in adult sympathetic neurons grown without neurotrophic factors and this was lost upon PI 3-kinase inhibition. The neurons died by a caspase-dependent mechanism after inhibition of PI 3-kinase, and were also killed by antisense Bcl-xL and antisense Bcl-2 or by overexpression of Bcl-xS, Bad, and Bax. These results demonstrate that PI 3-kinase/Akt signaling and the expression of antiapoptotic members of the Bcl-2 family are required to sustain the survival of adult sympathetic neurons.     

Octapeptide repeat region of prion protein (PrP) is required at an early stage for production of abnormal prion protein in PrP-deficient neuronal cell line

An abnormal isoform of prion protein (PrP^Sc), which is composed of the same amino acids as cellular PrP (PrP^C) and has proteinase K (PK)-resistance, hypothetically converts PrP^C into PrP^Sc. To investigate the region important for PrP^Sc production, we examined the levels of PrP^Sc in PrP gene-deficient cells (HpL3-4) expressing PrP^C deleted of various regions including the octapeptide repeat region (OR) or hydrophobic region (HR). After Chandler or Obihiro prion infection, PrP^Sc was produced in HpL3-4 cells expressing wild-type PrP^C or PrP^C deleted of HR at an early stage and further reduced to below the detectable level, whereas cells expressing PrP^C deleted of OR showed no PrP^Sc production. The results suggest that OR of PrP^C is required for the early step of efficient PrP^Sc production.     

Spatial and temporal regulation of the homeotic selector gene Antennapedia is required for the establishment of leg identity in Drosophila

Antennapedia is one of the homeotic selector genes required for specification of segment identity in Drosophila. Dominant mutations that ectopically express Antennapedia cause transformation of antenna to leg. Loss-of-function mutations cause partial transformation of leg to antenna. Here we examine the role of Antennapedia in the establishment of leg identity in light of recent advances in our understanding of antennal development. In Antennapedia mutant clones in the leg disc, Homothorax and Distal-less are coexpressed and act via spineless to transform proximal femur to antenna. Antennapedia is negatively regulated during leg development by Distal-less, spineless, and dachshund and this reduced Antennapedia expression is needed for the proper development of distal leg elements. These findings suggest that the temporal and spatial regulation of the homeotic selector gene Antennapedia in the leg disc is necessary for normal leg development in Drosophila.     

Gabrb3 is required for the functional integration of pyramidal neuron subtypes in the somatosensory cortex

1. Download : Download high-res image (143KB)
2. Download : Download full-size image