Homeodomain-interacting protein kinase-2 (HIPK2) regulates apoptosis in developing sensory and sympathetic neurons

AbstractExcess neurons in the developing nervous system are eliminated by apoptosis, an ordered cascade of proteolytic events orchestrated by the caspase family of proteases [1]. The apoptotic machinery is tightly regulated by a variety of extracellular signals that either activate or suppress apoptosis after binding to receptors on neurons [2]. These signals are integrated in neurons by a complex network of protein-protein interactions that bring about transcriptional and posttranslational changes in key regulators of the apoptotic machinery; such regulators include members of the Bcl-2 family [3]. Homeodomain-interacting protein kinase-2 (HIPK2) is a recently identified nuclear serine-threonine kinase that interacts with homeodomain transcription factors [4, 5] and participates in the regulation of cell growth [6] and genotoxic stress-induced apoptosis [7, 8]. Here we show that overexpression of HIPK2 in developing neurotrophin-dependent sensory and sympathetic neurons promotes apoptosis of these neurons grown with neurotrophins. HIPK2-induced apoptosis is caspase-dependent, is inhibited by overexpression of Bcl-2 and Bcl-W, and fails to occur in Bax-deficient neurons. Trigeminal sensory neurons, which are especially susceptible to HIPK2-induced apoptosis, express the highest levels of HIPK2 during the peak of apoptosis in vivo. Knockdown of endogenous HIPK2 with antisense oligonucleotides substantially reduces and delays apoptosis after neurotrophin deprivation in vitro. These findings identify HIPK2 as a novel participant in programmed cell death in the developing peripheral nervous system

Depolarisation causes reciprocal changes in GFR alpha-1 and GFR alpha-2 receptor expression and shifts responsiveness to GDNF and neurturin in developing neurons

GDNF and neurturin are structurally related neurotrophic factors that promote the survival of many different kinds of neurons and influence axonal and dendritic growth and synaptic function. These diverse effects are mediated via multicomponent receptors consisting of the Ret receptor tyrosine kinase plus one of two structurally related GPI-linked receptors, GFR alpha-1 and GFR alpha-2. To ascertain how the expression of these receptors is regulated during development, we cultured embryonic neurons under different experimental conditions and used competitive RT/PCR to measure the levels of the mRNAs encoding these receptors. We found that depolarisiug levels of KCl caused a marked increase in GFR alpha-1 mRNA and a marked decrease in GFR alpha-2 mRNA in sympathetic, parasympathetic and sensory neurons. These changes were accompanied by increased responsiveness to GDNF and decreased responsiveness to neurturin, and were inhibited by L-type Ca2+ channel antagonists, suggesting that they were due to elevated intracellular free-Ca2+. There was no consistent effect of depolarising levels of KCI on ret mRNA expression, and neither GDNF nor neurturin significantly affected receptor expression. These results show that depolarisation has marked and opposing actions on the expression of GFR alpha-1 and GFR alpha-2, which are translated into corresponding changes in neuronal responsiveness to GDNF and neurturin. This provides evidence for a mechanism of regulating the neurotrophic factor responses of neurons by neural activity that has important implications for structural and functional plasticity in the developing nervous system.</p

HAND transcription factors are required for neonatal sympathetic neuron survival

Expression of the basic helix–loop–helix transcription factor HAND2 begins early in sympathetic neuron development and is essential for the differentiation of noradrenergic neurons. Here, we show that the expression of HAND2 and related HAND1 are maintained in sympathetic neurons throughout fetal and postnatal development when these neurons depend on target-derived nerve growth factor (NGF) for survival. Short interfering RNA knockdown of endogenous HAND2 and, to a lesser extent, HAND1 in neonatal sympathetic neurons cultured with NGF, reduced the expression of the NGF receptor tyrosine kinase TrkA (tropomyosin-related kinase A), as well as neuronal survival. Chromatin immunoprecipitation analysis showed that NGF promotes HAND2 binding to the TrkA minimal enhancer and that transfection of sympathetic neurons with a TrkA expression plasmid rescued the neurons from HAND knockdown. These findings show that HAND transcription factors have a crucial function in sustaining the survival of neonatal sympathetic neurons with NGF by a feed-forward loop that maintains the expression of TrkA

Selective regulation of trkC expression by NT3 in the developing peripheral nervous system

We have studied the influence of neurotrophin-3 (NT3) on the expression of its receptor tyrosine kinase, trkC, in embryonic mice. The expression of trkC transcripts encoding full-length and kinase-deficient receptors was almost entirely restricted to neurons in the trigeminal ganglion and increased markedly throughout development. InNT3+/−embryos, the level of trkC mRNA in the trigeminal ganglion was much lower than that in wild-type embryos, although there was no significant reduction in the total number of neurons in the ganglion. This demonstrates that endogenous NT3 regulates trkC expression in trigeminal neurons independently of changes in population size. InNT3−/−embryos, the number of neurons in the trigeminal ganglion was much lower than in wild-type embryos, and there was a further reduction in the mean neuronal level of trkC mRNA. Direct regulation of trkC mRNA expression in cultured trigeminal neurons was also observed, although the finding that trkC mRNA levels were sustained better in explant cultures than in dissociated cultures irrespective of the presence of NT3 suggests that trkC mRNA expression is regulated by additional factors within the ganglion. In contrast to trigeminal neurons, the level of trkC mRNA was sustained at normal levels in neurons of the sympathetic chain ofNT3−/−embryos and was not increased by NT3 in sympathetic neuron cultures. TrkC mRNA expression in developing cutaneous tissues was also unaffected by the NT3 null mutation. In summary, our findings provide the first clear evidence that the expression of a trk receptor, tyrosine kinase, is regulated by physiological levels of its ligandin vivoand show that regulation by NT3 is cell type-specific.</jats:p

MicroRNAs and the regulation of neuronal plasticity under stress conditions.

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