Molecular Cloning of a Novel Polypeptide, DP5, Induced during Programmed Neuronal Death

Abstract

To study the molecular mechanisms underlying neuronal programmed cell death (PCD), we performed differential display screening for genes, the expression of which was induced during PCD in the sympathetic neuron culture model deprived of NGF. We cloned a gene encoding a novel polypeptide (DP5) which consisted of 92 amino acids. DP5 polypeptide had no homology with any other known protein and contained no motif that would indicate its putative biochemical functions. DP5 mRNA levels peaked at 15 h after nerve growth factor withdrawal, concurrent with the time at which neurons were committed to die. The induction of DP5 gene expression was blocked when cell death was rescued by treatment with cycloheximide, KCl, or the cyclic AMP analogue CPTcAMP. Overexpression of the full-length DP5 in cultured sympathetic neurons was in itself sufficient to induce apoptosis. These results suggest that DP5 plays a role in programmed neuronal death. Programmed cell death (PCD)1 is an indispensable phenomenon for proper development of the nervous system. Roughly half of all neurons produced by neurogenesis die during development of the mammalian nervous system (1). Neuronal survival is determined largely by neurotrophic factors such as NGF produced by target cells, and neurons that do not obtain an adequate supply of survival factors undergo apoptosis (2). Dissociated sympathetic neurons from rat superior cervical ganglia (SCG) are mainly used as an in vitro model of neuronal PCD and have been characterized extensively (3, 4). In this culture system, the majority of neurons die after removal of NGF from the culture medium. The characteristics of this neuronal death such as shrinkage of the neuronal soma with intact organelles, nuclear condensation, fragmentation of the DNA into oligonucleosomes, and blebbing of the plasma membrane are hallmarks of apoptosis (5, 6). Neuronal death in this system can be prevented by inhibitors of RNA or protein synthesis, suggesting that this phenomenon is controlled by a genetic program (7).In the search for constituents of the genetic program, several genes have been identified using this in vitro model. Immediate early genes such as c-fos and c-jun and cell cycle-related genes such as c-myb and cyclin D1 (8, 9) are these candidates. Especially cell cycle-related genes stimulate the postmitotic neurons to attempt re-entry into the cell cycle, and these comflicting growth-regulatory signals are thought to cause neurons to undergo apoptosis. On the other hand, several anti-apoptotic proteins such as Bcl-2 (10, 11), adenovirus E1B19k (12), or cowpox virus CrmA gene products (13) have been reported. Overexpression of the bcl-2 gene products in sympathetic neurons has been shown to protect neurons from apoptosis (10). Anti-apoptotic properties of Bcl-2 have been reported to prevent the loss of mitochondrial function (14). CrmA gene products also prevent neuronal death induced by NGF deprivation. This activity is attributed to interleukin 1␤-converting enzyme protease inhibition (13, 15).Re-entry into the...

BH3-only proteins: key regulators of neuronal apoptosis

“…In rodents, the expression of the dp5 mRNA is largely restricted to the nervous system. 16 The dp5 mRNA and DP5 protein increase in level in sympathetic neurons and neuronallydifferentiated PC12 cells deprived of NGF, and reach a peak by 15 h after the removal of NGF. 16 The induction of the dp5 mRNA by NGF withdrawal may require the JNK pathway because it is reduced by approximately 75% by the neuroprotective compound CEP-1347, a mixed lineage kinase (MLK) inhibitor that inhibits JNK activation in sympathetic neurons.…”

“…16 The dp5 mRNA and DP5 protein increase in level in sympathetic neurons and neuronallydifferentiated PC12 cells deprived of NGF, and reach a peak by 15 h after the removal of NGF. 16 The induction of the dp5 mRNA by NGF withdrawal may require the JNK pathway because it is reduced by approximately 75% by the neuroprotective compound CEP-1347, a mixed lineage kinase (MLK) inhibitor that inhibits JNK activation in sympathetic neurons. 27 However, this hypothesis will need to be confirmed by other approaches because CEP-1347 and related MLK inhibitors can also activate the PI3-kinase pathway under certain conditions.…”

“…Both sympathetic neurons and CGNs express multiple proapoptotic Bcl-2 family proteins. [15][16][17][18] Of the multidomain proapoptotic proteins (Bax and Bak), Bax is essential for the release of mitochondrial cytochrome c in sympathetic neurons and for NGF withdrawal-induced death, and for the KCl/serum deprivationinduced death of CGNs, whereas Bak is not required. [18][19][20] This is an interesting observation because in other cell types the expression of both Bax and Bak must be lost to prevent cell death induced by survival factor withdrawal.…”

“…DP5 was the first BH3-only protein to be found to be induced by NGF withdrawal and was identified in NGF-deprived sympathetic neurons by the differential display technique. 16 DP5 is the rodent homologue of human Harakiri (Hrk) 26 and encodes a 92 amino-acid protein with a BH3 domain and carboxyterminal hydrophobic membrane insertion sequence. In rodents, the expression of the dp5 mRNA is largely restricted to the nervous system.…”

“…Overexpression of DP5 in microinjected sympathetic neurons induces apoptosis and this can be inhibited by coexpression of antiapoptotic Bcl-2. 16 Similarly, overexpression of DP5 can induce apoptosis in CGNs and this death requires the expression of Bax. 27 Dp5 À/À knockout mice have been generated, and these survive development, are viable and have no major anatomical defects in the nervous system.…”