Programmed cell death during neuronal development: the sympathetic neuron model

Abstract

Developing sympathetic neurons of the superior cervical ganglion are one of the best studied models of neuronal apoptosis. These cells require nerve growth factor (NGF) for survival at the time that they innervate their final target tissues during late embryonic and early postnatal development. In the absence of NGF, developing sympathetic neurons die by apoptosis in a transcription-dependent manner. Molecular studies of sympathetic neuron apoptosis began in the 1980s. We now know that NGF withdrawal activates the mitochondrial (intrinsic) pathway of apoptosis in sympathetic neurons cultured in vitro, and the roles of caspases, Bcl-2 (B-cell CLL/lymphoma 2) family proteins and XIAP (X-linked inhibitor of apoptosis protein) have been extensively studied. Importantly, a considerable amount has also been learned about the intracellular signalling pathways and transcription factors that regulate programmed cell death in sympathetic neurons. In this article, we review the key papers published in the past few years, covering all aspects of apoptosis regulation in sympathetic neurons and focusing, in particular, on how signalling pathways and transcription factors regulate the cell death programme. We make some comparisons with other models of neuronal apoptosis and describe possible future directions for the field.

Figure 1

Morphological and biochemical changes that occur in sympathetic neurons undergoing programmed cell death in vivo or following NGF withdrawal in vitro. (a) Morphology of sympathetic neurons isolated from one-day-old rats and cultured for 6 days in vitro and then in the presence and absence of NGF for 48 h. Bar, 25 μm. (b) Apoptotic chromatin condensation and DNA fragmentation in cultured sympathetic neurons visualised by Hoechst 33342 staining and TUNEL analysis. The neurons were isolated from one-day-old rats, cultured for 6 days in vitro and then in the presence and absence of NGF for 24 h. Bar, 25 μm. (c) TUNEL analysis of apoptosis in the superior cervical ganglia of one-day-old wild-type and mkp1−/− mice. The mkp1−/− knockout mutation significantly increases the number of TUNEL-positive cells per ganglion. Scale bar, 100 μm. (d) NGF withdrawal activates caspase-3 in sympathetic neurons. Neurons were cultured in the presence or absence of NGF for 48 h. The cleaved form of caspase-3 and nuclear morphology were visualised by staining the neurons with an anti-active caspase-3 antibody and Hoechst 33342. Bar, 25 μm. (e) Distribution of cytochrome c in normal and apoptotic sympathetic neurons visualised by immunocytochemistry with an anti-cytochrome c antibody. In the presence of NGF, cytochrome c immunoreactivity is excluded from the nuclear space and has a punctate pattern. In the absence of NGF, a fainter, diffuse staining pattern that occurs throughout the whole cell is observed. Bar, 25 μm

Figure 2

Key events following NGF withdrawal in sympathetic neurons. Sympathetic neurons undergo death 24–48 h after NGF withdrawal. The N-terminal phosphorylation of c-Jun, the release of cytochrome c from the mitochondria and the activation of caspase-3 are key biochemical changes seen in NGF-deprived sympathetic neurons. By ∼16 h after NGF withdrawal, only 50% of the neurons can be rescued by the addition of inhibitors of transcription or protein synthesis (the transcriptional commitment point) and by ∼22 h after NGF withdrawal only 50% of the neurons can be rescued by the readdition of NGF (the commitment point for NGF withdrawal-induced death). By 48 h, almost all of the neurons have undergone apoptosis and the nuclear and morphological changes typical of apoptosis are apparent. Images represent snapshots of NGF-deprived sympathetic neurons at the timepoints shown. Scale bars, 20 μm

Figure 3

NGF withdrawal activates the mitochondrial (intrinsic) pathway of apoptosis in sympathetic neurons. After NGF withdrawal, the expression of the BH3-only proteins Dp5, Bim, Puma and Bmf increases.^{, ,} These promote MOMP by binding to and antagonising the antiapoptotic Bcl-2 and Bcl-x_L proteins or by directly activating Bax. The multidomain proapoptotic Bax protein is activated by NGF withdrawal and essential for MOMP and mitochondrial cytochrome c release. Cytosolic cytochrome c interacts with Apaf-1 and procaspase-9 to form the apoptosome complex, which then cleaves and activates the effector caspase, caspase-3. Bim, Puma, Bax, cytochrome c, Apaf-1, caspase-9 and caspase-3 have all been shown to be essential for normal NGF withdrawal-induced death in experiments using sympathetic neurons isolated from specific knockout mice or, in the case of cytochrome c, a neutralising anti-cytochrome c antibody.^{, , , , , , ,} RAIDD and caspase-2 may function upstream of the mitochondrial pathway in sympathetic neurons and promote cell death by increasing the expression of the BH3-only protein Bim. In the presence of NGF, XIAP inhibits caspases in sympathetic neurons, but after NGF withdrawal, the level of the XIAP protein significantly decreases, allowing the intrinsic pathway to activate caspase-3 and induce cell death. During the later postnatal development of sympathetic neurons, the level of the MiR-29b microRNA increases (from P13 onwards) and this inhibits expression of the BH3-only proteins and contributes to the resistance of late postnatal (P28) sympathetic neurons to NGF deprivation-induced death^,

Figure 4

Survival pathways activated by the binding of NGF to TrkA. The binding of NGF to its receptor TrkA can activate the PI3K-Akt signalling pathway, which can inhibit apoptosis and promote cell survival. The binding of NGF to TrkA triggers the activation of the small GTP-binding protein Ras. The subsequent activation of Akt through PI3K can inhibit apoptosis by phosphorylating, and therefore inactivating, proapoptotic proteins such as the BH3-only protein Bad and the transcription factor FOXO. The binding of NGF to its receptor TrkA can also activate the Raf-MEK-ERK signalling pathway. This pathway promotes survival by inhibiting the expression of Bim, and by activating Rsk, which phosphorylates and activates the transcription factor CREB, which can activate the transcription of the bcl-2 gene

Figure 5

Proapoptotic signalling pathways activated in NGF-deprived sympathetic neurons. When sympathetic neurons are deprived of NGF, the MLK-JNK-c-Jun pathway is activated. MLKs phosphorylate MAP kinase kinases such as MKK4/7, which in turn phosphorylate JNKs. JNK activity increases leading to the phosphorylation of the AP-1 transcription factors c-Jun and ATF2. This increases the ability of c-Jun to activate the transcription of target genes such as bim, dp5 and c-jun itself. c-Jun can also bind to AP-1 sites in the promoter of mkp1, which encodes a MAPK phosphatase that acts as a negative regulator of the JNK pathway. The MLK inhibitor CEP-11004, which prevents JNK activation, and dominant-negative c-Jun reduce the induction of c-Jun, Bim and Dp5 and block cell death