Robert S. Freeman

Phosphatidylinositol 3-kinase and Akt protein kinase are necessary and sufficient for the survival of nerve growth factor-dependent sympathetic neurons.

Recent studies have suggested a role for phosphatidylinositol (PI) 3-kinase in cell survival, including the survival of neurons. We used rat sympathetic neurons maintained in vitro to characterize the potential survival signals mediated by PI 3-kinase and to test whether the Akt protein kinase, a putative effector of PI 3-kinase, functions during nerve growth factor (NGF)-mediated survival. Two PI 3-kinase inhibitors, LY294002 and wortmannin, block NGF-mediated survival of sympathetic neurons. Cell death caused by LY294002 resembles death caused by NGF deprivation in that it is blocked by a caspase inhibitor or a cAMP analog and that it is accompanied by the induction of c-jun, c-fos, andcyclin D1 mRNAs. Treatment of neurons with NGF activates endogenous Akt protein kinase, and LY294002 or wortmannin blocks this activation. Expression of constitutively active Akt or PI 3-kinase in neurons efficiently prevents death after NGF withdrawal. Conversely, expression of dominant negative forms of PI 3-kinase or Akt induces apoptosis in the presence of NGF. These results demonstrate that PI 3-kinase and Akt are both necessary and sufficient for the survival of NGF-dependent sympathetic neurons.

Analysis of cell cycle-related gene expression in postmitotic neurons: Selective induction of cyclin D1 during programmed cell death

Sympathetic neurons undergo RNA and protein synthesis-dependent programmed cell death when deprived of nerve growth factor. To test the hypothesis that neuronal programmed cell death is a consequence of conflicting growth signals which cause the inappropriate activation of cell cycle genes, we have analyzed cell cycle-related genes for their expression in postmitotic neurons. Surprisingly, many of these genes are expressed in neurons, although cdc2, cdk2, and cyclin A are not. During programmed cell death, the expression of most of these genes, including several cyclins and the Rb and p53 tumor suppressor genes, decreases similar to that of neuronal genes. In contrast, cyclin D1 expression is selectively induced in dying neurons. Cyclin D1 mRNA levels peak 15-20 hr after nerve growth factor withdrawal, concurrent with the time that neurons become committed to die. These results provide an extensive characterization of cell cycle gene expression in postmitotic neurons and provide the evidence for a gene induced during neuronal programmed cell death.

Glycogen Synthase Kinase-3β Activity Is Critical for Neuronal Death Caused by Inhibiting Phosphatidylinositol 3-Kinase or Akt but Not for Death Caused by Nerve Growth Factor Withdrawal

Numerous studies reveal that phosphatidylinositol (PI) 3-kinase and Akt protein kinase are important mediators of cell survival. However, the survival-promoting mechanisms downstream of these enzymes remain uncharacterized. Glycogen synthase kinase-3β (GSK-3β), which is inhibited upon phosphorylation by Akt, was recently shown to function during cell death induced by PI 3-kinase inhibitors. In this study, we tested whether GSK-3β is critical for the death of sympathetic neurons caused by the withdrawal of their physiological survival factor, the nerve growth factor (NGF). Stimulation with NGF resulted in PI 3-kinase-dependent phosphorylation of GSK-3β and inhibition of its protein kinase activity, indicating that GSK-3β is targeted by PI 3-kinase/Akt in these neurons. Expression of the GSK-3β inhibitor Frat1, but not a mutant Frat1 protein that does not bind GSK-3β, rescued neurons from death caused by inhibiting PI 3-kinase. Similarly, expression of Frat1 or kinase-deficient GSK-3β reduced death caused by inhibiting Akt. In NGF-maintained neurons, overexpression of GSK-3β caused a small but significant decrease in survival. However, expression of neither Frat1, kinase-deficient GSK-3β, nor GSK-3-binding protein inhibited NGF withdrawal-induced death. Thus, although GSK-3β function is required for death caused by inactivation of PI 3-kinase and Akt, neuronal death caused by NGF withdrawal can proceed through GSK-3β-independent pathways.

Oxygen-Regulated β2-Adrenergic Receptor Hydroxylation by EGLN3 and Ubiquitylation by pVHL

Hypoxia reduces proline hydroxylation and ubiquitylation of a G protein–coupled receptor, preventing down-regulation. Oxygen-Regulated GPCR Down-Regulation Adrenergic signaling through β-adrenergic receptors regulates cardiovascular and pulmonary function, and dysfunction of β-adrenergic receptor signaling is associated with diseases such as heart failure and asthma. The responsiveness of a cell to adrenergic signaling depends substantially on the abundance and location of the receptors and is controlled by the processes of desensitization, a transient decrease in responsiveness of the receptor, and down-regulation, a prolonged decrease in responsiveness through internalization and subsequent degradation of the receptors. Xie et al. now show that oxygen regulates the stability of the β2 type of adrenergic receptor, which mediates the integrated physiological response to hypoxic conditions by enhancing cardiac contractility; peripheral vasodilation, which increases O2 delivery; and alveolar fluid clearance, which increases O2 uptake. Furthermore, they show that oxygen-regulated down-regulation of the receptors occurs through receptor proline hydroxylation by the dioxygenase EGLN3 and ubiquitylation by the von Hippel–Lindau tumor suppressor protein (pVHL)–E3 ligase complex, which also controls hypoxia-inducible factor stability, and that this process is inhibited by hypoxia. Agonist-induced ubiquitylation and degradation of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) play an essential role in surface receptor homeostasis, thereby tuning many physiological processes. Although β-arrestin and affiliated E3 ligases mediate agonist-stimulated lysosomal degradation of the β2-adrenergic receptor (β2AR), a prototypic GPCR, the molecular cues that mark receptors for ubiquitylation and the regulation of receptor degradation by the proteasome remain poorly understood. We show that the von Hippel–Lindau tumor suppressor protein (pVHL)–E3 ligase complex, known for its regulation of hypoxia-inducible factor (HIF) proteins, interacts with and ubiquitylates the β2AR, thereby decreasing receptor abundance. We further show that the interaction of pVHL with β2AR is dependent on proline hydroxylation (proline-382 and -395) and that the dioxygenase EGLN3 interacts directly with the β2AR to serve as an endogenous β2AR prolyl hydroxylase. Under hypoxic conditions, receptor hydroxylation and subsequent ubiquitylation decrease dramatically, thus attenuating receptor degradation and down-regulation. Notably, in both cells and tissue, the abundance of endogenous β2AR is shown to reflect constitutive turnover by EGLN3 and pVHL. Our findings provide insight into GPCR regulation, broaden the functional scope of prolyl hydroxylation, and expand our understanding of the cellular response to hypoxia.

SM-20 Is a Novel Mitochondrial Protein That Causes Caspase-dependent Cell Death in Nerve Growth Factor-dependent Neurons

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.

Expression of the SM-20 Gene Promotes Death in Nerve Growth Factor-Dependent Sympathetic Neurons

Abstract: Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM‐20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM‐20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3‐kinase inhibitor LY294002. In addition, SM‐20 protein synthesis is elevated in NGF‐deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM‐20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM‐20 may function to regulate cell death in neurons.

JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death.

Glaucoma is a neurodegenerative disease characterized by the apoptotic death of retinal ganglion cells (RGCs). The primary insult to RGCs in glaucoma is thought to occur to their axons as they exit the eye in the optic nerve head. However, pathological signaling pathways that exert central roles in triggering RGC death following axonal injury remain unidentified. It is likely that the first changes to occur following axonal injury are signal relay events that transduce the injury signal from the axon to the cell body. Here we focus on the c-Jun N-terminal kinase (JNK1-3) family, a signaling pathway implicated in axonal injury signaling and neurodegenerative apoptosis, and likely to function as a central node in axonal injury-induced RGC death. We show that JNK signaling is activated immediately after axonal injury in RGC axons at the site of injury. Following its early activation, sustained JNK signaling is observed in axonally-injured RGCs in the form of JUN phosphorylation and upregulation. Using mice lacking specific Jnk isoforms, we show that Jnk2 and Jnk3 are the isoforms activated in injured axons. Combined deficiency of Jnk2 and Jnk3 provides robust long-term protection against axonal injury-induced RGC death and prevents downregulation of the RGC marker, BRN3B, and phosphorylation of JUN. Finally, using Jun deficient mice, we show that JUN-dependent pathways are important for axonal injury-induced RGC death. Together these data demonstrate that JNK signaling is the major early pathway triggering RGC death after axonal injury and may directly link axon injury to transcriptional activity that controls RGC death.

NGF deprivation-induced gene expression: after ten years, where do we stand?

Nerve growth factor (NGF) is required for the survival of developing sympathetic and sensory neurons. In the absence of NGF, these neurons undergo protein synthesis-dependent apoptosis. Ten years have gone by since the first reports of specific genes being upregulated during NGF deprivation-induced cell death. Over the last decade, a few additional genes (DP5, Bim, SM-20) have been added to a list that began with cyclin D1 and c-jun. In this chapter, we discuss the evidence that these genes act as regulators of neuronal cell death. We also suggest a hypothesis for how one gene, SM-20, may function to suppress a self-protection mechanism in NGF-deprived neurons.

EGLN3 prolyl hydroxylase regulates skeletal muscle differentiation and myogenin protein stability

EGLN3, a member of the EGLN family of prolyl hydroxylases, has been shown to catalyze hydroxylation of the α subunit of hypoxia-inducible factor-α, which targets hypoxia-inducible factor-α for ubiquitination by a ubiquitin ligase complex containing the von Hippel-Lindau (VHL) tumor suppressor. We now report that EGLN3 levels increase during C2C12 skeletal myoblast differentiation. EGLN3 small interference RNAs and EGLN3 antisense oligonucleotides blocked C2C12 differentiation and decreased levels of myogenin, a member of the MyoD family of myogenic regulatory factors, which plays a critical role in myogenic differentiation. We also report that EGLN3 interacts with and stabilizes myogenin protein, whereas VHL associates with and destabilizes myogenin via the ubiquitin-proteasome system. The effect of VHL on myogenin stability and ubiquitination can be reversed, at least in part, by overexpression of EGLN3, suggesting that its binding to myogenin may prevent VHL-mediated degradation. These data demonstrate a novel role for EGLN3 in regulating skeletal muscle differentiation and gene expression. In addition, this report provides evidence for a novel pathway that regulates myogenin expression and skeletal muscle differentiation.

The survival of sympathetic neurons promoted by potassium depolarization, but not by cyclic AMP, requires phosphatidylinositol 3-kinase and Akt.

Abstract : Phosphatidylinositol (PI) 3‐kinase and Akt protein kinase mediate trophic factor‐dependent survival in certain neurons. However, a role for these enzymes in neuronal survival promoted by other agents is unclear. We have tested PI 3‐kinase and Akt for their role in survival promoted by membrane‐depolarizing concentrations of extracellular potassium and the cell‐permeable cyclic AMP analogue 8‐(4‐chlorophenylthio)cyclic AMP (cpt‐cAMP). Depolarization of sympathetic neurons resulted in an increase in the activities of both PI 3‐kinase and Akt. In addition, the PI 3‐kinase inhibitor LY294002 was a potent inducer of cell death in depolarized neurons. Stimulation with cpt‐cAMP resulted in relatively small increases in PI 3‐kinase and Akt activities, and neurons maintained with cpt‐cAMP were more resistant to LY294002‐induced death than were depolarized neurons. Expression of either dominant‐negative PI 3‐kinase or dominant‐negative Akt blocked survival promoted by depolarization but not by cpt‐cAMP. These results indicate that a PI 3‐kinase/Akt pathway is required for survival of sympathetic neurons mediated by depolarization but not by cpt‐cAMP. Thus, the survival of sympathetic neurons can be maintained through PI 3‐kinase/Akt‐dependent and‐independent pathways.