Extracellular proteases and neuronal cell death.

Neuronal cell death occurs during development of the central nervous system as well as in pathological situations such as acute injury and progressive degenerative diseases. For instance, granule cells in the developing cerebellum and neuronal precursor cells in the cortex undergo programmed cell death, or apoptosis. There is currently strong debate conceming the mechanism of death in many degenerative events such as ischemia, blunt head trauma, excitotoxicity and neurodegenerative diseases, i.e. Alzheimer's disease. Neurons can die a necrotic death when the initial insult is too great; apoptosis requires "planning." For example, the cell death seen in the core of an ischemic infarct is necrotic, while in the surrounding penumbra region the death is probably apoptotic. Regardless of the degenerative pathway, damaged or dead neurons are a hallmark of many diseases including Alzheimer's, Parkinson's, glaucoma, ischemia and multiple sclerosis. Molecules such as cytokines, chemokines, reactive nitrogen/oxygen species, and proteases play an important role in promoting and/or mediating neurodegeneration. Proteases have been implicated in both physiological and pathological events, suggesting their intervention in key points when things go awry. In this review we will summarize recent findings linking extracellular proteases with neuronal cell death in both human diseases and their animal models.     

p63 is an essential proapoptotic protein during neural development

The p53 family member p63 is required for nonneural development, but has no known role in the nervous system. Here, we define an essential proapoptotic role for p63 during naturally occurring neuronal death. Sympathetic neurons express full-length TAp63 during the developmental death period, and TAp63 levels increase following NGF withdrawal. Overexpression of TAp63 causes neuronal apoptosis in the presence of NGF, while cultured p63-/- neurons are resistant to apoptosis following NGF withdrawal. TAp63 is also essential in vivo, since embryonic p63-/- mice display a deficit in naturally occurring sympathetic neuron death. While both TAp63 and p53 induce similar apoptotic signaling proteins and require BAX expression and function for their effects, TAp63 induces neuronal death in the absence of p53, but p53 requires coincident p63 expression for its proapoptotic actions. Thus, p63 is essential for developmental neuronal death, likely functioning both on its own, and as an obligate proapoptotic partner for p53.     

Ribosomal Protein S3, a New Substrate of Akt,Serves as a Signal Mediator between Neuronal Apoptosis and DNA Repair

RPS3, a conserved, eukaryotic ribosomal protein of the 40 S subunit, is required for ribosome biogenesis. Because ribosomal proteins are abundant and ubiquitous, they may have additional extraribosomal functions. Here, we show that human RPS3 is a physiological target of Akt kinase and a novel mediator of neuronal apoptosis. NGF stimulation resulted in phosphorylation of threonine 70 of RPS3 by Akt, and this phosphorylation was required for Akt binding to RPS3. RPS3 induced neuronal apoptosis, up-regulating proapoptotic proteins Dp5/Hrk and Bim by binding to E2F1 and acting synergistically with it. Akt-dependent phosphorylation of RPS3 inhibited its proapoptotic function and perturbed its interaction with E2F1. These events coincided with nuclear translocation and accumulation of RPS3, where it functions as an endonuclease. Nuclear accumulation of RPS3 results in an increase in DNA repair activity to some extent, thereby sustaining neuronal survival. Abolishment of Akt-mediated RPS3 phosphorylation through mutagenesis accelerated apoptotic cell death and severely compromised nuclear translocation of RPS3. Thus, our findings define an extraribosomal role of RPS3 as a molecular switch that accommodates apoptotic induction to DNA repair through Akt-mediated phosphorylation.     

P53 Is Essential for Developmental Neuron Death as Regulated by the TrkA and p75 Neurotrophin Receptors

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/− or p53−/− mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.     

Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.     

A dp53/JNK-dependant feedback amplification loop is essential for the apoptotic response to stress in Drosophila

Programmed cell death (apoptosis) is a conserved process aimed to eliminate unwanted cells. The key molecules are a group of proteases called caspases that cleave vital proteins, which leads to the death of cells. In Drosophila, the apoptotic pathway is usually represented as a cascade of events in which an initial stimulus activates one or more of the proapoptotic genes (hid, rpr, grim), which in turn activate caspases. In stress-induced apoptosis, the dp53 (Drosophila p53) gene and the Jun N-terminal kinase (JNK) pathway function upstream in the activation of the proapoptotic genes. Here we demonstrate that dp53 and JNK also function downstream of proapoptotic genes and the initiator caspase Dronc (Drosophila NEDD2-like caspase) and that they establish a feedback loop that amplifies the initial apoptotic stimulus. This loop plays a critical role in the apoptotic response because in its absence there is a dramatic decrease in the amount of cell death after a pulse of the proapoptotic proteins Hid and Rpr. Thus, our results indicate that stress-induced apoptosis in Drosophila is dependant on an amplification loop mediated by dp53 and JNK. Furthermore, they also demonstrate a mechanism of mutual activation of proapoptotic genes.     

Siah1 interacts with the scaffold protein POSH to promote JNK activation and apoptosis

Siah proteins are ubiquitin-protein isopeptide ligases (E3) that have been implicated in a variety of cellular actions, including promotion of apoptotic death. Here, we show that Siah1 is a binding partner for POSH (plenty of SH3s), a scaffold component of the apoptotic JNK pathway, and that Siah contributes to death of neurons and other cell types by activating the JNK pathway. Such proapoptotic activity requires the E3 ligase activity of Siah1. Moreover, apoptotic stimuli markedly elevate cellular Siah1 levels by a mechanism reliant on Siah1 protein stabilization. This stabilization requires JNK pathway activation and interaction with POSH and is enhanced by phosphorylation of SIAH1 at tyrosines 100 and 126. Depletion of intracellular Siah proteins via small interference RNA partially protects cells from death evoked by apoptotic stimuli such as trophic factor deprivation and DNA damage. These findings thus reveal a "loop" mechanism in which the JNK pathway promotes SIAH1 stabilization and in which SIAH1 in turn activates the JNK pathway and, ultimately, contributes to cell death.     

Semaphorin 3A and neurotrophins: a balance between apoptosis and survival signaling in embryonic DRG neurons

Large numbers of neurons are eliminated by apoptosis during nervous system development. For instance, in the mouse dorsal root ganglion (DRG), the highest incidence of cell death occurs between embryonic days 12 and 14 (E12-E14). While the cause of cell death and its biological significance in the nervous system is not entirely understood, it is generally believed that limiting quantities of neurotrophins are responsible for neuronal death. Between E12 and E14, developing DRG neurons pass through tissues expressing high levels of axonal guidance molecules such as Semaphorin 3A (Sema3A) while navigating to their targets. Here, we demonstrate that Sema3A acts as a death-inducing molecule in neurotrophin-3 (NT-3)-, brain-derived neurotrophic factor (BDNF)- and nerve growth factor (NGF)-dependent E12 and E13 cultured DRG neurons. We show that Sema3A most probably induces cell death through activation of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway, and that this cell death is blocked by a moderate increase in NGF concentration. Interestingly, increasing concentrations of other neurotrophic factors, such as NT-3 or BDNF, do not elicit similar effects. Our data suggest that the number of DRG neurons is determined by a fine balance between neurotrophins and Semaphorin 3A, and not only by neurotrophin levels.     

Phagocytic Removal of Neuronal Debris by Olfactory Ensheathing Cells Enhances Neuronal Survival and Neurite Outgrowth via p38MAPK Activity

Compelling evidence from animal models and clinical studies suggest that transplantation of olfactory ensheathing cells (OECs), specialized glia in the olfactory system, combined with specific training may be therapeutically useful in the central nervous system (CNS) injuries and neurodegenerative diseases. The unique function of OECs could mainly attribute to both production of cell adhesion molecules and secretion of growth factors in OECs, which support neuron survival and neurite outgrowth. However, little is known about whether engulfment of neuronal degenerative debris by OECs also equally contributes to neuronal survival and neurite outgrowth. Furthermore, the molecular mechanisms responsible for neuronal degenerative corpses' removal remain elusive. Here, we used an in vitro model of primary culture of spinal cord neurons to investigate the effect of engulfment of degenerative neuron debris by OECs on neuronal survival and neurite outgrowth and the possible molecular mechanisms. Our results showed that OECs can engulf an amount of degenerated neuron debris, and this phagocytosis can make a substantial contribution to neuron growth, as demonstrated by increased number of neurons with longer neurite length and richer neurite branches when compared with the combination of neuron debris and OEC conditioned medium (OECCM). Moreover, p38 mitogen-activated protein kinase (p38MAPK) signaling pathway may mediate the OEC engulfment of debris because the p38MAPK-specific inhibitor, SB203580, can abrogate all the positive effects of OECs, including clearance of degenerated neuron debris and generation of bioactive molecules, indicating that p38MAPK is required for the process of phagocytosis of the neuron debris. In addition, the OEC phagocytic activity had no influence on its generation of bioactive molecules. Therefore, these findings provide new insight into further investigations on the OEC role in the repair of traumatic CNS injury and neurodegenerative diseases.     

Role of DAPK in neuronal cell death

Neuronal cell death happens as a result of the normal physiological process that occurs during development, or as part of the pathological process that occurs during disease. Death-associated protein kinase (DAPK) is an intracellular protein that mediates cell death by its serine/threonine kinase activity, and transmits apoptotic cell death signals in various cells, including neurons. DAPK is elevated in injured neurons in acute models of injury such as ischemia and seizure. The absence of DAPK has been shown to protect neurons from a wide variety of acute toxic insults. Moreover, DAPK also regulates neuronal cell death during central nervous system development. Neurons are initially overproduced in the developing nervous system, following which approximately one-half of the original cell population dies. This “naturally-occurring” or “programmed” cell death is essential for the construction of the developing nervous system. In this review, we focus on the role of DAPK in neuronal cell death after neuronal injury. The participation of DAPK in developmental neuronal death is also explained.     

Siva is an apoptosis-selective p53 target gene important for neuronal cell death

p53 plays a central role in neuronal cell death resulting from acute injury or disease. To define the pathway by which p53 triggers apoptosis, we used microarray analysis to identify p53 target genes specifically upregulated during apoptosis but not cell cycle arrest. This analysis identified a small subset of targets highly selective for the p53 apoptotic response, including Siva, a proapoptotic protein whose function is not well understood. Siva's expression pattern suggests that it plays an instructive role in apoptosis, and accordingly, we demonstrate that Siva is essential for p53-dependent apoptosis in cerebellar granule neurons. In addition, we determine that endogenous Siva is associated with the plasma membrane and that Caspase-8 and Bid are important for neuronal apoptosis. Our studies highlight the participation of membrane signaling events in p53's apoptotic program in primary neurons and have significant implications for understanding the mechanisms underlying pathogenesis after neuronal injury and in neurodegenerative diseases.     

Caspase-2 cleavage of BID is a critical apoptotic signal downstream of endoplasmic reticulum stress

The accumulation of misfolded proteins stresses the endoplasmic reticulum (ER) and triggers cell death through activation of the multidomain proapoptotic BCL-2 proteins BAX and BAK at the outer mitochondrial membrane. The signaling events that connect ER stress with the mitochondrial apoptotic machinery remain unclear, despite evidence that deregulation of this pathway contributes to cell loss in many human degenerative diseases. In order to "trap" and identify the apoptotic signals upstream of mitochondrial permeabilization, we challenged Bax-/- Bak-/- mouse embryonic fibroblasts with pharmacological inducers of ER stress. We found that ER stress induces proteolytic activation of the BH3-only protein BID as a critical apoptotic switch. Moreover, we identified caspase-2 as the premitochondrial protease that cleaves BID in response to ER stress and showed that resistance to ER stress-induced apoptosis can be conferred by inhibiting caspase-2 activity. Our work defines a novel signaling pathway that couples the ER and mitochondria and establishes a principal apoptotic effector downstream of ER stress.     

In vivo regulation of cell death by embryonic (pro)insulin and the insulin receptor during early retinal neurogenesis

Programmed cell death is an established developmental process in the nervous system. Whereas the regulation and the developmental role of neuronal cell death have been widely demonstrated, the relevance of cell death during early neurogenesis, the cells affected and the identity of regulatory local growth factors remain poorly characterized. We have previously described specific in vivo patterns of apoptosis during early retinal neurogenesis, and that exogenous insulin acts as survival factor (Díaz, B., Pimentel, B., De Pablo, F. and de la Rosa, E. J. (1999) Eur. J. Neurosci. 11, 1624-1632). Proinsulin mRNA was found to be expressed broadly in the early embryonic chick retina, and decreased later between days 6 and 8 of embryonic development, when there was increased expression of insulin-like growth factor I mRNA, absent or very scarce at earlier stages. Consequently, we studied whether proinsulin and/or insulin ((pro)insulin) action in prevention of cell death has physiological relevance during early neural development. In ovo treatment at day 2 of embryonic development with specific antibodies against (pro)insulin or the insulin receptor induced apoptosis in the neuroretina. The distribution of apoptotic cells two days after the blockade was similar to naturally occurring cell death, as visualized by TdT-mediated dUTP nick end labeling. The apoptosis induced by the insulin receptor blockade preferentially affected to the Islet1/2 positive cells, that is, the differentiated retinal ganglion cells. In parallel, the insulin survival effect on cultured retinas correlated with the activation of Akt to a greater extent than with the activation of MAP kinase. These results suggest that the physiological cell death occurring in early stages of retinal development is regulated by locally produced (pro)insulin through the activation of the Akt survival pathway.