Erythropoietin (EPO) promotes neuronal survival after hypoxia and other metabolic insults by largely unknown mechanisms. Apoptosis and necrosis have been proposed as mechanisms of cellular demise, and either could be the target of actions of EPO. This study evaluates whether antiapoptotic mechanisms can account for the neuroprotective actions of EPO. Systemic administration of EPO (5,000 units͞kg of body weight, i.p.) after middle-cerebral artery occlusion in rats dramatically reduces the volume of infarction 24 h later, in concert with an almost complete reduction in the number of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling of neurons within the ischemic penumbra. In both pure and mixed neuronal cultures, EPO (0.1-10 units͞ml) also inhibits apoptosis induced by serum deprivation or kainic acid exposure. Protection requires pretreatment, consistent with the induction of a gene expression program, and is sustained for 3 days without the continued presence of EPO. EPO (0.3 units͞ml) also protects hippocampal neurons against hypoxia-induced neuronal death through activation of extracellular signal-regulated kinases and protein kinase Akt-1͞protein kinase B. The action of EPO is not limited to directly promoting cell survival, as EPO is trophic but not mitogenic in cultured neuronal cells. These data suggest that inhibition of neuronal apoptosis underlies short latency protective effects of EPO after cerebral ischemia and other brain injuries. The neurotrophic actions suggest there may be longer-latency effects as well. Evaluation of EPO, a compound established as clinically safe, as neuroprotective therapy in acute brain injury is further supported. E rythropoietin (EPO) was first characterized as a hematopoietic growth factor (1) and has been in clinical use by millions of patients over the last decade for the treatment of anemia. The observation that EPO and its receptor are expressed in rodent and human brain tissue (2-4), as well as by cultured neurons (5-8) and astrocytes (3,7,9), and that EPO has effects on neuronal cells (5), expanded the biological role of EPO beyond hematopoiesis. EPO gene expression in the brain is regulated by hypoxia-inducible factor-1 (1) that is activated by a variety of stressors, including hypoxia. Several independent research groups have reported that EPO protects cultured neurons against glutamate toxicity (6, 10) and reduces ischemic neuronal damage and neurological dysfunction in rodent models of stroke (6,(11)(12)(13). We recently reported that systemic administration of EPO is neuroprotective not only in animal models of cerebral ischemia, but also for mechanical trauma, excitotoxins, and neuroinflammation (11). Marked changes in EPO and EPOreceptor (EPOR) gene expression have been reported to occur in brain tissue after ischemic injury (6, 12). Specificity and biological relevance of these changes have been demonstrated by the observation that neutralization of endogenous EPO with soluble EPOR augments ischemic brain damage (13). Thus, it seems that E...

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Ras is a universal eukaryotic intracellular protein integrating extracellular signals from multiple receptor types. To investigate its role in the adult central nervous system, constitutively activated V12-Ha-Ras was expressed selectively in neurons of transgenic mice via a synapsin promoter. Ras-transgene protein expression increased postnatally, reaching a four- to fivefold elevation at day 40 and persisting at this level, thereafter. Neuronal Ras was constitutively active and a corresponding activating phosphorylation of mitogen-activated kinase was observed, but there were no changes in the activity of phosphoinositide 3-kinase, the phosphorylation of its target kinase Akt/PKB, or expression of the anti-apoptotic proteins Bcl-2 or Bcl-XL. Neuronal Ras activation did not alter the total number of neurons, but induced cell soma hypertrophy, which resulted in a 14.5% increase of total brain volume. Choline acetyltransferase and tyrosine hydroxylase activities were increased, as well as neuropeptide Y expression. Degeneration of motorneurons was completely prevented after facial nerve lesion in Ras-transgenic mice. Furthermore, neurotoxin-induced degeneration of dopaminergic substantia nigra neurons and their striatal projections was greatly attenuated. Thus, the Ras signaling pathway mimics neurotrophic effects and triggers neuroprotective mechanisms in adult mice. Neuronal Ras activation might become a tool to stabilize donor neurons for neural transplantation and to protect neuronal populations in neurodegenerative diseases.

RAS is frequently mutated in human cancers and has opposing effects on autophagy and tumorigenesis. Identifying determinants of the cellular responses to RAS is therefore vital in cancer research. Here, we show that autophagic activity dictates the cellular response to oncogenic RAS. N-terminal Apoptosis-stimulating of p53 protein 2 (ASPP2) mediates RAS-induced senescence and inhibits autophagy. Oncogenic RAS-expressing ASPP2 (Δ3/Δ3) mouse embryonic fibroblasts that escape senescence express a high level of ATG5/ATG12. Consistent with the notion that autophagy levels control the cellular response to oncogenic RAS, overexpressing ATG5, but not autophagy-deficient ATG5 mutant K130R, bypasses RAS-induced senescence, whereas ATG5 or ATG3 deficiency predisposes to it. Mechanistically, ASPP2 inhibits RAS-induced autophagy by competing with ATG16 to bind ATG5/ATG12 and preventing ATG16/ATG5/ATG12 formation. Hence, ASPP2 modulates oncogenic RAS-induced autophagic activity to dictate the cellular response to RAS: to proliferate or senesce.A ctive mutations of RAS, one of the first oncogenes identified, occur in about 20% of human tumors (1). Oncogenic RAS can transform cells and promote tumorigenesis, although it can also induce senescence and suppress tumor growth (2). Senescent cells are arrested and incapable of responding to mitogens, although they are viable and metabolically active. Senescence is characterized by dramatic cellular remodelling, which is energetically demanding. Autophagy, a genetically regulated process responsible for the turnover of cellular proteins and damaged or superfluous organelles, is a stress response involved in energy homeostasis (3). It was shown that autophagy is a critical mediator of oncogenic RAS-induced senescence, suggesting a negative role of autophagy in tumorigenesis (4). In contrast, a number of recent studies showed that active RAS requires autophagy to maintain its oncogenic function in tumorigenesis, arguing for a positive role of autophagy in tumorigenesis (5-8). The underlying reason for the conflicting observations remains unclear.RAS activation inhibits autophagy by activating the PI3K/ AKT/mammalian target of rapamycin (mTOR) pathway (9), and rapamycin, an mTOR inhibitor, is a potent inducer of autophagy. RAS also inhibits autophagy by reducing Beclin-1 expression in intestinal epithelial cells, an important mediator of autophagy (10). However, RAS was reported to induce autophagy by increasing the expression of key components of the autophagy machinery, such as ATG5 (11) and Beclin-1 (12). These reports suggest that RAS signaling performs a finely regulated balancing act to control autophagy. Identification of switching molecules that determine the cellular responses to RAS is thus needed urgently.The tumor suppressor p53 is one of the most well-established pathways by which RAS mediates cellular senescence. A recent study also showed that p53 is able to regulate autophagic activity by inducing the expression of LC3 (13). However, it remains unknown whether the abilit...

Rheb is a homolog of Ras GTPase that regulates cell growth, proliferation, and regeneration via mammalian target of rapamycin (mTOR). Because of the well established potential of activated Ras to promote survival, we sought to investigate the ability of Rheb signaling to phenocopy Ras. We found that overexpression of lipid-anchored Rheb enhanced the apoptotic effects induced by UV light, TNFα, or tunicamycin in an mTOR complex 1 (mTORC1)-dependent manner. Knocking down endogenous Rheb or applying rapamycin led to partial protection, identifying Rheb as a mediator of cell death. Ras and c-Raf kinase opposed the apoptotic effects induced by UV light or TNFα but did not prevent Rheb-mediated apoptosis. To gain structural insight into the signaling mechanisms, we determined the structure of Rheb-GDP by NMR. The complex adopts the typical canonical fold of RasGTPases and displays the characteristic GDP-dependent picosecond to nanosecond backbone dynamics of the switch I and switch II regions. NMR revealed Ras effector-like binding of activated Rheb to the c-Raf-Ras-binding domain (RBD), but the affinity was 1000-fold lower than the Ras/RBD interaction, suggesting a lack of functional interaction. shRNA-mediated knockdown of apoptosis signal-regulating kinase 1 (ASK-1) strongly reduced UV or TNFα-induced apoptosis and suppressed enhancement by Rheb overexpression. In conclusion, Rheb-mTOR activation not only promotes normal cell growth but also enhances apoptosis in response to diverse toxic stimuli via an ASK-1-mediated mechanism. Pharmacological regulation of the Rheb/mTORC1 pathway using rapamycin should take the presence of cellular stress into consideration, as this may have clinical implications.

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Caspase inhibition can extend the survival of cells undergoing apoptosis beyond the point of mitochondrial outer membrane permeabilisation (MOMP), but this does not confer long-term protection because caspase-independent death pathways emerge. Here, we describe a novel mechanism of mitochondrial self-destruction in caspase-inhibited cells, whose hallmark is the degradation of Tim23, the essential pore-forming component of the TIM23 inner membrane translocase. We show that Tim23 degradation occurs in cycling and post-mitotic cells, it is caspase-independent but Bax/Bak dependent, and it follows cytochrome c release. The proteolytic degradation of Tim23 is induced by MOMP and is mitochondrion-autonomous, as it also occurs in isolated mitochondria undergoing permeability transition. Degradation of Tim23 is selective, as expression of several other inner membrane proteins that regulate respiratory chain function is unaffected, and is not autophagic, as it occurs similarly in autophagy-proficient and -deficient (Atg-5 knockout) cells. Depleting Tim23 with siRNA is sufficient to inhibit cell proliferation and prevent long-term survival, while expression of degradation-resistant Tim23-GFP in mitochondria delays caspaseindependent cell death. Thus, mitochondrial autodigestion of Tim23 joins the array of processes contributing to caspaseindependent cell death. Because mitochondrial biogenesis requires a functional protein-import machinery, preventing Tim23 degradation might, therefore, be essential for repairing damaged mitochondria in chronic degenerative diseases. Apart from providing cellular ATP, mitochondria are also integral components of proapoptotic signalling. During apoptosis, mitochondrial outer membrane permeabilisation (MOMP) causes release of apoptogenic factors such as cytochrome c from the intermembrane space (IMS) to the cytoplasm. Cytochrome c-mediated activation of caspases via Apaf-1 is a dominant regulator of apoptosis.2 Although pan-specific caspase inhibitors can extend survival considerably depending on the cell type and death stimulus, caspaseindependent mechanisms of death execution take over in the long term. This has been ascribed to the action of other mitochondrial apoptogenic factors, such as AIF or EndoG.2 In addition, autophagy, which is co-activated by many apoptotic stimuli, has pro-death activity when apoptosis execution is impaired. 3,4 Exploiting mechanisms that suppress mitochondrial dysfunction and its deleterious follow-on effects is an obvious starting point for the development of treatments against loss of irreplaceable cells during neurodegenerative diseases, stroke, or myocardial infarction. However, therapeutic strategies should also address ways to mend defective mitochondria, thereby hopefully achieving long-term protection. Therefore, it is important to assess whether damaged mitochondria retain a functional import machinery so that the supply of cytoplasmic proteins is maintained.Here we focus on Tim23, the eponymous constituent of the TIM23 complex that forms the crucia...

Proteins containing extended polyglutamine repeats cause at least nine neurodegenerative disorders, but the mechanisms of diseaserelated neuronal death remain uncertain. We show that sympathetic neurons containing cytoplasmic inclusions formed by 97 glutamines expressed within human huntingtin exon1-enhanced green fluorescent protein (Q97) undergo a protracted form of nonapoptotic death that is insensitive to Bax deletion or caspase inhibition but is characterized by mitochondrial dysfunction. By treating the neurons with combined cytosine arabinoside and NGF withdrawal, we demonstrate that Q97 confers a powerful resistance to apoptosis at multiple levels: despite normal proapoptotic signaling (elevation of P-ser15-p53 and BimEL), there is no increase of Puma mRNA or Bax activation, both necessary for apoptosis. Even restoration of Bax translocation with overexpressed Puma does not activate apoptosis. We demonstrate that this robust inhibition of apoptosis is caused by Q97-mediated accumulation of Hsp70, which occurs through inhibition of proteasomal activity. Thus, apoptosis is reinstated by short hairpin RNA-mediated knockdown of Hsp70. These findings explain the rarity of apoptotic death in Q97-expressing neurons. Given the proteasomal blockade, we test whether enhancing lysosomal-mediated degradation with rapamycin reduces Q97 accumulation. Rapamycin reduces the amount of nonpathological Q25 by 70% over 3 d, but Q97 accumulation is unaffected. Interestingly, Q47 inclusions form more slowly as a result of constitutive lysosomal degradation, but fasterforming Q97 inclusions escape lysosomal control. Thus, cytoplasmic Q97 inclusions are refractory to clearance by proteasomal and lysosomal systems, leading to a toxicity that dominates over neuroprotective Hsp70. Our findings may explain the rarity of apoptosis but the inevitable cell death associated with polyQ inclusion diseases.