Search

Globally, developed nations spend a significant amount of their resources on health care initiatives that poorly translate into increased population life expectancy. As an example, the United States devotes 16% of its gross domestic product to health care, the highest level in the world, but falls behind other nations that enjoy greater individual life expectancy. These observations point to the need for pioneering avenues of drug discovery to increase life span with controlled costs. In particular, innovative drug development for metabolic disorders such as diabetes mellitus becomes increasingly critical given that the number of diabetic people will increase exponentially over the next 20 years. This article discusses the elucidation and targeting of novel cellular pathways that are intimately tied to oxidative stress in diabetes mellitus for new treatment strategies. Pathways that involve wingless, β-nicotinamide adenine dinucleotide (NAD(+)) precursors, and cytokines govern complex biological pathways that determine both cell survival and longevity during diabetes mellitus and its complications. Furthermore, the role of these entities as biomarkers for disease can further enhance their utility irrespective of their treatment potential. Greater understanding of the intricacies of these unique cellular mechanisms will shape future drug discovery for diabetes mellitus to provide focused clinical care with limited or absent long-term complications.

Background and purpose: Clinical indications for erythropoietin (EPO) in the vascular system reach far beyond the treatment of anemia, but the development of EPO as a non-toxic agent rests heavily upon the cellular pathways controlled by EPO that require elucidation. Experimental approach: We modulated gene activity and examined cellular trafficking of critical pathways during oxidative stress that may work in concert with EPO to protect primary cerebral endothelial cells (ECs) during oxidative stress, namely protein kinase B (Akt1), 14-3-3 protein, the Forkhead transcription factor FOXO3a. Key results: Here, we show that preservation of ECs by EPO during oxygen-glucose deprivation (OGD) required the initial activation of the phosphatidylinositol 3-kinase (PI-3K) pathway through Akt1, since specific pharmacological blockade of Akt1 activity or gene silencing of Akt1 prevented EC protection by EPO. EPO subsequently involved a series of anti-apoptotic pathways to activate STAT3, STAT5, and ERK 1/2. Furthermore, EPO maintained the inhibitory phosphorylation and integrity of the ‘pro-apoptotic’ transcription factor FOXO3a, promoted the binding of FOXO3a to 14-3-3 protein and regulated the intracellular trafficking of FOXO3a. Additionally, gene silencing of FOXO3a during OGD significantly increased EC survival, but did not synergistically improve cytoprotection by EPO, illustrating that EPO relied upon the blockade of the FOXO3a pathway. Conclusions and implications: Our work defines a novel cytoprotective pathway in ECs that involves PI-3 K, STAT3, STAT5, ERK 1/2, 14-3-3 protein and FOXO3a, which can be targeted for the development of EPO as a clinically effective and safe agent in the vascular system. British Journal of Pharmacology (2007) 150, 839–850. doi:10.1038/sj.bjp.0707161

In several cell systems, protein kinase B (Akt1) can promote cell growth and development, but the "antiapoptotic" pathways of this kinase that may offer protection against cellular inflammatory demise have not been defined. Given that early cellular membrane phosphatidylserine exposure is a critical component of apoptosis, we investigated the role of Akt1 during neuronal apoptotic injury. By employing differentiated SH-SY5Y neuronal cells that overexpress a constitutively active form of Akt1 (myristoylated Akt1), free radical-induced cell injury was assessed through trypan blue dye exclusion, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, and mitochondrial membrane potential. Membrane phosphatidylserine exposure was both necessary and sufficient for microglial activation, insofar as cotreatment with an antiphosphatidylserine receptor-neutralizing antibody could prevent microglial activity following neuronal loss of membrane asymmetry. Furthermore, expression of myristoylated Akt1 not only prevented cell injury through the prevention of membrane phosphatidylserine exposure and genomic DNA fragmentation but also inhibited microglial activation and proliferation that required the inhibition of caspase 9-, caspase 3-, and caspase 1-like activities linked to cytochrome c release. Interestingly, Akt1 modulation of membrane phosphatidylserine exposure was primarily through caspase 1 activity. Removal of Akt1 activity abolished neuronal protection, suggesting that Akt1 functions as a critical pathway for the maintenance of cellular integrity and the prevention of phagocytic cellular removal during neurodegenerative insults.

Erythropoietin (EPO) plays a significant role in the hematopoietic system, but the function of EPO as a neuroprotectant and anti-inflammatory mediator requires further definition. We therefore examined the cellular mechanisms that mediate protection by EPO during free radical injury in primary neurons and cerebral microglia. Neuronal injury was evaluated by trypan blue, DNA fragmentation, phosphatidylserine (PS) exposure, Akt1 phosphorylation, Bad phosphorylation, mitochondrial membrane potential, and cysteine protease activity. Microglial activation was assessed through proliferating cell nuclear antigen and PS receptor expression. EPO provides intrinsic neuronal protection that is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent membrane PS exposure, since protection by EPO is completely abolished by cotreatment with an anti-EPO neutralizing antibody. Extrinsic protection by EPO is offered through the inhibition of cerebral microglial activation and the suppression of microglial PS receptor expression for the prevention of neuronal phagocytosis. In regards to microglial chemotaxis, EPO modulates neuronal poptotic membrane PS exposure necessary for microglial activation primarily through the regulation of caspase 1. EPO increases Akt1 activity, phosphorylates Bad, and maintains neuronal nuclear DNA integrity through the downstream modulation of mitochrondrial membrane potential, cytochrome c release, and caspase 1, 3, and 8-like activities. Elucidating the intrinsic and extrinsic protective pathways of EPO that mediate both neuronal integrity and inflammatory microglial activation may enhance the development of future therapies directed against acute neuronal injury. Keywords: Apoptosis, Bad, cytochrome c, cysteine proteases, erythropoietin, microglial activation, mitochondrial membrane potential, phosphatidylserine exposure, protein kinase B Introduction Initially considered to mediate primarily the proliferation and differentiation of erythroid progenitors, erythropoietin (EPO) has emerged as a versatile growth factor that may play a significant role in the nervous system. Both EPO and the erythropoietin receptor EPOR are expressed throughout the nervous system in neurons, endothelial cells, and astrocytes in the cerebral cortex, hippocampus, and the amygdala (Morishita et al., 1997; Nagai et al., 2001; Chong et al., 2002b). In neuronal injury paradigms, EPO has been shown to provide protection against toxic insults, such as ischemia and free radical injury (Bernaudin et al., 1999; Chong et al., 2002a; Wen et al., 2002). As a result, EPO has been identified as a possible candidate in the formulation of therapeutic strategies against neurodegenerative diseases. To further the development of EPO as a novel neuroprotectant against both acute and chronic neurodegenerative disease, it is first critical to understand the cellular pathways that may mediate neuronal injury and are subsequently susceptible to modulation by EPO. Oxygen-free radicals, such as nitric oxide (NO), have been established as significant precipitants of neuronal degeneration (Maiese & Vincent, 2000; Anderson et al., 2001). NO can trigger the induction of two independent apoptotic pathways that consist of nuclear DNA degradation and the exposure of membrane phosphatidylserine (PS) residues (Maiese & Vincent, 2000; Dumont et al., 2001; Lin & Maiese, 2001). Although DNA degradation in neurons may immediately impact cellular integrity (Jessel et al., 2002), the exposure of membrane PS residues in neurons can precipitate a latent cellular inflammation (Dombroski et al., 2000) and microglial phagocytosis of viable neurons (Maiese & Vincent, 2000; Hoffmann et al., 2001). Several downstream cellular pathways may ultimately determine the protective role of EPO. In particular, the serine/threonine kinase Akt1, a key determinant of cell survival, appears to be necessary for EPO to prevent apoptosis of erythroid progenitors (Uddin et al., 2000). Once activated through phosphorylation, Akt1 can inhibit the activity of several substrates that promote apoptosis, such as Bad (Blume-Jensen et al., 1998), IκB kinase α (IKKβ) (Romashkova & Makarov, 1999), the forkhead transcription factor (FHKRL1) (Brunet et al., 1999), and the glycogen synthase kinase-3β (Cross et al., 1995). The protective role of Akt1 also may be dependent upon the preservation of mitochondrial membrane integrity and the modulation of cysteine protease activity through cytochrome c. The protein Akt1 may serve to stabilize mitochondrial membrane potential and prevent the release of cytochrome c (Kennedy et al., 1999). Cellular release of NO can directly lead to mitochondrial membrane depolarization and the opening of mitochondrial permeability transition pores (Bal-Price & Brown, 2000; Chong et al., 2002a). As a result, cytochrome c is released from mitochondria and subsequently leads to the activation of a family of cysteine proteases (caspases) that include caspase 8, caspase 1, and caspase 3. Together, these cysteine proteases can lead to both DNA fragmentation and membrane PS exposure (Lin & Maiese, 2001; Mandal et al., 2002). Given the strong neuroprotective potential of EPO, we investigated the underlying cellular mechanisms controlled by EPO that may determine both the maintenance of neuronal cellular integrity and the inhibition of microglial activation to gain greater insight for the development of future neurodegenerative therapeutic strategies.

Erythropoietin (EPO) modulates primarily the proliferation of immature erythroid precursors, but little is known of the potential protective mechanisms of EPO in the central nervous system. We therefore examined the ability of EPO to modulate a series of death-related cellular pathways during anoxia and free radical induced neuronal degeneration. Neuronal injury was evaluated by trypan blue, DNA fragmentation, membrane phosphatidylserine exposure, protein kinase B phosphorylation, cysteine protease activity, mitochondrial membrane potential, and mitogen-activated protein (MAP) kinase phosphorylation. We demonstrate that constitutive neuronal EPO is insufficient to prevent cellular injury, but that signaling through the EPO receptor remains biologically responsive to exogenous EPO administration. Exogenous EPO is both necessary and sufficient to prevent acute genomic DNA destruction and subsequent phagocytosis through membrane PS exposure, because neuronal protection by EPO is completely abolished by co-treatment with an anti-EPO neutralizing antibody. Through pathways that involve the initial activation of protein kinase B, EPO maintains mitochondrial membrane potential. Subsequently, EPO inhibits caspase 8-, caspase 1-, and caspase 3-like activities linked to cytochrome c release through mechanisms that are independent from the MAP kinase systems of p38 and JNK. Elucidating some of the novel neuroprotective pathways employed by EPO may further the development of new therapeutic strategies for neurodegenerative disorders.

The successful treatment for multiple disease entities can rest heavily upon the ability to elucidate the intricate relationships that govern cellular proliferation, metabolism, survival, and inflammation. Here we discuss the therapeutic potential of the mammalian forkhead transcription factors predominantly in the O class, FoxO1, FoxO3, FoxO4, and FoxO6, which play a significant role during normal cellular function as well as during progressive disease. These transcription factors are integrated with several signal transduction pathways, such as Wnt proteins, that can regulate a broad array of cellular process that include stem cell proliferation, aging, and malignancy. FoxO transcription factors are attractive considerations for strategies directed against human cancer in light of their pro-apoptotic effects and ability to lead to cell cycle arrest. Yet, FoxO proteins can be associated with infertility, cellular degeneration, and unchecked cellular proliferation. As our knowledge continues to develop for this novel family of proteins, potential clinical applications for the FoxO family should heighten our ability to limit disease progression without clinical compromise.