Mitochondrial Trafficking by Prohibitin-Kinesin-Myosin- Cadherin Complex in the Eye

Disruption of the mitochondrial-nuclear network leads to accelerated aging and age-related diseases, including age-related macular degeneration. The current study tested the hypothesis that mitochondrial morphology could be demonstrated quantitatively using a mathematic model and mitochondrial trafficking complex under stress conditions. To test our hypothesis, normal and aberrant mitochondria were examined quantitatively based on mitochondrial size, shape, position, composition, and dynamics. Adaptation of the mitochondrial network to changes in the intracellular oxidation and reduction milieu is critical for the survival of retinal pigment epithelial cells. Our mitochondrial interactome mapping demonstrated that a positive correlation may exist between oxidative stress-mediated phosphorylation and age-related disease progression. The current interactome may provide a potential therapeutic approach to treat mitochondria-induced neurodegeneration, including age-related macular degeneration

Mechanistic Dissection of Macular Degeneration Using the Phosphorylation Interactome

In the current study, we suggest that phosphorylation reactions of specific proteins in mitochondria and the nucleus are a key step in the progression of age-related macular degeneration (AMD). To determine the molecular mechanism of AMD, we examined proteomic changes under oxidative stress to establish the protein interaction map using in vitro and in vivo models that mimic the complex and progressive characteristics of AMD. We postulated that apoptosis can be initiated by phosphorylation reactions under chronic oxidative stress in a region-specific and tissue-specific manner. The analysis of AMD interactome and oxidative biomarker network demonstrated that the presence of tissue- and region-dependent post-translational mechanisms may contribute toward AMD progression through the mitochondrial-nuclear communication. The AMD interactome suggests that new therapeutic targets, including prohibitin, erythropoietin, vitronectin, crystalline, nitric oxide synthase, ubiquitin, and complement inhibition may exist as a proteome network. Further, immunocytochemistry demonstrated that mitochondria could enter the nucleus in the retinal pigment epithelium (RPE) under oxidative stress. The current interactome map implies that a positive correlation may exist between oxidative stress-mediated phosphorylation and AMD progression. The unbiased proteome network provides a basis for understanding oxidative stress-induced mitochondrial dysfunction in AMD and exploring effective therapeutic approaches to treat age-related neurodegeneration

Generating Aptamers by Cell-SELEX for Applications in Molecular Medicine

Aptamers are single-stranded oligonucleotides of DNA or RNA that bind to target molecules with high affinity and specificity. Typically, aptamers are generated by an iterative selection process, called systematic evolution of ligands by exponential enrichment (SELEX). Recent advancements in SELEX technology have extended aptamer selection from comparatively simple mixtures of purified proteins to whole living cells, and now cell-based SELEX (or cell-SELEX) can isolate aptamers that bind to specific target cells. Combined with nanotechnology, microchips, microfluidic devices, RNAi and other advanced technologies, cell-SELEX represents an integrated platform providing ultrasensitive and highly specific tools for clinical medicine. In this review, we describe the recent progress made in the application of cell-SELEX for diagnosis, therapy and biomarker discovery

Mitochondrial dysfunction and biogenesis: do ICU patients die from mitochondrial failure?

Mitochondrial functions include production of energy, activation of programmed cell death, and a number of cell specific tasks, e.g., cell signaling, control of Ca2+ metabolism, and synthesis of a number of important biomolecules. As proper mitochondrial function is critical for normal performance and survival of cells, mitochondrial dysfunction often leads to pathological conditions resulting in various human diseases. Recently mitochondrial dysfunction has been linked to multiple organ failure (MOF) often leading to the death of critical care patients. However, there are two main reasons why this insight did not generate an adequate resonance in clinical settings. First, most data regarding mitochondrial dysfunction in organs susceptible to failure in critical care diseases (liver, kidney, heart, lung, intestine, brain) were collected using animal models. Second, there is no clear therapeutic strategy how acquired mitochondrial dysfunction can be improved. Only the benefit of such therapies will confirm the critical role of mitochondrial dysfunction in clinical settings. Here we summarized data on mitochondrial dysfunction obtained in diverse experimental systems, which are related to conditions seen in intensive care unit (ICU) patients. Particular attention is given to mechanisms that cause cell death and organ dysfunction and to prospective therapeutic strategies, directed to recover mitochondrial function. Collectively the data discussed in this review suggest that appropriate diagnosis and specific treatment of mitochondrial dysfunction in ICU patients may significantly improve the clinical outcome

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia

587-P: miR34a-Induced Loss of Nicotinamide Phosphoribosyltransferase Mediates Diabetes-Induced Retinal Vascular Senescence

Stress-associated premature senescence (SAPS) has been implicated in diabetes-induced vascular dysfunction. We have shown that microRNA34a (miR-34a) is involved in promoting senescence of the retinal microvasculature. Loss of nicotinamide phosphoribosyltransferase (NAMPT), a rate-limiting enzyme of NAD+ biosynthesis from nicotinamide mononucleotide (NMN), has been implicated in aging and metabolic diseases. Interestingly NAMPT is a target of miR34a, therefore, herein, we investigated the role and reciprocal relationship of miR34a and NAMPT on diabetes-induced senescence of the retinal vasculature. NAMPT expression and NAD+ content were significantly reduced in retina of streptozotocin-induced diabetic rats (STZ-rats, 8-12 weeks of hyperglycemia) and in human postmortem diabetic retinas. On the contrary, miR34a expression was found increased and positively correlated with augmented levels of senescence markers. Exposure of human retinal endothelial cells (HuREC) to glucidic stress (25mM) also decreased NAMPT and NAD+ levels while augmented the expression of miR34a and of senescence markers. Transfection of HuREC with miR-34a mimic, but not a scramble construct, in normal glucose conditions inhibited the expression of NAMPT and reduced NAD+ levels while promoting the expression of senescence markers. These effects of miR34a mimic on HuREC were halted by supplementation with NMN (0.05-1mM), but without modifying NAMPT expression. Lastly, NMN supplementation also prevented high glucose-induced loss of NAD+ and elevation of senescence markers. Our data show that high glucose/diabetes-induced SAPS in the retinal microvasculature involves miR34a-mediated blockade of NAMPT and consequent loss of NAD+. Moreover, our data suggest that NMN supplementation could be a viable therapeutic strategy to improve endothelial dysfunction in diabetic retinopathy. Disclosure M. Bartolli: None. R. Jadeja: None. M. Thounaojam: None. D. Gutsaeva: None. P. Martin: None. Funding National Institutes of Health (EY022416, EY028714) </jats:sec

Hydroxyurea Induces Fetal Hemoglobin Expression by Activating cAMP Signaling Pathways In a cAMP- and cGMP-Dependent Manner; New Hypothesis to Account for a Role of Non-Erythroid Cells In Fetal Hemoglobin Induction

Abstract Abstract 1622 Despite considerable concerns and efforts, the mechanism of action of hydroxyurea (HU) for the induction of fetal hemoglobin (HbF) remains elusive. For example, clinical studies with HU suggest that bone marrow reserve is critical for HbF response to HU, but the underlying mechanism remains unknown. We and others have demonstrated that HU activates the cGMP signaling pathway in erythroid cells, which plays a role in HbF induction. However, the mechanisms by which intracellular signals are transduced to downstream cascades of cyclic nucleotide-dependent pathways in erythroid cells treated with HU remain to be established. Here we present evidence that HU induces HbF expression by activating the cAMP signaling pathway through two independent mechanisms: cAMP and cGMP. To study signal transduction by HU in cyclic nucleotide-dependent pathways, we initially focused on identifying substrates for cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) that are expressed in erythroid-lineage cells. We found that vasodilator-stimulated phosphoprotein (VASP), which is a 46/50-kDa phosphoprotein expressed in platelets at high levels, is also expressed in erythroid-lineage cells. VASP can be phosphorylated by cyclic nucleotide-elevating agents such as forskolin (activator of adenylate cyclase) and nitric oxide donors (activator of soluble guanylate cyclase). Interestingly, cAMP and cGMP phosphorylate distinct serine residues of VASP; Ser157 is phosphorylated by cAMP-elevating agents, while cGMP-elevating agents phosphorylated Ser239. Although HU increased both intracellular cAMP and cGMP levels in CD34+-derived erythroblasts, we found that Ser157, but not Ser239, is phosphorylated in adult erythroid cells treated with HU, suggesting activation of the cAMP signaling pathway. However, HU-induced HbF expression was down-regulated by inhibiting the activity of adenylate cyclase or soluble guanylate cyclase, suggesting that both enzymes are involved in HU-induced HbF expression. Our studies found that HU decreased the expression of cGMP-inhibitable phosphodiesterase 3B in a manner dependent on soluble guanylate cyclase, resulting in activation of the cAMP signaling pathway. Although a recent study showed that HU directly activates soluble guanylate cyclase, our studies showed that HU is unable to directly stimulate the enzyme activity of adenylate cyclase. Furthermore, HU induced the expression of cyclooxygenase-1 (COX-1) and increased the production of prostaglandin E2 (PGE2) that activates adenylate cyclase through G protein-coupled E-prostanoid receptors. Plasma PGE2 levels were also elevated in sickle cell patients upon HU therapy. These results demonstrate that HU induces HbF expression by activating the cAMP pathway by cAMP- and cGMP-dependent mechanisms, producing redundancy in the response of HbF to HU. Both cAMP and cGMP may represent major molecules that transduce signals from HU to the fetal globin gene. It is known that non-erythroid cells such as leukocytes and monocytes produce a large amount of PGE2. Thus, the involvement of PGE2 in HU-induced HbF expression may suggest an important role of non-erythroid cells as well as bone marrow reserve in the induction of HbF expression. More interestingly, several single nucleotide polymorphisms with amino acid changes have been demonstrated for COX-1; some genetic variants exhibit reduced COX activities. If SCD patients have some mutations in the COX-1 gene, such patients might be resistant to HU therapy. Disclosures: No relevant conflicts of interest to declare. </jats:sec