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The role played by autophagy after ischemia/reperfusion (I/R) in the retina remains unknown. Our study investigated whether ischemic injury in the retina, which causes an energy crisis, would induce autophagy. Retinal ischemia was induced by elevation of the intraocular pressure and modulation of autophagic markers was analyzed at the protein levels in an early and late phase of recovery. Following retinal ischemia an increase in LC3BII was first observed in the early phase of recovery but did not stay until the late phase of recovery. Post-ischemic induction of autophagy by intravitreal rapamycin administration did not provide protection against the lesion induced by the ischemic stress. On the contrary, an increase in the number of apoptotic cells was observed following I/R in the rapamycin treated retinas. [ABSTRACT FROM AUTHOR]

Sphingolipids are essential to cell membrane structure and the development and maintenance of neural tissues. The role of bioactive sphingolipids has been established in numerous cellular events, including cell survival, growth, and apoptosis. Ocular inflammatory and autoimmune diseases involve activation and migration of endothelial cells, neovascularization, and infiltration of immune cells into various tissues. Clinically, the impact and role of sphingolipid-mediated signaling is increasingly being appreciated in the pathogenesis and treatment of diseases ranging from multiple sclerosis to neovascularization in age-related macular degeneration and diabetic retinopathy. In this review, we discuss our current knowledge and understanding of sphingolipid metabolism and signaling associated with the pathogenesis of ocular diseases. [ABSTRACT FROM AUTHOR]

The duration of cell death may allow deducing the underlying degenerative mechanism. To find out how long a photoreceptor takes to die, we used the rd1 mouse model for retinal neurodegeneration, which is characterized by phosphodiesterase-6 (PDE6) dysfunction and photoreceptor death triggered by high cGMP levels. Based on cellular data on the progression of cGMP accumulation, cell death, and survival, we created a mathematical model to simulate the temporal development of the degeneration and the clearance of dead cells. Both cellular data and modelling suggested that at the level of the individual cell, the degenerative process was rather slow, taking around 80 h to complete. Organotypic retinal explant cultures derived from wild-type animals and exposed to the selective PDE6 inhibitor zaprinast, confirmed the surprisingly long duration of an individual photoreceptor cell's death. We briefly discuss the possibility to link different cell death stages and their temporal progression to specific enzymatic activities known to be causally connected to cell death. This in turn opens up new perspectives for the treatment of inherited retinal degeneration, both in terms of therapeutic targets and temporal windows-of-opportunity. [ABSTRACT FROM AUTHOR]

The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect. [ABSTRACT FROM AUTHOR]

Müller cells are major macroglia and play many essential roles as a supporting cell in the retina. As Müller cells only constitute a small portion of retinal cells, investigating the role of Müller glia in retinal biology and diseases is particularly challenging. To overcome this problem, we first generated a Cre/lox-based conditional gene targeting system that permits the genetic manipulation and functional dissection of gene of interests in Müller cells. To investigate diabetes-induced alteration of Müller cells, we recently adopted methods to analyze Müller cells survival/death in vitro and in vivo. We also used normal and genetically altered primary cell cultures to reveal the mechanistic insights for Müller cells in biological and disease processes. In this article, we will discuss the applications and limitations of these methodologies, which may be useful for research in retinal Müller cell biology and pathophysiology. [ABSTRACT FROM AUTHOR]

Glucocorticoids (GCs) have a wide range of functions on several mammalian cell types, most of which are aimed at boosting survival, which is the raison d'être of the acute stress response. The role GCs play in the survival and viability of neurons is incongruous, as studies have revealed neuroprotective as well as neurodegenerative effects. These effects seem to depend on multiple factors amongst which are; the cell type involved, the mode of injury or underlying cause of cell death, likewise the concentration and or duration of GC exposure. In this mini review, we discuss mechanisms of GC action and their effect on neurodegeneration in general, and specifically review the effect of GCs on retinal neurons, in animal models of retinal degeneration or acute neuronal damage. Finally, we summarize potential protective and harmful GC-mediated mechanisms, which might be involved in the determination of neuronal fate in the retina following injury or during degeneration. [ABSTRACT FROM AUTHOR]

Genetic and immunohistochemical studies have identified the alternative complement pathway as an important component of age-related macular degeneration (AMD). The objective of this chapter is to review the impact of complement regulators on complement activation in the macula as it relates to AMD. Our laboratory and other investigators have identified CD46 and CD59 as important retinal pigment epithelium (RPE) cell membrane complement regulators, which are decreased in AMD. Using oxidized low-density lipoproteins (oxLDLs), which are found in Bruch's membrane in AMD, we found that CD46 and CD59 were decreased in RPE cells in part, by their release in exosomes and apoptotic particles. The release of complement regulators could potentially impair complement regulation on RPE cells and contribute to lesion formation in the outer retina and Bruch's membrane during the development of AMD. [ABSTRACT FROM AUTHOR]

Similar to other parts of the central nervous system, there are two types of programmed cell death during retinal development. In early development, the neuronal progenitor population is affected. In the mouse eye, this kind of programmed cell death begins at around embryonic day (E) 12.5 and peaks between E14.5 and E16.5. The second phase of programmed cell death occurs during synaptogenesis within the first 2 postnatal weeks. Important signaling mechanisms that induce programmed cell death of retinal progenitors appear to involve nerve growth factor acting on the proapoptotic receptor to p75 neurotrophin receptor (p75NTR) and transforming growth factor-β. [ABSTRACT FROM AUTHOR]

The sortilin family of Vps10p-domain receptors includes sortilin, SorLA, and SorCS1–3. These type-I transmembrane receptors predominate in distinct neuronal tissues, but expression is also present in certain specialized non-neuronal cell populations including hepatocytes and cells of the immune system. The biology of sortilins is complex as they participate in both cell signaling and in intracellular protein sorting. Sortilins function physiologically in signaling by pro- and mature neurotrophins in neuronal viability and functionality. Recent genome-wide association studies have linked members to neurological disorders such as Alzheimer's disease and bipolar disorder and outside the nervous system to development of coronary artery disease and type-2 diabetes. Particularly well described are the receptor functions in neuronal signaling by pro- (proNT) and mature (NT) neurotrophins and in the processing/metabolism of amyloid precursor protein (APP). [ABSTRACT FROM AUTHOR]

The neurotrophins play crucial roles regulating survival and apoptosis in the developing and injured nervous system. The four neurotrophins exert profound and crucial survival effects on developing peripheral neurons, and their expression and action is intimately tied to successful innervation of peripheral targets. In the central nervous system, they are dispensable for neuronal survival during development but support neuronal survival after lesion or other forms of injury. Neurotrophins also regulate apoptosis of both peripheral and central neurons, and we now recognize that there are regulatory advantages to having the same molecules regulate life and death decisions. This chapter examines the biological contexts in which these events take place and highlights the specific ligands, receptors, and signaling mechanisms that allow them to occur. [ABSTRACT FROM AUTHOR]

The tropomyosin-related tyrosine kinase (Trk) receptors were initially described as a family of growth factor receptors required for neuronal survival. They have since been shown to influence many aspects of neuronal development and function, including differentiation, outgrowth, and synaptic plasticity. This chapter will give an overview on the biology of Trk receptors within the nervous system. The structure and downstream signaling pathways of the full-length receptors will be described, as well as the biological functions of their truncated isoforms. Finally, the role of Trk receptors in the nervous system in health and disease will be discussed. [ABSTRACT FROM AUTHOR]

Like most growth factors, neurotrophins are initially synthesized as precursors that are cleaved to release C-terminal mature forms. The well-characterized mature neurotrophins bind to Trk receptors to initiate survival and differentiative responses. More recently, the precursor forms or proneurotrophins have been found to act as distinct ligands by binding to an unrelated receptor complex consisting of the p75 neurotrophin receptor (p75) and sortilin to initiate cell death. Induction of proNGF and p75 has been observed in preclinical injury models and in pathological states in the central nervous system, and strategies that block the proNGF/p75 interaction are effective in limiting neuronal apoptosis. In contrast, the mechanisms that regulate expression of other proneurotrophins, including proBDNF and proNT-3, are less well understood. Here, recent findings on the biological actions, regulation of expression, and pathophysiological effects of proneurotrophins will be reviewed. [ABSTRACT FROM AUTHOR]

Many toxins that individuals are exposed to cause DNA damage. Cells that have sustained DNA damage may attempt to repair the damage prior to replication. However, if a cell has sustained serious damage it cannot repair, it will commit suicide through a genetically regulated programmed cell death (PCD) pathway. Crucial to the ultimate execution of PCD is a family of cysteine proteases called caspases. Activation of these enzymes occurs late enough in the PCD pathway that a cell can no longer avoid cell death, but still earlier than PCD-associated morphological changes or DNA fragmentation. This protocol details a method for using fluorochrome-conjugated caspase inhibitors for the detection of activated caspases in intact cells. The analysis and documentation is performed using fluorescence microscopy. [ABSTRACT FROM AUTHOR]

We present here the application of a novel assay that measures the absolute amount of PS externalized on the surface of cells. While based on the same annexin binding principle as the fluorescent flow cytometry assay, we use paramagnetic iron as the ultimate reporter molecule, establishing a linear relationship between signal amplitude and amount of PS on the cell surface, allowing a quantitative assay of PS externalization over a wide dynamic range. The application of this technique, alone and in concert with the PS oxidation method presented in the previous chapter, will greatly aid in studying the mechanistic connection between lipid peroxidation and translocation events during apoptosis. [ABSTRACT FROM AUTHOR]

Membrane phospholipids are gaining increasing attention as important mediators in a variety of signal transduction processes. Oxidation and changes in membrane topography of lipids are likely important elements in the regulation of phospholipid-dependent signaling. Phosphatidylserine (PS), in particular, is implicated in the regulation of macrophage-dependent clearance of apoptotic cell ˵corpses″ in a pathway likely mediated by selective oxidation and translocation of PS in the plasma membrane. Here we describe our highly sensitive and specific assay to measure differential lipid peroxidation in individual phospholipid classes in live cells using metabolic integration of the fluorescent oxidation-sensitive fatty acid analog, cis- parinaric acid and resolution of specific phospholipids by high-pressure liquid chromatography. These experimental approaches can provide insight into the roles and mechanisms of PS oxidation in the identification and clearance of apoptotic cells. [ABSTRACT FROM AUTHOR]

Current techniques for examining the global creation and repair of DNA double-strand breaks are restricted in their sensitivity, and such techniques mask any site-dependent variations in breakage and repair rate or fidelity. We present here a system for analyzing the fate of documented DNA breaks, using the MLL gene as an example, through application of ligation-mediated PCR. Here, a simple asymmetric double-stranded DNA adapter molecule is ligated to experimentally induced DNA breaks and subjected to seminested PCR using adapter- and gene-specific primers. The rate of appearance and loss of specific PCR products allows detection of both the break and its repair. Using the additional technique of inverse PCR, the presence of misrepaired products (translocations) can be detected at the same site, providing information on the fidelity of the ligation reaction in intact cells. Such techniques may be adapted for the analysis of DNA breaks and rearrangements introduced into any identifiable genomic location. We have also applied parallel sequencing for the high-throughput analysis of inverse PCR products to facilitate the unbiased recording of all rearrangements located at a specific genomic location. [ABSTRACT FROM AUTHOR]

This chapter contains sections titled: Introduction, Inflammation and Gastrointestinal Function, Nutritional Implications and Strategies, Summary, References [ABSTRACT FROM AUTHOR]

Surgery is needed for non-obstetric conditions in 0.3 to 2% of pregnancies in the United States. Drug selection is important as some may affect the developing fetus, although the vast majority are safe. This chapter reviews the commonly used anesthetic agents and their effect on the developing fetus. [ABSTRACT FROM AUTHOR]

Relatively enriched pre-clinical data suggest that commonly used general anesthetics can cause widespread neuronal apoptosis with potentially long-term neurocognitive deficits in animals exposed at the stage of brain development. A number of potential mechanisms have been demonstrated in various animal studies but further investigation is still needed. Possible preventative measures have been sought and several neuroprotective agents such as xenon were found, with more emerging. However, extrapolating experimental data obtained from animals to humans is difficult and under debate. This chapter provides an overview of most research in the field of anesthetic-induced neuroapoptosis. Readers are encouraged to be vigilant and receptive to new information. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) channels affect several physiological and pathological processes. In particular, TRP channels have been recently involved in the triggering of enhanced proliferation, aberrant differentiation, and resistance to apoptotic cell death leading to the uncontrolled tumor invasion. About thirty TRPs have been identified to date, and are classified in seven different families: TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like). Among these channel families, the TRPC, TRPM, and TRPV families have been mainly correlated with malignant growth and progression. The aim of this review is to summarize data reported so far on the expression and the functional role of TRPV channels during cancer growth and progression. TRPV channels have been found to regulate cancer cell proliferation, apoptosis, angiogenesis, migration and invasion during tumor progression, and depending on the stage of the cancer, up- and down-regulation of TRPV mRNA and protein expression have been reported. These changes may have cancer promoting effects by increasing the expression of constitutively active TRPV channels in the plasma membrane of cancer cells by enhancing Ca2+-dependent proliferative response; in addition, an altered expression of TRPV channels may also offer a survival advantage, such as resistance of cancer cells to apoptotic-induced cell death. However, recently, a role of TRPV gene mutations in cancer development, and a relationship between the expression of specific TRPV gene single nucleotide polymorphisms and increased cancer risk have been reported. We are only at the beginning, a more deep studies on the physiopathology role of TRPV channels are required to understand the functional activity of these channels in cancer, to assess which TRPV proteins are associated with the development and progression of cancer and to develop further knowledge of TRPV proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting in cancer. [ABSTRACT FROM AUTHOR]

It has long been appreciated that the apoptotic activity of TNFα is context dependent and requires inhibition of NFΚB signaling or de novo protein synthesis to be manifested in most normal cells in culture. Recent studies have uncovered an unexpected pro-apoptotic synergism between TNF cytokines and the CCN family of extracellular matrix proteins, which are dynamically expressed at sites of injury repair and inflammation. The presence of CCN1, CCN2, or CCN3 allows TNFα to induce apoptosis with high efficacy without perturbation of NFΚB signaling or de novo protein synthesis, thus converting TNFα from a proliferation-promoting protein into an apoptotic inducer. CCN proteins also enhance the cytotoxicity of other TNF family cytokines including LTα, FasL, and TRAIL. CCN proteins synergize with TNF cytokines through binding to integrin α6β1 and the heparan sulfate proteoglycan (HSPG) syndecan-4 to induce reactive oxygen species (ROS) accumulation. Knockin mice that express a CCN1 mutant defective for binding α6β1-HSPG are severely blunted in TNFα- and Fas-mediated apoptosis, indicating that CCN1 is a physiologic regulator of these processes. Thus, CCN proteins in the extracellular matrix microenvironment can provide the contextual cues for the cytotoxicity of TNFα and related cytokines and profoundly influence their activity. [ABSTRACT FROM AUTHOR]

Apoptosis is a complex and highly regulated process with numerous and varied biological consequences, it is typically described as a sequence of morphological events that can be easily recognized histologically. In fact, the initial identification and subsequent characterization of apoptosis were based on microscopic observations of its occurrence in vivo. In the early 1970΄s, an experimental pathologist recognized variations in the morphology of dead cells. He deduced from these observations that the mechanisms for cell death could likewise differ. This Australian pathologist, Professor John Kerr, made these seminal observations and with his colleagues also devised the name apoptosis to distinguish the process from necrosis. Kerr΄s sound morphological observations and interpretations based on those observations are the foundations for the explosion of apoptosis research that has occurred since his original observations. As will be detailed in this chapter, apoptosis can be quantified as a response of normal and tumor tissues to various cancer therapies in specimens from animals and patients treated in vivo. [ABSTRACT FROM AUTHOR]

Apoptosis is a well-conserve cellular disassembly process, which has been implicated in a variety of diseases. Unlike cells with a single nucleus, apoptotic signaling can target individual nuclei in multi-nucleated skeletal muscle cells without necessarily eliminating the entire cell (muscle fiber). This targeted apoptosis or ˵nuclear apoptosis″ appears to have a role in regulating aging-induced muscle loss (sarcopenia) by reducing the myofiber volume (i.e. cytoplasm) that can be supported in a single muscle fibre. Recent investigations indicate that apoptotic signaling in aged skeletal muscles occurs through three apoptotic pathways. The intrinsic or mitochondria apoptotic pathway has been most widely studied in muscle. Mitochondria dysfunction and increased mitochondria permeability lead to activation of cysteine-aspartic acid proteases (caspases) and eventually DNA fragmentation in sarcopenia. The death receptor (extrinsic) apoptotic pathway has been strongly implicated in sarcopenia and other conditions of muscle loss with aging or disuse. TNF-α is thought to initiate apoptotic signaling via the death receptor, and this can proceed to activate the effort proteases (e.g., caspase 3) independent from mitochondria signaling. Nevertheless, there is some cross-talk between the intrinsic and the extrinsic apoptotic pathways. Finally, a few studies have shown data to suggest that the endoplasmic reticulum-stress apoptotic pathway may also have a role in sarcopenia, although the importance of this pathway relative to the other two pathways is less clear. Both myonuclei and satellite cells appear to be susceptible to nuclear apoptosis in sarcopenia. [ABSTRACT FROM AUTHOR]

There is an abundance of studies examining the involvement of mitochondria in aging, including their role in the functional and structural deterioration of skeletal muscle with aging. Despite years of study, the precise involvement of mitochondria in the aging of skeletal muscle remains to be fully understood. This chapter provides some context for the current knowledge in this area and areas that will be refined through further study. It will examine the issue of ˵mitochondrial dysfunction″ in aging; why it occurs and the functional consequences. The potential impact of three important age-related changes in mitochondria will be considered here: a reduced capacity for generating cellular energy in the form of adenosine triphosphate (ATP); an increased susceptibility to apoptosis; and an increase in reactive oxygen species (ROS) production with aging. The chapter considers the extent to which the mitochondrial content may be up-regulated in response to muscle activity as a means of assessing the malleability of the age-related impairments in mitochondria. Given the central importance of mitochondrial biology to so many facets of normal cell function, particularly in tissues with a wide metabolic scope like skeletal muscle, new discoveries about the significance of changes in mitochondria for aging skeletal muscles, and their potential remedy through lifestyle modification (e.g., exercise training, diet) and/or medical intervention (e.g., pharmaceuticals, gene therapy), will remain at the forefront of our quest to promote healthy aging. [ABSTRACT FROM AUTHOR]

Following exposure to X-radiation (10 cGy), human mesenchymal stem cells (MSCs) appear to exhibit transposition of 1q12 loci of the homologous chromosomes from the perimembrane regions deep into the nucleus, their approximation and activation of chromosomal nucleolus-forming regions – the initial stages of the adaptive response (AR) development. Transfer of the culture medium containing irradiated MSCs to the intact bystander cells induced in the latter all three reactions being studied, i.e., the bystander effect (BE). Previously, similar results concerning radiation-induced AR and BE were obtained for human G0-lymphocytes. The cumulative findings obtained suggest a universal nature of the chromosomal-homolog loci-transposition reaction revealed during the development of AR in the differentiated (lymphocytes) or undifferentiated (MSCs) human cells, and the possibility of the development of the radiation-induced BE in both suspended and monolayer-cell cultures during transfer of stress-signalling factors through the incubation medium. We believe that these factors are the fragments of extracellular genomic DNA of apoptotic cells. [ABSTRACT FROM AUTHOR]

CpG-enriched rDNA accumulating in human cfDNA significantly stimulate gene transcription in mesenchymal stem cells by activating TLR9 and MyD88-dependent signaling pathways and inhibiting differentiation of mesenchymal stem cells into adipocytes. These data are important for stem cell therapy. [ABSTRACT FROM AUTHOR]

The plasma levels of cell-free DNA have been considered as a potential marker of the biocompatibility of membranes used for hemodialysis. Some studies demonstrated, but not fully elucidated, the elevations of cfDNA concentrations in plasma during hemodialysis. Therefore, we focused on the comparison of plasma cfDNA levels and the expression of selected genes in peripheral blood cells during hemodialysis. The concentrations of plasma cfDNA have been measured in patients before, throughout and after HD and compared with the cfDNA levels of healthy volunteers. The expressions of selected genes with known functions in inflammation and apoptosis were measured (before and immediately after hemodialysis) using mRNA and real-time PCR. We found significant elevations of cfDNA in plasma during hemodialysis sessions with the highest values at the end of hemodialysis and slow decline within 30 min after hemodialysis in most patients. In comparison with healthy controls, the cfDNA levels in patients were significantly higher in the interdialytic interval. We found remarkable individual differences in expression profiles both among hemodialysis patients and in comparison to the control group. [ABSTRACT FROM AUTHOR]

Low-molecular-weight DNA of blood plasma is supposed to be a universal quantitative indicator of apoptosis, which allows the distinction between fundamentally different conditions of organisms. Cerebrovascular accidents are characterized by the increase of this DNA concentration in the course of 3 days after acuity with a maximum after 3 h in the case of hemorrhage and after 24 h in the case of ischemia. An analogous DNA fraction in the same quantity appears in the spinal fluid of patients with ischemia. Chronic obstructive pulmonary disease is detected for the first time by the decrease of the content of this DNA. [ABSTRACT FROM AUTHOR]

A comparative study of extracellular DNA versus genomic or apoptotic DNA was executed by fluorescent in situ hybridization (FISH) analysis. Extracellular DNA from culture medium and bound to the cell surface of human primary endotheliocytes, human primary fibroblasts and HeLa cells were investigated. There were not any specific peculiarities found in extracellular DNA isolated from the culture medium of endotheliocytes and HeLa cells. We revealed an overrepresentation of chromosome 9 fragments and the regions of the short arms of chromosomes 13, 14, 15, 21, 22 in DNA isolated from the culture medium of primary fibroblasts. The pericentromeric region of chromosome 9 is also overrepresented in cell surface bound DNA isolated from endotheliocytes, fibroblasts and HeLa cells. The data obtained allow a rational selection of DNA targets for the investigation of extracellular DNA generation and circulating DNA-based diagnostics. [ABSTRACT FROM AUTHOR]

Thyroiditis consists of a number of different inflammatory processes in thyroid and is caused by a variety of factors. Among the various inflammatory thyroid lesions, Hashimoto΄s thyroiditis is the most common, and we will focus on the mechanisms involved in this disease. While extensive studies have been performed to elucidate the immunopathogenesis of Hashimoto΄s thyroiditis, the processes underlying this disease are still not fully understood. However, significant progress has been made in areas related to the roles that iodine, CD4+CD25+ regulatory T cells, and apoptosis play in autoimmune-mediated thyroid damage, the defining feature of Hashimoto΄s thyroiditis. [ABSTRACT FROM AUTHOR]