Prognostic Impact of BRAF and KRAS Mutation in Patients with Colorectal and Appendiceal Peritoneal Metastases Scheduled for CRS and HIPEC.

BACKGROUND: KRAS and BRAF mutations are prognostic and predictive tools in metastatic colorectal cancer, but little is known about their prognostic value in patients scheduled for cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Therefore, we analyzed the prognostic impact of KRAS and BRAF mutations in patients with peritoneal metastases scheduled for CRS and HIPEC. PATIENTS AND METHODS: In a consecutive series of 399 patients scheduled for CRS and HIPEC between 2009 and 2017, 111 subjects with peritoneal metastases from primaries of the appendix, colon, or rectum were analyzed for KRAS mutation and 92 for BRAF mutation. RESULTS: Mutation in KRAS was present in 51/111 (46%), and mutated BRAF was found in 10/92 (11%). There was no difference in overall survival between KRAS mutation tumors and KRAS wild type, whereas BRAF mutation was associated with short survival. No subject with BRAF mutation survived 2 years. On multivariate analysis, completeness of cytoreduction score (CCS, p = 0.000001), presence of signet cell differentiation (p = 0.000001), and BRAF mutation (p = 0.0021) were linked with poor prognosis. CONCLUSIONS: BRAF mutation is a marker of poor prognosis in patients with appendiceal and colorectal peritoneal metastases scheduled for CRS and HIPEC, whereas survival outcome in subjects with mutated KRAS does not differ from wild-type KRAS. This finding suggests that those with BRAF mutation should be considered for alternative treatment options.

Gains of Chromosome 1p and 15q are Associated with Poor Survival After Cytoreductive Surgery and HIPEC for Treating Colorectal Peritoneal Metastases.

PURPOSE: Genetic alterations in colorectal peritoneal metastases (PM) are largely unknown. This study was designed to analyze whole-genome copy number alterations (CNA) in colorectal PM and to identify alterations associated with prognosis after cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). METHODS: All patients with PM, originating from a colorectal adenocarcinoma, who were treated with CRS and HIPEC in Uppsala Sweden, between 2004 and 2015, were included (n = 114). DNA derived from formalin-fixed paraffin-embedded (FFPE) specimens were analyzed for CNA using molecular inversion probe arrays. RESULTS: There were extensive but varying degrees of CNA, ranging from minimal CNA to total aneuploidy. In particular, gain of parts of chromosome 1p and major parts of 15q were associated with poor survival. A combination of gains of 1p and 15q was associated with poor survival, also after adjustment for differences in peritoneal cancer index and completeness of cytoreduction score [hazard ratio (HR) 5.96; 95% confidence interval (CI) 2.19-16.18]. These patients had a mean copy number (CN) of 3.19 compared with 2.24 in patients without gains. Complete CN analysis was performed in 53 patients. Analysis was unsuccessful for the remaining patients due to insufficient amounts of DNA and signals caused by interstitial components and normal cells. There was no difference in survival between patients with successful and unsuccessful CN analysis. CONCLUSIONS: This study shows that gains of parts of chromosome 1p and of major parts of chromosome 15q were significantly associated with poor survival after CRS and HIPEC, which could represent future prognostic biomarkers.

A Preliminary Report: Radical Surgery and Stem Cell Transplantation for the Treatment of Patients With Pancreatic Cancer.

We examined the immunologic effects of allogeneic hematopoietic stem cell transplantation (HSCT) in the treatment of pancreatic ductal adenocarcinoma, a deadly disease with a median survival of 24 months for resected tumors and a 5-year survival rate of 6%. After adjuvant chemotherapy, 2 patients with resected pancreatic ductal adenocarcinoma underwent HSCT with HLA-identical sibling donors. Comparable patients who underwent radical surgery, but did not have a donor, served as controls (n=6). Both patients developed humoral and cellular (ie, HLA-A*01:01-restricted) immune responses directed against 2 novel tumor-associated antigens (TAAs), INO80E and UCLH3 after HSCT. Both TAAs were highly expressed in the original tumor tissue suggesting that HSCT promoted a clinically relevant, long-lasting cellular immune response. In contrast to untreated controls, who succumbed to progressive disease, both patients are tumor-free 9 years after diagnosis. Radical surgery combined with HSCT may cure pancreatic adenocarcinoma and change the cellular immune repertoire capable of responding to clinically and biologically relevant TAAs.

Intracellular localization of amyloid-ß peptide in SH-SY5Y neuroblastoma cells.

Amyloid-ß peptide (Aß), the main component of Alzheimer's disease (AD) senile plaques, has been found to accumulate within the lysosomal compartment of AD neurons. We have previously shown that in differentiated SH-SY5Y neuroblastoma cells cultured under normal conditions, the majority of Aß is localized extralysosomally, while oxidative stress significantly increases intralysosomal Aß content through activation of macroautophagy. It is, however, not clear which cellular compartments contain extralysosomal Aß in intact SH-SY5Y cells, and how oxidative stress influences the distribution of extralysosomal Aß. Using confocal laser scanning microscopy and immunoelectron microscopy, we showed that in differentiated neuroblastoma cells cultured under normal conditions Aß (Aß40, Aß42, and Aß oligomers) is colocalized with both membrane-bound organelles (endoplasmic reticulum, Golgi complexes, multivesicular bodies/late endosomes, lysosomes, exocytotic vesicles and mitochondria) and non-membrane-bound cytosolic structures. Neuroblastoma cells stably transfected with AßPP Swedish KM670/671NL double mutation showed enlarged amount of Aß colocalized with membrane compartments. Suppression of exocytosis by 5 nM tetanus toxin resulted in a significant increase of the amount of cytosolic Aß as well as Aß colocalized with exocytotic vesicles, endoplasmic reticulum, Golgi complexes, and lysosomes. Hyperoxia increased Aß localization in the endoplasmic reticulum, Golgi apparatus, mitochondria, and lysosomes, but not in the secretory vesicles. These results indicate that in SH-SY5Y neuroblastoma cells intracellular Aß is not preferentially localized to any particular organelle and, to a large extent, is secreted from the cells. Challenging cells to hyperoxia, exocytosis inhibition, or Aß overproduction increased intracellular Aß levels but did not dramatically changed its localization pattern.

Lysosomal iron, iron chelation, and cell death.

SIGNIFICANCE: Lysosomes are acidic organelles containing more than fifty hydrolases that provide for the degradation of intracellular and endocytosed materials by autophagy and heterophagy, respectively. They digest a variety of macromolecules, as well as all organelles, and their integrity is crucial. As a result of the degradation of iron-containing macromolecules (e.g., ferritin and mitochondrial components) or endocytosed erythrocytes (by macrophages), lysosomes can accumulate large amounts of iron. This iron occurs often as Fe(II) due to the acidic and reducing lysosomal environment. Fe(II) is known to catalyze Fenton reactions, yielding extremely reactive hydroxyl radicals that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Lysosomes play key roles not only in apoptosis and necrosis but also in neurodegeneration, aging, and atherosclerosis. RECENT ADVANCES: The damaging effect of intralysosomal iron can be hampered by endogenous or exogenous iron chelators that enter the lysosomal compartment by membrane permeation, endocytosis, or autophagy. CRITICAL ISSUES: Cellular sensitivity to oxidative stress is enhanced by lysosomal redox-active iron or by lysosomal-targeted copper chelators binding copper (from degradation of copper-containing macromolecules) in redox-active complexes. Probably due to higher copper levels, lysosomes of malignant cells may be specifically sensitized by such chelators. FUTURE DIRECTIONS: By increasing lysosomal redox-active iron or exposing cells to lysosomal-targeted copper chelators, it should be possible to enhance the sensitivity of cancer cells to radiation-induced oxidative stress or treatment with cytostatics that induce such stress.

Intracellular distribution of amyloid beta peptide and its relationship to the lysosomal system.

BACKGROUND: Amyloid beta peptide (Aß) is the main component of extraneuronal senile plaques typical of Alzheimer's disease (AD) brains. Although Aß is produced by normal neurons, it is shown to accumulate in large amounts within neuronal lysosomes in AD. We have recently shown that under normal conditions the majority of Aß is localized extralysosomally, while oxidative stress significantly increases intralysosomal Aß content through activation of macroautophagy. It is also suggested that impaired Aß secretion and resulting intraneuronal increase of Aß can contribute to AD pathology. However, it is not clear how Aß is distributed inside normal neurons, and how this distribution is effected when Aß secretion is inhibited. METHODS: Using retinoic acid differentiated neuroblastoma cells and neonatal rat cortical neurons, we studied intracellular distribution of Aß by double immunofluorescence microscopy for Aß40 or Aß42 and different organelle markers. In addition, we analysed the effect of tetanus toxin-induced exocytosis inhibition on the intracellular distribution of Aß. RESULTS: Under normal conditions, Aß was found in the small cytoplasmic granules in both neurites and perikarya. Only minor portion of Aß was colocalized with trans-Golgi network, Golgi-derived vesicles, early and late endosomes, lysosomes, and synaptic vesicles, while the majority of Aß granules were not colocalized with any of these structures. Furthermore, treatment of cells with tetanus toxin significantly increased the amount of intracellular Aß in both perikarya and neurites. Finally, we found that tetanus toxin increased the levels of intralysosomal Aß although the majority of Aß still remained extralysosomally. CONCLUSION: Our results indicate that most Aß is not localized to Golgi-related structures, endosomes, lysosomes secretory vesicles or other organelles, while the suppression of Aß secretion increases intracellular intra- and extralysosomal Aß.

Macroautophagy-generated increase of lysosomal amyloid ß-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells.

Increasing evidence suggests the toxicity of intracellular amyloid ß-protein (Aß) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aß within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aß monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aß production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aß resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aß accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondrial-lysosomal axis theory of aging.

It is now generally accepted that aging and eventual death of multicellular organisms is to a large extent related to macromolecular damage by mitochondrially produced reactive oxygen species, mostly affecting long-lived postmitotic cells, such as neurons and cardiac myocytes. These cells are rarely or not at all replaced during life and can be as old as the whole organism. The inherent inability of autophagy and other cellular-degradation mechanisms to remove damaged structures completely results in the progressive accumulation of garbage, including cytosolic protein aggregates, defective mitochondria, and lipofuscin, an intralysosomal indigestible material. In this review, we stress the importance of crosstalk between mitochondria and lysosomes in aging. The slow accumulation of lipofuscin within lysosomes seems to depress autophagy, resulting in reduced turnover of effective mitochondria. The latter not only are functionally deficient but also produce increased amounts of reactive oxygen species, prompting lipofuscinogenesis. Moreover, defective and enlarged mitochondria are poorly autophagocytosed and constitute a growing population of badly functioning organelles that do not fuse and exchange their contents with normal mitochondria. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and collapse of the cellular catabolic machinery, which eventually is incompatible with survival.

Oxidative stress induces macroautophagy of amyloid beta-protein and ensuing apoptosis.

There is increasing evidence for the toxicity of intracellular amyloid beta-protein (Abeta) to neurons and the involvement of lysosomes in this process in Alzheimer disease (AD). We have recently shown that oxidative stress, a recognized determinant of AD, enhances macroautophagy and leads to intralysosomal accumulation of Abeta in cultured neuroblastoma cells. We hypothesized that oxidative stress promotes AD by stimulating macroautophagy of Abeta that further may induce cell death by destabilizing lysosomal membranes. To investigate such possibility, we compared the effects of hyperoxia (40% ambient oxygen) in cultured HEK293 cells that were transfected with an empty vector (Vector), wild-type APP (APPwt), or Swedish mutant APP (APPswe). Exposure to hyperoxia for 5 days increased the number of cells with Abeta-containing lysosomes, as well as the number of apoptotic cells, compared to normoxic conditions. The rate of apoptosis in all three cell lines demonstrated dependence on intralysosomal Abeta content (Vector

Lysosomes and oxidative stress in aging and apoptosis.

The lysosomal compartment consists of numerous acidic vesicles (pH approximately 4-5) that constantly fuse and divide. It receives a large number of hydrolases from the trans-Golgi network, while their substrates arrive from both the cell's outside (heterophagy) and inside (autophagy). Many macromolecules under degradation inside lysosomes contain iron that, when released in labile form, makes lysosomes sensitive to oxidative stress. The magnitude of generated lysosomal destabilization determines if reparative autophagy, apoptosis, or necrosis will follow. Apart from being an essential turnover process, autophagy is also a mechanism for cells to repair inflicted damage, and to survive temporary starvation. The inevitable diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow oxidative formation of lipofuscin in long-lived postmitotic cells, where it finally occupies a substantial part of the volume of the lysosomal compartment. This seems to result in a misdirection of lysosomal enzymes away from autophagosomes, resulting in depressed autophagy and the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. This scenario might put aging into the category of autophagy disorders.

Lysosomes in iron metabolism, ageing and apoptosis.

The lysosomal compartment is essential for a variety of cellular functions, including the normal turnover of most long-lived proteins and all organelles. The compartment consists of numerous acidic vesicles (pH approximately 4 to 5) that constantly fuse and divide. It receives a large number of hydrolases ( approximately 50) from the trans-Golgi network, and substrates from both the cells' outside (heterophagy) and inside (autophagy). Many macromolecules contain iron that gives rise to an iron-rich environment in lysosomes that recently have degraded such macromolecules. Iron-rich lysosomes are sensitive to oxidative stress, while 'resting' lysosomes, which have not recently participated in autophagic events, are not. The magnitude of oxidative stress determines the degree of lysosomal destabilization and, consequently, whether arrested growth, reparative autophagy, apoptosis, or necrosis will follow. Heterophagy is the first step in the process by which immunocompetent cells modify antigens and produce antibodies, while exocytosis of lysosomal enzymes may promote tumor invasion, angiogenesis, and metastasis. Apart from being an essential turnover process, autophagy is also a mechanism by which cells will be able to sustain temporary starvation and rid themselves of intracellular organisms that have invaded, although some pathogens have evolved mechanisms to prevent their destruction. Mutated lysosomal enzymes are the underlying cause of a number of lysosomal storage diseases involving the accumulation of materials that would be the substrate for the corresponding hydrolases, were they not defective. The normal, low-level diffusion of hydrogen peroxide into iron-rich lysosomes causes the slow formation of lipofuscin in long-lived postmitotic cells, where it occupies a substantial part of the lysosomal compartment at the end of the life span. This seems to result in the diversion of newly produced lysosomal enzymes away from autophagosomes, leading to the accumulation of malfunctioning mitochondria and proteins with consequent cellular dysfunction. If autophagy were a perfect turnover process, postmitotic ageing and several age-related neurodegenerative diseases would, perhaps, not take place.

Giant mitochondria do not fuse and exchange their contents with normal mitochondria.

Giant mitochondria accumulate within aged or diseased postmitotic cells as a consequence of insufficient autophagy, which is normally responsible for mitochondrial degradation. We report that giant mitochondria accumulating in cultured rat myoblasts due to inhibition of autophagy have low inner membrane potential and do not fuse with each other or with normal mitochondria. In addition to the low inner mitochondrial membrane potential in giant mitochondria, the quantity of the OPA1 mitochondrial fusion protein in these mitochondria was low, but the abundance of mitofusin-2 (Mfn2) remained unchanged. The combination of these factors may explain the lack of mitochondrial fusion in giant mitochondria and imply that the dysfunctional giant mitochondria cannot restore their function by fusing and exchanging their contents with fully functional mitochondria. These findings have important implications for understanding the mechanisms of accumulation of age-related mitochondrial damage in postmitotic cells.

The involvement of lysosomes in myocardial aging and disease.

The myocardium is mainly composed of long-lived postmitotic cells with, if there is any at all, a very low rate of replacement through the division and differentiation of stem cells. As a consequence, cardiac myocytes gradually undergo pronounced age-related alterations which, furthermore, occur at a rate that inversely correlates with the longevity of species. Basically, these alterations represent the accumulation of structures that have been damaged by oxidation and that are useless and often harmful. These structures (so-called 'waste' materials), include defective mitochondria, aberrant cytosolic proteins, often in aggregated form, and lipofuscin, which is an intralysosomal undegradable polymeric substance. The accumulation of 'waste' reflects the insufficient capacity for autophagy of the lysosomal compartment, as well as the less than perfect functioning of proteasomes, calpains and other cellular digestive systems. Senescent mitochondria are usually enlarged, show reduced potential over their inner membrane, are deficient in ATP production, and often produce increased amounts of reactive oxygen species. The turnover of damaged cellular structures is hindered by an increased lipofuscin loading of the lysosomal compartment. This particularly restricts the autophagic turnover of enlarged, defective mitochondria, by diverting the flow of lysosomal hydrolases from autophagic vacuoles to lipofuscin-loaded lysosomes where the enzymes are lost, since lipofuscin is not degradable by lysosomal hydrolases. As a consequence, aged lipofuscin-rich cardiac myocytes become overloaded with damaged mitochondria, leading to increased oxidative stress, apoptotic cell death, and the gradual development of heart failure. Defective lysosomal function also underlies myocardial degeneration in various lysosomal storage diseases, while other forms of cardiomyopathies develop due to mitochondrial DNA mutations, resulting in an accumulation of abnormal mitochondria that are not properly eliminated by autophagy. The degradation of iron-saturated ferritin in lysosomes mediates myocardial injury in hemochromatosis, an acquired or hereditary disease associated with iron overload. Lysosomes then become sensitized to oxidative stress by the overload of low mass, redox-active iron that accumulates when iron-saturated ferritin is degraded following autophagy. Lysosomal destabilization is of importance in the induction and/or execution of programmed cell death (either classical apoptotic or autophagic), which is a common manifestation of myocardial aging and a variety of cardiac pathologies.

Autophagy, ageing and apoptosis: the role of oxidative stress and lysosomal iron.

As an outcome of normal autophagic degradation of ferruginous materials, such as ferritin and mitochondrial metalloproteins, the lysosomal compartment is rich in labile iron and, therefore, sensitive to the mild oxidative stress that cells naturally experience because of their constant production of hydrogen peroxide. Diffusion of hydrogen peroxide into the lysosomes results in Fenton-type reactions with the formation of hydroxyl radicals and ensuing peroxidation of lysosomal contents with formation of lipofuscin that amasses in long-lived postmitotic cells. Lipofuscin is a non-degradable polymeric substance that forms at a rate that is inversely related to the average lifespan across species and is built up of aldehyde-linked protein residues. The normal accumulation of lipofuscin in lysosomes seems to reduce autophagic capacity of senescent postmitotic cells--probably because lipofuscin-loaded lysosomes continue to receive newly formed lysosomal enzymes, which results in lack of such enzymes for autophagy. The result is an insufficient and declining rate of autophagic turnover of worn-out and damaged cellular components that consequently accumulate in a way that upsets normal metabolism. In the event of a more substantial oxidative stress, enhanced formation of hydroxyl radicals within lysosomes jeopardizes the membrane stability of particularly iron-rich lysosomes, specifically of autophagolysosomes that have recently participated in the degradation of iron-rich materials. For some time, the rupture of a limited number of lysosomes has been recognized as an early upstream event in many cases of apoptosis, particularly oxidative stress-induced apoptosis, while necrosis results from a major lysosomal break. Consequently, the regulation of the lysosomal content of redox-active iron seems to be essential for the survival of cells both in the short- and the long-term.

Mitochondrial damage and intralysosomal degradation in cellular aging.

Normal mitochondrial respiration is associated with a continuous production of superoxide and hydrogen peroxide, inevitably resulting in minor macromolecular damage. Damaged cellular components are not completely turned over by autophagy and other cellular repair systems, leading to a progressive age-related accumulation of biological "garbage" material, such as defective mitochondria, cytoplasmic protein aggregates and an intralysosomal undegradable material, lipofuscin. These changes primarily affect neurons, cardiac myocytes and other long-lived postmitotic cells that neither dilute this "garbage" by mitotic activity, nor are replaced by newly differentiated cells. Defective mitochondria are insufficient in ATP production and often generate increased amounts of reactive oxygen species, further enhancing oxidative stress. Lipofuscin-loaded lysosomes, in turn, poorly turn over mitochondria that gradually leads to the overload of long-lived postmitotic cells with "garbage" material, decreased adaptability and eventual cell death.

Oxidative stress and Alzheimer disease: the autophagy connection?

Intraneuronal accumulation of amyloid beta-protein (Abeta) is believed to be responsible for degeneration and apoptosis of neurons and consequent senile plaque formation in Alzheimer disease (AD), the main cause of senile dementia. Oxidative stress, an early determinant of AD, has been recently found to induce intralysosomal Abeta accumulation in cultured differentiated neuroblastoma cells through activation of macroautophagy. Because Abeta is known to destabilize lysosomal membranes, potentially resulting in apoptotic cell death, this finding suggests the involvement of oxidative stress-induced macroautophagy in the pathogenesis of AD.

Catabolic insufficiency and aging.

Cellular degradative processes, which include lysosomal (autophagic) and proteasomal degradation, as well as the activity of cytosolic and mitochondrial proteases, provide for a continuous turnover of damaged and obsolete biomolecules and organelles. Inherent insufficiency of these degradative processes results in progressive accumulation within long-lived postmitotic cells of biological "garbage" ("waste" material), such as indigestible protein aggregates, defective mitochondria, and lipofuscin (age pigment), an intralysosomal, polymeric, undegradable material. Intracellular "garbage" is neither completely catabolized, nor exocytosed to any considerable extent. Heavy lipofuscin loading of lysosomes, typical of old age, seems to pronouncedly decrease autophagic potential. As postulated in the mitochondrial-lysosomal axis theory of aging, this occurs on account of the transport of newly synthesized lysosomal enzymes to lipofuscin-loaded lysosomes rather than to active lysosomes/late endosomes, making the enzyme content of autophagolysosomes insufficient for proper degradation. Consequently, the turnover of mitochondria progressively declines, resulting in decreased ATP synthesis and enhanced formation of reactive oxygen species, inducing further mitochondrial damage and additional lipofuscin formation. With advancing age, lipofuscin-loaded lysosomes and defective mitochondria occupy increasingly larger parts of long-lived postmitotic cells, leaving less and less capability for normal turnover and ATP production, finally resulting in cell death.

Oxidative stress induces intralysosomal accumulation of Alzheimer amyloid beta-protein in cultured neuroblastoma cells.

Oxidative stress is considered important for the pathogenesis of Alzheimer's disease (AD), which is characterized by the formation of extracellular senile plaques, mainly composed of amyloid beta-protein (Abeta). Abeta also accumulates within AD neurons and is believed to exert cellular toxicity through lysosomal labilization. We report that the exposure of human neuroblastoma cells to hyperoxia (40% vs. 8% ambient oxygen) induced the accumulation of large (over 1 microM) Abeta-containing lysosomes, which were not typical of control cells, showing a distinct localization of Abeta and lysosomal markers. An inhibitor of autophagy, 3-methyladenine, suppressed the effect of hyperoxia. The results suggest a link between the involvement of oxidative stress and lysosomes in AD.

The lysosomal-mitochondrial axis theory of postmitotic aging and cell death.

Aging (senescence) is characterized by a progressive accumulation of macromolecular damage, supposedly due to a continuous minor oxidative stress associated with mitochondrial respiration. Aging mainly affects long-lived postmitotic cells, such as neurons and cardiac myocytes, which neither divide and dilute damaged structures, nor are replaced by newly differentiated cells. Because of inherent imperfect lysosomal degradation (autophagy) and other self-repair mechanisms, damaged structures (biological "garbage") progressively accumulate within such cells, both extra- and intralysosomally. Defective mitochondria and aggregated proteins are the most typical forms of extralysosomal "garbage", while lipofuscin that forms due to iron-catalyzed oxidation of autophagocytosed or heterophagocytosed material, represents intralysosomal "garbage". Based on findings that autophagy is diminished in lipofuscin-loaded cells and that cellular lipofuscin content positively correlates with oxidative stress and mitochondrial damage, we have proposed the mitochondrial-lysosomal axis theory of aging, according to which mitochondrial turnover progressively declines with age, resulting in decreased ATP production and increased oxidative damage. Due to autophagy of ferruginous material, lysosomes contain a pool of redox-active iron, which makes these organelles particularly susceptible to oxidative damage. Oxidant-mediated destabilization of lysosomal membranes releases hydrolytic enzymes to the cytosol, eventuating in cell death (either apoptotic or necrotic depending on the magnitude of the insult), while chelation of the intralysosomal pool of redox-active iron prevents these effects. In relation to the onset of oxidant-induced apoptosis, but after the initiating lysosomal rupture, cytochrome c is released from mitochondria and caspases are activated. Mitochondrial damage follows the release of lysosomal hydrolases, which may act either directly or indirectly, through activation of phospholipases or pro-apoptotic proteins such as Bid. Additional lysosomal rupture seems to be a consequence of a transient oxidative stress of mitochondrial origin that follows the attack by lysosomal hydrolases and/or phospholipases, creating an amplifying loop system.

Oxidative stress, accumulation of biological 'garbage', and aging.

Normal metabolism is associated with unavoidable mild oxidative stress resulting in biomolecular damage that cannot be totally repaired or removed by cellular degradative systems, including lysosomes, proteasomes, and cytosolic and mitochondrial proteases. Consequently, irreversibly damaged and functionally defective structures (biological 'garbage') accumulate within long-lived postmitotic cells, such as cardiac myocytes and neurons, leading to progressive loss of adaptability and increased probability of death and characterizing a process called aging, or senescence. Intralysosomal 'garbage' is represented by lipofuscin (age pigment), an undegradable autophagocytosed material, while extralysosomal 'garbage' involves oxidatively modified cytosolic proteins, altered biomembranes, defective mitochondria and other organelles. In aged postmitotic cells, heavily lipofuscin-loaded lysosomes perform poorly, resulting in the enhanced accumulation of defective mitochondria, which in turn produce more reactive oxygen species causing additional damage (the mitochondrial-lysosomal axis theory). Potential anti-aging strategies may involve not only overall reduction of oxidative stress, but also the use of intralysosomal iron chelators hampering Fenton-type chemistry as well as the stimulation of cellular degradative systems.