In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro-inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence-like state in mature postmitotic neurons in vivo. About 40–80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl/6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL-6 production, heterochromatinization and senescence-associated β-galactosidase activity. Frequencies of these senescence-like neurons increased with age. Short-term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late-generation TERC−/− mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late-generation TERC−/−CDKN1A−/− mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence-like phenotype in neurons, as in senescing fibroblasts and other proliferation-competent cells. We conclude that a senescence-like phenotype is possibly not restricted to proliferation-competent cells. Rather, dysfunctional telomeres and/or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence-like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging.

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Biomarker panels for frailty would be of high value and better than single markers. Based on our search we would propose a core panel of frailty biomarkers consisting of (1) CXCL10 (C-X-C motif chemokine ligand 10), IL-6 (interleukin 6), CX3CL1 (C-X3-C motif chemokine ligand 1), (2) GDF15 (growth differentiation factor 15), FNDC5 (fibronectin type III domain containing 5), vimentin (VIM), (3) regucalcin (RGN/SMP30), calreticulin, (4) PLAU (plasminogen activator, urokinase), AGT (angiotensinogen), (5) BDNF (brain derived neurotrophic factor), progranulin (PGRN), (6) α-klotho (KL), FGF23 (fibroblast growth factor 23), FGF21, leptin (LEP), (7) miRNA (micro Ribonucleic acid) panel (to be further defined), AHCY (adenosylhomocysteinase) and KRT18 (keratin 18). An expanded panel would also include (1) pentraxin (PTX3), sVCAM/ICAM (soluble vascular cell adhesion molecule 1/Intercellular adhesion molecule 1), defensin α, (2) APP (amyloid beta precursor protein), LDH (lactate dehydrogenase), (3) S100B (S100 calcium binding protein B), (4) TGFβ (transforming growth factor beta), PAI-1 (plasminogen activator inhibitor 1), TGM2 (transglutaminase 2), (5) sRAGE (soluble receptor for advanced glycosylation end products), HMGB1 (high mobility group box 1), C3/C1Q (complement factor 3/1Q), ST2 (Interleukin 1 receptor like 1), agrin (AGRN), (6) IGF-1 (insulin-like growth factor 1), resistin (RETN), adiponectin (ADIPOQ), ghrelin (GHRL), growth hormone (GH), (7) microparticle panel (to be further defined), GpnmB (glycoprotein nonmetastatic melanoma protein B) and lactoferrin (LTF). We believe that these predicted panels need to be experimentally explored in animal models and frail cohorts in order to ascertain their diagnostic, prognostic and therapeutic potential.

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU's biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H 2 O 2 , respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Replicative senescence in human fibroblasts is accompanied with alterations of various biological processes, including the impaired function of the proteasome. The proteasome is responsible for the removal of both normal and damaged proteins. Due to its latter function, proteasome is also considered a representative secondary antioxidant cellular mechanism. Nrf2 is a basic transcription factor responsible for the regulation of the cellular antioxidant response that has also been shown to regulate several proteasome subunits in mice. We have established in this study the proteasome-related function of Nrf2 in human fibroblasts undergoing replicative senescence. We demonstrate that Nrf2 has a declined function in senescence, whereas its silencing leads to premature senescence. However Normal human fibroblasts undergo a limited number of divisions in culture and progressively reach a state of irreversible growth arrest, a process termed replicative senescence. Senescence has been attributed to a combination of genetic and environmental factors (1). Senescent fibroblasts are viable and fully functional, however, they exhibit several morphologic and biochemical changes as compared with their young/proliferating counterparts (2). We and others (3) have reported loss of proteasome function upon aging of several human tissues as well as in senescent primary cultures. Our previous work in human embryonic fibroblast cultures undergoing replicative senescence has shown that the reduced levels of proteasome activities are accompanied by lower proteasome content due to the down-regulation of its ␤-catalytic subunits (4). The fundamental link between cellular senescence and proteasome function is further supported by our additional studies (5, 6) demonstrating that when the proteasome is partially inhibited in young fibroblasts cultures, an irreversible senescence-like phenotype is triggered. Importantly, reconstitution of the levels of the "down-regulated" ␤-subunits by genetic means in human embryonic fibroblasts, results in enhanced rates of assembled, and functional proteasome and delays senescence by ϳ15% (7).The proteasome is the major cellular non-lysosomal threonine protease, implicated in the removal of normal as well as abnormal, denatured, or otherwise damaged proteins (8). It is a well organized protein complex that contains a proteolytic active 20 S core, composed of 28 subunits, 7 different ␣-and 7 different ␤-subunits, arranged as ␣ 1-7 ␤ 1-7 ␤ 1-7 ␣ 1-7 structure (9). The ␤ 5, ␤ 1 , and ␤ 2 subunits are the catalytic centers of chymotrypsin-like (CT-L), 3 caspase-like/PGPH (peptidylglutamyl-peptide hydrolyzing), and trypsin-like activities, respectively, cleaving peptide bonds after hydrophobic, acidic, and basic residues, respectively (10). The 20 S proteasome is also central to the ATP/ubiquitin-dependent intracellular protein degradation pathway where it represents the proteolytic core of the 26 S complex (11).Although the regulation of human proteasome is not yet entirely elucidated, reports in mice indica...

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The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of ␤-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the ␤ 5 subunit. Stable overexpression of the ␤ 5 subunit in WI38/T and HL60 cells resulted in elevated levels of other ␤-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) ␣-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in ␤ 5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that ␤ 5 -overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the ␤ 5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4 -5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.