CELLULAR SENESCENE RAVAL NIRAVSINH, ANANYA TRIPATHI , MS.VIDISHA JANI ABSTRACT:- The term cellular senescence was introduced more than five decades ago to describe the state of growth arrest observed in aging cells. Since this initial discovery, the phenotypes associated with cellular senescence have expanded beyond growth arrest to include alterations in cellular metabolism, secreted cytokines, epigenetic regulation and protein expression. Recently, senescence has been shown to play an important role in vivo not only in relation to aging, but also during embryonic development. Thus, cellular senescence serves different purposes and comprises a wide range of distinct phenotypes across multiple cell types. Whether all cell types, including post-mitotic neurons, are capable of entering into a senescent state remains unclear. In this review we examine recent data that suggest that cellular senescence plays a role in brain aging and, notably, may not be limited to glia but also neurons. We suggest that there is a high level of similarity between some of the pathological changes that occur in the brain in Alzheimer’s and Parkinson’s diseases and those phenotypes observed in cellular senescence, leading us to propose that neurons and glia can exhibit hallmarks of senescence previously documented in peripheral tissues. 1.WHAT IS SENESCENE ? Cellular senescence was originally identified as a stable exit from the cell cycle caused by the finite proliferative capacity of cultured human fibroblasts . Currently, senescence is considered a stress response that can be induced by a wide range of intrinsic and extrinsic insults, including oncogenic activation, oxidative and genotoxic stress, mitochondrial dysfunction, irradiation, or chemotherapeutic agents . While the defining characteristic of senescence is the estab_lishment of a stable growth arrest that limits the replication of damaged and old cells, many other phenotypic alterations associ_ated with the senescent program are relevant to understanding the pathophysiological functions of senescent cells . For example, senescent cells undergo morphology changes, chromatin remod_eling, and metabolic reprogramming, and secrete a complex mix of mostly proinflammatory factors termed the senescence-associated secretory phenotype (SASP) Here, we review the molecular mechanisms controlling cellular senescence with a special focus on their translational relevance and suitability for identifying and characterizing senescent cells in vivo. 2. PHENOTYPIC CHARECTERISTICS OF SENESCENE Figure 1. Phenotypic characteristics of senescent cells. Diagram depicting some of the phenotypic alterations associated with senescence initiation, early senescence, and late phases of senescence. 3. MORPHOLOGICAL CHANGES In cell culture, senescence is normally accompanied by significant morphological changes. Senescent cells become flat, enlarged, and vacuolized, and sometimes appear with multiple or enlarged nuclei. Changes in shape rely on the status of the scaffolding protein caveolin 1 and the Rho GTPases Rac1 and CDC42 , and vacuolation has been associated with ER stress caused by the unfolded protein response . Senescent cells also form cytoplasmic bridges that allow them to signal to neighboring cells via direct intercellular protein transfer . Beyond these examples, the functional significance of most morphological changes asso_ciated with senescence is unclear. In vivo, senescent cells appear to preserve the morphology dictated by the architecture of the tissue. However, recent studies have discovered that SA β-gal+ cells in aged mice increase in size . 4. A SIMPLIFIED MODEL Senescence has been traditionally considered as a defined, stat_ic cell fate. However, it is now recognized that senescence is a dynamic multistep process. suggests that although the initial senescence-inducing signals are sufficient to initiate cell cycle exit, this merely constitutes an early step in the senescence process. Senescent cells progressively remodel their chromatin and start to sequentially implement other aspects of the senescence program, such as the SASP, to enter into a second step of “full senescence.” If these senescent cells persist for extended periods of time, they continue evolving and can be categorized as entering into a third step of “late senescence,” which can involve adaptation and diversification of the senescent phenotype. It is tempting to suggest that the concept of senescence progression may help account for the heterogeneity of senescent cells and their associated phenotypes in vivo. Indeed, the senescent responses occurring in vivo can be categorized into two types. Acute senescence seems to be a programmed process that is triggered in response to discrete stressors, is established with fast kinetics, and normally contributes to tissue homeostasis. In contrast, chronic senescence may result from long-term unscheduled damage, and it is often associated with detrimental processes such as aging. 4.1.Cell cycle arrest One of the defining features of senescent cells is their stable cell cycle arrest. This cell cycle exit is controlled by activation of the p53/p21CIP1 and p16INK4a/Rb tumor suppressor pathways (Figure 2). Unlike quiescent cells, senescent cells are nonresponsive to mito_genic or growth factor stimuli; thus, they are unable to reenter the cell cycle even in advantageous growth conditions. Figure 2. Molecular pathways controlling growth arrest during senescence. A variety of stressors induce senescence-associated growth arrest. Cell cycle exit is regulated by induc_tion of the p16INK4a/Rb and p53/p21CIP1 pathways. Figure reproduced with permission from McHugh and Gil (126). 4.2.DNA Damage response The senescence growth arrest is often triggered by a persistent DNA damage response (DDR) caused by either intrinsic (oxida_tive damage, telomere attrition, hyperproliferation) or external insults (ultraviolet, γ-irradiation, chemotherapeutic drugs) . 4.3.Non–cell-autonomous effects of senescence Cellular senescence was initially considered to be a cell-intrinsic program. Increasing evidence, however, has shown that senescent cells have the ability to signal and influence their surrounding environment. Senescent cells produce a complex mixture of soluble and insoluble factors that are collectively termed senescence-associated secretory phenotype (SASP) or senescence-messaging secretome . SASP is the general term given to the combination of cytokines, che_mokines, extracellular matrix proteases, growth factors, and other signaling molecules secreted by senescent cells. 4.4. SA β-galactosidase activity The most widely used senescence marker is senescence-associ_ated β-galactosidase (SA β-gal) activity. This enzymatic activity, which is found in many normal cells under physiological conditions (pH 4.0–4.5), is significantly amplified in senescent cells as a result of increased lysosomal content . 4.5.Chromatin reorganization Senescence is associated with large-scale chromatin rearrangements . Besides the already described DDR and the formation of PML bodies (a type of matrix-associated nuclear domain) , the most striking chromatin change observed in senescent cells is the formation of senescence-associated hetero_chromatic foci (SAHFs), which are more prominent in human cells undergoing OIS . These foci can be identified by DAPI staining and are characterized by enrichment of repressive marks such trimethylated H3K9 and heterochromatic protein 1 (HP1), accumulation of high-mobility group HMGA proteins, and loss of linker histone H1 . 4.6.Resistance to apoptosis Senescence and apoptosis are alternative cell fates that often can be triggered by the same stressors. While we do not have a full understanding of what makes the cell decide between one and another program, mechanisms must be in place to lock those decisions. In this regard, senescent cells are resistant to extrinsic and intrinsic apoptosis . Recent studies have suggested that this is a result of the upregulation of BCL-2 family proteins such as BCL-W and BCL-XL . This is of extraordinary practical rel_evance since inhibiting BCL-2 family proteins induces apoptosis on senescent cells . 5. MARKERS OF CELLULAR SENESCENE 5.1.Telomere theory Telomere theory depends on three specific principles, the three pillars the Telomere theory stands on: first, that aging is programmed; the second being that irreversible cell cycle arrest happens in response to the telomere shortening; and lastly, that the total number of cell divisions in the absence of telomerase activity cannot exceed a particular limit termed the Hayflick limit . 5.2 Senescence-associated changes in cells isolated from aged subjects Cell factor Changes Cell volume Increased cell volume and surface area in senescence Cell adhesion More focal adhesions present in senescence (ILK activity is associated with the expression of senescence phenotype Lysosome content Dramatic increase in number in senescence SA-b-Gal activity The most widely cited marker of senescence Glycogen granule content High in cells undergoing replicative or induced senescence Mitochondria morphology Increased mitochondria mass in senescence SAHF One of the most prominent cellular traits of senescence TIF A key factor responsible for irreversible growth arrest in senescence HGPS-like nucleus Fraction of cells with the HGPS-like nucleus increases with passage in vitro and the slope is steeper during the passage of fibroblasts from an old individual 6.Future research areas Our review of the literature has revealed that only indirect evidence exists to suggest the possibility of CS in post-mitotic cells such as neurons. We feel that experiments addressing four critical questions will shed light on the contribution of CS to CNS aging and neurodegenerative disease: Does inhibiting cellular senescence in the brain in either neuronal or glial populations attenuate age-related cognitive decline and progression of neurodegeneration? Conversely, does artificially inducing senescence in either neuronal or glial populations produce enhanced cognitive decline and accelerated neurodegeneration? Are the common phenotypes observed in both classical CS and neurodegenerative disease representative of a common mechanism of senescence or simply indicators of cellular stress and dysfunction? What is the occurrence of neuronal senescence in vivo under normal cognitive aging and in neurological disease? Recent developments have now enabled researchers to answer these critical questions. Progress in single cell analysis techniques now enables researchers to examine neuronal populations expressing SA -gal using techniques like single-cell PCR to determine similarities between neuronal senescence and senescence in mitotic populations. A more thorough understanding of the mechanisms involved in CNS senescence will be critical to understanding the contribution of CS to age-related neurodegenerative disease. Such work will contribute to understanding of age-related cognitive decline and could help in the development of novel therapeutic interventions in age-related neurodegenerative disease. 7.CONCLUSIONS It has been accepted that cellular senescence is induced by a number of cellular stresses such as oncogene activation, oxidative stress, and DNA damage in vitro and in vivo through elevated levels of ROS. Unlike programmed differentiation, cellular senescence is likely to be a stochastic event that is induced by a variety of genotoxic stresses. Recently, we developed a real-time in vivo imaging system for visualizing the expression of senescence_related genes, such as p21Waf1/Cip1 in mice . Visualizing the dynamics of cellular senescence responses in vivo in the context of living animals is likely to be a useful tool in the identification of the location and timing of gene expression and hence their likely roles in cellular senescence in vivo. Fig. 4. Real-time in vivo imaging of p21Waf1/Cip1 gene expression after doxorubicin (DXR) treatment. We established a transgenic mouse line (p21-p-luc) expressing firefly luciferase under control of the p21Waf1/Cip1 gene promoter. The 8-week-old p21-p-luc mouse was injected intraperitoneally with DXR (20 mg/kg) and was subjected to non-invasive bioluminescene imaging 24 h after DXR treatment under anesthesia. DXR treatment (lower panels) and its control (untreated mice) (upper panels).The color bar indicates photons with minimal and maximal threshold values. 8.REFERENCES - Hwang, E. 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