Role of RN Autophagy Mediated ERV Suppression in Cellular Senescence

Cellular senescence is a permanent state of cell cycle arrest in response to stress, including DNA damage, telomeric erosion, oncogenic activation, mitochondrial dysfunction and replicative stress [1,2]. Accumulation of senescent cells contribute to decline of the regenerative potential and function of tissues, accelerates aging. Senescence have been implicated in aging as well as in aging related pathologies including cancer, metabolic syndrome, neurological disorders like Alzheimer’s and Parkinson disease [3]. Hence, senescence is viewed as an essential process to eliminate damaged cells by inducing tissue remodeling [3]. Hallmarks of senescent cells include enhanced senescence associated βgal (SA-β gal) activity, expression of senescence markers (p16, p21, p19 and pRB) and secretion of a plethora of factors including cytokines, chemokines, matrix metalloproteinases, popularly known as senescence associated secretory phenotype (SASP) [4]. SASP, aid in clearance of senescent cells through both innate and adaptive immune system, termed as senescence surveillance [5].


Introduction
Cellular senescence is a permanent state of cell cycle arrest in response to stress, including DNA damage, telomeric erosion, oncogenic activation, mitochondrial dysfunction and replicative stress [1,2]. Accumulation of senescent cells contribute to decline of the regenerative potential and function of tissues, accelerates aging. Senescence have been implicated in aging as well as in aging related pathologies including cancer, metabolic syndrome, neurological disorders like Alzheimer's and Parkinson disease [3]. Hence, senescence is viewed as an essential process to eliminate damaged cells by inducing tissue remodeling [3]. Hallmarks of senescent cells include enhanced senescence associated β-gal (SA-β gal) activity, expression of senescence markers (p16, p21, p19 and pRB) and secretion of a plethora of factors including cytokines, chemokines, matrix metalloproteinases, popularly known as senescence associated secretory phenotype (SASP) [4]. SASP, aid in clearance of senescent cells through both innate and adaptive immune system, termed as senescence surveillance [5].
Transposable elements (TEs) comprise ~50% of human genome and often considered as detrimental, because of their inherent mobile nature [6]. Endogenous retrovirus (ERV) are specialized transposable elements that constitute 8% of human genome and have the ability to transpose through DNA or RNA intermediates [7,8]. Though majority of ERVs have lost their ability to actively move across the genome, some may still impact their host by modulating nearby genes. Several human diseases have been linked to ERVs, including cancer, amyotropic lateral sclerosis (ALS), multiple sclerosis (MS) and schizophrenia [9].

Insights in Biomedicine ISSN 2572-5610
ERV suppression as a potential mechanism in modulating immunogenicity regarding AML development [6].
Current study aimed in identifying the role of ERVs in prolonged senescence. Interestingly, results indicate that during prolonged senescence, ERVs are suppressed via specialized autophagy, RNautophagy. Hence, suggesting ERV suppression as a prospective mechanism to evade immune surveillance.

Case Study
All animal procedures were approved by the Institutional Animal Care and Use Committee at New York University School of Medicine. All mice studied were male, had free access to water and were subjected to 12 h light/dark cycles. WT C57BL/6 male that were 2 months old, were used for the study. For primary skin fibroblasts, 2 mm ear lobe tissues were minced in DMEM buffer containing Type III Collagenase overnight and passed through 50µm filter to obtain single cell suspension. The attached cells were then cultured and passaged in DMEM supplemented with 10% fetal bovine serum (FBS). HCT116 and IMR-90 cells were purchased from ATCC and maintained in McCoy5A and EMEM medium, supplemented with 10% FBS. Media and FBS were purchased from GIBCO.
Mitomycin C, was purchased from SigmaAldrich. Cells were treated with and without 10 µM MMC for 4hrs, washed and incubated in fresh media. Cells were analyzed 4 or 12 days post treatment.
RNA was isolated using Qiagen RNAesy Kit (Qiagen). cDNA was synthesized using superscript cDNA synthesis kit (Invitrogen). qPCR performed using FastSybr green mastermix (Applied biosystems) and were normalized against β-actin. Table 1 lists the primer sequences used.
Data were analyzed using Graphpad Prism software. All data are expressed as mean± standard error mean (SEM). Results were analyzed using two-tailed unpaired t-test. P values <0.05 were considered significant.

Results and Discussion
Primary dermal fibroblasts, isolated from 2 months old WT C57BL/6 mice were used in this study. Primary mice fibroblasts were treated with either vehicle or mitomycin C (MMC), to induce senescence [17]. Cells were analyzed 4 and 12 days post treatment. qPCR for senescence markers (p21 and p16), revealed increased expression of both the markers studied ( Figure 1A). Results indicate the induction of senescence upon MMC treatment. Further, ERVs (Merv-k, MusD, MuLV and Linc9) were examined via qPCR in mock vs senescent fibroblasts. As can be seen in (Figure 1B), ERVs were upregulated in 4 days post senescence (Sen 4 d), which then were then downregulated in 12 days post senescence (sen 12 d).
To verify the results, the same experiment was repeated in two different human cell lines-IMR-90 (human diploid fibroblasts) and HCT116 (colorectal cancer cell line). Both the cell lines were induced senescence using MMC and senescence as well as ERV expression were analyzed via qPCR. The results consistently showed ERV downregulation at 12 days post senescence, senescent post 12 days, compared to 4 days post senescence, despite no decrease in the senescence markers, p16, p21 (Figures  2A-2D).
To further examine, whether autophagy is responsible for ERV suppression, cells were treated with chloroquine (CQ), an autophagy inhibitor 12 d post treatment. When compared to vehicle treated post senescent 12-day fibroblasts, CQ treatment drastically enhances the ERV expression ( Figure 3B). This data clearly demonstrates the role of autophagy in ERV suppression.
Senescence is viewed as an essential process for tissue remodeling. While acute senescence aids in wound healing and tissue repair, under chronic settings, senescence contributes to functional decline and aging [3]. Senescent cells, through ERVs and SASP, recruit immune cells, to eliminate them. However, prolonged senescence is the result of evasion of surveillance. This study demonstrates ERV suppression via RNautophagy, as an escape mechanism for immune surveillance.
Increased ERV expression can lead to genomic instability, resulting in cancer development and cancer occurs when cells bypass senescence [8]. However, our results suggest ERV suppression aids in evading surveillance, resulting in senescent cells accumulation, which seems to be detrimental. Nonetheless, just like senescence, ERV expression under acute settings, assists in clearing damaged cells, while chronic expression leads to genomic instability and cancer. Like most of the biological phenomenon, senescence and ERV regulation seems to be a 'double edged' sword.
Autophagy is a catabolic process, where macromolecules are degraded by lysosomes. Autophagy occurs at basal level in normal condition but is accelerated during stress and is essential for biological homeostasis. Apart from the macroautophagy, microautophagy and chaperone mediated autophagy, RNA/ DNAutophagy forms the fourth type, where DNA and RNA are degraded by lysosomes in ATP dependent manner [18,20]. Loss of autophagy has been shown to cause premature aging in many species [21,22]. Treatment with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis has shown to extend life span in worms and lessen the metabolic syndrome in mice [23].

Conclusion
Several evidences are in favor of autophagy activation to extend longevity. Autophagy activation have been shown to induce senescence [24]. Our results show, that autophagy suppression might help the senescent cells to escape surveillance and contribute to aging. Nevertheless, it is the type of autophagy cargo, which determines the outcome in terms of longevity and aging. This study is in lieu with the observations in AML patients, where ERV regulation correlates with Lamp2, RNautophagy marker [6]. Senolytic drugs under development targets these surveillance resistant senescent cells. Hence, mechanism mediating evasion from senescence surveillance is essential for designing better senolytic drugs and this study provides a possible direction.