SIRT6 regulates protein synthesis and folding through nucleolar remodeling

SIRT6 regulates protein synthesis and folding through nucleolar remodeling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SIRT6 regulates protein synthesis and folding through nucleolar remodeling Debra Toiber, Daniel Stein, Miguel Portillo, Shai Kaluski- Kopatch, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4215918/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract An important hallmark of aging – and particularly of neurodegeneration – is the loss of proteostasis, which often leads to cellular stress responses and even cell death. However, the causal mechanisms driving proteostasis are unclear. Here, we show that SIRT6 has a critical role in maintaining proteostasis. It negatively regulates global translation by controlling ribosomal genes, nucleolar function and TIP5 chromatin localization. SIRT6 deletion dramatically increases nucleolar size, rRNA production and protein translation. However, the expression of protein-folding genes remains unchanged, failing to compensate for excessive translation, hence leading to reduced protein folding capacity and the production of aggregates. In vivo , we establish a C. elegans model ( sir-2.4 KO) that shows reduced heat shock resistance and an accelerated age-dependent reduction in motility. Sir-2.4 depletion in a neuron-specific protein aggregation-prone polyQ strain led to premature motility loss indicative of motor neuron dysfunction. These results point to proteostasis-stress intolerance in the absence of the SIRT6 ortholog that can be rescued by pharmacologically reducing protein translation rates. Together, our data suggest that SIRT6 deficiency in aging and neurodegeneration contributes to proteostasis loss through gene dysregulation of nucleolar function and the translation machinery. These results highlight that deficient proteostasis is the consequence of chromatin dysregulation that ultimately leads to neurodegeneration. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Molecular biology/Nuclear organization Biological sciences/Molecular biology/Protein folding/Protein aggregation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Aging is considered to be a major risk factor for developing a neurodegenerative disease, where about 95% of the cases are sporadic and age-related. Therefore, understanding the molecular processes that occur during human aging is critical to prevent and treat age-related neurodegenerative diseases. Neurodegeneration is one of the most predominant age-related pathologies, with chances of developing a neurodegenerative condition doubling every 5 years after the age of 65 1 . For more than 30 years the focus in neurodegeneration research has majorly been on the formation of toxic protein aggregates. However, this seems to be a late event in the development of the pathology, and therapies targeting aggregates have failed to improve patients’ cognitive capacity and to prevent memory loss 2 . On the other hand, DNA damage accumulation and epigenetic changes are the key drivers of aging 3 , 4 , yet their implications on the loss of proteostasis are elusive. In this work, we use SIRT6 as a model of accelerated aging, particularly in the brain, and observe how its loss results in nucleolar misfunction that results in the loss of proteostasis. SIRT6 is a nuclear deacetylase and mono-ADP ribosyltransferase, which plays a critical role in many cellular processes. It represses gene expression through histone deacetylation 5 – 7 and is involved in the DNA damage response, preventing genomic instability 8 – 13 . SIRT6 is essential especially in mammals: SIRT6 knock-out (KO) mice show postnatal lethality and genomic instability 8 , 14 – 16 . In monkeys, SIRT6 deletion leads to prenatal developmental retardation, delayed neuronal differentiation and to death a few hours postnatally, pointing to a crucial role of SIRT6 in the brain of primates 17 . In contrast, SIRT6-overexpressing mice show increased physical activity and extended lifespan 18 , 19 . Together, these data suggest that SIRT6 is essential, and its roles are tightly linked to the aging process. Interestingly, brain-specific SIRT6-deficient mice (brSIRT6KO) survive to adulthood but present behavioral defects with major hippocampus-dependent learning impairments by 4 months of age 20 . Moreover, the brains of these mice show increased signs of DNA damage, cell death, and hyperphosphorylated and acetylated Tau – critical marks in many neurodegenerative diseases 20 , 21 . Thus, brSIRT6KO phenotypes resemble animal models of Alzheimer’s Disease and pathological characteristics of brains from patients suffering from age-related neurodegeneration 20 . Indeed, SIRT6 levels and activity decrease in aging brains and furthermore in Alzheimer’s Disease patients 20 , 22 – 25 . Although neurodegenerative diseases have each a different phenotype, there are many commonalities that we also found when comparing them with SIRT6-deficient models 20 , 26 . For example, we discovered an additional link between SIRT6 and Alzheimer’s, Parkinson’s, Huntington diseases and amyotrophic lateral sclerosis (AD, PD, HD and ALS, respectively) through mitochondrial dysfunction 27 . The SIRT6-neurodegeneration link is especially intriguing because another common trait in many age-related brain pathologies is the extreme form of proteostasis loss, manifested in aggregation of specific proteins – suggesting a role for SIRT6 in proteostasis. Among these conditions are AD 28 (with Tau and Amyloid-beta), PD 29 , 30 (with alpha-synuclein, parkin and Tau) and HD 31 , 32 (with Huntingtin). While the protein aggregation in AD and PD seems to be a downstream product of a covert age-dependent malfunction, Huntingtin aggregation in HD is clearly the driver of the disease 2 . Still, HD develops only as the person gets older, and not during development or early adulthood – implying that as the person ages, the capacity to cope with the polyglutamine-driven aggregation is lost. Proteostasis includes several layers of regulation ranging from ribosome formation to protein folding and degradation. Translation is done by the ribosomes, which are composed of specialized RNA and protein subunits. While the ribosomal proteins are critical for the regulation of mRNA preference by the ribosome, most of the ribosomal structure – the ribosomal RNA (rRNA) – is expressed and pre-formed in the nucleolus: a sub-nuclear membrane-less compartment, separated by liquid-liquid phase separation 33 . The nucleolus contains the ribosomal DNA (rDNA) that is transcribed to the 45S pre-rRNA, which is later processed and assembled – together with ribosomal proteins – into pre-ribosomal subunits. Importantly, nucleolar physiology is impaired in aging and progerias, with increased nucleolar size and number 34 . Furthermore, nucleolar size negatively correlates with longevity and its premature enlargement can be seen in progeroid syndromes 34 , 35 . All these age-related changes in the nucleolus might lead to altered ribosomal content and translation capacity downstream 34 . One of the main regulators of the pre-rRNA transcription is the Nucleolar Remodeling Complex (NoRC), which one of its components is SNF2H – a chromatin remodeler that was previously found to be recruited by SIRT6 to the chromatin at DNA double strand break sites 9 . Here, we reveal the critical role of SIRT6 in maintaining proteostasis from ribosomal production to protein folding. We show that SIRT6 regulates proteostasis through regulation of nuclear and nucleolar genes. SIRT6 negatively regulates translation through the nucleolus: knocking out SIRT6 resulted in enlarged nucleoli; elevated rRNA production; and increased amino acid production and transport to cope with the demand. While this resulted in elevated translation rate, the molecular chaperone family of proteins did not change, leading to reduced protein folding, leaving the cells vulnerable to aggregate formation. Furthermore, we developed a SIRT6-ortholog KO model in C. elegans to show that the role in translation regulation and proteostasis is conserved. We show that the polyQ-mediated motor neuron dysfunction is exacerbated in sir-2.4 KO animals and could be reverted by the FDA-approved weak protein translation inhibitor and chemical chaperone 4-phenylbutyric acid (4PBA) 36 . Collectively, these findings establish SIRT6 as critical regulator of proteostasis through nucleolar activity and emphasize its relevance in aging and neurodegenerative processes. Results SIRT6 deficiency affects pathways of neurodegeneration, with common association to ribosomal genes. We first analyzed brain transcriptomics of brSIRT6KO compared to age-matched control mice. Strikingly, we found 18 significantly changing categories in KEGG enrichment analysis, out of which 6 categories belong to neurodegenerative pathologies. Interestingly, however, the most significant category was the Ribosome (ribosomal proteins) (Fig. 1A). Next, to further elucidate the ribosome-neurodegeneration connection in brSIRT6KO mice, we used Gene Ontology (GO) analysis. We found 431 significantly upregulated and 68 significantly downregulated categories. Out of these categories, 79 upregulated and 14 downregulated categories are ribosome-, proteostasis- and brain-related (Fig. S1A), including biogenesis and assembly of the ribosome subunits; amino acid import; protein localization; cognition, trans-synaptic signaling and action potential. AD is the most common age-related neurodegenerative disease; since SIRT6 levels decrease in AD 20 , we compared Geneset Enrichment Analysis (GSEA) results of AD patients and brSIRT6KO brains and found a positive correlation between the two groups of genesets (Fig. 1B). Again, the Ribosome (ribosomal proteins) category as well as Alzheimer’s disease and Parkinson’s disease categories appear to be co-regulated in the brains of both AD patients and brSIRT6KO mice (Fig. 1B). Thus, these results emphasize the importance of SIRT6 in both neurodegeneration and ribosomal regulation, as well as the tight correlation between ribosome structure and regulation and pathological brain aging. Lack of SIRT6 results in nucleolar dysregulation and expansion. SIRT6 could be involved in ribosomal changes through its known interaction with SNF2H 9 and the role of the latter in NoRC. This complex is directed to the rDNA by its largest component: TIP5 (also known as BAZ2A). While TIP5 RNA levels are increased in brSIRT6KO mice brains (Fig. 2A), its protein levels and chromatin recruitment are reduced (Fig. 2B, S2A-B). When observing SNF2H – NoRC chromatin remodeling subunit – the brSIRT6KO brains show no significant difference in total protein levels, while its chromatin recruitment has a clear trend of reduction, as expected by previous reports 9 (Fig. S2A-B). Moreover, knocking-out SIRT6 in combination with SNF2H knockdown fully abolished TIP5 from the chromatin in HeLa cells (Fig. 2C), supporting the connection between SIRT6 and NoRC. A reduced NoRC recruitment to chromatin could lead to the lack of repression at rDNA loci and to possibly increase pre-rRNA production. Therefore, we tested nucleolar transcription with 5-fluorouridine labeling and found that SIRT6-deficient cells have elevated rRNA production in the nucleolus (Fig. 2D). These results suggested that SIRT6 regulates the ribosomal genes at the transcription level directly in the nucleolus. Since nucleolar size and function are biomarkers of aging, we hypothesized that reduced NoRC regulation in the nucleolus and increased nucleolar transcription could affect nucleolar size and function. We tested 2 nucleolar markers – Nucleolin and Fibrillarin (NCL and FBL, respectively) – that play a critical role in rRNA post-transcriptional processing inside the nucleolus. In brSIRT6KO mouse brains, we saw a significant increase of the mRNA levels of NCL but not of FBL (Fig. 3A). Next, we tested our cellular models. In SIRT6KO SH-SY5Y, we find increased nucleoli number and area per nucleus, as well as elevated intensity of both nucleolar factors (Fig. 2D, 3B). These results were recapitulated in FBL immunofluorescence and NCL protein blot in HeLa cells as well (Fig. S3A-B), suggesting a different regulation from mRNA and protein levels for FBL. Hence, SIRT6 deficiency not only increases rRNA production, but also its processing in the nucleolus. Moreover, to confirm this happens in post-mitotic neurons and to understand the age-related changes, we isolated nuclei from NeuN-positive cortical cells of adult WT and brSIRT6KO mice (see scheme on Fig. 3C). Neurons from brSIRT6KO mice presented NCL overexpression (Fig. 3D-E). Importantly, the number of nucleoli per nucleus followed a similar trend: brSIRT6KO brains present significantly more nucleoli than their WT counterparts (Fig. 3D, F). Thus, our results in cell lines are also recapitulated in cortical neurons of adult mice. Our combined results from mouse brains and different cell lines support the notion that lack of SIRT6 and aging compromises the nucleolus, by affecting pre-rRNA synthesis and nucleolar function and physiology. This, in turn, plays an important role in the nucleolar expansion and overproduction of rRNA. SIRT6 deficient cells have increased protein synthesis. We suspected that the enhanced production and processing of rRNA could lead to elevated translation. To test this, we directly measured translation using the SUnSET technique 37 . We found an increased protein synthesis rate in SIRT6KO HeLa, SH-SY5Y and HEK293T cells (Fig. 4A-C, S4A). Importantly, this elevation in translation was independent of 4EBP1 phosphorylation (Fig. S4B), in contrast to previous reports 38 . To validate that the SIRT6KO hyper-translation is mediated by nucleolar dysregulation, we repeated the SUnSET assay with TIP5 overexpression, and found that indeed, it partially rescues the elevated translation rates in SIRT6KO HeLa cells (Fig. 4D). However, the rescue effect is relatively mild, probably since the overexpression only partially compensates for the reduced capacity to recruit TIP5 protein (and hence NoRC) to the chromatin. Thus, our data suggests that the above-identified nucleolar dysregulation resulting from SIRT6 deficiency leads to elevated rRNA and protein biosynthesis. To support the increased protein synthesis upon SIRT6 deficiency, the intracellular amino acid content should be elevated. The brSIRT6KO brain RNA-seq data reveals that indeed, there is a significant increase in many amino acid transporters, that together cover all the amino acids (Fig. S4C). Analyzing brSIRT6KO brain metabolomics revealed that out of the top 25 enriched categories, 10 were related to amino acid and protein metabolism (Fig. 4E, marked in red arrows) – pointing on the dramatic metabolic changes that occur in SIRT6-deficient brains. We then co-analyzed the RNA-seq with the metabolomics data to obtain a joint pathway enrichment and found 6 significantly changing pathways (FDR < 0.05), one of them is ‘Aminoacyl-tRNA biosynthesis’ (Fig. 4F, S4D). Analyzing SIRT6KO SHSY5Y cells metabolomics supported the mouse data, as intracellular content was significantly elevated for most detected amino acids (Fig. S4E). We conclude, then, that the hyper-translation in SIRT6 deficiency is accompanied by a metabolic shift in amino acid content that supports the increased protein synthesis rates. SIRT6 deficiency impairs protein refolding. To be functional, proteins must be properly folded. As folding is very delicate, it often requires the aid of a specialized group of proteins – molecular chaperones. While our results show elevated protein synthesis rates, chaperones and folding machinery were lacking in the differentially expressed categories (Fig. 1A, S1A). Therefore, we manually inspected the brSIRT6KO RNA-seq for proteostasis categories and found that the 33 proteostasis- and protein folding-related categories in the analysis were not changed significantly (Fig. S5A). Indeed, when verifying the protein levels of several chaperones, no considerable increase was found, even though various translation- and protein folding-related stresses are usually accompanied by increased chaperone machinery (Fig. 5A). Thus, we speculated that while there is a large mass of newly translated proteins, the support of the folding machinery is insufficient, rendering the accumulation of misfolded proteins. To test that, we measured the cellular capacity of refolding misfolded proteins, using a luciferase-based heat shock-recovery assays (LHS): Luciferase is very sensitive to heat, and inducing a heat shock (HS) reduces its activity. Cells with competent proteostasis machinery will manage to refold it over time – a property measured by its activity (see Fig. S5B for a scheme). Our results indicate that while HEK293T WT cells managed to recover ~ 60% of Renilla Luciferase activity, SIRT6KO cells could recover only ~ 20% (Fig. 5B). To validate that the reduced recovery is driven by the hyper-translation, we treated the cells with a translation attenuator. We previously demonstrated that 4-phenylbutyric acid (4PBA) is a conserved protein synthesis inhibitor, and contrary to the literature – has little or no effect as a chemical chaperone 36 . In concert with our hypothesis, translation attenuation helped to fully recover the folding capacity of the SIRT6KO cells (Fig. 5C). As expected, this treatment reduced the translation rate in SIRT6KO cells to similar levels as WT in all tested cell lines (Fig. 5D, S5C-E), further supporting that the 4PBA rescue is through translation attenuation 36 . Thus, we see that the hyper-translation upon SIRT6KO is not accompanied by a proper folding-promoting environment in the cell, which results in a low capability to properly fold newly translated proteins or to refold misfolded proteins. Lack of SIRT6 results in increased protein aggregates. Un- or misfolded proteins that are not eliminated from the cell, might accumulate into toxic protein aggregates, as often seen in neurodegeneration. To test the role of SIRT6 in aggregation, we used an aggregation-prone polyglutamine (polyQ) vector fused to EGFP (Q74-EGFP), which is based on the structural motif in the base of the aggregation mechanism in HD. Briefly, HD patients have CAG trinucleotide repetitions in HTT gene, which give rise to a polyQ chain in the translated protein N-terminus. Unlike healthy individuals, HD patients have more than 35 consecutive repeats, which form β-sheet structures that tend to aggregate in cells and become toxic; the longer the polyQ chain – the harsher and earlier the pathology appears. First, we measured aggregation in SIRT6KO SH-SY5Y cells. SIRT6-deficient cells have increased levels of EGFP-Q74 aggregates that cannot migrate through the SDS gel and hence are stuck in the wells, compared to WT controls (Fig. 5E). Thus, the hyper-translation upon SIRT6KO is also accompanied by increased aggregate formation. Next, we observed the EGFP-positive cells in a microscope and measured the Q74 aggregation through the texture of the EGFP signal (how smooth/rough the texture is), by calculating the contrast between neighboring pixels in an unbiased manner. We found a significant increase in texture contrast in SIRT6KO (Fig. 5F), indicative of more aggregation upon SIRT6 deficiency. Last, we manually classified the EGFP-positive cells into 3 score groups: S0 – no foci nor granulation; S1 – up to 5 foci or medium granulation; S2 – more than 5 foci or heavily granulated cells. Our data show that SIRT6KO leads to a larger portion of S2 on expanse of the S0 population (Fig. 5G). Overall, SIRT6-deficient cells translate more of the toxic polyQ protein, leading to its elevated aggregation. C. elegans sir-2.4 KO model recapitulates loss of proteostasis sensitivity and neurotoxicity but can be rescued by 4PBA. To test SIRT6 effects on proteostasis on the physiological level, we developed a new SIRT6-deficient model in C. elegans , by knocking out sir-2.4 – SIRT6 ortholog in nematodes ( sir-2.4 KO; Fig. S6). This model shows enhanced translation rate compared to the wildtype N2 genetic background strain (Fig. 6A), recapitulating our results in cell lines (Fig. 4A-C, S4A). To test proteostasis capacity, we stressed the nematodes with a heat shock and measured paralysis after 24h of recovery. We found that the WT (N2 genetic background) worms presented, as expected, a dramatic heat shock-driven increase in paralysis only after the reported collapse of proteostasis at day 2 of adulthood (Fig. 6B) 39 . However, the sir-2.4 KO worms have a poor resistance to heat shock even at day 1 of adulthood – with a high rate of paralysis before the expected collapse of proteostasis (Fig. 6B). These data suggest that the roles of SIRT6/ sir-2.4 in translation regulation and proteostasis maintenance are well conserved, and loss of proteostasis occurs prematurely in the sir-2.4 -deficient animals. Moreover, we conducted a thrashing-based motility assay, and found that the KO strain presented a more rapid age-dependent reduction in motility rate compared to WT (Fig. 6C, full lines), suggestive of faster decline of motor neuron function in sir-2.4 deletion. To further elucidate sir-2.4 role in neuronal proteostasis specifically, we adopted a polyQ-based proteostatic stress model in nematodes: we crossed the KO worms with a neuron-specific polyQ-expressing strain with 40 glutamine repeats (Q40n). We found an earlier collapse of motility compared to the genetic background-matching strain (Fig. 6C, day 1, dotted lines). Interestingly, both strains reached similar motility rates on day 3 of adulthood. These data suggests that in sir-2.4 deficiency, loss of proteostasis becomes harsher and manifests a deteriorated neurodegenerative phenotype and premature aging. Last, we tested the ability of translation attenuation to rescue the Q40n motility impairment using 4PBA treatment. Since sir-2.4 KO affects proteostasis-stressed worms already at day 1 of adulthood (Fig. 6B-C), we started the 4PBA treatment at larval stage 4 (L4). WT and sir-2.4 KO strains presented no effect through 4PBA treatment (Fig. 6D). However, 4PBA treatment significantly alleviated the motility decline in both Q40n and sir-2.4 KO;Q40n strains (Fig. 6D), pinpointing the critical role of translation rates in polyQ aggregation. Moreover, 4PBA rescued the sir-2.4 KO;Q40n worms nearly to the level of the non-treated Q40n strain, but kept on a constant gap from the 4PBA-treated Q40n worms (Fig. 6D). Thus, these results support our hypothesis that the proteostatic collapse upon sir-2.4 deficiency is driven by the hyper-translation. These results indicate that pharmacological reduction of protein synthesis could alleviate HD-related decline of neuronal function. Together, our results establish that SIRT6 and its orthologs regulate proteostasis by controlling nucleolar gene expression. Once chromatin is more open and allows the expression of ribosomal genes and translation-required machinery, the cells enter a pathological mode of hyper-translation with no enhancement in protein folding capacity. The ensuing accumulation of misfolded proteins and aggregates becomes toxic to the neurons, impairing movement of the organism. Thus, our data suggest that loss of SIRT6, that occurs naturally upon aging, provokes increased protein synthesis, but reduces protein folding capacity – thus leading to pathological aging and neurodegeneration. Discussion Loss of proteostasis is a hallmark of aging and neurodegenerative diseases. Still, in the absence of mutations that result in protein aggregation, it is poorly understood why proteostasis capacity is diminished in aging. To the best of our knowledge, we are the first to show that loss of SIRT6, as it occurs in aging and neurodegeneration, is causal of the proteostasis loss by altering the balance between translation and folding capacity of the cells. Moreover, the SIRT6 reduction can also explain the appearance of nucleolar expansion and aggregates in aging and neurodegeneration. Decreased levels of SIRT6 disrupt the recruitment of chromatin remodelers and regulators, resulting in an elevated transcription and imbalanced gene expression, particularly within the nucleolus. In addition to the elevated nucleolar transcription, the nucleolar rDNA repeats are also especially prone to DNA damage. Consequently, upon SIRT6 deficiency, the elevated transcription rates – coupled with nucleolar expansion – might enhance the vulnerability to DNA breaks. Furthermore, compromised DNA repair mechanisms in the absence of the sensor SIRT6 13 perpetuate a harmful cycle of DNA damage, nucleolar expansion, and increased nucleolar transcription. This cycle significantly impacts protein synthesis and homeostasis both in quantity, by affecting ribosome production, and in quality, by the introduction of mutations. We observed that SIRT6 regulates not only cap-dependent translation as previously reported 38 , but the entire regulation of ribosomes – both through their synthesis and processing, and through the expression of ribosomal proteins. The dysregulation of ribosomal activity results in dramatically higher translation rates. Although translation is increased, the chaperone- and protein folding-related genes nearly do not change, leading to a deteriorated ability to properly fold proteins – a critical machinery in every cell. Importantly, the polyglutamine models reveal that upon SIRT6 deficiency, aggregation-prone stress results in higher aggregation. These roles of SIRT6 in proteostasis are well conserved from nematodes to humans. As we demonstrated, the observed reduced proteostasis is rescuable using translation attenuation, by allowing the system to regain the balance between protein synthesis and folding machinery. Importantly, the efficacy of the FDA-approved drug 4PBA suggests a clinically implementable pharmacological strategy to alleviate SIRT6 deficiency-driven protein misfolding conditions, whether in SIRT6-deficient patients or during the normal decline of SIRT6 during aging. The role of SIRT6 in regulating proteostasis is evolutionarily conserved – its C. elegans ortholog, sir-2.4 , also has a critical role in translation and maintaining thermal resistance and motility in worms as they age. In reproducing nematodes, proteostasis is known to collapse between day 1 and day 2 of adulthood 39 . Here, we show that combining sir-2.4 deletion and proteostatic stress precedes the collapse to day 1, pinpointing its critical role in proteostasis and health. From another evolutionary perspective, the different aspects of ribosome biogenesis and function can be coregulated by several master regulators – one of them is SIRT6. Previous work found that DNA damage leads to a temporary transcription-translation stalling, followed by an increased protein synthesis after DNA damage was repaired – lowering the chance of accumulating mutated RNA and proteins 40 . Here, we see that SIRT6KO leads to accelerated translation, bypassing this important regulatory mechanism of translation attenuation, even though DNA damage is known to be elevated in SIRT6 deficiency 8 , 20 . We speculate that SIRT6 deletion is so deleterious to the organism, precisely because of the harsh imbalance it causes: SIRT6-deficient cells are in a state of chronic DNA damage, but as SIRT6 is a critical sensor of DNA double strand breaks, its absence impairs the repair 13 . Moreover, the absence of SIRT6 compromises also its role as a gene regulator, leading to epigenetic dysregulation and to nucleolar hyperactivity. Thus, the combination of DNA damage and hyper-translation could also result in the synthesis of mutated RNA molecules (from damaged DNA molecules), which further pushes the loss of proteostasis apparent in SIRT6KO. As SIRT6 activity declines with normal aging, and even further in AD patients, this implies a mechanism which feeds the loss of proteostasis – in several ways – in aging and age-related brain pathologies. To conclude, we show a novel role of SIRT6 in nucleolar regulation and proteostasis. This role explains how loss of proteostasis begins with nucleolar dysregulation, and how this regulation widely affects translation and proteostasis downstream, leading to the aggregates seen in age-related neurodegenerative diseases. We suggest that the FDA-approved 4PBA could provide a clinically accessible route for reversing the protein folding stress by mildly reducing protein translation and subsequently alleviating the neurodegenerative consequence of protein aggregates. Methods Generation of brain-specific SIRT6KO mice and SIRT6KO cells Brain-specific SIRT6KO C57BL/6 mice were generated according to the protocol described in Sebastián et al. (2012) 41 . SH-SY5Y, HeLa and HEK293T SIRT6KO or WT control cells were generated according to the protocol described in Kaluski et al. (2017) 20 . brSIRT6KO brain transcriptomics and Alzheimer’s Disease patients’ correlation The brSIRT6KO mouse brain transcriptomic dataset was already published in GSE221077. The analysis was as previously described in Smirnov et al. (2023) 27 . The brSIRT6KO-AD transcriptomics GSEA correlation was conducted using the above-mentioned brSIRT6KO mouse brain transcriptomics dataset and the supplementary portrait published in Hill & Gammie (2022) 42 . Cell culture All cells were grown in DMEM (catalog number: 41965039, Thermo-Fisher Gibco®, MA), supplemented with 1% L-glutamine (catalog number: 25030024, Thermo-Fisher Gibco®, MA), 1% Penicillin/Streptomycin antibiotics mix (catalog number: 15140122, Thermo-Fisher Gibco®, MA) and 10% FBS (catalog number: 12657-029, Thermo-Fisher Gibco®, MA). Incubation was 37°C, 5% CO 2 . Chromatin-bound protein acid extraction The chromatin-bound proteins were extracted from brain tissues and cell culture dry pellets according to the following protocol: Brain tissues and cell culture pellets were resuspended in cytoplasmic protein lysis buffer (described below), in a volume equivalent to 3 times the volume of the tissue. For tissues, the samples were homogenized for 2 cycles of 30 seconds in an electric homogenizer; for cell pellets, the samples were homogenized by thorough pipetting. Once homogenized, samples were kept on ice for 20 minutes, then centrifuged for 10 minutes, 21100g, 4°C. The supernatant, which contains the non-chromatin-bound fraction of proteins, was transferred into new tubes. The pellets (which contain the chromatin and cell leftovers) were washed twice with the same volume of cytoplasmic protein lysis buffer, incubated 5 minutes on ice and centrifuged for 5 minutes, 21100g, 4°C. then, Add 0.2N HCl solution to the dry pellet (equivalent of 1/8-1/10 of the original cytoplasmic protein lysis buffer volume) and pipette thoroughly. Samples were incubated on ice for 20 minutes with occasional vortexes, then centrifuged for 10 minutes, 21100g, 4°C. Supernatants (contain the chromatin-bound proteins) were transferred to new tubes and neutralized by adding 1M Tris pH8 (the same volumes as the 0.2N HCl), then vortexed. Protein concentrations were determined using Bradford assays. cytoplasmic protein lysis buffer: 10 mM HEPES pH7.4, 10mM KCl, 0.05% NP-40, phosphatase inhibitor cocktail X1 (APExBIO K1013), 0.2mM PMSF, 0.0015mM trichostatin A. Total protein extraction Total protein extraction from brain tissues and cell culture dry pellets was done according to the following protocol: Brain tissues and cell culture pellets were resuspended in RIPA lysis buffer, in a volume equivalent to 3 times the volume of the tissue. For tissues, the samples were homogenized for 2 cycles of 30 seconds in an electric homogenizer; for cell pellets, the samples were homogenized by thorough pipetting. Once homogenized, samples were kept on ice for 20 minutes, then centrifuged for 30 minutes, 21100g, 4°C. The supernatant, which contains the extracted proteins, was transferred to a new tube and protein concentrations were determined using Bradford assays. Antibody List Antibody Company Catalog number Dilution TIP5 Abcam ab195278 1:1000 H3K56ac Active Motif 39281 1:3000 H3 Santa Cruz Biotechnology sc-10809 1:3000 BrdU (FUrd) Sigma-Aldrich B8434-100UL 1:1000 Nucleolin Abcam ab22758 1:1000 SNF2H Novus Biologicals NB100-55310 1:1000 Fibrillarin Abcam ab4566 1:1000 NeuN Sigma-Aldrich FCMAB317PE 1:1000 Puromycin Millipore MABE343 1:12,000 4EBP1-phosphorylated Cell Signaling Technology CST9451 1:1000 HSPA9 Santa Cruz Biotechnology sc-133137 1:1000 HSPD1 Santa Cruz Biotechnology sc-13115 1:1000 HSP90β Developmental Studies Hybridoma Bank H90-10 1:1000 CRYAB Santa Cruz Biotechnology sc-22744 1:1000 DnaJB11 Proteintech 15484-1-AP 1:1000 HSPA2 Sigma-Aldrich HPA000798-100UL 1:1000 GFP Sigma-Aldrich 11814460001 1:1000 SIRT6 Abcam ab88494 1:1000 Rabbit anti-mouse IgG H&L (HRP) Abcam ab97046 1:10,000 Goat anti rabbit IgG H&L (HRP) Abcam ab6721 1:10,000 Alexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L) Jackson ImmunoResearch 715-545-150 1:250 Alexa-Fluor 555 Donkey Anti-Mouse IgG H&L Abcam ab150110 1:250 Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) Jackson ImmunoResearch 711-545-152 1:250 Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) Jackson ImmunoResearch 711-585-152 1:250 Plasmids and transfections TIP5-GFP plasmid – addgene plasmid #65373 - https://www.addgene.org/65373/ Renilla Luciferase vector – Promega pRL-null. Q74-GFP plasmid – addgene plasmid #40262 - https://www.addgene.org/40262/ Q0-GFP plasmid – pEGFP-C1 backbone vector. All other plasmids were homemade: shGFP, shSNF2H, empty vector (CMV-flag backbone). Transfections were conducted using PolyJet™ In Vitro DNA Transfection Reagent (catalog number: SL100688, SignaGen® Laboratories, MD), according to manufacturer’s protocol. 4PBA treatment Media with 4PBA were prepared as previously described in Stein et al. (2022) 36 . 5-fluorouridine nucleolar transcription labelling Nucleolar transcription labelling was conducted as previously described in Portillo et al. (2021) 21 . SUnSET puromycin labelling The SUnSET 37 experiment was done as previously described in Stein et al. (2022) 36 . Briefly, Cells were grown in regular growth conditions (see above) and plated in 6-well plates. On the following day, puromycin was added to a final concentration of 10 μg/ml directly to the cell media, then the cells were re-incubated in 37°C for 10-30 minutes, depending on cell line. Once puromycin labelling was done, cells were collected for total protein extraction (see above). In case 4PBA or transfections were conducted, they were done as previously described (stein et al. 36 ; see above), and the puromycin labelling was done 24-48h after the treatment/transfection. Cycloheximide was used as a negative control, and was added 5 minutes before the puromycin labelling initiation, in a final concentration of 1-10 μg/ml. Luciferase assays Heat shock Luciferase-based assays 20k cells were plated in 48-well plates and 24h post-plating, transfected with Renilla plasmid (10%), supplemented with an empty vector (see above). 24h after transfections, cells were put in 42°C, 5% CO2 incubator for an hour, then in 37°C recovery for 90-120 minutes. Lysis, luciferase substrates and luminescence readings were conducted using Promega Renilla Luciferase Assay System kit (Promega E2820), according to manufacturer’s protocol. Protein refold capacity was calculated as the division of the Renilla activity after heat shock-recovery in non-shocked activity. Importantly, since SIRT6-deficient cells present elevated translation, WT and KO cells were normalized to their own non-heat-shocked samples. Statistics Analyses were done as described in the supplementary statistics table. Declarations The Ben Gurion University of the Negev, together with the Israel animal ethics committee, has approved the animal protocol Brains specific SIRT6-KO effect on aging and neurodegeneration, authorization number IL-55-09-2023C. Acknowledgements and funding The study was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 849029), the David and Inez Myers foundation, the Israeli Ministry of Science and Technology (MOST), the High-tech, Bio-tech and Negev fellowships of Kreitman School of Advanced Research of Ben-Gurion University. References Alzheimer’s Association. Alzheimer’s disease facts and figures. Alzheimers Dement. 17 , 327–406 (2021). Espay, A. J. et al. Revisiting protein aggregation as pathogenic in sporadic Parkinson and Alzheimer diseases. Neurology 92 , 329–337 (2019). Soto-Palma, C., Niedernhofer, L. J., Faulk, C. D. & Dong, X. Epigenetics, DNA damage, and aging. J. Clin. Invest. 132 , e158446 (2022). Schumacher, B., Pothof, J., Vijg, J. & Hoeijmakers, J. H. J. The central role of DNA damage in the ageing process. Nature 592 , 695–703 (2021). Michishita, E. et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature (2008) doi:10.1038/nature06736. Michishita, E. et al. Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6. Cell Cycle Preprint at https://doi.org/10.4161/cc.8.16.9367 (2009). Yang, B., Zwaans, B. M. M., Eckersdorff, M. & Lombard, D. B. The sirtuin SIRT6 deacetylates H3 K56Ac in vivo to promote genomic stability. Cell Cycle Preprint at https://doi.org/10.4161/cc.8.16.9329 (2009). Mostoslavsky, R. et al. Genomic instability and aging-like phenotype in the absence of mammalian SIRT6. Cell (2006) doi:10.1016/j.cell.2005.11.044. Toiber, D. et al. SIRT6 recruits SNF2H to DNA break sites, preventing genomic instability through chromatin remodeling. Mol. Cell (2013) doi:10.1016/j.molcel.2013.06.018. McCord, R. A. et al. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair. Aging (2009) doi:10.18632/aging.100011. Mao, Z. et al. SIRT6 promotes DNA repair under stress by activating PARP1. Science (2011) doi:10.1126/science.1202723. Kugel, S. & Mostoslavsky, R. Chromatin and beyond: The multitasking roles for SIRT6. Trends in Biochemical Sciences Preprint at https://doi.org/10.1016/j.tibs.2013.12.002 (2014). Onn, L. et al. SIRT6 is a DNA double-strand break sensor. eLife 9 , e51636 (2020). Kim, H. S. et al. Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis. Cell Metab. (2010) doi:10.1016/j.cmet.2010.06.009. Xiao, C. et al. SIRT6 deficiency results in severe hypoglycemia by enhancing both basal and insulin-stimulated glucose uptake in mice. J. Biol. Chem. (2010) doi:10.1074/jbc.M110.168039. Garcia-Venzor, A. & Toiber, D. SIRT6 Through the Brain Evolution, Development, and Aging. Front. Aging Neurosci. 13 , 747989 (2021). Zhang, W. et al. SIRT6 deficiency results in developmental retardation in cynomolgus monkeys. Nature 560 , 661–665 (2018). Kanfi, Y. et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature (2012) doi:10.1038/nature10815. Roichman, A. et al. SIRT6 overexpression improves various aspects of mouse healthspan. J. Gerontol. - Ser. Biol. Sci. Med. Sci. (2017) doi:10.1093/gerona/glw152. Kaluski, S. et al. Neuroprotective Functions for the Histone Deacetylase SIRT6. Cell Rep. (2017) doi:10.1016/j.celrep.2017.03.008. Portillo, M. et al. SIRT6-CBP-dependent nuclear Tau accumulation and its role in protein synthesis. Cell Rep. 35 , 109035 (2021). Braidy, N. et al. Differential expression of sirtuins in the aging rat brain. Front. Cell. Neurosci. 9 , (2015). Yaku, K., Okabe, K. & Nakagawa, T. NAD metabolism: Implications in aging and longevity. Ageing Res. Rev. 47 , 1–17 (2018). Fang, E. F. et al. NAD + in Aging: Molecular Mechanisms and Translational Implications. Trends Mol. Med. 23 , 899–916 (2017). Chini, C. C. S., Tarragó, M. G. & Chini, E. N. NAD and the aging process: Role in life, death and everything in between. Mol. Cell. Endocrinol. 455 , 62–74 (2017). Stein, D. et al. Aging and pathological aging signatures of the brain: through the focusing lens of SIRT6. Aging 13 , 6420–6441 (2021). Smirnov, D. et al. SIRT6 is a key regulator of mitochondrial function in the brain. Cell Death Dis. 14 , 35 (2023). Ittner, L. M. & Götz, J. Amyloid-β and tau - A toxic pas de deux in Alzheimer’s disease. Nat. Rev. Neurosci. (2011) doi:10.1038/nrn2967. Shulman, J. M., De Jager, P. L. & Feany, M. B. Parkinson’s Disease: Genetics and Pathogenesis. Annu. Rev. Pathol. Mech. Dis. (2011) doi:10.1146/annurev-pathol-011110-130242. Dawson, T. M. & Dawson, V. L. The Role of Parkin in Familial and Sporadic Parkinson ’ s Disease. Mov. Disord. 25 , 32–39 (2010). Bates, G. Huntingtin aggregation and toxicity in Huntington’s disease. The Lancet 361 , 1642–1644 (2003). DiFiglia, M. et al. Aggregation of Huntingtin in Neuronal Intranuclear Inclusions and Dystrophic Neurites in Brain. Science 277 , 1990–1993 (1997). Feric, M. et al. Coexisting Liquid Phases Underlie Nucleolar Subcompartments. Cell 165 , 1686–1697 (2016). Buchwalter, A. & Hetzer, M. W. Nucleolar expansion and elevated protein translation in premature aging. Nat. Commun. 8 , 328 (2017). Tiku, V. & Antebi, A. Nucleolar Function in Lifespan Regulation. Trends Cell Biol. 28 , 662–672 (2018). Stein, D. et al. 4‐phenylbutyric acid—Identity crisis; can it act as a translation inhibitor? Aging Cell 21 , (2022). Schmidt, E. K., Clavarino, G., Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nat. Methods 6 , 275–277 (2009). Ravi, V. et al. SIRT6 transcriptionally regulates global protein synthesis through transcription factor Sp1 independent of its deacetylase activity. Nucleic Acids Res. gkz648 (2019) doi:10.1093/nar/gkz648. Shemesh, N., Shai, N. & Ben-Zvi, A. Germline stem cell arrest inhibits the collapse of somatic proteostasis early in Caenorhabditis elegans adulthood. Aging Cell 12 , 814–822 (2013). Wang, S., Meyer, D. H. & Schumacher, B. H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage. Nat. Struct. Mol. Biol. 27 , 1165–1177 (2020). Sebastián, C. et al. The Histone Deacetylase SIRT6 Is a Tumor Suppressor that Controls Cancer Metabolism. Cell 151 , 1185–1199 (2012). Hill, M. A. & Gammie, S. C. Alzheimer’s disease large-scale gene expression portrait identifies exercise as the top theoretical treatment. Sci. Rep. 12 , 17189 (2022). Additional Declarations There is NO Competing Interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4215918","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":291897485,"identity":"f2130989-1cfa-4f46-b7be-7180e29cdaa2","order_by":0,"name":"Debra 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05:52:20","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":240096,"visible":true,"origin":"","legend":"","description":"","filename":"SupFig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4215918/v1/c6b90f54805e6dd486081e9e.jpg"},{"id":57319742,"identity":"351a64dd-46b7-4d65-b242-533aed5084ab","added_by":"auto","created_at":"2024-05-29 05:52:20","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":189452,"visible":true,"origin":"","legend":"","description":"","filename":"SupFig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4215918/v1/00c779cc9d48a7ef3acb3efd.jpg"},{"id":57320288,"identity":"21946393-6db3-46c5-a547-e5de30562d48","added_by":"auto","created_at":"2024-05-29 06:00:20","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":62133,"visible":true,"origin":"","legend":"","description":"","filename":"SupFig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4215918/v1/3263167577078de06f7dd98b.jpg"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"SIRT6 regulates protein synthesis and folding through nucleolar remodeling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAging is considered to be a major risk factor for developing a neurodegenerative disease, where about 95% of the cases are sporadic and age-related. Therefore, understanding the molecular processes that occur during human aging is critical to prevent and treat age-related neurodegenerative diseases. Neurodegeneration is one of the most predominant age-related pathologies, with chances of developing a neurodegenerative condition doubling every 5 years after the age of 65\u003csup\u003e1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor more than 30 years the focus in neurodegeneration research has majorly been on the formation of toxic protein aggregates. However, this seems to be a late event in the development of the pathology, and therapies targeting aggregates have failed to improve patients\u0026rsquo; cognitive capacity and to prevent memory loss\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. On the other hand, DNA damage accumulation and epigenetic changes are the key drivers of aging\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, yet their implications on the loss of proteostasis are elusive. In this work, we use SIRT6 as a model of accelerated aging, particularly in the brain, and observe how its loss results in nucleolar misfunction that results in the loss of proteostasis.\u003c/p\u003e \u003cp\u003eSIRT6 is a nuclear deacetylase and mono-ADP ribosyltransferase, which plays a critical role in many cellular processes. It represses gene expression through histone deacetylation\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and is involved in the DNA damage response, preventing genomic instability\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. SIRT6 is essential especially in mammals: SIRT6 knock-out (KO) mice show postnatal lethality and genomic instability\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In monkeys, SIRT6 deletion leads to prenatal developmental retardation, delayed neuronal differentiation and to death a few hours postnatally, pointing to a crucial role of SIRT6 in the brain of primates\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. In contrast, SIRT6-overexpressing mice show increased physical activity and extended lifespan\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Together, these data suggest that SIRT6 is essential, and its roles are tightly linked to the aging process.\u003c/p\u003e \u003cp\u003eInterestingly, brain-specific SIRT6-deficient mice (brSIRT6KO) survive to adulthood but present behavioral defects with major hippocampus-dependent learning impairments by 4 months of age\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Moreover, the brains of these mice show increased signs of DNA damage, cell death, and hyperphosphorylated and acetylated Tau \u0026ndash; critical marks in many neurodegenerative diseases\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Thus, brSIRT6KO phenotypes resemble animal models of Alzheimer\u0026rsquo;s Disease and pathological characteristics of brains from patients suffering from age-related neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Indeed, SIRT6 levels and activity decrease in aging brains and furthermore in Alzheimer\u0026rsquo;s Disease patients\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Although neurodegenerative diseases have each a different phenotype, there are many commonalities that we also found when comparing them with SIRT6-deficient models\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. For example, we discovered an additional link between SIRT6 and Alzheimer\u0026rsquo;s, Parkinson\u0026rsquo;s, Huntington diseases and amyotrophic lateral sclerosis (AD, PD, HD and ALS, respectively) through mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The SIRT6-neurodegeneration link is especially intriguing because another common trait in many age-related brain pathologies is the extreme form of proteostasis loss, manifested in aggregation of specific proteins \u0026ndash; suggesting a role for SIRT6 in proteostasis. Among these conditions are AD\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e (with Tau and Amyloid-beta), PD\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e (with alpha-synuclein, parkin and Tau) and HD\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e (with Huntingtin).\u003c/p\u003e \u003cp\u003eWhile the protein aggregation in AD and PD seems to be a downstream product of a covert age-dependent malfunction, Huntingtin aggregation in HD is clearly the driver of the disease\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Still, HD develops only as the person gets older, and not during development or early adulthood \u0026ndash; implying that as the person ages, the capacity to cope with the polyglutamine-driven aggregation is lost.\u003c/p\u003e \u003cp\u003eProteostasis includes several layers of regulation ranging from ribosome formation to protein folding and degradation. Translation is done by the ribosomes, which are composed of specialized RNA and protein subunits. While the ribosomal proteins are critical for the regulation of mRNA preference by the ribosome, most of the ribosomal structure \u0026ndash; the ribosomal RNA (rRNA) \u0026ndash; is expressed and pre-formed in the nucleolus: a sub-nuclear membrane-less compartment, separated by liquid-liquid phase separation\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The nucleolus contains the ribosomal DNA (rDNA) that is transcribed to the 45S pre-rRNA, which is later processed and assembled \u0026ndash; together with ribosomal proteins \u0026ndash; into pre-ribosomal subunits. Importantly, nucleolar physiology is impaired in aging and progerias, with increased nucleolar size and number\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Furthermore, nucleolar size negatively correlates with longevity and its premature enlargement can be seen in progeroid syndromes\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. All these age-related changes in the nucleolus might lead to altered ribosomal content and translation capacity downstream\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. One of the main regulators of the pre-rRNA transcription is the Nucleolar Remodeling Complex (NoRC), which one of its components is SNF2H \u0026ndash; a chromatin remodeler that was previously found to be recruited by SIRT6 to the chromatin at DNA double strand break sites\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we reveal the critical role of SIRT6 in maintaining proteostasis from ribosomal production to protein folding. We show that SIRT6 regulates proteostasis through regulation of nuclear and nucleolar genes. SIRT6 negatively regulates translation through the nucleolus: knocking out SIRT6 resulted in enlarged nucleoli; elevated rRNA production; and increased amino acid production and transport to cope with the demand. While this resulted in elevated translation rate, the molecular chaperone family of proteins did not change, leading to reduced protein folding, leaving the cells vulnerable to aggregate formation. Furthermore, we developed a SIRT6-ortholog KO model in \u003cem\u003eC. elegans\u003c/em\u003e to show that the role in translation regulation and proteostasis is conserved. We show that the polyQ-mediated motor neuron dysfunction is exacerbated in \u003cem\u003esir-2.4\u003c/em\u003e KO animals and could be reverted by the FDA-approved weak protein translation inhibitor and chemical chaperone 4-phenylbutyric acid (4PBA)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings establish SIRT6 as critical regulator of proteostasis through nucleolar activity and emphasize its relevance in aging and neurodegenerative processes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eSIRT6 deficiency affects pathways of neurodegeneration, with common association to ribosomal genes.\u003c/p\u003e \u003cp\u003eWe first analyzed brain transcriptomics of brSIRT6KO compared to age-matched control mice. Strikingly, we found 18 significantly changing categories in KEGG enrichment analysis, out of which 6 categories belong to neurodegenerative pathologies. Interestingly, however, the most significant category was the \u003cem\u003eRibosome (ribosomal proteins)\u003c/em\u003e (Fig.\u0026nbsp;1A). Next, to further elucidate the ribosome-neurodegeneration connection in brSIRT6KO mice, we used Gene Ontology (GO) analysis. We found 431 significantly upregulated and 68 significantly downregulated categories. Out of these categories, 79 upregulated and 14 downregulated categories are ribosome-, proteostasis- and brain-related (Fig. S1A), including biogenesis and assembly of the ribosome subunits; amino acid import; protein localization; cognition, trans-synaptic signaling and action potential.\u003c/p\u003e \u003cp\u003eAD is the most common age-related neurodegenerative disease; since SIRT6 levels decrease in AD\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, we compared Geneset Enrichment Analysis (GSEA) results of AD patients and brSIRT6KO brains and found a positive correlation between the two groups of genesets (Fig.\u0026nbsp;1B). Again, the \u003cem\u003eRibosome (ribosomal proteins)\u003c/em\u003e category as well as \u003cem\u003eAlzheimer\u0026rsquo;s disease\u003c/em\u003e and \u003cem\u003eParkinson\u0026rsquo;s disease\u003c/em\u003e categories appear to be co-regulated in the brains of both AD patients and brSIRT6KO mice (Fig.\u0026nbsp;1B). Thus, these results emphasize the importance of SIRT6 in both neurodegeneration and ribosomal regulation, as well as the tight correlation between ribosome structure and regulation and pathological brain aging.\u003c/p\u003e \u003cp\u003eLack of SIRT6 results in nucleolar dysregulation and expansion.\u003c/p\u003e \u003cp\u003eSIRT6 could be involved in ribosomal changes through its known interaction with SNF2H\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and the role of the latter in NoRC. This complex is directed to the rDNA by its largest component: TIP5 (also known as BAZ2A). While TIP5 RNA levels are increased in brSIRT6KO mice brains (Fig.\u0026nbsp;2A), its protein levels and chromatin recruitment are reduced (Fig.\u0026nbsp;2B, S2A-B). When observing SNF2H \u0026ndash; NoRC chromatin remodeling subunit \u0026ndash; the brSIRT6KO brains show no significant difference in total protein levels, while its chromatin recruitment has a clear trend of reduction, as expected by previous reports\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e (Fig. S2A-B). Moreover, knocking-out SIRT6 in combination with SNF2H knockdown fully abolished TIP5 from the chromatin in HeLa cells (Fig.\u0026nbsp;2C), supporting the connection between SIRT6 and NoRC. A reduced NoRC recruitment to chromatin could lead to the lack of repression at rDNA loci and to possibly increase pre-rRNA production. Therefore, we tested nucleolar transcription with 5-fluorouridine labeling and found that SIRT6-deficient cells have elevated rRNA production in the nucleolus (Fig.\u0026nbsp;2D). These results suggested that SIRT6 regulates the ribosomal genes at the transcription level directly in the nucleolus.\u003c/p\u003e \u003cp\u003eSince nucleolar size and function are biomarkers of aging, we hypothesized that reduced NoRC regulation in the nucleolus and increased nucleolar transcription could affect nucleolar size and function. We tested 2 nucleolar markers \u0026ndash; Nucleolin and Fibrillarin (NCL and FBL, respectively) \u0026ndash; that play a critical role in rRNA post-transcriptional processing inside the nucleolus. In brSIRT6KO mouse brains, we saw a significant increase of the mRNA levels of NCL but not of FBL (Fig.\u0026nbsp;3A). Next, we tested our cellular models. In SIRT6KO SH-SY5Y, we find increased nucleoli number and area per nucleus, as well as elevated intensity of both nucleolar factors (Fig.\u0026nbsp;2D, 3B). These results were recapitulated in FBL immunofluorescence and NCL protein blot in HeLa cells as well (Fig. S3A-B), suggesting a different regulation from mRNA and protein levels for FBL. Hence, SIRT6 deficiency not only increases rRNA production, but also its processing in the nucleolus.\u003c/p\u003e \u003cp\u003eMoreover, to confirm this happens in post-mitotic neurons and to understand the age-related changes, we isolated nuclei from NeuN-positive cortical cells of adult WT and brSIRT6KO mice (see scheme on Fig.\u0026nbsp;3C). Neurons from brSIRT6KO mice presented NCL overexpression (Fig.\u0026nbsp;3D-E). Importantly, the number of nucleoli per nucleus followed a similar trend: brSIRT6KO brains present significantly more nucleoli than their WT counterparts (Fig.\u0026nbsp;3D, F). Thus, our results in cell lines are also recapitulated in cortical neurons of adult mice.\u003c/p\u003e \u003cp\u003eOur combined results from mouse brains and different cell lines support the notion that lack of SIRT6 and aging compromises the nucleolus, by affecting pre-rRNA synthesis and nucleolar function and physiology. This, in turn, plays an important role in the nucleolar expansion and overproduction of rRNA.\u003c/p\u003e \u003cp\u003eSIRT6 deficient cells have increased protein synthesis.\u003c/p\u003e \u003cp\u003eWe suspected that the enhanced production and processing of rRNA could lead to elevated translation. To test this, we directly measured translation using the SUnSET technique\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We found an increased protein synthesis rate in SIRT6KO HeLa, SH-SY5Y and HEK293T cells (Fig.\u0026nbsp;4A-C, S4A). Importantly, this elevation in translation was independent of 4EBP1 phosphorylation (Fig. S4B), in contrast to previous reports\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. To validate that the SIRT6KO hyper-translation is mediated by nucleolar dysregulation, we repeated the SUnSET assay with TIP5 overexpression, and found that indeed, it partially rescues the elevated translation rates in SIRT6KO HeLa cells (Fig.\u0026nbsp;4D). However, the rescue effect is relatively mild, probably since the overexpression only partially compensates for the reduced capacity to recruit TIP5 protein (and hence NoRC) to the chromatin. Thus, our data suggests that the above-identified nucleolar dysregulation resulting from SIRT6 deficiency leads to elevated rRNA and protein biosynthesis.\u003c/p\u003e \u003cp\u003eTo support the increased protein synthesis upon SIRT6 deficiency, the intracellular amino acid content should be elevated. The brSIRT6KO brain RNA-seq data reveals that indeed, there is a significant increase in many amino acid transporters, that together cover all the amino acids (Fig. S4C). Analyzing brSIRT6KO brain metabolomics revealed that out of the top 25 enriched categories, 10 were related to amino acid and protein metabolism (Fig.\u0026nbsp;4E, marked in red arrows) \u0026ndash; pointing on the dramatic metabolic changes that occur in SIRT6-deficient brains. We then co-analyzed the RNA-seq with the metabolomics data to obtain a joint pathway enrichment and found 6 significantly changing pathways (FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05), one of them is \u0026lsquo;Aminoacyl-tRNA biosynthesis\u0026rsquo; (Fig.\u0026nbsp;4F, S4D). Analyzing SIRT6KO SHSY5Y cells metabolomics supported the mouse data, as intracellular content was significantly elevated for most detected amino acids (Fig. S4E). We conclude, then, that the hyper-translation in SIRT6 deficiency is accompanied by a metabolic shift in amino acid content that supports the increased protein synthesis rates.\u003c/p\u003e \u003cp\u003eSIRT6 deficiency impairs protein refolding.\u003c/p\u003e \u003cp\u003eTo be functional, proteins must be properly folded. As folding is very delicate, it often requires the aid of a specialized group of proteins \u0026ndash; molecular chaperones. While our results show elevated protein synthesis rates, chaperones and folding machinery were lacking in the differentially expressed categories (Fig.\u0026nbsp;1A, S1A). Therefore, we manually inspected the brSIRT6KO RNA-seq for proteostasis categories and found that the 33 proteostasis- and protein folding-related categories in the analysis were not changed significantly (Fig. S5A). Indeed, when verifying the protein levels of several chaperones, no considerable increase was found, even though various translation- and protein folding-related stresses are usually accompanied by increased chaperone machinery (Fig.\u0026nbsp;5A). Thus, we speculated that while there is a large mass of newly translated proteins, the support of the folding machinery is insufficient, rendering the accumulation of misfolded proteins. To test that, we measured the cellular capacity of refolding misfolded proteins, using a luciferase-based heat shock-recovery assays (LHS): Luciferase is very sensitive to heat, and inducing a heat shock (HS) reduces its activity. Cells with competent proteostasis machinery will manage to refold it over time \u0026ndash; a property measured by its activity (see Fig. S5B for a scheme). Our results indicate that while HEK293T WT cells managed to recover\u0026thinsp;~\u0026thinsp;60% of \u003cem\u003eRenilla\u003c/em\u003e Luciferase activity, SIRT6KO cells could recover only\u0026thinsp;~\u0026thinsp;20% (Fig.\u0026nbsp;5B). To validate that the reduced recovery is driven by the hyper-translation, we treated the cells with a translation attenuator. We previously demonstrated that 4-phenylbutyric acid (4PBA) is a conserved protein synthesis inhibitor, and contrary to the literature \u0026ndash; has little or no effect as a chemical chaperone\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In concert with our hypothesis, translation attenuation helped to fully recover the folding capacity of the SIRT6KO cells (Fig.\u0026nbsp;5C). As expected, this treatment reduced the translation rate in SIRT6KO cells to similar levels as WT in all tested cell lines (Fig.\u0026nbsp;5D, S5C-E), further supporting that the 4PBA rescue is through translation attenuation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Thus, we see that the hyper-translation upon SIRT6KO is not accompanied by a proper folding-promoting environment in the cell, which results in a low capability to properly fold newly translated proteins or to refold misfolded proteins.\u003c/p\u003e \u003cp\u003eLack of SIRT6 results in increased protein aggregates.\u003c/p\u003e \u003cp\u003eUn- or misfolded proteins that are not eliminated from the cell, might accumulate into toxic protein aggregates, as often seen in neurodegeneration. To test the role of SIRT6 in aggregation, we used an aggregation-prone polyglutamine (polyQ) vector fused to EGFP (Q74-EGFP), which is based on the structural motif in the base of the aggregation mechanism in HD. Briefly, HD patients have CAG trinucleotide repetitions in HTT gene, which give rise to a polyQ chain in the translated protein N-terminus. Unlike healthy individuals, HD patients have more than 35 consecutive repeats, which form β-sheet structures that tend to aggregate in cells and become toxic; the longer the polyQ chain \u0026ndash; the harsher and earlier the pathology appears.\u003c/p\u003e \u003cp\u003eFirst, we measured aggregation in SIRT6KO SH-SY5Y cells. SIRT6-deficient cells have increased levels of EGFP-Q74 aggregates that cannot migrate through the SDS gel and hence are stuck in the wells, compared to WT controls (Fig.\u0026nbsp;5E). Thus, the hyper-translation upon SIRT6KO is also accompanied by increased aggregate formation.\u003c/p\u003e \u003cp\u003eNext, we observed the EGFP-positive cells in a microscope and measured the Q74 aggregation through the texture of the EGFP signal (how smooth/rough the texture is), by calculating the contrast between neighboring pixels in an unbiased manner. We found a significant increase in texture contrast in SIRT6KO (Fig.\u0026nbsp;5F), indicative of more aggregation upon SIRT6 deficiency.\u003c/p\u003e \u003cp\u003eLast, we manually classified the EGFP-positive cells into 3 score groups: S0 \u0026ndash; no foci nor granulation; S1 \u0026ndash; up to 5 foci or medium granulation; S2 \u0026ndash; more than 5 foci or heavily granulated cells. Our data show that SIRT6KO leads to a larger portion of S2 on expanse of the S0 population (Fig.\u0026nbsp;5G). Overall, SIRT6-deficient cells translate more of the toxic polyQ protein, leading to its elevated aggregation.\u003c/p\u003e \u003cp\u003e \u003cem\u003eC. elegans sir-2.4\u003c/em\u003eKO model recapitulates loss of proteostasis sensitivity and neurotoxicity but can be rescued by 4PBA.\u003c/p\u003e \u003cp\u003eTo test SIRT6 effects on proteostasis on the physiological level, we developed a new SIRT6-deficient model in \u003cem\u003eC. elegans\u003c/em\u003e, by knocking out \u003cem\u003esir-2.4\u003c/em\u003e \u0026ndash; SIRT6 ortholog in nematodes (\u003cem\u003esir-2.4\u003c/em\u003eKO; Fig. S6). This model shows enhanced translation rate compared to the wildtype N2 genetic background strain (Fig.\u0026nbsp;6A), recapitulating our results in cell lines (Fig.\u0026nbsp;4A-C, S4A).\u003c/p\u003e \u003cp\u003eTo test proteostasis capacity, we stressed the nematodes with a heat shock and measured paralysis after 24h of recovery. We found that the WT (N2 genetic background) worms presented, as expected, a dramatic heat shock-driven increase in paralysis only after the reported collapse of proteostasis at day 2 of adulthood (Fig.\u0026nbsp;6B)\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, the \u003cem\u003esir-2.4\u003c/em\u003eKO worms have a poor resistance to heat shock even at day 1 of adulthood \u0026ndash; with a high rate of paralysis before the expected collapse of proteostasis (Fig.\u0026nbsp;6B). These data suggest that the roles of SIRT6/\u003cem\u003esir-2.4\u003c/em\u003e in translation regulation and proteostasis maintenance are well conserved, and loss of proteostasis occurs prematurely in the \u003cem\u003esir-2.4\u003c/em\u003e-deficient animals.\u003c/p\u003e \u003cp\u003eMoreover, we conducted a thrashing-based motility assay, and found that the KO strain presented a more rapid age-dependent reduction in motility rate compared to WT (Fig.\u0026nbsp;6C, full lines), suggestive of faster decline of motor neuron function in \u003cem\u003esir-2.4\u003c/em\u003e deletion. To further elucidate \u003cem\u003esir-2.4\u003c/em\u003e role in neuronal proteostasis specifically, we adopted a polyQ-based proteostatic stress model in nematodes: we crossed the KO worms with a neuron-specific polyQ-expressing strain with 40 glutamine repeats (Q40n). We found an earlier collapse of motility compared to the genetic background-matching strain (Fig.\u0026nbsp;6C, day 1, dotted lines). Interestingly, both strains reached similar motility rates on day 3 of adulthood. These data suggests that in \u003cem\u003esir-2.4\u003c/em\u003e deficiency, loss of proteostasis becomes harsher and manifests a deteriorated neurodegenerative phenotype and premature aging.\u003c/p\u003e \u003cp\u003eLast, we tested the ability of translation attenuation to rescue the Q40n motility impairment using 4PBA treatment. Since \u003cem\u003esir-2.4\u003c/em\u003eKO affects proteostasis-stressed worms already at day 1 of adulthood (Fig.\u0026nbsp;6B-C), we started the 4PBA treatment at larval stage 4 (L4). WT and \u003cem\u003esir-2.4\u003c/em\u003eKO strains presented no effect through 4PBA treatment (Fig.\u0026nbsp;6D). However, 4PBA treatment significantly alleviated the motility decline in both Q40n and \u003cem\u003esir-2.4\u003c/em\u003eKO;Q40n strains (Fig.\u0026nbsp;6D), pinpointing the critical role of translation rates in polyQ aggregation. Moreover, 4PBA rescued the \u003cem\u003esir-2.4\u003c/em\u003eKO;Q40n worms nearly to the level of the non-treated Q40n strain, but kept on a constant gap from the 4PBA-treated Q40n worms (Fig.\u0026nbsp;6D). Thus, these results support our hypothesis that the proteostatic collapse upon \u003cem\u003esir-2.4\u003c/em\u003e deficiency is driven by the hyper-translation. These results indicate that pharmacological reduction of protein synthesis could alleviate HD-related decline of neuronal function.\u003c/p\u003e \u003cp\u003eTogether, our results establish that SIRT6 and its orthologs regulate proteostasis by controlling nucleolar gene expression. Once chromatin is more open and allows the expression of ribosomal genes and translation-required machinery, the cells enter a pathological mode of hyper-translation with no enhancement in protein folding capacity. The ensuing accumulation of misfolded proteins and aggregates becomes toxic to the neurons, impairing movement of the organism. Thus, our data suggest that loss of SIRT6, that occurs naturally upon aging, provokes increased protein synthesis, but reduces protein folding capacity \u0026ndash; thus leading to pathological aging and neurodegeneration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eLoss of proteostasis is a hallmark of aging and neurodegenerative diseases. Still, in the absence of mutations that result in protein aggregation, it is poorly understood why proteostasis capacity is diminished in aging. To the best of our knowledge, we are the first to show that loss of SIRT6, as it occurs in aging and neurodegeneration, is causal of the proteostasis loss by altering the balance between translation and folding capacity of the cells. Moreover, the SIRT6 reduction can also explain the appearance of nucleolar expansion and aggregates in aging and neurodegeneration.\u003c/p\u003e \u003cp\u003eDecreased levels of SIRT6 disrupt the recruitment of chromatin remodelers and regulators, resulting in an elevated transcription and imbalanced gene expression, particularly within the nucleolus. In addition to the elevated nucleolar transcription, the nucleolar rDNA repeats are also especially prone to DNA damage. Consequently, upon SIRT6 deficiency, the elevated transcription rates \u0026ndash; coupled with nucleolar expansion \u0026ndash; might enhance the vulnerability to DNA breaks. Furthermore, compromised DNA repair mechanisms in the absence of the sensor SIRT6\u003csup\u003e13\u003c/sup\u003e perpetuate a harmful cycle of DNA damage, nucleolar expansion, and increased nucleolar transcription. This cycle significantly impacts protein synthesis and homeostasis both in quantity, by affecting ribosome production, and in quality, by the introduction of mutations.\u003c/p\u003e \u003cp\u003eWe observed that SIRT6 regulates not only cap-dependent translation as previously reported\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, but the entire regulation of ribosomes \u0026ndash; both through their synthesis and processing, and through the expression of ribosomal proteins. The dysregulation of ribosomal activity results in dramatically higher translation rates. Although translation is increased, the chaperone- and protein folding-related genes nearly do not change, leading to a deteriorated ability to properly fold proteins \u0026ndash; a critical machinery in every cell. Importantly, the polyglutamine models reveal that upon SIRT6 deficiency, aggregation-prone stress results in higher aggregation. These roles of SIRT6 in proteostasis are well conserved from nematodes to humans.\u003c/p\u003e \u003cp\u003eAs we demonstrated, the observed reduced proteostasis is rescuable using translation attenuation, by allowing the system to regain the balance between protein synthesis and folding machinery. Importantly, the efficacy of the FDA-approved drug 4PBA suggests a clinically implementable pharmacological strategy to alleviate SIRT6 deficiency-driven protein misfolding conditions, whether in SIRT6-deficient patients or during the normal decline of SIRT6 during aging.\u003c/p\u003e \u003cp\u003eThe role of SIRT6 in regulating proteostasis is evolutionarily conserved \u0026ndash; its \u003cem\u003eC. elegans\u003c/em\u003e ortholog, \u003cem\u003esir-2.4\u003c/em\u003e, also has a critical role in translation and maintaining thermal resistance and motility in worms as they age. In reproducing nematodes, proteostasis is known to collapse between day 1 and day 2 of adulthood\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Here, we show that combining \u003cem\u003esir-2.4\u003c/em\u003e deletion and proteostatic stress precedes the collapse to day 1, pinpointing its critical role in proteostasis and health. From another evolutionary perspective, the different aspects of ribosome biogenesis and function can be coregulated by several master regulators \u0026ndash; one of them is SIRT6.\u003c/p\u003e \u003cp\u003ePrevious work found that DNA damage leads to a temporary transcription-translation stalling, followed by an increased protein synthesis after DNA damage was repaired \u0026ndash; lowering the chance of accumulating mutated RNA and proteins\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Here, we see that SIRT6KO leads to accelerated translation, bypassing this important regulatory mechanism of translation attenuation, even though DNA damage is known to be elevated in SIRT6 deficiency\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. We speculate that SIRT6 deletion is so deleterious to the organism, precisely because of the harsh imbalance it causes: SIRT6-deficient cells are in a state of chronic DNA damage, but as SIRT6 is a critical sensor of DNA double strand breaks, its absence impairs the repair\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Moreover, the absence of SIRT6 compromises also its role as a gene regulator, leading to epigenetic dysregulation and to nucleolar hyperactivity. Thus, the combination of DNA damage and hyper-translation could also result in the synthesis of mutated RNA molecules (from damaged DNA molecules), which further pushes the loss of proteostasis apparent in SIRT6KO. As SIRT6 activity declines with normal aging, and even further in AD patients, this implies a mechanism which feeds the loss of proteostasis \u0026ndash; in several ways \u0026ndash; in aging and age-related brain pathologies.\u003c/p\u003e \u003cp\u003eTo conclude, we show a novel role of SIRT6 in nucleolar regulation and proteostasis. This role explains how loss of proteostasis begins with nucleolar dysregulation, and how this regulation widely affects translation and proteostasis downstream, leading to the aggregates seen in age-related neurodegenerative diseases. We suggest that the FDA-approved 4PBA could provide a clinically accessible route for reversing the protein folding stress by mildly reducing protein translation and subsequently alleviating the neurodegenerative consequence of protein aggregates.\u003c/p\u003e "},{"header":"Methods","content":"\u003ch2\u003eGeneration of brain-specific SIRT6KO mice and SIRT6KO cells\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eBrain-specific SIRT6KO C57BL/6 mice were generated according to the protocol described in Sebasti\u0026aacute;n et al. (2012)\u003csup\u003e41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eSH-SY5Y, HeLa and HEK293T SIRT6KO or WT control cells were generated according to the protocol described in Kaluski et al. (2017)\u003csup\u003e20\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003ebrSIRT6KO brain transcriptomics and Alzheimer\u0026rsquo;s Disease patients\u0026rsquo; correlation\u003c/h2\u003e\n\u003cp\u003eThe brSIRT6KO mouse brain transcriptomic dataset was already published in GSE221077. The analysis was as previously described in Smirnov et al. (2023)\u003csup\u003e27\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe brSIRT6KO-AD transcriptomics GSEA correlation was conducted using the above-mentioned brSIRT6KO mouse brain transcriptomics dataset and the supplementary portrait published in Hill \u0026amp; Gammie (2022)\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eCell culture\u003c/h2\u003e\n\u003cp\u003eAll cells were grown in DMEM (catalog number: 41965039, Thermo-Fisher Gibco\u0026reg;, MA), supplemented with 1% L-glutamine (catalog number: 25030024, Thermo-Fisher Gibco\u0026reg;, MA), 1% Penicillin/Streptomycin antibiotics mix (catalog number: 15140122, Thermo-Fisher Gibco\u0026reg;, MA) and 10% FBS (catalog number: 12657-029, Thermo-Fisher Gibco\u0026reg;, MA). Incubation was 37\u0026deg;C, 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003ch2\u003eChromatin-bound protein acid extraction\u003c/h2\u003e\n\u003cp\u003eThe chromatin-bound proteins were extracted from brain tissues and cell culture dry pellets according to the following protocol:\u003c/p\u003e\n\u003cp\u003eBrain tissues and cell culture pellets were resuspended in cytoplasmic protein lysis buffer (described below), in a volume equivalent to 3 times the volume of the tissue. For tissues, the samples were homogenized for 2 cycles of 30 seconds in an electric homogenizer; for cell pellets, the samples were homogenized by thorough pipetting. Once homogenized, samples were kept on ice for 20 minutes, then centrifuged for 10 minutes, 21100g, 4\u0026deg;C. The supernatant, which contains the non-chromatin-bound fraction of proteins, was transferred into new tubes.\u003c/p\u003e\n\u003cp\u003eThe pellets (which contain the chromatin and cell leftovers) were washed twice with the same volume of cytoplasmic protein lysis buffer, incubated 5 minutes on ice and centrifuged for 5 minutes, 21100g, 4\u0026deg;C. then, Add 0.2N HCl solution to the dry pellet (equivalent of 1/8-1/10 of the original cytoplasmic protein lysis buffer volume) and pipette thoroughly. Samples were incubated on ice for 20 minutes with occasional vortexes, then centrifuged for 10 minutes, 21100g, 4\u0026deg;C. Supernatants (contain the chromatin-bound proteins) were transferred to new tubes and neutralized by adding 1M Tris pH8 (the same volumes as the 0.2N HCl), then vortexed. Protein concentrations were determined using Bradford assays.\u003c/p\u003e\n\u003cp\u003ecytoplasmic protein lysis buffer: 10 mM HEPES pH7.4, 10mM KCl, 0.05% NP-40, phosphatase inhibitor cocktail X1 (APExBIO K1013), 0.2mM PMSF, 0.0015mM trichostatin A.\u003c/p\u003e\n\u003ch2\u003eTotal protein extraction\u003c/h2\u003e\n\u003cp\u003eTotal protein extraction from brain tissues and cell culture dry pellets was done according to the following protocol:\u003c/p\u003e\n\u003cp\u003eBrain tissues and cell culture pellets were resuspended in RIPA lysis buffer, in a volume equivalent to 3 times the volume of the tissue. For tissues, the samples were homogenized for 2 cycles of 30 seconds in an electric homogenizer; for cell pellets, the samples were homogenized by thorough pipetting. Once homogenized, samples were kept on ice for 20 minutes, then centrifuged for 30 minutes, 21100g, 4\u0026deg;C. The supernatant, which contains the extracted proteins, was transferred to a new tube and protein concentrations were determined using Bradford assays.\u003c/p\u003e\n\u003ch2\u003eAntibody List\u003c/h2\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"662\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eAntibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eCompany\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eCatalog number\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003eDilution\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eTIP5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab195278\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eH3K56ac\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eActive Motif\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e39281\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:3000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eH3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003esc-10809\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:3000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eBrdU (FUrd)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eB8434-100UL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eNucleolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab22758\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eSNF2H\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eNovus Biologicals\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eNB100-55310\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eFibrillarin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab4566\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eNeuN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eFCMAB317PE\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003ePuromycin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eMillipore\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eMABE343\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:12,000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003e4EBP1-phosphorylated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eCell Signaling Technology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eCST9451\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eHSPA9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003esc-133137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eHSPD1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003esc-13115\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eHSP90\u0026beta;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eDevelopmental Studies Hybridoma Bank\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eH90-10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eCRYAB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSanta Cruz Biotechnology\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003esc-22744\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eDnaJB11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eProteintech\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e15484-1-AP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eHSPA2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eHPA000798-100UL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eGFP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eSigma-Aldrich\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e11814460001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eSIRT6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab88494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:1000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eRabbit anti-mouse IgG H\u0026amp;L (HRP)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab97046\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:10,000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eGoat anti rabbit IgG H\u0026amp;L (HRP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab6721\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:10,000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eAlexa Fluor 488 AffiniPure Donkey Anti-Mouse IgG (H+L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e715-545-150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eAlexa-Fluor 555 Donkey Anti-Mouse IgG H\u0026amp;L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eAbcam\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003eab150110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eAlexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e711-545-152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.64954682779456%\" valign=\"top\"\u003e\n \u003cp\u003eAlexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.54984894259819%\" valign=\"top\"\u003e\n \u003cp\u003eJackson ImmunoResearch\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"24.3202416918429%\" valign=\"top\"\u003e\n \u003cp\u003e711-585-152\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.48036253776435%\" valign=\"top\"\u003e\n \u003cp\u003e1:250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003ePlasmids and transfections\u003c/h2\u003e\n\u003cp\u003eTIP5-GFP plasmid \u0026ndash; addgene plasmid #65373 - https://www.addgene.org/65373/\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRenilla\u0026nbsp;\u003c/em\u003eLuciferase vector \u0026ndash; Promega pRL-null.\u003c/p\u003e\n\u003cp\u003eQ74-GFP plasmid \u0026ndash; addgene plasmid #40262 - https://www.addgene.org/40262/\u003c/p\u003e\n\u003cp\u003eQ0-GFP plasmid \u0026ndash; pEGFP-C1 backbone vector.\u003c/p\u003e\n\u003cp\u003eAll other plasmids were homemade: shGFP, shSNF2H, empty vector (CMV-flag backbone).\u003c/p\u003e\n\u003cp\u003eTransfections were conducted using PolyJet\u0026trade; In Vitro DNA Transfection Reagent (catalog number: SL100688, SignaGen\u0026reg; Laboratories, MD), according to manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\n\u003ch2\u003e4PBA treatment\u003c/h2\u003e\n\u003cp\u003eMedia with 4PBA were prepared as previously described in Stein et al. (2022)\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003e5-fluorouridine nucleolar transcription labelling\u003c/h2\u003e\n\u003cp\u003eNucleolar transcription labelling was conducted as previously described in Portillo et al. (2021)\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eSUnSET puromycin labelling\u003c/h2\u003e\n\u003cp\u003eThe SUnSET\u003csup\u003e37\u003c/sup\u003e experiment was done as previously described in Stein et al. (2022)\u003csup\u003e36\u003c/sup\u003e. Briefly, Cells were grown in regular growth conditions (see above) and plated in 6-well plates. On the following day, puromycin was added to a final concentration of 10 \u0026mu;g/ml directly to the cell media, then the cells were re-incubated in 37\u0026deg;C for 10-30 minutes, depending on cell line. Once puromycin labelling was done, cells were collected for total protein extraction (see above).\u003c/p\u003e\n\u003cp\u003eIn case 4PBA or transfections were conducted, they were done as previously described (stein et al.\u003csup\u003e36\u003c/sup\u003e; see above), and the puromycin labelling was done 24-48h after the treatment/transfection.\u003c/p\u003e\n\u003cp\u003eCycloheximide was used as a negative control, and was added 5 minutes before the puromycin labelling initiation, in a final concentration of 1-10 \u0026mu;g/ml.\u003c/p\u003e\n\u003ch2\u003eLuciferase assays\u003c/h2\u003e\n\u003ch2\u003eHeat shock Luciferase-based assays\u003c/h2\u003e\n\u003cp\u003e20k cells were plated in 48-well plates and 24h post-plating, transfected with Renilla plasmid (10%), supplemented with an empty vector (see above). 24h after transfections, cells were put in 42\u0026deg;C, 5% CO2 incubator for an hour, then in 37\u0026deg;C recovery for 90-120 minutes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLysis, luciferase substrates and luminescence readings were conducted using Promega \u003cem\u003eRenilla\u003c/em\u003e Luciferase Assay System kit (Promega E2820), according to manufacturer\u0026rsquo;s protocol. Protein refold capacity was calculated as the division of the \u003cem\u003eRenilla\u003c/em\u003e activity after heat shock-recovery in non-shocked activity. Importantly, since SIRT6-deficient cells present elevated translation, WT and KO cells were normalized to their own non-heat-shocked samples.\u003c/p\u003e\n\u003ch2\u003eStatistics\u003c/h2\u003e\n\u003cp\u003eAnalyses were done as described in the supplementary statistics table.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe Ben Gurion University of the Negev, together with the Israel animal ethics committee, has approved the animal protocol Brains specific SIRT6-KO effect on aging and neurodegeneration, authorization number IL-55-09-2023C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements and funding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 849029), the David and Inez Myers foundation, the Israeli Ministry of Science and Technology (MOST), the High-tech, Bio-tech and Negev fellowships of Kreitman School of Advanced Research of Ben-Gurion University.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlzheimer\u0026rsquo;s Association. 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Rep.\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e, 17189 (2022).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4215918/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4215918/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAn important hallmark of aging \u0026ndash; and particularly of neurodegeneration \u0026ndash; is the loss of proteostasis, which often leads to cellular stress responses and even cell death. However, the causal mechanisms driving proteostasis are unclear. Here, we show that SIRT6 has a critical role in maintaining proteostasis. It negatively regulates global translation by controlling ribosomal genes, nucleolar function and TIP5 chromatin localization. SIRT6 deletion dramatically increases nucleolar size, rRNA production and protein translation. However, the expression of protein-folding genes remains unchanged, failing to compensate for excessive translation, hence leading to reduced protein folding capacity and the production of aggregates. \u003cem\u003eIn vivo\u003c/em\u003e, we establish a \u003cem\u003eC. elegans\u003c/em\u003e model (\u003cem\u003esir-2.4\u003c/em\u003e KO) that shows reduced heat shock resistance and an accelerated age-dependent reduction in motility. \u003cem\u003eSir-2.4\u003c/em\u003e depletion in a neuron-specific protein aggregation-prone polyQ strain led to premature motility loss indicative of motor neuron dysfunction. These results point to proteostasis-stress intolerance in the absence of the SIRT6 ortholog that can be rescued by pharmacologically reducing protein translation rates. Together, our data suggest that SIRT6 deficiency in aging and neurodegeneration contributes to proteostasis loss through gene dysregulation of nucleolar function and the translation machinery. These results highlight that deficient proteostasis is the consequence of chromatin dysregulation that ultimately leads to neurodegeneration.\u003c/p\u003e","manuscriptTitle":"SIRT6 regulates protein synthesis and folding through nucleolar remodeling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-29 05:52:15","doi":"10.21203/rs.3.rs-4215918/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b92046ce-de34-4b64-b20f-5e73e1cb599d","owner":[],"postedDate":"May 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":30751634,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":30751635,"name":"Biological sciences/Molecular biology/Nuclear organization"},{"id":30751636,"name":"Biological sciences/Molecular biology/Protein folding/Protein aggregation"}],"tags":[],"updatedAt":"2024-05-29T05:52:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-29 05:52:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4215918","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4215918","identity":"rs-4215918","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}