DNA double-strand breaks (DSB) are the most deleterious type of DNA damage. In this work, we show that SIRT6 directly recognizes DNA damage through a tunnel-like structure that has high affinity for ...DSB. SIRT6 relocates to sites of damage independently of signaling and known sensors. It activates downstream signaling for DSB repair by triggering ATM recruitment, H2AX phosphorylation and the recruitment of proteins of the homologous recombination and non-homologous end joining pathways. Our findings indicate that SIRT6 plays a previously uncharacterized role as a DNA damage sensor, a critical factor in initiating the DNA damage response (DDR). Moreover, other Sirtuins share some DSB-binding capacity and DDR activation. SIRT6 activates the DDR before the repair pathway is chosen, and prevents genomic instability. Our findings place SIRT6 as a sensor of DSB, and pave the road to dissecting the contributions of distinct DSB sensors in downstream signaling.
During an organism’s lifespan, two main phenomena are critical for the organism’s survival. These are (1) a proper embryonic development, which permits the new organism to function with high fitness, ...grow and reproduce, and (2) the aging process, which will progressively undermine its competence and fitness for survival, leading to its death. Interestingly these processes present various similarities at the molecular level. Notably, as organisms became more complex, regulation of these processes became coordinated by the brain, and failure in brain activity is detrimental in both development and aging. One of the critical processes regulating brain health is the capacity to keep its genomic integrity and epigenetic regulation—deficiency in DNA repair results in neurodevelopmental and neurodegenerative diseases. As the brain becomes more complex, this effect becomes more evident. In this perspective, we will analyze how the brain evolved and became critical for human survival and the role Sirt6 plays in brain health. Sirt6 belongs to the Sirtuin family of histone deacetylases that control several cellular processes; among them, Sirt6 has been associated with the proper embryonic development and is associated with the aging process. In humans, Sirt6 has a pivotal role during brain aging, and its loss of function is correlated with the appearance of neurodegenerative diseases such as Alzheimer’s disease. However, Sirt6 roles during brain development and aging, especially the last one, are not observed in all species. It appears that during the brain organ evolution, Sirt6 has gained more relevance as the brain becomes bigger and more complex, observing the most detrimental effect in the brains of
Homo sapiens
. In this perspective, we part from the evolution of the brain in metazoans, the biological similarities between brain development and aging, and the relevant functions of Sirt6 in these similar phenomena to conclude with the evidence suggesting a more relevant role of Sirt6 gained in the brain evolution.
The histone deacetylase SIRT6 promotes DNA repair, but its activity declines with age with a concomitant accumulation of DNA damage. Furthermore, SIRT6 knockout mice exhibit an accelerated aging ...phenotype and die prematurely. Here, we report that brain-specific SIRT6-deficient mice survive but present behavioral defects with major learning impairments by 4 months of age. Moreover, the brains of these mice show increased signs of DNA damage, cell death, and hyperphosphorylated Tau—a critical mark in several neurodegenerative diseases. Mechanistically, SIRT6 regulates Tau protein stability and phosphorylation through increased activation of the kinase GSK3α/β. Finally, SIRT6 mRNA and protein levels are reduced in patients with Alzheimer’s disease. Taken together, our results suggest that SIRT6 is critical to maintain genomic stability in the brain and that its loss leads to toxic Tau stability and phosphorylation. Therefore, SIRT6 and its downstream signaling could be targeted in Alzheimer’s disease and age-related neurodegeneration.
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•SIRT6 loss causes pathological changes in behavior, DNA damage, apoptosis, and Tau•Tau is stabilized and hyperphosphorylated by GSK3 in the absence of SIRT6•Phenotypes in SIRT6 knockout cells can be rescued by GSK3 or ATM inhibition•Patients with Alzheimer’s disease show a severe decrease in SIRT6 levels
Kaluski et al. show that lack of SIRT6 in the brain provokes neurodegeneration by increasing DNA damage, apoptosis, and toxic Tau phosphorylation. DNA damage or lack of SIRT6 activates GSK3, resulting in Tau phosphorylation and stability. GSK3 inhibition rescues this phenotype. Importantly, AD patients show reduced SIRT6 in the brain.
The SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to ...neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.
Neurodegenerative diseases are caused by the loss of neuro- nal cells. As neurons deteriorate, a patient may experience relatively mild symptoms such as failing to name objects and people. However, ...with the progression of the disease, symp- toms worsen resulting in loss of independence and a sense of＂loss of oneself＂. Finally, the patient dies from a variety of complications.
Signs of pervasive developmental disorder and social deficits were reported in toddlers and children whose mothers were exposed to organophosphate pesticides during pregnancy. Deficits in social ...preference were reported in adult male mice exposed to chlorpyrifos on gestational days 12-15. This study aimed (a) to test the hypothesis that adult female and male mice that were exposed prenatally to subtoxic doses of chlorpyrifos would be impaired in social behavior and (b) to determine if prenatal chlorpyrifos altered the expression of transcripts for oxytocin in the hypothalamus. Pregnant mice were treated by gavage with corn oil vehicle or 2.5 mg/kg or 5 mg/kg of CPF on gestational days 12-15. Social preference, social and non-social conditioned place preference tasks were tested in adults. Expression of oxytocin transcripts in hypothalamus was measured by qPCR.
Chlorpyrifos (5 mg/kg on GD 12-15) reduced the innate preference for a conspecific in a dose and sex dependent manner. Adult males exposed prenatally to 5 mg/kg CPF showed a reduction in social preference. Socially conditioned place preference was impaired in offspring of dams treated with either dose of CPF. Non-social appetitive place conditioning was impaired in offspring of dams exposed to 2.5 mg/kg, but not to 5 mg/kg chlorpyrifos. Prenatal chlorpyrifos treatment did not alter the expression of the oxytocin mRNA in the hypothalamus, although expression was significantly lower in females.
Prenatal chlorpyrifos induced innate and learned social deficits and non-specific conditioning deficits in adult mice in a sex-dependent manner. Males showed specific social deficits following the higher dose whereas both males and females showed a more generalized conditioning deficit following the intermediate dose.
Alzheimer's disease (AD) involves loss of cholinergic neurons and Tau protein hyper-phosphorylation. Here, we report that overexpression of an N-terminally extended "synaptic" acetylcholinesterase ...variant, N-AChE-S is causally involved in both these phenomena.
In transfected primary brain cultures, N-AChE-S induced cell death, morphological impairments and caspase 3 activation. Rapid internalization of fluorescently labeled fasciculin-2 to N-AChE-S transfected cells indicated membranal localization. In cultured cell lines, N-AChE-S transfection activated the Tau kinase GSK3, induced Tau hyper-phosphorylation and caused apoptosis. N-AChE-S-induced cell death was suppressible by inhibiting GSK3 or caspases, by enforced overexpression of the anti-apoptotic Bcl2 proteins, or by AChE inhibition or silencing. Moreover, inherent N-AChE-S was upregulated by stressors inducing protein misfolding and calcium imbalances, both characteristic of AD; and in cortical tissues from AD patients, N-AChE-S overexpression coincides with Tau hyper-phosphorylation.
Together, these findings attribute an apoptogenic role to N-AChE-S and outline a potential value to AChE inhibitor therapeutics in early AD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Retinal Müller glial cells (MGs) are among the first to demonstrate metabolic changes during retinal disease and are a potential source of regenerative cells. In response to a harmful stimulus, they ...can dedifferentiate acquiring neural stem cells properties, proliferate and migrate to the damaged retinal layer and differentiate into lost neurons. However, it is not yet known how this reprogramming process is regulated in mammals. Since glucose and oxygen are important regulatory elements that may help directing stem cell fate, we aimed to study the effect of glucose variations and oxidative stress in Müller cells reprogramming capacity and analyze the participation the histone deacetylase SIRT6, as an epigenetic modulator of this process. We found that the combination of high glucose and oxidative stress induced a decrease in the levels of the marker glutamine synthetase, and an increase in the migration capacity of the cells suggesting that these experimental conditions could induce some degree of dedifferentiation and favor the migration ability. High glucose induced an increase in the levels of the pluripotent factor SOX9 and a decrease in SIRT6 levels accompanied by the increase in the acetylation levels of H3K9. Inhibiting SIRT6 expression by siRNA rendered an increase in SOX9 levels. We also determined SOX9 levels in retinas from mice with a conditional deletion of SIRT6 in the CNS. To further understand the mechanisms that regulate MGs response under metabolic impaired conditions, we evaluated the gene expression profile and performed Gene Ontology enrichment analysis of Müller cells from a murine model of Diabetes. We found several differentially expressed genes and observed that the transcriptomic change involved the enrichment of genes associated with glucose metabolism, cell migration, development and pluripotency. We found that many functional categories affected in cells of diabetic animals were directly related to SIRT6 function. Transcription factors enrichment analysis allowed us to predict several factors, including SOX9, that may be involved in the modulation of the differential expression program observed in diabetic MGs. Our results underline the heterogeneity of Müller cells response and the challenge that the study of metabolic impairment
in vivo
represents.
Abstract Down syndrome (DS) associates with impaired brain functions, but the underlying mechanism(s) are yet unclear. The “gene dosage” hypothesis predicts that in DS, overexpression of a single ...gene can impair multiple brain functions through a signal amplification effect due to impaired regulatory mechanism(s). Here, we report findings attributing to impairments in the splicing process such a regulatory role. We have used DS fetal brain samples in search for initial evidence and employed engineered mice with MMU16 partial trisomy (Ts65Dn) or direct excess of the splicing-associated nuclear kinase Dyrk1A, overdosed in DS for further analyses. We present specific albeit modest changes in the DS brain's splicing machinery with subsequently amplified effects in target transcripts; and we demonstrate that engineered excess of Dyrk1A can largely recapitulate these changes. Specifically, in both the fetal DS brains and the Dyrk1A overdose models, we found ample modestly modified splicing-associated transcripts which apparently induced secondary enhancement in exon inclusion of key synaptic transcripts. Thus, DS-reduced levels of the dominant-negative TRKBT1 transcript, but not other TRKB mRNA transcripts, were accompanied by corresponding decreases in BDNF. In addition, the DS brains and Dyrk1A overdosage models showed selective changes in the transcripts composition of neuroligin mRNAs as well as reductions in the “synaptic” acetylcholinesterase variant AChE-S mRNA and corresponding increases in the stress-inducible AChE - R mRNA variant, yielding key synaptic proteins with unusual features. In cotransfected cells, Dyrk1A overdosage caused parallel changes in the splicing pattern of an AChE mini-gene, suggesting that Dyrk1A overdosage is both essential and sufficient to induce the observed change in the composition of AChE mRNA variants. Furthermore, the Dyrk1A overdosage animal models showed pronounced changes in the structure of neuronal nuclear speckles, where splicing events take place and in SR proteins phosphorylation known to be required for the splicing process. Together, our findings demonstrate DS-like brain splicing machinery malfunctioning in Dyrk1A overexpressing mice. Since individual splicing choices may alter cell fate determination, axon guidance, and synaptogenesis, these findings suggest the retrieval of balanced splicing as a goal for DS therapeutic manipulations early in DS development.
The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated ...with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).
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•Isolation of nuclei from mouse cortical tissue•Immunolabeling of nuclei and sorting by FACS•Fixed nuclei protocol•Preparation of ATAC-seq and Hi-C libraries
The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq and chromatin conformation by Hi-C.
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