INDUCTION OF AUTOPHAGY BY SPERMIDINE PROMOTES LONGEVITY PDF

Metrics details Abstract Ageing results from complex genetically and epigenetically programmed processes that are elicited in part by noxious or stressful events that cause programmed cell death. Here, we report that administration of spermidine, a natural polyamine whose intracellular concentration declines during human ageing, markedly extended the lifespan of yeast, flies and worms, and human immune cells. In addition, spermidine administration potently inhibited oxidative stress in ageing mice. In ageing yeast, spermidine treatment triggered epigenetic deacetylation of histone H3 through inhibition of histone acetyltransferases HAT , suppressing oxidative stress and necrosis.

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Natural products Abstract Spermidine, a natural polyamine presented widely in mammalian cells, has been implicated to extend the lifespan of several model organisms by inducing autophagy. However, the effect of spermidine against neuronal damage has not yet been fully determined. We found that STS-induced cell injury could be efficiently attenuated by pretreatment with 1 mM spermidine. Spermidine inhibited the caspase 3 activation induced by STS. Moreover, STS incubation resulted in autophagic degradation failure, which could be attenuated by the pretreatment of spermidine.

Knocking down the expression of Beclin 1 efficiently suppressed autophagosome and autolysosome accumulation, and abolished the protective effects of spermidine against STS-induced neurotoxicity.

Increased Beclin 1 cleavage and partial nuclear translocation of Beclin 1 fragment was detected in STS-treated cells, which could be blocked by spermidine, pan-caspase inhibitor or caspase 3-specific inhibitor. The nuclear translocation of Beclin 1 fragment universally occurs in damaged neurons. Beclin 1 mutation at the sites of and prevented the intracellular re-distribution of Beclin 1 induced by STS.

Our findings suggest that caspase 3-mediated Beclin 1 cleavage occurs in acute neuronal cell injury both in vitro and in vivo. The neuroprotective effect of spermidine may be related to inhibition of the caspase 3-mediated Beclin 1 cleavage and restoration of the Beclin 1-dependent autophagy. Download PDF Main Spermidine, a naturally formed polyamine in mammalian living cells, has crucial roles in various cellular processes under pathophysiological conditions.

Autophagy and apoptosis are well-characterized processes that contribute to the maintenance of cellular and tissue homeostasis. The crosstalk between autophagy and apoptosis has been documented in various physiological and pathological conditions; in agreement with this notion, several crucial molecules have been identified as the points of convergence between two pathways.

In this study, staurosporine STS was adopted to induce acute neuronal injury in cultured PC12 cells and cortical neurons, and the potential neuroprotective effects of spermidine against STS were clarified. Our findings indicate that spermidine may represent a promising natural compound for the treatment of neurological diseases. A profound loss of mitochondrial membrane potential was detected in cells exposed to STS Figures 1a and c. Moreover, treatment with spermidine alone at the dose of 1 mM for 2 h had no significant influences on cells.

Similar to that seen in PC12 cells, spermidine exerted prominent neuroprotection in primary cortical cultures against STS treatment Figures 1a and c. Figure 1 Spermidine prevented STS-induced neuronal cell death. Untreated cells were used as control Ctrl. Cells were stained with Rhodamine Rh for analyzing the mitochondrial membrane potential.

For each group, 60 cells were utilized for analysis and data were calculated from three independent experiments. The black and blue markers indicate the statistical significance of the data when comparing neuritic length and nuclear diameter, respectively.

For each group, 15 images were used for analysis and data were calculated from three independent experiments. Immunostaining studies, using an anti-cleaved activated caspase 3 antibody, showed strong immunoreactivity in cells exposed to STS, in comparison with the weak staining in other experimental groups Figure 2e.

Pretreatment with spermidine significantly declined the percentage of cells expressing cleaved caspase 3 STS, The endogenous protein levels of the pro-caspase 3 remained unchanged through all treatments Figure 2f. However, increased caspase 3 cleavage was detected in STS-treated cells, and administration of spermidine or spermidine plus an pan-caspase inhibitor z-VAD-fmk blocked the caspase 3 activation. Figure 2 Spermidine inhibits caspase 3 activity. Representative data were presented.

Data were calculated from six independent experiments. Data were calculated from three independent experiments. Nuclei were counterstained with Hoechst GAPDH was used as internal control. At least three independent experiments were conducted and representative immunoblots were presented. CasI, caspase inhibitor z-VAD-fmk.

Second, autophagic flux was traced by transfecting cells with an mRFP-GFP-LC3 plasmid according to updated guidelines for the use and interpretations of assays for monitoring autophagy. Incubation with STS for 0. Combination of STS and an autophagy blocker CQ or BafA1 also resulted in autophagic degradation failure, which could be attenuated by the pretreatment of spermidine. The LC3-II level remained high in the presence of STS and spermidine, which may be explained by the autophagic flux promotion capacity of spermidine.

Interestingly, Beclin 1 cleavage occurred in cells exposed to STS, which could be reversed by the addition of spermidine. To illustrate whether or not STS-induced autophagy activation depends on Beclin 1, RNA interference was used to knockdown the intracellular Beclin 1 expression. Knocking down of Beclin 1 not only decreased baseline autophagy activation, but also strikingly suppressed STS-induced accumulation of LC3-positive puncta Figures 4a and b , indicating the autophagic activity depends on Beclin 1.

However, deficient in Beclin 1 expression could not suppress cell injury and mitochondrial membrane potential loss resulted from STS exposure Figure 4c. Moreover, the protective effect of spermidine seems to be dependent on autophagy, as silencing of Beclin 1 abolished the neuroprotection of spermidine against STS. Collectively, these data suggest that spermidine prevents STS-induced cell death possibly through augmentation of Beclin 1-dependent autophagic flux. For each group, 15 cells were utilized for analysis and data were calculated from three independent experiments.

At least three independent experiments were conducted and representative immunoblots were presented Full size image Figure 4 Influences of Beclin 1 silencing on STS-induced autophagy. After 48 h, total protein was extracted for western blotting analysis. After 48 h, some of the cells were exposed to STS. Cells were exposed to STS in the presence or absence of spermidine.

The caspase cleavage sites in Beclin 1 are highly conserved in diverse species Supplementary Figure S2. The iCasper was reported to become infrared fluorescent when cells underwent apoptotic cell death, and the number of cells expressing infrared fluorescent increased with time following STS incubation 20 see also Supplementary Figures S5. When the cleavage sequence is recognized and cleaved, infrared fluorescent would be detected. Data were calculated from five independent experiments.

The fluorescence distribution was examined under fluorescence microscope equipped with a live cell station. Images of single cell before or after STS treatment were presented.

For each group, 10 images were used for analysis and data were calculated from three independent experiments. Cells with infrared fluorescence signals were indicated by arrows. However, pretreatment with spermidine abolished the intracellular re-distribution of Beclin 1, possibly resulted from the blockage of Beclin 1 cleavage.

Conversely, immunocytochemical studies using an antibody against the C-terminal Beclin 1 showed an apparently different expression pattern of C-terminus in cells exposed to STS, as C-terminal Beclin 1 was restricted in cytoplasm of most STS-treated cells.

Note that inhibition of caspase 3 also blocked the nuclear translocation of Beclin 1 in cells treated with STS Supplementary Figures S6. The nuclear translocation of Beclin 1 in damaged neurons appears to be a universal event among diversity of experimental paradigms. Figure 7 Nuclear translocation of Beclin 1 in damaged neurons. The nuclei were counterstained with Hoechst Curves indicated the colocalization between Beclin 1 green and Hoechst blue and correlated to the lines drawn in the enlarged images.

The x axis represented the distance inches along the line and the y axis indicated the pixel intensity. Brain slides were immunostained with anti-Beclin 1 N terminal antibody and the nuclei were counterstained with hematoxylin. Administration of spermidine relieved neuronal damage in both hippocampus and cortex.

These data were in accordance with in vitro studies and collectively suggest that the neuroprotective function of spermidine may be yielded through inhibiting caspase 3-mediated Beclin 1 cleavage. Brain samples were removed from Sham-operated animals at 3 days after surgery. For each group, at least six animals were included; for each animal, at least four brain slices were used for analysis. Black arrows indicate the c-Cas3-positive cells, and white arrows indicate cells with nuclear translocation of Beclin 1 Full size image Discussion In this study, we investigated the involvement of caspase 3-mediated Beclin 1 cleavage during neuronal cell injury using cultured neuronal cells and rodents brain samples.

Our results showed that Beclin 1 cleavage followed by partial nuclear translocation of N-terminal fragments occurred universally in damaged neuronal cells. Spermidine exerts neuroprotection in diverse experimental paradigms The endogenous levels of polyamines, especially spermidine, decline continuously with age. Although the blood—brain barrier transport of spermidine is reported to be quite limited under physiological condition, 24 Gilad et al.

Such phenomenon may be owned to the ability of spermidine in increasing the blood—brain barrier permeability under pathological states.

One limitation of this study is the absence of spermidine treatment group on Sham-operated control rats, and therefore we could not speculate the potential effects of spermidine on normal animals. Spermidine restores autophagic flux Emerging lines of evidence highlight the autophagy enhancing capacity of spermidine.

To our knowledge, we here for the first time demonstrated that spermidine prevented caspase 3-mediated Beclin 1 cleavage and favored neuronal cell survival in vitro and in vivo. Silencing of Beclin 1 abolished the neuroprotective action of spermidine against STS incubation. These results were in contrast with previous report, which shows a functional role of Beclin 1-independent autophagy in sensitizing cortical neurons to STS-induced apoptosis. However, we could not exclude the possibility that spermidine excerts its neuroprotective function via preserving the cleavage of other caspase 3 substrates apart from Beclin 1, including autophagy proteins, such as Atg7.

It is reported that spermine induces caspase 3 activation and triggers cell death through evoking cytochrome C release from mitochondria. Here, caspase 3 activation, an early event of cell apoptosis, occurred in PC12 cells at the early stage of STS incubation, whereas no abundant pycnotic nuclei were detected.

In accordance with this result, it has been demonstrated that STS incubation led to early autophagy induction followed by apoptosis in HeLa cells. Cell-free in vitro studies reveal that Beclin 1 can be cleaved by several cell death proteases, such as caspase 3, caspase 6, caspase 8 and calpain 1.

Moreover, given that administration of pan-caspase inhibitor or caspase 3-specific inhibitor efficiently suppressed Beclin 1 cleavage, we speculate the caspase 3-induced Beclin 1 cleavage may have a pivotal role in damaged PC12 cells. Characterization of the crystal structure of the Beclin 1 sequence comprising DQLD may offer insights into the preference of caspase cleavage in this site. Nuclear translocation of Beclin 1N terminal fragment during neuronal injury Beclin 1 is mainly expressed in the cytoplasm of healthy neuronal cells.

In cultured cells, as well as rodent brain tissues, a partial nuclear localization of Beclin 1 was coincidently detected in damaged neuronal cells. One possible explanation is the cleavage occurs at the site before aa, as Beclin 1 contains a leucine-rich nuclear export signal at amino acids —

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Induction of autophagy by spermidine promotes longevity

Nat Cell Biol. Epub Oct 4. Induction of autophagy by spermidine promotes longevity. Comment in Nat Cell Biol.

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Spermidine Promotes Cardioprotective Autophagy

Natural products Abstract Spermidine, a natural polyamine presented widely in mammalian cells, has been implicated to extend the lifespan of several model organisms by inducing autophagy. However, the effect of spermidine against neuronal damage has not yet been fully determined. We found that STS-induced cell injury could be efficiently attenuated by pretreatment with 1 mM spermidine. Spermidine inhibited the caspase 3 activation induced by STS. Moreover, STS incubation resulted in autophagic degradation failure, which could be attenuated by the pretreatment of spermidine. Knocking down the expression of Beclin 1 efficiently suppressed autophagosome and autolysosome accumulation, and abolished the protective effects of spermidine against STS-induced neurotoxicity. Increased Beclin 1 cleavage and partial nuclear translocation of Beclin 1 fragment was detected in STS-treated cells, which could be blocked by spermidine, pan-caspase inhibitor or caspase 3-specific inhibitor.

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Induction of autophagy by spermidine promotes longevity.

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