Decreased cognitive performance is a hallmark of brain aging, but the underlying mechanisms and potential therapeutic avenues remain poorly understood. Recent studies have revealed health-protective and lifespan-extending effects of dietary spermidine, a natural autophagy-promoting polyamine. Here, we show that dietary spermidine passes the blood-brain barrier in mice and increases hippocampal eIF5A hypusination and mitochondrial function. Spermidine feeding in aged mice affects behavior in homecage environment tasks, improves spatial learning, and increases hippocampal respiratory competence. In a Drosophila aging model, spermidine boosts mitochondrial respiratory capacity, an effect that requires the autophagy regulator Atg7 and the mitophagy mediators Parkin and Pink1. Neuron-specific Pink1 knockdown abolishes spermidine-induced improvement of olfactory associative learning. This suggests that the maintenance of mitochondrial and autophagic function is essential for enhanced cognition by spermidine feeding. Finally, we show large-scale prospective data linking higher dietary spermidine intake with a reduced risk for cognitive impairment in humans.

As our society ages, neurodegenerative disorders like Parkinson`s disease (PD) are increasing in pandemic proportions. While mechanistic understanding of PD is advancing, a treatment with well tolerable drugs is still elusive. Here, we show that administration of the naturally occurring polyamine spermidine, which declines continuously during aging in various species, alleviates a series of PD-related degenerative processes in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology. In the fruit fly, simple feeding with spermidine inhibited loss of climbing activity and early organismal death upon heterologous expression of human α-synuclein, which is thought to be the principal toxic trigger of PD. In this line, administration of spermidine rescued α-synuclein-induced loss of dopaminergic neurons, a hallmark of PD, in nematodes. Alleviation of PD-related neurodegeneration by spermidine was accompanied by induction of autophagy, suggesting that this cytoprotective process may be responsible for the beneficial effects of spermidine administration.

Spermidine and other polyamines alleviate oxidative stress, yet excess spermidine seems toxic to Escherichia coli unless it is neutralized by SpeG, an enzyme for the spermidine N-acetyl transferase function. Thus, wild-type E. coli can tolerate applied exogenous spermidine stress, but ΔspeG strain of E. coli fails to do that. Here, using different reactive oxygen species (ROS) probes and performing electron paramagnetic resonance spectroscopy, we provide evidence that although spermidine mitigates oxidative stress by lowering overall ROS levels, excess of it simultaneously triggers the production of superoxide radicals, thereby causing toxicity in the ΔspeG strain. Furthermore, performing microarray experiment and other biochemical assays, we show that the spermidine-induced superoxide anions affected redox balance and iron homeostasis. Finally, we demonstrate that while RNA-bound spermidine inhibits iron oxidation, free spermidine interacts and oxidizes the iron to evoke superoxide radicals directly. Therefore, we propose that the spermidine-induced superoxide generation is one of the major causes of spermidine toxicity in E. coli.

Background The protective effects of polyamines on cardiovascular disease have been demonstrated in many studies. However, the roles of spermidine, a natural polyamine, in abdominal aortic aneurysm (AAA) disease have not been studied. In this study, we investigated the influence and potential mechanisms of spermidine treatment on experimental AAA disease. Methods and Results Experimental AAAs were induced in 8- to 10-week-old male C57BL/6J mice by transient intra-aortic infusion of porcine pancreatic elastase. Spermidine was administered via drinking water at a concentration of 3 mmol/L. Spermidine treatment prevented experimental AAA formation with preservation of medial elastin and smooth muscle cells. In immunostaining, macrophages, T cells, neutrophils, and neovessels were significantly reduced in aorta of spermidine-treated, as compared with vehicle-treated elastase-infused mice. Additionally, flow cytometric analysis showed that spermidine treatment reduced aortic leukocyte infiltration and circulating inflammatory cells. Furthermore, we demonstrated that spermidine treatment promoted autophagy-related proteins in experimental AAAs using Western blot analysis, immunostaining, and transmission electron microscopic examination. Autophagic function was evaluated for human abdominal aneurysmal and nonaneurysmal adjacent aortae from AAA patients using Western blot analysis and immunohistochemistry. Dysregulated autophagic function, as evidenced by increased SQSTM1/p62 protein and phosphorylated mTOR, was found in aneurysmal, as compared with nonaneurysmal, aortic segments. Conclusions Our results suggest that spermidine supplementation limits experimental AAA formation associated with preserved aortic structural integrity, attenuated aortic inflammatory infiltration, reduced circulating inflammatory monocytes, and increased autophagy-related proteins. These findings suggest that spermidine may be a promising treatment for AAA disease.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3, is a fatal neurodegenerative disease that causes loss of balance and motor co-ordination, eventually leading to paralysis. It is caused by the autosomal dominant inheritance of a long CAG trinucleotide repeat sequence within the ATXN3 gene, encoding for an expanded polyglutamine (polyQ) repeat sequence within the ataxin-3 protein. Ataxin-3 containing an expanded polyQ repeat is known to be highly prone to intraneuronal aggregation, and previous studies have demonstrated that protein quality control pathways, such as autophagy, are impaired in MJD patients and animal models of the disease. In this study, we tested the therapeutic potential of spermidine on zebrafish and rodent models of MJD to determine its capacity to induce autophagy and improve functional output. Spermidine treatment of transgenic MJD zebrafish induced autophagy and resulted in increased distances swum by the MJD zebrafish. Interestingly, treatment of the CMVMJD135 mouse model of MJD with spermidine added to drinking water did not produce any improvement in motor behaviour assays, neurological testing or neuropathology. In fact, wild type mice treated with spermidine were found to have decreased rotarod performance when compared to control animals. Immunoblot analysis of protein lysates extracted from mouse cerebellar tissue found little differences between the groups, except for an increased level of phospho-ULK1 in spermidine treated animals, suggesting that autophagy was indeed induced. As we detected decreased motor performance in wild type mice following treatment with spermidine, we conducted follow up studies into the effects of spermidine treatment in zebrafish. Interestingly, we found that in addition to inducing autophagy, spermidine treatment also induced apoptosis, particularly in wild type zebrafish. These findings suggest that spermidine treatment may not be therapeutically beneficial for the treatment of MJD, and in fact warrants caution due to the potential negative side effects caused by induction of apoptosis.

Previous studies have highlighted that spermidine has the ability to trigger the important process of dissolving amyloid-beta plaques by autophagy. This manuscript focuses on the correlation of serum spermidine levels between age and between performance in mini-mental state examinations. It will serve as a premise for an ongoing multicentric placebo-controlled study, which focuses on the effect of oral spermidine supplementation on memory performance. Memory tests were carried out on 80 subjects aged 60-96 years old in 6 nursing homes in Styria. Blood samples were taken for the determination of spermidine concentration. The results showed a significant correlation between the spermidine concentration and the mini-mental state examination score (p = 0.025). On the basis of the dependence demonstrated it can be concluded that spermidine might be suitable as a biomarker for the diagnosis of neurocognitive changes (senile dementia or Alzheimer's disease).

The hallmark of cisplatin-induced acute kidney injury is the necrotic cell death in the kidney proximal tubules. However, an effective approach to limit cisplatin nephrotoxicity remains unknown. Spermidine is a polyamine that protects against oxidative stress and necrosis in aged yeasts, and the present study found that exogenous spermidine markedly attenuated tubular necrosis and kidney dysfunction, but not apoptosis, during cisplatin nephrotoxicity. In addition, exogenous spermidine potently inhibited oxidative/nitrative DNA damage, poly(ADP-ribose) polymerase 1 (PARP1) activation and ATP depletion after cisplatin injection. Conversely, inhibition of ornithine decarboxylase (ODC) via siRNA transfection in vivo significantly increased DNA damage, PARP1 activation and ATP depletion, resulting in acceleration of tubular necrosis and kidney dysfunction. Finally, exogenous spermidine removed severe cisplatin injury induced by ODC inhibition. In conclusion, these data suggest that spermidine protects kidneys against cisplatin injury through DNA damage and tubular necrosis, and this finding provides a novel target to prevent acute kidney injury including nephrotoxicity.

Asthma is a heterologous disease that is influenced by complex interactions between multiple environmental exposures, metabolism, and host immunoregulatory processes. Specific metabolites are increasingly recognized to influence respiratory inflammation. However, the role of protein-derived metabolites in regulating inflammatory responses in the lung are poorly described. The aims of the present study were to quantify polyamine levels in bronchoalveolar lavages (BALs) from healthy volunteers and asthma patients, and to evaluate the impact of each polyamine on inflammatory responses using in vitro models and in a house dust mite (HDM)-induced respiratory allergy model. Spermidine levels were decreased, while cadaverine levels were increased in BALs from asthma patients compared to healthy controls, using Ultra Performance Liquid Chromatography (UPLC). Both spermine and spermidine inhibit lipopolysaccharide (LPS)-induced cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs) in vitro. In addition, oral gavage with spermine or spermidine modulate HDM-induced cell infiltration, cytokine secretion, and epithelial cell tight junction expression in murine models. Spermidine also reduces airway hyper-responsiveness. These results suggest that modulation of polyamine metabolism, in particular spermidine, is associated with respiratory inflammation and these molecules and pathways should be further explored as biomarkers of disease and potential targets for novel therapies.

1. A sensitive and specific assay for spermidine oxidase is described. The method involves the separation of [14C]spermidine (substrate) from [14C]putrescine (product) and other 14C-labelled products on a Dowex 50 cation-exchange column: 92% of the putrescine applied to the column was eluted by 2.3 M-HCl, but this treatment left 96% of the spermidine bound to the column. Unchanged spermidine could be removed from the column by elution with 6 M-HCl. 2. By means of this assay, foetal and adult bovine serum were each shown to contain spermidine oxidase activity, putrescine being a major product of the oxidation of spermidine by the serum enzymes. 3. In foetal bovine serum, spermidine oxidase activity is separable from putrescine oxidase activity by chromatography on a cadaverine-Sephadex column, by gel filtration and by ion-exchange column chromatography. Putrescine oxidase was purified 1900-fold and spermidine oxidase 130-fold by these procedures. The former oxidized putrescine but not spermidine, and spermidine oxidase exhibited no activity with putrescine as substrate.

The cross-talk between the host and its microbiota plays a key role in the promotion of health. The production of metabolites such as polyamines by intestinal-resident bacteria is part of this symbiosis shaping host immunity. The polyamines putrescine, spermine, and spermidine are abundant within the gastrointestinal tract and might substantially contribute to gut immunity.

Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT.

Spermidine is a polyamine molecule that performs various cellular functions, such as DNA and RNA stabilization, autophagy modulation, and eIF5A formation, and is generated from putrescine by aminopropyltransferase spermidine synthase (SpdS). During synthesis, the aminopropyl moiety is donated from decarboxylated S-adenosylmethionine to form putrescine, with 5'-deoxy-5'-methylthioadenosine being produced as a byproduct. Although the molecular mechanism of SpdS function has been well-established, its structure-based evolutionary relationships remain to be fully understood. Moreover, only a few structural studies have been conducted on SpdS from fungal species. Here, we determined the crystal structure of an apo-form of SpdS from Kluyveromyces lactis (KlSpdS) at 1.9 Å resolution. Structural comparison with its homologs revealed a conformational change in the α6 helix linked to the gate-keeping loop, with approximately 40° outward rotation. This change caused the catalytic residue Asp170 to move outward, possibly due to the absence of a ligand in the active site. These findings improve our understanding of the structural diversity of SpdS and provide a missing link that expands our knowledge of the structural features of SpdS in fungal species.

Globally, there are concerns over the risks associated with radiation exposure, so it is important to understand the biological effects of radiation exposure. Driven by the need to detect the presence of radiation exposure, biomarkers to monitor potential exposure after radiological accidents can be developed and would be extremely valuable for biological response. In this study, the behavior of spermidine as a biomarker was investigated in a C57BL/6 mouse model exposed to an acute whole-body sublethal dose of 6 Gy. The spermidine content values in serum increased for up to two days after 6 Gy irradiation. However, the enhanced spermidine content observed on day +3 in irradiated mice returned to normal levels on the subsequent five days. The result indicates that spermidine can be used as a biomarker of biological response to radiation exposure.

Retinal pigment epithelium (RPE) degeneration is a crucial event in dry age-related macular degeneration and gyrate atrophy. The polyamine spermidine has been shown to induce RPE cell death in vitro. The present study aimed to establish a novel in vivo model of spermidine-induced RPE degeneration and to determine whether spermidine-induced RPE cell death involves oxidative mechanisms. In this study, spermidine caused ARPE-19 cell death in a concentration-dependent manner. This effect was prevented by removal of serum from the culture medium or treatment with amine oxidase inhibitors, N-acetylcysteine (NAC), or aldehyde dehydrogenase (ALDH). Intravitreal injection of spermidine into rats significantly increased the permeability of the blood-retinal barrier and decreased the amplitudes of scotopic electroretinogram a- and b-waves. Histological analysis revealed that spermidine induced vacuolation, atrophy, and dropout of RPE cells, leading to the disruption of photoreceptor outer segments. Simultaneous intravitreal administration of NAC and ALDH with spermidine prominently inhibited the functional and morphological changes induced by spermidine. In conclusion, this study demonstrated that the intravitreal administration of spermidine induced RPE cell dysfunction and death followed by photoreceptor degeneration in rats. These effects of spermidine are thought to be mediated by oxidative stress and a toxic aldehyde generated during spermidine oxidation.

Because inflammatory bowel disease is increasing worldwide and can lead to colitis-associated carcinoma (CAC), new interventions are needed. We have shown that spermine oxidase (SMOX), which generates spermidine (Spd), regulates colitis. Here we determined whether Spd treatment reduces colitis and carcinogenesis.

We have earlier reported antileishmanial activity of hypericin by spermidine starvation. In the current report, we have used label free proteome quantitation approach to identify differentially modulated proteins after hypericin treatment. A total of 141 proteins were found to be differentially regulated with ANOVA P value less than 0.05 in hypericin treated Leishmania promastigotes. Differentially modulated proteins have been broadly classified under nine major categories. Increase in ribosomal protein S7 protein suggests the repression of translation. Inhibition of proteins related to ubiquitin proteasome system, RNA binding protein and translation initiation factor also suggests altered translation. We have also observed increased expression of Hsp 90, Hsp 83-1 and stress inducible protein 1. Significant decreased level of cyclophilin was observed. These stress related protein could be cellular response of the parasite towards hypericin induced cellular stress. Also, defective metabolism, biosynthesis and replication of nucleic acids, flagellar movement and signalling of the parasite were observed as indicated by altered expression of proteins involved in these pathways. The data was analyzed rigorously to get further insight into hypericin induced parasitic death.

Spermidine is important for the hypothalamic control of pituitary secretion of hormones involved in neuroendocrine functions in mammals. In this study, the effect of exogenous spermidine on the expression of genes and proteins related to polyamine metabolism and polyamine levels was examined. The results indicated that treatment with spermidine at 0.05 mg/g (BW) significantly increased the levels of Oaz1 mRNA and protein expression and decreased putrescine content in mouse hypothalamus (p < 0.05). The administration with spermidine at 0.10 mg/g significantly increased the levels of Oaz1, Oaz2, and Odc expression in mouse hypothalamus (p < 0.05). Treatment with spermidine at 0.05 mg/g significantly increased the levels of Ssat mRNA expression and reduced the level of Smo mRNA expression in mouse hypothalamus (p < 0.05). Putrescine concentrations in the hypothalamus after the administration of spermidine at 0.10 and 0.15 mg/g were significantly higher than those in the control group (p < 0.05). The concentration of both spermidine and spermine in the hypothalamus after the administration of spermidine at 0.15 mg/g was decreased significantly (p < 0.05). In summary, our results indicate that exogenous spermidine affects polyamine homeostasis in the mouse hypothalamus by modulating the expression of genes and proteins related to polyamine metabolism.

Multiple sclerosis (MS) is a chronic and debilitating autoimmune disease, characterized by chronic inflammatory demyelination in the nervous tissue and subsequent neurological dysfunction. Spermidine, a natural polyamine, has been shown to affect inflammation in some experimental models. We show here that spermidine could alleviate experimental autoimmune encephalomyelitis (EAE), a model for MS, through regulating the infiltration of CD4+ T cells and macrophages in central nervous system. Unexpectedly, we found that spermidine treatment of MOG-specific T cells did not affect their pathogenic potency upon adaptive transfer; however, spermidine diminished the ability of macrophages in activating MOG-specific T cells ex vivo. Depletion of macrophages in diseased mice completely abolished the therapeutic effect of spermidine, indicating a critical role of spermidine-activated macrophages. Mechanistically, spermidine was found to specifically suppress the expression of interleukin-1beta (IL-1β), IL-12 and CD80 while enhance the expression of arginase 1 in macrophages. Interestingly, macrophages from spermidine-treated mice could also reverse EAE progression, while pretreatment of those macrophages with the arginase 1 inhibitor abrogated the therapeutic effect. Therefore, our studies revealed a critical role of macrophages in spermidine-mediated treatment on EAE and provided novel information for better management of MS.

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy.

Kidney metabolism may be greatly altered in chronic kidney disease. Here we report that arginine metabolism is the most altered in unilateral ureteral obstruction (UUO)-induced fibrosis of the kidneys in metabolomic analysis. Spermidine is the most increased metabolite of arginine. In human glomerulonephritis, the amount of spermidine shown by immunostaining is associated with the amount of fibrosis. In human proximal tubule cells, spermidine induces nuclear factor erythroid 2-related factor 2 (Nrf2). Subsequently, fibrotic signals, such as transforming growth factor β1 secretion, collagen 1 mRNA, and oxidative stress, represented by a decrease in the mitochondrial membrane potential is suppressed by spermidine. UUO kidneys of Arg2 knockout mice show less spermidine and significantly exacerbated fibrosis compared with wild-type mice. Nrf2 activation is reduced in Arg2 knockout UUO kidneys. Spermidine treatment prevents significant fibrotic progression in Arg2 knockout mice. Spermidine is increased in kidney fibrosis, but further increases in spermidine may reduce fibrosis.