Introduction

Exploration of the molecular basis underlying the pathology of age-related neurodegenerative diseases has revealed an intricate interplay between several cellular processes that differentially contribute to continual neuronal demise, including mitochondrial (dys)function and an impairment of autophagy.1-4 Neurons are mainly quiescent cells surviving for decades; therefore, they might be particularly vulnerable to the slow but progressive accumulation of organellar and molecular damage, a characteristic feature of aging, as well as to defects in continuous organelle and protein turnover, which is governed by the ubiquitin proteasome system (UPS) and macroautophagy (henceforth called autophagy). A pathological hallmark of various chronic neurodegenerative disorders including Amyotrophic lateral sclerosis, Alzheimer’s (AD), Huntington’s (HD), and Parkinson’s (PD) disease is the accumulation of large, intracellular inclusions composed of misfolded (and often ubiquitinated) proteins in disease-specific regions of the brain.5,6 The important involvement of the UPS in the degradation of these protein aggregates is for instance highlighted by the fact that the occurrence of an elongated and stable ubiquitin protein variant termed UBBC 1 inhibits the UPS and is tightly connected to AD and HDassociated pathologies.7-10 The UBBC 1 protein is generated by molecular misreading of the ubiquitin B mRNA11 and decreases the proteasomal activity (at least in part) via inhibition of specific deubiquitinating enzymes.

12 Besides the UPS, autophagy as the other main system of the protein quality control has emerged as a decisive factor in the pathology of numerous age-associated neurodegenerative diseases, including AD, HD and PD.13-20 Spermidine is a naturally occurring polyamine that counteracts age-associated cell death (at least in part) via an activation of the autophagic machinery. It belongs to an ubiquitous family of organic polycations, the polyamines, which exert diverse roles in cell proliferation, differentiation, survival and death.21 An agedependent decrease of polyamine levels has been described in many organisms and tissues,22 including the brain of Drosophila, rodents and humans.23-25 External administration of spermidine prolonged the lifespan of yeast, flies, worms and human PBMCs (peripheral blood mononuclear cells) via induction of autophagy. 26 Interestingly, dietary spermidine supplementation as well as genetically enforced biosynthesis of spermidine protected against cognitive aging in Drosophila, preventing age-induced memory loss through activation of the autophagic machinery.23  Spermidine (as well as other activators of autophagy) was able to reduce neurodegeneration in a mouse model for TDP-43- pathology.27 Besides this cytoprotective activity related to aging, spermidine conferred a higher resistance toward environmental stress, leading to increased heat tolerance in yeast, protection against oxidative stress in yeast and flies, and reduction of agerelated oxidative damage in mice.26

 Drosophila subjected to oxidative stress exhibited improved motor function and survival upon supplementation of food with spermidine.28 Notably, this beneficial effect was autophagy-dependent upon challenge with the superoxide generator paraquat but not upon hydrogen peroxide treatment.28 Thus, depending on the specific scenario, spermidine-mediated cytoprotection seems to engage alternative mechanisms other than autophagy. In this study, we show that spermidine is able to alleviate PDassociated cellular demise using heterologous expression of human a-synuclein (aSyn) in the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, two established model systems for PD pathology.29-32 The protein aSyn is thought to play a pivotal role in the degenerative processes during PD, as it (i) represents the main protein component of the intracellular protein aggregates called Lewy bodies, a pathological hallmark of PD, and (ii) point mutations in as well as duplication/ triplication of the gene locus coding for aSyn are linked to familial PD.33 Here, we demonstrate that supplementation of food with spermidine protects against aSyn-facilitated motor dysfunction and organismal death in flies and reduces dopaminergic neuron loss in nematodes while causing an activation of autophagy.

Results

Spermidine treatment is able to extend the life- and healthspan of various model organisms. We therefore hypothesized that its age-protective function might as well be effective during typical age-related neurodegenerative disorders.

 By using flies expressing human aSyn under the control of the UAS-GAL4 system,34 an established model for PD-associated neurotoxicity, we evaluated the effect of spermidine supplementation on organismal survival. The sole expression of aSyn per se only caused a very modest decrease in viability during regular aging (data not shown). This result prompted us to combine this genetic trigger of PD (aSyn) with the administration of manganese, which represents a known environmental risk factor for PD.35 Employing this experimental setup,31,32 pan-neuronal elav-GAL4-driven expression of aSyn killed the flies within 4–6 days (Fig. 1A). Simultaneous supplementation of food with spermidine could largely protect from aSyn-induced organismal death, extending both the mean and the maximum lifespan (Fig. 1A and B). In fact, while the mean lifespan in flies expressing aSyn decreased by 20% upon manganese treatment, spermidine supplementation led to an almost complete restoration of mean lifespan (Fig. 1B). Of note, we could not detect any effect of spermidine on the expression levels of aSyn after 24 h of manganese treatment (Fig. 1C ). In addition, we monitored the effect of spermidine supplementation on motor dysfunction, a typical pathological feature of PD. The expression of aSyn caused a significant defect in motor function, which was already detectable after 48 h of manganese treatment as indicated by a significant reduction in climbing ability (Fig. 1D). Again, spermidine supplementation was able to completely inhibit this pathological consequence of aSyn expression, suggesting that spermidine not only protects against organismal death induced by neurotoxicity but also prevents typical symptoms prior to final demise (Fig. 1D).

 As mentioned above, accumulating data point to an involvement of autophagy in neuronal demise during PD in general and the toxic consequences of aSyn in particular. In addition, spermidine-mediated lifespan prolongation in several model organisms has been shown to largely depend on an intact autophagic machinery. Thus, we tested whether the neuroprotective function of spermidine involves an activation of autophagy, as well. To this end, we analyzed the protein levels of Atg8a (autophagyrelated gene 8a) in brain lysates of flies expressing aSyn. Atg8a is a Drosophila member of the Atg8/LC3 protein family, which is cleaved and lipidated during early autophagosome formation. The level of Atg8a-II, which represents the lipidated, autophagosome-associated form of this protein, significantly increased upon spermidine administration (Fig. 1E), indicating an enhanced autophagosome formation. Thus, the neuroprotective effect of spermidine might well be exerted via induction of autophagy in the fly brain. We next tested the ability of spermidine to protect against aSyn–associated neurodegeneration in a C. elegans model of PD.36 For that purpose, we analyzed nematodes expressing human aSyn under the control of a dopamine-specific neuronal promoter (Pdat-1) for survival of the anterior CEP (cephalic) dopaminergic neurons, which were visualized via co-expression of GFP driven by the dat-1 gene promoter. 

This phenotype most probably results from lysosomal depletion and defective lysosomal clearance.2,40 Several genetic factors implicated in the pathology of PD, including aSyn, the leucine-rich repeat kinase LRRK2, the ubiquitin ligase parkin or the PTEN-induced putative kinase PINK1, have been shown to differentially influence autophagic processes. Vice versa, modulation of autophagy alters the cellular consequences of these factors’ toxic gain-of-function or loss-of-function, though exact mechanisms remain yet to be elucidated.4,13,14,41 In mouse models for AD and PD, overexpression of the proautophagic regulator Beclin-1 (Atg6) ameliorated signs of neurodegeneration,42,43 and mice conditionally lacking neuronal Atg5 or Atg7 displayed severe behavior and motor deficits, abundant cellular protein inclusions, and neurodegeneration in several brain regions.44,45 These results affirm the importance of basal autophagy to maintain neuronal homeostasis. In the same lines, the pharmacological induction of autophagy is thought to represent a potential strategy to ameliorate neurodegenerative demise. Treatment with rapamycin, for example, which induces autophagy via inactivation of mTOR, has been demonstrated to be neuroprotective in several cell culture and animal models of PD, HD and AD.1,46,47 Nevertheless, though autophagy has been demonstrated to be a pro-survival process in most studies, and genetic or chemical induction of autophagy generally provided neuroprotection, excessive autophagy has also been suggested to contribute to non-apoptotic neuronal cell death.4,40 

 For instance, neurodegeneration after brain injury could be prevented by conditional Atg7 deficiency in mice,48 and Beclin-1 silencing or chemical inhibition of autophagy protected from cell death in cell culture models of AD.49 Similarly, pharmacological induction of autophagy via rapamycin turned out to be neurotoxic in alternative scenarios than those where protection was observed.50,51 These data indicate that a tightly controlled balance of autophagic processes is mandatory for neuronal survival. The herein presented data show that spermidine-mediated neuroprotection in both D. melanogaster and C. elegans models for aSyn-toxicity is accompanied by an induction of autophagy. Even though the causal involvement of autophagic processes in this protection remains to be elucidated, these results are in line with studies reporting an impairment of autophagy upon high gene doses of aSyn52-54 and enhanced neuroprotection upon induction of autophagy by pharmacological and genetic means such as treatment with resveratrol, trehalose, metformin, or rapamycin or overexpression of Beclin-1 or TFEB, the major transcriptional regulator of the autophagic pathway.43,55-58 Altogether these studies place the fine-tuning of autophagy regulation rather than autophagy itself, which as a degradative process is not intrinsically protective, at the core of neuronal viability during PD. Our results describe a scenario relevant at the organismal level that may help unveil active regulatory elements involved in protective neuroautophagy upon aSyn-toxicity.

 TE is recipient of an APART fellowship of the Austrian Academy of Sciences at the Institute of Molecular Biosciences, University of Graz. In addition, this work was supported by grants to GK from the Ligue Nationale contre le Cancer (Equipes labellisee), Agence Nationale pour la Recherche (ANR), the Longevity Research Chair of the AXA Foundation, Association pour la Recherche sur le Cancer, European Commission (ArtForce), European Research Council (Advanced Investigator Award), Fondation pour la Recherche Medicale, Institut National du Cancer, Cancerop^ole Ile-de-France, Fondation Bettencourt-Schueller, the LabEx Onco-Immunology, and the Paris Alliance of Cancer Research Institutes.