Targeting mTORs by omega-3 fatty acids: a possible novel therapeutic strategy for neurodegeneration?

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/326257025 Targeting mTORs by omega-3 fatty acids: A possible novel therapeutic strategy for neurodegeneration? ArticleinPharmacological Research · July 2018 DOI: 10.1016/j.phrs.2018.07.004 CITATIONS 8 READS 361 12 authors, including: Some of the authors of this publication are also working on these related projects: Bioactive secondary metabolites from cameroonian medicinal plants View project A study on the function of calcium-parathormone-vitamin D axis and the role of IL-6 in BDL cirrhotic rats and the effect of Naltrexone and L-NAME View project Ahmad reza Dehpour Tehran University of Medical Sciences 811 PUBLICATIONS10,474 CITATIONS SEE PROFILE Tarun Belwal Zhejiang University 66 PUBLICATIONS397 CITATIONS SEE PROFILE All content following this page was uploaded by Tarun Belwal on 23 November 2018. The user has requested enhancement of the downloaded file. Accepted Manuscript Title: Targeting mTORs by omega-3 fatty acids: a possible novel therapeutic strategy for neurodegeneration? Authors: Samira Shirooie, Seyed Fazel Nabavi, Ahmad R. Dehpour, Tarun Belwal, Solomon Habtemariam, Sandro Arg¨uelles, Antoni Sureda, Maria Daglia, Michał Tomczyk, Eduardo Sobarzo-Sanchez, Suowen Xu, Seyed Mohammad Nabavi PII: S1043-6618(18)30531-0 DOI: https://doi.org/10.1016/j.phrs.2018.07.004 Reference: YPHRS 3941 To appear in: Pharmacological Research Received date: 15-4-2018 Revised date: 3-7-2018 Accepted date: 5-7-2018 Please cite this article as: Shirooie S, Nabavi SF, Dehpour AR, Belwal T, Habtemariam S, Arg¨uelles S, Sureda A, Daglia M, Tomczyk M, Sobarzo-Sanchez E, Xu S, Nabavi SM, Targeting mTORs by omega-3 fatty acids: a possible novel therapeutic strategy for neurodegeneration?, Pharmacological Research (2018), https://doi.org/10.1016/j.phrs.2018.07.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Targeting mTORs by omega-3 fatty acids: a possible novel therapeutic strategy for neurodegeneration? Samira Shirooie 1, Seyed Fazel Nabavi 2, Ahmad R. Dehpour 3, 4, Tarun Belwal 5, Solomon Habtemariam 6, Sandro Argüelles 7, Antoni Sureda 8, Maria Daglia 9, Michał Tomczyk 10, Eduardo Sobarzo-Sanchez 11, 12, Suowen Xu 13, Seyed Mohammad Nabavi 2* 1- Department of Pharmacology, Faculty of Pharmacy, Kermanshah University of Medical Sciences, Kermanshah, Iran 2- Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran 14359-16471, Iran 3- Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran 4- Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran 5- G.B. Pant National Institute of Himalayan Environment and Sustainable Development, Kosi Katarmal, Almora, Uttarakhand, India 6- Pharmacognosy Research Laboratories & Herbal Analysis Services UK, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK 7- Department of Physiology, Faculty of Pharmacy, University of Seville, Seville, Spain 8- Research Group on Community Nutrition and Oxidative Stress (NUCOX) and CIBEROBN (Physiopathology of Obesity and Nutrition CB12/03/30038), University of Balearic Islands, Palma de Mallorca E-07122, Balearic Islands, Spain ACCEPTED MANUSCRIPT 2 9- Department of Drug Sciences, Medicinal Chemistry and Pharmaceutical Technology Section, University of Pavia, Italy 10- Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Białystok, ul. Mickiewicza 2a, 15-230, Białystok, Poland 11- Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Santiago de Compostela 15782, Spain 12- Instituto de Investigación en Innovación en Salud, Facultad de Ciencias de la Salud, Universidad Central de Chile, Santiago, Chile 13. Aab Cardiovascular Research Institute, University of Rochester, Rochester, NY, 14623 *Corresponding to: SM Nabavi, [email protected] Graphical abstract ACCEPTED MANUSCRIPT 3 Abstract Neurodegenerative diseases (NDs) such as Parkinson's (PD), Alzheimer's (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) cause significant world-wide morbidity and mortality. To date, there is no drug of cure for these, mostly age-related diseases, although approaches in delaying the pathology and/or giving patients some symptomatic relief have been adopted for the last few decades. Various studies in recent years have shown the beneficial effects of omega-3 poly unsaturated fatty acids (PUFAs) through diverse mechanisms including anti-inflammatory effects. This review now assesses the potential of this class of compounds in NDs therapy through specific action against the mammalian target of rapamycin (mTOR) signaling pathway. The role of mTOR in neurodegenerative diseases and targeted therapies by PUFAs are discussed. Keywords: Neurodegenerative diseases, mTOR, omega-3, docosahexaenoic acid, eicosapentaenoic acid. ACCEPTED MANUSCRIPT 4 1. Introduction The progressive neuronal cell functional losses that arise from some kind of abnormal biochemical and structural changes or cell death is called neurodegeneration; while the disease associated with the processes are known as neurodegenerative diseases (NDs). The classical examples of the NDs are Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) and that occur through the progressive neuronal degeneration accumulated over a period of time. Extensive researches in recent decades have been devoted to understanding the pathophysiological changes and risk factors of these chronic NDs [1]. One of the common risk factors is age and, in fact, these group of diseases are called age-relate neurodegenerative pathologies. Taking into account recent reports suggesting a significant increase in life expectancy in the very near future [2], a higher incidence of chronic NDs together with a lack of effective therapy would be a real concern. Even though the molecular mechanisms responsible for NDs are not fully elucidated, dysregulation of several signal transduction pathways such as autophagy and endosomallysosomal [3], AMPK [4], p53 [5], P38 MAPK [6], JAK/STAT [7], PI3K/AKT [8], mTOR [9] etc., have been shown to be associated with these disorders. Within these pathways, mammalian target of rapamycin (mTOR), has been extensively studied in the context of aging and agerelated NDs. The mTOR has a key role in important cellular processes including cell survival, protein synthesis, mitochondrial biogenesis, proliferation, apoptosis, autophagy, etc. [10,11], and whose dysregulation can promote the initiation and progression of NDs. Since classical NDs has special features of accumulating abnormal misfolded or toxic proteins which regulates autophagy and cell fate, targeting mTOR may have important consequences on therapy against NDs. mTOR pathway in normal conditions inhibits autophagic mechanisms, which are necessary to degrade ACCEPTED MANUSCRIPT 5 and eliminate abnormal or aggregated proteins [71]. Therefore, mTOR inhibitors may represent an interesting therapy for this kind of diseases by inducing autophagy or regulating apoptotic processes (Bove et al., 2011). In fact, classic mTOR inhibitors such as rapamycin or metformin have been reported to improve the neuronal damage in human and animal models of NDs [12]. Another important approach to fight against NDs are the use of natural products/compound including antioxidants [13], flavonoids [14], polyphenols [15], and polyunsaturated fatty acids (PUFAs) [16]. The omega-3 PUFAs represent a wide-range of unsaturated fatty acids which are often claimed to have numerous health benefits [17]. The best studied omega-3 FAs are docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and -Linolenic acid (ALA). Structurally, the omega-3 group of FAs (Fig 1, drawn following the gguidelines for preparing color figures [18] is characterized by having the first double bond at position 3 of the terminal carbon or methyl group which is designated as omega ( or n). However, the most common nomenclature of these compounds is the numbering starting from the carbonyl acid carbon assigned as carbon-1 and as shown in Fig. 1. Omega-3 FAs differ from each other by the total number of carbon groups in the molecule and amount and position where double bonds are formed. The essential oils of the higher plants are the main source of ALA while fish oils and other marine animal fats are the main sources of DHA and EPA. Hence, the Mediterranean dietary pattern, rich in vegetables and fish oils/products along with algae as the primary producers of EPA and DHA, have been advocated as a healthy diet option in recent years [19,20]. Omega-3 PUFAs have a central role in the maintenance of cerebral functions and brain development contributing to neurogenesis and neuroplasticity. Moreover, these PUFAs also exert significant protective effects against inflammatory damage to the neurons and glial cells. Among ACCEPTED MANUSCRIPT 6 the omega-3 fatty acids, the EPA has been reported to show the best promising effects as antidepressant agent [21,22]. Omega-3 PUFAs also have beneficial effects on cardiovascular diseases, as lipid lowering [23], antihypertensive [24] and antiinflammatory effect via modulation of cytokines and adhesion molecules expression [25]. These antiinflammatory properties have been well demonstrated in various animal models [26-29]. Moreover, omega-3 PUFAs reduce the risk or incidence of autoimmune diseases such as type 1 diabetes [30], multiple sclerosis [31], lupus erythematosus [32], psoriasis [33] and rheumatoid arthritis [34,35]. Mounting evidence has shown that omega-3 PUFAs can improve mental disorders such as schizophrenia and bipolar disorder [36]. The negative correlation between omega-3 PUFAs consumption and risk for cancer including pancreatic [37], colon [38], breast cancer [39] have been documented. Previous review articles also showed the potential of omega-3 PUFAs in diabetes retinopathy and other pathologies [40,41]. Omega-3 PUFAs, particularly DHA, have protective role in retina [42]. In addition, to the modulation of cytokine and adhesion molecules, the antiiflamatory effects of omega-3 PUFAs are associated to their incorporation in cell membrane phospholipids, contributing to the decrease in the production of proinflammatory omega-6 mediators [43]. Omega-3 PUFA are also important for the generation of antiinflammatory factors, such as neuroprotection D1 (NPD1) [44]. Recent evidences reported that omega-3 PUFAs can act as negative regulators of PI3K/Akt pathway which, in turn, results in mTORC1 inhibition [45]. In this sense omega-3 PUFAs could be interesting agents to prevent the initiation and progression of NDs. The aim of this review is to overview the potential of omega-3 PUFAs as therapy for NDs through a possible specific action against the mTOR signaling pathway. Here, we summarize the ACCEPTED MANUSCRIPT 7 main concepts of neurodegeneration, the role of mTOR in ND, and therapeutic implications of targeting mTOR pathway with omega-3 PUFAs. 2. Hallmark of Neurodegeneration: Protein misfolding and precipitation. Neurodegenerative diseases (NDs) such as PD, AD, HD, and ALS cause significant world-wide morbidity and mortality. Hence, accumulation of deleterious changes through aging process is the main factor associated with many NDs [46]. The most common form of NDs is AD, which is characterized by a loss of memory in the older age group and account to over 60% of all forms of dementia cases [47]. As with many other NDs, protein misfolding and precipitation are the hallmark of AD, the key player being the amyloid β (Aβ) peptides [48]. In addition to the presence of extracellular amyloid plaques composed of aggregated peptides, AD is characterized by intracellular neurofibrillary tangles comprising the microtubule-associated protein tau [49]. Aβ peptides derive from the proteolytic cleavage of the amyloid precursor protein (APP) by aspartyl-proteases and secretases [50]. The link between Aβ peptides aggregation and inflammation and oxidative stress in the Alzheimer’s brain has been established [51-54]. The main group affected in AD appear to be those of the cholinergic type that are closely linked to memory storage and retrieval [55,56]. PD is a cognitive and motor dysfunction characterized by the degeneration or loss of dopaminergic neurons [57,58]. The key pathological hallmark of PD is the appearance of Lewy bodies which derive from the progressive deposition of protein inclusions of α-synuclein and ubiquitin leading to cell death (apoptosis or necrosis). ALS is a complex disorder which has the special feature of degenerated motor neurons, followed by muscle weakness, paralysis, and finally, death [59]. The basis of ALS is multifactorial and comprises genetic mutations with more than 20 genes involved, and epigenetic and ACCEPTED MANUSCRIPT 14 Extensive studies have shown that in AD, functional impairments of synaptic plasticity normally develops before neurodegeneration resulting in neuronal damage and/or lost [108,109]. mTOR integrates multiple inputs associated with nutrients, growth factors and the cellular energy status that result in its activation or inhibition being a crucial regulator between anabolic and catabolic processes [110,111]. Diverse investigations have evidenced that soluble oligomers of Aβ, derived from APP, are able to inhibit long-term potentiation (LTP) and cause deficits in learning and memory [112-114]. Ma and colleagues studied the association of mTOR signaling pathway with Aβ-induced synaptic dysfunction. The authors reported that in the hippocampus of animal models of AD, the activity of mTOR was decreased, and when mTOR signaling was activated, the inhibition of LTP associated with Aβ was recovered. In 3xTg mice model of AD without evidence of amyloid plaques and tangles, the mTOR-induction of autophagy with rapamycin have been reported to ameliorate cognitive deficits [115]. The chronic treatment with rapamycin reduced the progression of AD and increased autophagy which, in turn, reduced Aβ levels in the mouse model of the human amyloid precursor protein (PDAPP) [116]. Thus, mTOR pathway regulates Aβ-related synaptic dysfunction in AD [113]. Other studies also reported that mTOR activation induces tau hyperphosphorylation contributing to the progress of AD [117,118]. Rapamycin treatment reduces the phosphorylation of tau (at Ser214) via regulating cAMP- dependent kinase. Also, autophagy induction could improve the clearance of the hyperphosphorylated tau [119,120]. 4.2. Down syndrome (DS) DS is regarded as a frequently-occurring chromosomal abnormality which leads to intellectual disorder or disability [121,122]. The neurophysiology of AD with respect to DS is multifaceted ACCEPTED MANUSCRIPT 15 and includes impaired functions of mitochondria [123], defects in neurogenesis [124], high generation of oxidative species [125], impaired autophagy networks and proteostasis [126] due to triplication of chromosome 21. Gene products at Chromosome 21 are recognized as principal neuropathogenic moieties in the development of DS [127,128]. Moreover, DS after age 40-50 has considerable AD-like neuropathology and individuals present notable signs of dementia. Thus, two principal biomarkers of the etiology of AD (senile plaques that are mostly composed of fibrillar Aß peptides and hyperphosphorylated tau protein) appear to operate through the mTOR signaling system. Moreover, it has been reported that inhibition of mTOR decreases the tau pathology [106]. 4.3. Parkinson’s disease In PD, the pathology is associated with deletion of dopaminergic cells in central and peripheral neurons, associated to the presence of lewy bodies formation with the major toxic protein component of α-synuclein (α-syn), that is phosphorylated in serine residue at position 129 [129]. In vitro studies have shown that metformin reduces phospho-Ser129 α-syn through activation of the AMPK which inhibits mTOR activity and decreases the levels of phosphorylated forms of mTOR downstream targets including p-S6K [69]. Inhibition of mTOR leads to PP2A activation, a neuroprotective protein phosphatase, which dephosphorylates phospho-Ser129 α-syn. Rapamycin has also shown to display similar effect to metformin reducing the formation of lewy bodies [130]. In animal models of PD, rapamycin mediated inhibition of mTOR also exerts neuroprotection through blockade of expression of stress response protein RTP801 and survivalpromoting kinase Akt [131]. ACCEPTED MANUSCRIPT 16 Moreover, it has been observed that the mTOR pathway showed central role in dopaminergic neurons activity in PD [132,133] [134]. There is increasing evidence to suggest that activation of PI3K/Akt/mTOR pathway can regulate neuronal development, survival and differentiation, and it also played a crucial role in synaptic plasticity [9,135]. Nakasoand colleagues observed in a cellular model of PD that the neuroprotective effect of caffeine was caused by the activation of the PI3K/Akt pathway [136,134]. Additionally, administration of MPP+ (1-methyl-4- phenylpyridinium) decreased the phosphorylation of mTOR and its canonical substrate, p70S6K, in control and G2019S LRRK2-mutant skin fibroblasts [137,134]. The PI3K/Akt/mTOR genes in the SH-SY5Y dopaminergic cells also provide a reference gene framework for researchers working in PD. The expression pattern will further offer useful clues about the function of the signaling pathway during PD pathogenesis and possible neuroprotection options [138,134,139]. However, mTOR has a controversial sort of role as various results presented neuroprotective as well as neurotoxic effects in different studies. For instance, rotenone, a known PD toxin, caused downstream regulation of mTOR and decreased cell viability in different PD models [140,141]. Lysosomal dysfunction appears to contributes to rotenone’s neurotoxicity [142,143] and could be antagonized by pretreatment with rosuvastatin [144]. On the other hand, there are evidences suggesting that upstream regulation of mTOR causes partial prevention in neuronal cell loss provoked by PD toxin [145,146]. Therefore, future studies are warranted to explore the exact role of mTOR signaling pathway in PD. 4.4. Huntington’s disease (HD) HD is an autosomal dominant neurodegenerative disease, which is characterized by neurodegeneration of the basal ganglia and cortex linked to the aggregation of huntingtin protein. ACCEPTED MANUSCRIPT 17 Mutant huntingtin protein has expansion of CAG (cytosine-adenine-guanine) triplet repeats, which is not cleared from cells and leads to neural death. In vivo studies has indicated that rapamycin mediated inhibition of mTOR can relieve the symptoms of HD by increasing autophagy and clearance of mutant huntingtin [147,91]. Evidence also supports that mTOR inhibitors have protective roles in neurodegenerative diseases in animal models, but more studies are needed using mTOR inhibitors. 4.5. Amyotrophic lateral sclerosis (ALS) ALS is associated to the progressive loss of functional motor neurons, and to the formation of misfolded protein inclusions in neurons [148]. Diverse studies using SOD1G93A mice (which mimics the most usual familial cause of ALS), evidenced that the treatment with rapamycin failed to protect against the disease progression even aggravated motor neuron loss [149,150]. Similar results were obtained using rapamycin in a mouse model of ALS induced by mutant valosin protein [151]. On the contrary, the treatment with trehalose, an mTOR-independent activator of autophagy prolonged motor neuron survival and reduced the autophagic flux defect in a SOD1G93A mouse model of ALS [152]. Taking together these results suggest that in ALS mTOR seems not to be an adequate target. 5. Omega-3 fatty acids and neurodegeneration The broad protective effects of omega-3 PUFAs in the CNS function have been well recognized (Table 2 and Table 3). As described above, AD is a devastating neurodegenerative disease with over-expression of Aβ peptide, extensive inflammation and neuronal cell death [153,154]. Omega-3 PUFAs have been shown to improve mental health and cognition including in ACCEPTED MANUSCRIPT 18 Alzheimer’s disease [155]. They also attenuates the symptoms of attention-deficit/hyperactivity disorder (ADHD) and improve attention and the ability to complete tasks in children [156]. Multiple reports have clearly demonstrated that omega-3 PUFA can prevent or treat psychopathologies such as “emotional depression,” “bipolar disorder,” “schizophrenia,” and AD [36]. The DHA and EPA treatment have significant anti-inflammatory effects in AD [157]. The EPA showed effect through BDNF over expression while DHA caused down regulation of TNF-α [158,159] leading to phagocytosis and anti-inflammatory effects. The potential role of PUFAs in immunoregulation has also been reported in patients with AD suplemented with omega-3 PUFAs [160,161]. Both A and tau aggregates results in increasing neuronal death, stress and defects in neuronal pathways, and leads to neurodegenerative disease. Omega-3 was found to attenuate the disease condition and enhances neuroprotection by increasing the expression of BDNF and also high affinity BDNF receptor (TrkB). During neurodegeneration disease such as AD and PD, the omega-3 concentration were shown to decrease in the brain and resulted in more serious consequences. In an experiment where three transgenic AD animal model were used to evaluate the impact of omega-3 on neurological disorders, adding omega-3 in diet was found to revert the condition via inhibitory accumulation of A, hyperphosphorylation of tau and improve the cognitive deficits [162]. A accumulation is the major cause of AD causing oxidative damage, inflammation of neurons and also phosphorylating tau protein, [163]. In a study, a transgenic animal model of memory loss and A depletion (Tg 2576 mouse) exposed to PUFA (1g/kg/day), dietary intake for 16-20 months, reduces the A accumulation [162,164] and exert neuroprotective activity. DHA ACCEPTED MANUSCRIPT 19 treatment (0.98g/kg/day) for 6 to 10 months in transgenic animal model (APPs we/PSIdE9) decreases A accumulation [165]. In similar study, the intraneuronal A concentration along with tau accumulation and phosphorylation decreases, when DHA incorporated to the diet (2.3g/kg/day) was given for 9 months in the 3xTg-AD mouse model [166]. Also, NPDI derived from omega-3 was found to protect neurons from A direct apoptosis in HN culture cells [167,168]. Kusat and colleagues studied the effect of omega-3 along with betaine for possible protection against alcohol induced neurodegeneration [169]. In their study, female rats in the prenatal and postnatal stages were exposed to high alcohol concentrations (25-75 mL ethanol) that cause fetal alcohol syndrome (FAS) [170-172]. When omega-3 PUFAs in modified diet were administered during pregnancy and after delivery, they provoked a significant reduction in brain necrosis induced by excessive ethanol intake. It is already known that neonatal rat brain is highly sensitive to ethanol and necrosis is regard as the primary death caused after acute neonatal head injury [173], which was also noted in histopathalogical studies [174]. Various studies have confirmed the role of omega-3 in preventing neurodegeneration [175-177], along with clinical trials proven its effectiveness [178-181]. A mechanistic activity of omega-3 PUFA in protecting NDs has been shown in Figure 3. PUFAs have been shown to have critical role in cerebral functions and brain development. For example, DHA in the brain contributes in gene expression, neurogenesis, neuroplasticity, neuron differentiation, survival, oxidative metabolism, learning and memory process [155,182]. The EPA and DHA diet in rats during post-natal development, can increase SOD [183].The EPA can also inhibit JNK activation, which phosphorylate and inhibit the antiapoptotic bcl-2 protein by proliferator activating receptors (PPAR- Aα) [184]. The DHA decreases the production and ACCEPTED MANUSCRIPT 20 aggregation of Aβ from amyloid precursor protein. The DHA diet for 8-10 months in Aged 3×Tg-AD mice also improved cognition and prevent the pathological effect of mpp+onnigral cells and striatum. Ethyl-EPA diet in MPTP-probencid mouse model of PD can also prevent some of the PD symptoms such as hypokinesia but it failed to prevent the decreation of nigrostriatal dopamine [185]. In mouse model of HD, oral administration of PUFAs can prevent motor deficits [182]. The Rthyl-EPA in YAC128 HD mice has also been shown to decrease motor deficits [182]. On the other hand, DHA-enriched diet can increase the number of dendrite spine in hippocampus of animals. Moreover, there is a negative association between homocyteine levels and DHA in plasma. Homocysteine can act as a glutamate agonist on NMDARs and has excitotoxicity effect and neurodegeneration [186]. 6. mTOR as a new omega-3 target in NDs therapies? As reported above, in section 4, mTOR seems to be an interesting candidate for some types of NDs, such as AD, SD, PD and HD. In scientific literature, as described in section 5, the omega-3 PUFAs role as neuroprotective agent is well recognized. Some references reported mTOR as an omega-3 target in different pathological conditions, but in NDs there aren’t studies that prove mTOR as an omega-3 target. Nevertheless, considering the promising evidence regarding the involvement of mTOR in other chronical pathologies, studies on the omega-3 beneficial activity in NDs through mTOR inhibition are strongly needed. 6.1. Omega-3 mechanism of action in NDs Omega-3 PUFAs protect neurons and related disorders through mTOR signaling pathway. Upstream of mTOR, i.e., PI3K/Akt pathway was activated by omega-3 PUFAs which is central ACCEPTED MANUSCRIPT 21 for cell survival. Accordingly, omega-3 PUFAs supplementation facilitates Akt translocation to the membrane for activation and this process is blocked under serine-depleted conditions, suggesting that omega-3 PUFAs-derived phosphatidylserine (PS) is necessary for this effect [187]. Omega-3 PUFAs are suggested to be the best substrate for PS synthesis which prevents normal cell death and is one of the potent neuroprotective agents. The serine/theonine Akt kinase is an important enzyme in cell survival [188] and it played important role in mediating the protective effect of omega3 in neuronal cells [189,190]. The reduction in activity of Akt kinase pathway leads to the apoptotic death, which can be prevented by omega-3 via phosphorylation/ activation of Akt kinase. The PS is also involved in signaling event of kinases such as protein kinase C, Raf-1 kinase [187] and Akt [190] . In addition to the effects via mTOR, diverse evidenced showed that the omega-3 also form neuroprotectin D1 (NPD1), a new DHA-derived 10,17S-docosatriene, which prevents the A secretion and, consequently, represses A triggered activation of proinflammatory genes and activation of antiapoptosis genes [191]. In AD mouse model and human neuroglial (HNG) cells cultured with A protein, the role of omega-3 derived NPD1 was also investigated [192]. The results showed that NPD1 treatment prevented the A driven expression of proinflammatory enzymes (COX-2 and B-94), downregulated apoptosis and reduced neuronal cell death. However, diverse studies using human retinal pigment epithelium cells reported that NPD1 leads to cell survival after challenged with oxidative stress, at least in part, via activating the PI3K/Akt and mTOR/p70S6K pathways [193,194]. Similar protective effects of omega-3 were recorded by Ma and coworkers in the survival in cultured hippocampal neurons by upregulating the expression genes with anti-apoptotic functions, repressed proinflammatory genes and inhibit the phosphorylation of IRS-1 by different pathways including PI3K/Akt/mTOR and tau by A [195]. ACCEPTED MANUSCRIPT 22 The mechanism of omega-3 was studied in some of experiments. For instance, omega-3 enriched diet which has direct effect on signaling pathway (PI3K/Akt/mTOR), increased the neurotrophin levels like NGF (Nerve growth factor) and BDNF and thus, improved cognition function [196]. These neurotrophins regulate/stimulate the transcription and release of the vascular endothelial growth factor (VEGF) [197]. VEGF represents a major pro-angiogenesis protein [198]. VEGF is regulated by HIF-1 alpha levels which, in turn, depend on PI3K/Akt/m+TOR pathway. Activation of mTOR pathway by BDNF and NGF neurotrophins, phosphorylates the two downstream effectors P7056K and 4E-BP1, which regulate and promote angiogenesis [199]. Autophagy is an important part of cell cycle and has a role in preventing cellular damage by elimination of toxic protein aggregates, which causes cell death. In case of neuronal death, these toxic proteins result in increased Aβ and tau concentration. The mTOR signaling pathway mainly regulates autophagic process, which is regulated by growth factors, cellular stress and nutritive deficiency. The PI3K/Akt pathway modulates the mTOR signaling pathway and thus played role in modulating the disorders. In one way, this pathway enhances the autophagic events of A, on the other side hyperactivation of pathway will also leads to neuronal death. Thus, the regulation of this process is important and omega-3 PUFAs play a crucial role as proved by several studies. 6.2. Targeting mTOR by omega-3 in pathological conditions Some studies showed that mTOR inhibition represent one major mechanisms whereby omega-3 PUFAs exert beneficial effects towards different types of pathologies. Both in vitro and in vivo studies, have reported mTOR as an omega-3 target in different chronical pathological conditions, such as glioblastoma [200], prostate cancer [173], lung cancer ACCEPTED MANUSCRIPT 23 [201], adrenocortical cancer [202]. Moreover, omega-3 PUFAs trough mTOR inhibition resulted to be active in renal ischemia reperfusion injury attenuation [183], in the protection against systemic inflammation and insulin resistance through the inhibiting the activation of NLRP3 inflammasome, as well as subsequent caspase-1 activation, and the secretion of cleaved IL-1β [203]. Previous study has shown that AMPK and TSC1/2 knockdown by siRNA slightly decrease the inhibitory effect of DHA on mTORC1. The DHA can stabilize the mTOR-raptor complex and decrease its activity. The omega-3 PUFAs decrease the IGF1R and increase PTEN levels, negative regulator of PI3K/Akt pathway which activates mTORC1 [204]. In an in vitro study performed on isolated CD4+ T cells from spleen tissues from NOD mice, while, omega-3 PUFAs treatment inhibited the differentiation of CD4+ T cells into Th1 cells by inhibiting TORC1, thus having beneficial effects in autoimmune diseases such as type 1 diabetes [205]. In one study, DHA has been shown to increase the level of intracellular ROS. It was proposed that ROS inactivate AKT/mTOR signaling and, consequently, both autophagy and apoptosis are activated in cancer cells [206]. The LKB1 gene expression and its activity are also stimulated by DHA. In mammary epithelial cells expressing LKB1, the treatment with for 24h evidenced increased AMPK phosphorylation which leads to the inhibition of the mTOR pathway [84]. 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Amminger, Effects of omega-3 PUFA on immune markers in adolescent individuals at ultra-high risk for psychosis – Results of the randomized controlled Vienna omega-3 study, Schizophr. Res. 188 (2017)110-117. ACCEPTED MANUSCRIPT 52 Figures legends: Figure 1. Structures of some common Omega-3 PUFAs Figure 2: Role and regulation of mTOR signaling pathway. mTOR is a major modulator of autophagy and it receives inputs from different signaling pathways. The kinase mTOR is a downstream target of PI3K and kinase AKT pathway, which is activated by receptors of neurotrophins and growth factors, and promotes cell growth, differentiation and survival, while downregulating apoptotic signals. AKT also promotes mTOR activity by suppressing the repressive action of TSC by phosphorylating TSC2. TSC2 phosphorylation promotes activation of Rheb and stimulation of mTOR activity. Rag A/B is also activated by the presence of RagGTPase, which further regulates mTOR signaling. Activation of mTOR leads to regulation of many target proteins including FoxO3, A, autophagy, synaptic plasticity, cell growth, ACCEPTED MANUSCRIPT 53 differentiation and survival. This figure has been prepared according to the guidelines as published in [217]. mTOR (mammalian target of rapamycin), PI3K (phosphatidylinositol 3 kinase), TSC (tuberous sclerosis complex), Rheb (Ras homolog enriched in brain), Rag (recombination activating gene), A (amyloid beta), ATG (autophagy related protein), ULK (serine/threonine protein kinase ULK), FoxO3 (forkhead box O3) Figure 3. Potential of omega-3 in neurodegenerative diseases therapy through inhibition of mammalian target of rapamycin (mTOR) signaling pathway. Aging, stress and inflammation are common risk factor for neurodegenerative diseases such as Alzheimer's (AD), Parkinson's (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD), in which defects in autophagy cause accumulation of toxic proteins in neurons which leads to neural damage and death. The mTOR has a key role in important cellular process such as cell survival, protein synthesis, mitochondrial biogenesis, proliferation, apoptosis, and is negative regulator of ACCEPTED MANUSCRIPT 54 autophagy. The mTOR signaling can be activated by PI3K/AKT and inhibited by AMPK. Omega-3 can inhibits mTOR activity through a mechanism involving activation of PTEN and AMPK. Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and -Linolenic acid (ALA), adenosine monophosphate-activated protein kinase (AMPK), tuberous sclerosis complex (TSC), ribosomal protein S6 kinase (S6K), initiation factor 4E-binding protein (4E-BP), eukaryotic initiation factor 4E (eIF4E), eukaryotic initiation factor 4B (eIF4B). Figure 4. Schematic report on literature evidence suggesting omega-3 as mTOR inhibitor in neurodegenerative diseases. Omega-3 PUFAs which are recognized as mTOR inhibitors in different chronic pathologies, might be also considered as mTOR inhibitory agents to develop ACCEPTED MANUSCRIPT 55 new therapeutic strategies for NDs. ACCEPTED MANUSCRIPT