Neuroprotection of multifunctional phytochemicals as novel therapeutic strategy for neurodegenerative disorders: antiapoptotic and antiamyloidogenic activities by modulation of cellular signal pathways

In our aging society, increase of patients with age-associated neurodegenerative disorders is an oppressive issue for medical care, society and economy. The etiology of two most common neurodegenerative disorders, Alzheimer's disease (AD) and Parkinson's disease (PD), has not been clarified. However, the common pathological hallmarks have been associated with these diseases: loss of distinct population of neurons and accumulation of disease-specific inclusion bodies. New therapeutic strategy is now proposed to target multiple pathogenic factors, including oxidative stress, mitochondrial dysfunction, impaired homeostasis of energy, transition metals (iron, copper), calcium and hormones (insulin, estrogen for women), deficits of neurotrophic factors (NTFs), inflammation, accumulation of modified proteins and activation of programmed cell death. These factors are regulated by interaction between the genetic and environmental factors, and the molecular mechanism has been intensively investigated.

Neuroprotection is aimed to prevent neuronal death, regenerate neuronal network and ameliorate the brain dysfunction as ‘disease-modifying’ therapy in AD, PD and depression. Quite a huge number of compounds, including inhibitors of monoamine oxidase (MAO) (selegiline rasagiline), antioxidants (vitamin E, C, transient metal chelators), bioenergetic compounds (coenzyme Q) and anti-inflammatory agents, have been reported to be neuroprotective, but clinical studies have not been able to prove fully the prevention of disease progression [1].

Epidemiological and clinical intervention studies have presented that diet habits, such as Mediterranean diet, show beneficial effects in patients with dementia, AD, mild cognitive impairment, PD and depressive disorders [2,3]. Components of Mediterranean diet, phytochemicals (the secondary metabolites of plants) and ω-3 polyunsaturated fatty acids, were reported to have maintained cognitive function in human studies. The total low level of urinary polyphenols has been correlated with the risk of cognitive decline in older adults [4].

Herbal preparation and phytochemicals isolated from plant food have been proposed as ‘herbal medicine’ for the treatment of PD [5] and AD [6]. Flavonoids found in high quantities in vegetables, fruits, tea, red wine and chocolate, and resveratrol enriched in red wine were reported to improve cognitive function in clinical trials, which, however, has been not fully established [7,8]. Flavonoids are strong antioxidants via direct scavenge of reactive oxygen and nitrogen species (ROS, RNS), upregulation of ROS-removing enzymes and downregulation of radical-producing ones. However, the neuroprotective activity does not directly depend on the antioxidant activity, and new molecular mechanisms are now proposed for promotion of neuronal survival.

This review presents the recent results on neuroprotective intervention by phytochemicals to the pathogenic factors of neurodegeneration: oxidative stress, neuroinflammation, mitochondrial dysfunction, deficit NTFs and accumulation of abnormal-modified protein. Phytochemicals can directly inhibit or promote mitochondrial apoptosis cascade, which might be associated with the ambivalent functions in decision of cell survival [9]. Phytochemicals modulate cellular signal pathways linking to the expression of antiapoptotic Bcl-2 and NTFs, such as brain-derived and glial cell line-derived NTF (BDNF, GDNF), and show NTF-like activities in the brain [10,11]. Phytochemicals activate the major proteolysis pathways in neuron, the ubiquitin-proteasome system (UPS) and autophagy–lysosome pathway (ALP), to prevent accumulation of disease-specific abnormal protein aggregates and production of toxic oligomer and fibril [12]. These multiple neuroprotective activities of phytochemicals are discussed in relation to the development of new therapeutic strategy for neurodegenerative disorders. Synthesis of novel multitargeted neuroprotective compounds based on the structure of phytochemicals is also discussed in order to develop novel therapeutic agents capable of inhibiting MAO and acetylcholinesterase (AChE), preventing protein aggregation and inducing NTF expression.

Phytochemicals: the classification, structure & antioxidative & anti-inflammatory functions

Polyphenols are present in plants, fruits and vegetables, and flavonoids are the largest groups of >6000 established species. Figure 1 shows the classification and chemical structure of major flavonoids and other phytochemicals discussed in this review. Oxidative stress is caused by excess production of ROS–RNS by mitochondria and NADPH oxidase, and decline in antioxidant activity in cells, and it is an early and critical event in PD, AD, amyotrophic lateral sclerosis and multiple sclerosis [13,14]. Free radicals oxidize protein, RNA and DNA, peroxide membrane lipids, damage membrane and induce proteins crosslinking. The antioxidant activity of catechol structure-containing flavonoids depends on the number of hydroxyl groups in the aromatic A and B rings, and the presence of 2,3-unsaturation and a 4-carbonyl in the C ring. Flavonoids donate a hydrogen molecule, form a phenoxyl radical and scavenge single oxygen, superoxide, hydroxyl and peroxyl radicals by release of another hydrogen. The diol group forms a complex with ferric iron, copper and other transition metal ions, and prevents ROS production [15]. A marine carotenoid astaxanthin scavenges singlet oxygen, hydroxyl and peroxy radicals and nitric oxide. It spans through the membrane lipid bilayers, traps radicals at the conjugated polyene chain and in the terminal ring moiety, and removes lipid peroxide products from the membrane [16]. The antioxidant potency of phytochemicals, especially flavonoids, has been proposed to contribute to neuroprotection primarily in vitro and in vivo. However, the in vivo antioxidant actions of polyphenols require the concentrations of 10–100 μM range, one or two order higher than those in the plasma. The structure required for neuroprotection is not always correlated with that for antioxidant function. The ortho-dihydroxy substitution in B ring of flavones is not necessary for neuroprotection, whereas the hydroxyl substitution in the position 3 on the C ring and in position 5 on the A ring is required for neuroprotective activity of quercetin [17]. These results suggest the involvement of antioxidant-independent mechanism in neuroprotection.

Polyphenols upregulate ROS-scavenging enzymes, like catalase, superoxide dismutase, glutathione peroxidase, glutathione S-transferases, glutathione reductase and NAD(P)H: quinone oxidoreductase (NQO1). The induction is mediated by activation of cellular signal pathways as discussed below. Flavonoids (apigenin, luteolin, kaempferol) inhibit ROS-producing xanthine oxidase, and polyphenols from blue berry and amentoflavone suppress the expression of inducible nitric oxide synthase. Flavonoids (baicalein, quercetin, myricetin, EGCG) and nonflavonoids (gallic, protocatechuic acids) were reported to chelate iron and copper ions, and reduce free radical production.

Chronic inflammation is one of the decisive pathogenic factors in AD and PD [18]. ROS plays a key role in inflammation by release of inflammatory signal molecules peroxiredoxin 2 (PRDX2) and activates macrophages to release IL-6. Flavonoids, curcumin, lycopene (a linear unsaturated hydrocarbon carotinoid present in tomatoes) and stilbenoids inhibited proinflammatory transcription factors (NF-κB, AP-1 to suppress synthesis and release of inflammatory TNF-α, IL-6, inducible nitric oxide synthase and MCP-1 [19,20]. In in vivo models, resveratrol, curcumin and flavonoids inhibited the activity of arachidonic acid-metabolizing enzymes, COX and lipoxygenase, and suppressed the production of inflammation mediators, arachidonic acid, prostaglandins and leukotrienes, through modulation of the second messengers (cAMP, cGMP, protein kinases and calcium).

Phytochemicals protect mitochondrial function & increase biogenesis

Mitochondria are the main energy producers in cells and essential for cellular functions. The defects cause oxidative stress, activate death signal pathways and induce neurodegeneration in aging, AD, PD, Huntington's disease, stroke and psychiatric disorders [21]. In AD, the pathogenic amyloid-β (Aβ) and tau, and in PD α-synuclein (αSyn) are accumulated in mitochondria, and Aβ inhibits complex IV in the electron transport chain (ETC) and αSyn deregulates complex I. In PD, the deficiency of complex I in the ETC was reported [22], and a dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) inhibited the complex I and induced parkinsonism in humans.

Phytochemicals regulate mitochondrial functions, apoptosis signaling, the ETC activity, ATP synthesis, ROS/RNS production, mitochondrial biogenesis and degradation by autophagy (mitophagy), and exhibit neuroprotection [23]. EGCG, quercetin, myricitrin, luteolin, hesperidin, huperzine (daidzen-8-C-glucoside isolated from Huperzia serrata), hesperetin-rutinoside (a flavanone glycoside found in citrus fruits) and proanthocyanidins improved the ETC activity [24].

Mitochondria are dynamic organelles and the quality control is carried out through sequencing damaged organelle by fission (division) and exchanging matrix protein by fusion. Anthocyanins and resveratrol protected complex I and modulated mitochondrial fission/fusion pathways in SH-SY5Y cells transfected with amyloid precursor protein (APP) Swedish K670N/M671L double mutation (APPswe) [25]. Liquiritigenin induced mitochondrial fusion and inhibited mitochondrial fragmentation and cytotoxicity induced by Aβ in SK-N-MC cells [26]. Resveratrol improved the expression of fusion-mediating optic atrophy protein (Opa1), mitofisin-1 and -2 (Mfn1, Mfn2), and fission-regulating dynamin-related protein 1 (Drp1) and mitochondrial fission protein 1 (Fis1), and protected PC12 cells against rotenone-induced cytotoxicity [27].

Phytochemicals increase mitochondrial biogenesis to protect neurons [28]. Mitochondrial biogenesis is regulated by the proliferator-activated receptor PGC-1α and NRF-1 and -2, whereas mitochondrial transcription factor A (TFAM) regulates mtDNA replication and transcription. PGC-1α activates NRF-1 and -2 transcription factors and estrogen-related receptor α and induces expression of nuclear DNA-encoded mitochondrial protein. PGC-1α activity is post-transcriptionally regulated by phosphorylation and acetylation. Sirt-1 (a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase) deacetylates PGC-1α and regulates the activity. EGCG and resveratrol were reported to upregulate Sirt-1 and 5′-AMP-activated protein kinase (AMPK), enhance mitochondrial biogenesis via PGC-1α, and protect neuronal cells [29]. Quercetin, hydroxytryrosol (3,4-dihydroxy-phenylethanol present in olives), daidzein, genistein, baicalein and wogonin also induced mitochondrial biogenesis. Quercetin was reported to increase the number of muscle mtDNA and promote exercise performance in adult men [30].

Phytochemicals intervene mitochondrial cell death cascades

In neurodegenerative disorders, apoptosis is a commonly observed type of cell death characterized by the morphological and biochemical changes: cell shrinkage, chromatin condensation and nucleosomal degradation. Apoptosis signal pathways in mitochondria are well conserved and one of the most promising targets of neuroprotection, as presented in Figure 2 and Box 1. Resveratrol, quercetin, rosmarinic acid (a dimer of caffeic acid), astaxanthin and black tea extract prevented membrane permeabilization and protected cells from apoptosis induced by MPTP, amyloid aggregates and ischemia [31]. Recently, we reported that phytochemicals directly prevented or promoted the pore formation at mitochondrial membrane in a cellular model of apoptosis induced by PK11195, a ligand of the outer membrane translocator (TSPO) [9]. Astaxanthin, lipophilic derivatives of ferulic acid and sesamolin inhibited formation of pore composed of adenine nucleotide translocator (ANT) and cyclophilin-D at the inner mitochondrial membrane, which was detected as the burst of superoxide production called ‘superoxide flash’. The aldehyde and alcohol derivative of ferulic acid suppressed the pore opening at the outer mitochondrial membrane and prevented formation of the mitochondrial permeability transition pore (mPTP), efflux of apoptogenic cytochrome c (Cyt c) and calcium, and apoptosis. On the other hand, hydrophilic ethyl ester of ferulic acid and sesamin promoted the mPTP formation and apoptosis. Phytochemicals, especially curcumin, directly interacted with voltage-dependent anion channel, prevented oxidative modification of the critical thiol residues in ANT and membrane protein, and phosphorylation of voltage-dependent anion channel by glycogen synthase kinase 3 (GSK3), and prevented the pore opening. Flavonoids (kaempferol, bailarein, quercetin, naringenin, EGCG) activated signal pathways to induce antiapoptotic Bcl-2 and Bcl-xL and suppress apoptogenic Bax and Bak, and modulated apoptosis pathway in mitochondria.

On the other hand, quercetin, luteolin, kaempferol, EGCG, theaflavins, baicalein, wogonin, resveratrol and curcumin opened the mPTP and induced apoptosis in cancer cells, through inhibition of Bcl-2 and Bcl-xL, induction of Bax oligomerization, and downregulation of NF-κB signal pathway [32]. Genistein oxidized the thiol groups and pyridine nucleotides and induced mitochondrial permeability transition [33]. The ambivalent functions of polyphenols in mitochondrial death signal pathways have been considered to depend on their concentrations, antioxidant or prooxidant activities, redox state and amphipathic properties [34].

Phytochemicals present NTF-like activity by binding to NTF receptors & activating signal pathways for neuroprotection

NTFs play a major role in the development, function and survival of neurons, and the deficiency and gene variation of BDNF and GDNF are associated with pathogenesis of AD, PD and psychiatric disorder (Box 2) [35]. BDNF and GDNF have been proposed as therapeutic targets in neurodegeneration. The administration of GDNF, a dopamine neuron-specific NTF, into the nigra-striatal system has been reported in patients with PD, and the beneficial results were reported in some trials [36]. However, these NTFs cannot penetrate the blood–brain barrier (BBB) and the delivery system has been required for transport of NTFs into the brain. As an alternative practical method, the activation of de novo biosynthesis of BDNF and GDNF by phytochemicals permeable across the BBB has been proposed. Flavonoids (EGCG, puerarin, hesperitin, quercetin, genistein, kaempferol, naringenin), curcumin and resveratrol can cross the BBB in agreement with the high lipophilicity, and present NTF-like function in the brain [37]. As summarized in Figure 3, phytochemicals show NTF-mimic activity by binding to NTF receptors, direct activation of downstream signal pathways and induction of NTF expression.

7,8-Dihydroxyflavone (7,8-DHF), 7,8,3′-trihydroxyflavone (7,8,3′-THF), fisetin, deoxygedunin (a derivative of gedunin isolated from Indian neem tree), diosmetin and curcumin were found to bind to BDNF-targeted tropomycin-related kinase B (TrkB) receptor and activate PI3K-Akt (protein kinase B), Ras-MEK (MAPK)-ERK pathways, and finally cAMP-response element-binding protein (CREB) in nuclei. Activation of these pathways has promoted survival of cultured motor neurons, cerebral cortical and hippocampal neuronal cells and spinal ganglion neurons, and also provided antidepressant effects [38]. Nonflavonoid polyphenols activated NGF-specific TrkA receptor. Hesperetin, huperzine, 4,6-dimethoxyphenyanthrene, spicoside A and quinic acid derivatives bound to TrkA receptor, increased neurite outgrowth and showed neuroprotective potency [39]. On the other hand, epicatechin, a selective inhibitor of tyrosine nitration, was reported to inhibit the expression of p75 neurotrophin receptor (p75NTR) and prevent retinal neurodegeneration in a rat model of diabetes [40]. GDNF binds to GDNF family receptor α, phosphorylates the receptor tyrosine kinase RET (rearranged during transfection) and activates PI3K and ERK-MAPK pathways, but the binding of polyphenols directly to this receptor has not been reported.

In vitro studies have suggested that polyphenols interact with other receptors, protein or enzymes, and activate signal pathways for neuroprotection. A specific binding site for polyphenols and resveratrol was isolated from plasma membrane in the rat brain [41]. EGCG was found to bind to cell surface-associated 67-kDa laminin receptors, and potentiate neuroprotective and neurogenic action [42]. However, the role of these phytochemical-specific binding proteins in neuroprotection has been only scarcely reported.

MAO is localized at the outer membrane of mitochondria, regulates the homeostasis of monoamine neurotransmitters in the brain, and also serves as the modulator of signal pathways linking to decision of neuronal survival and death [43]. Inhibitors of type A MAO (MAO-A) are applied as antidepressant and anxiolytic agents, whereas type B (MAO-B) inhibitors (selegiline, rasagiline) are associated with activation of neuroprotective signaling pathways. Flavonoids (catechin, quercetin, naringenin, EGb 761, ginkgolide) inhibited MAO, depending on the presence of a (para-OH-substituted) phenyl at position 2 and unsaturation at the positions 2–3 on the C ring [44]. Flavonoids with catechol structure (quercetin, ginkgolide B, EGCG) inhibited MAO-A and induced BDNF expression in neuroblastoma SH-SY5Y cells, whereas resveratrol and curcumin inhibited MAO-B functioning as a transcription repressor and increased GDNF in glioblastoma U118MG cells [12].

GABA receptors mediated neuroprotection by anthocyanins against ethanol toxicity in prenatal rat via Bcl-2 induction by activation of calcium/calmodulin-dependent protein kinase II (CaMKII)-CREB pathway [45], and that by baicalin (baicalein 7-O-glucuronide) against global ischemia/reperfusion injury in gerbil neurons [46]. Nicotinic acetylcholine receptor α4 and α7 subunits were associated with neuroprotection by EGCG against Aβ_1-42-induced cytotoxicity in cultured cortical neurons [47]. Curcumin induced serotonin-1A (5-HT_1A) receptor [48] and hesperidin κ-opioid receptor [49], and they increased BDNF expression and hippocampal neurogenesis and showed antidepressant-like effects. Flavonoids, hesperetin and resveratrol bound to ERβ and IGF-1 receptor, enhanced BDNF and GDNF expression and promoted cell survival in astrocytes [50].

Phytochemicals modulate cellular signal pathways for neuroprotection

Phytochemicals activate prosurvival signal pathways through a number of prosurvival MAPK pathways, including PI3K/Akt and PKC (Figure 3). These pathways affect cellular function, synaptic plasticity and memory formation by altering the phosphorylation state of target molecules and modulation of gene expression. Oxidative stress is a major stimulus for MAPK cascades, which activate prosurvival ERK and also apoptosis-inducing Jun N-terminal kinase and p38 MAPKs. ERK activation inhibits the death-inducing signaling complex formation and promotes cell survival via transcriptional upregulation of antiapoptotic Bcl-2 and Bcl-xL, and suppression of Fas-mediated apoptosis.

Hesperetin, naringenin, quercetin, luteolin, amentoflavone, resveratrol and bilobalide derived from Ginkgo biloba leaf have been reported to bind to the ATP-binding site on enzymes and receptors, activate PI3K/Akt and PKC, protect neurons from cell death and ameliorate cognition and memory deficits. Flavonoids activated upstream MAPK–kinase–kinase, prevented Jun N-terminal kinase activation and inhibited apoptosis induced by oxidative stress [51]. Resveratrol, curcumin and salvianolic acid derived from traditional Chinese herb Salvia miltiorrhiza protected neurons in vivo and in vitro by activation of PI3K/Akt pathways. Hesperetin activated Akt and downregulated proapoptotic proteins, such as apoptosis signal-regulating kinase 1 (ASK1), Bad, caspase-9 and -3 in hippocampus neurons [52]. Akt phosphorylates longevity transcription factor Forkhead box O3 (FOXO3) and inhibitors of NF-κB (IκB), and activates NF-κB to upregulate the expression of prosurvival IAP (inhibitor of apoptosis protein) and Bcl-2 protein family. EGCG, hesperetin, resveratrol, curcumin and caffeic acid activated PI3K/Akt- or ERK-CREB-BDNF cascade in vivo and in vitro, leading to protect neurons, promote memory and reverse depression-like behavior [53]. Resveratrol attenuated MPP⁺-induced mitochondrial dysfunction and apoptosis through inactivation of GSK-3β by phosphorylated Akt [54].

Polyphenols activate PKC, a member of a serine-threonine kinase superfamily, which regulates cell survival, apoptosis, neuronal differentiation, immune responses, learning and memory [55]. In the striatum of MPTP-treated mice, EGCG upregulated PKCα and PKCε, prevented deletion of PKC and degraded apoptogenic Bad by the UPS [56]. EGCG and resveratrol activated PKCγ and increased neuronal interconnection in the rat hippocampus [57].

Polyphenols regulate the expression of antioxidant enzymes through activation of Keep1/Nrf2/antioxidant response element (ARE) pathway. Keap-1 binds to transcription regulator nuclear factor erythroid-derived 2-related factor 2 (Nrf2) and prevents the signal activation. Astaxanthin, lutein, lycopene, baicalein, carnosic acid, carnosol and hydroxytyrosol have been reported to dissociate Keap-1 from the complex with Nrf2, induce translocation of phosphorylated Nrf2 into the nuclei and binding to ARE in the regulatory region of the target genes, and enhance expression of Phase II enzymes, such as glutathione S-transferases, glutathione peroxidase, glutathione reductase, superoxide dismutase and NQO1 [58]. Keap1/Nrf2/ARE pathway is activated by PKCα and PI3K/Akt signal pathways, and contributes the antioxidant, anti-inflammatory and neuroprotective functions of polyphenols.

Phytochemicals induce prosurvival NTFs & NTF receptors

Polyphenols have been reported to increase BDNF, GDNF and other NTFs in humans and animal and cellular models, and ameliorate neurochemical and behavioral changes. In humans, green tea catechin and coffee fruit extract increased plasma BDNF levels [59,60]. Ginkgo biloba supplement was reported to enhance serum BDNF and aerobic performance in physically active men [61], and curcumin to increase serum BDNF levels and ameliorate the syndrome in women with premenstrual syndrome [62]. In animal models of aging, depression, AD, PD, stroke and brain ischemia, flavonoids (EGCG, hesperidin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, naringen, ginkgolide Rd), curcumin, resveratrol, astaxanthin and piperine (a major alkaloid of black pepper) increased BDNF (Figure 3). In primarily and clonally cultured neurons and astrocytes, flavonoids, resveratrol, rosmarinic acid, and oleuropein (a polyphenol constituent of olive oil) induced BDNF and GDNF expression and showed neuroprotection via ERK/cAMP response element-binding protein (CREBS), or PI3K/Akt pathways. Naringen, catalpol derived from herb Rehmannia glutinosa, smilagenin (a sapogenin from Rhizoma anemoarrhence) and harpagoside purified from Scrophularia ningpoensis increased expression of GDNF in animal models of PD [63,64]. In general, BDNF is induced by flavonoids in vivo and in vitro, whereas GDNF is mostly by nonflavonoid phytochemicals like curcumin, resveratrol and catalpol [11].

Polyphenols increase Trk expression and neurogenesis to show neuroprotection. Olive polyphenols increased TrkB and TrkA in the hippocampus, but did not in the striatum and frontal cortex, and presented antidepressant-like effects in animal models of depression [65]. EGCG increased BDNF, TrkA and TrkB expression, and prevented the pathological changes in a mouse model of AD [66]. Deoxygedunin and ethanol extracts from Hemerocallis citrina, and nobiletin isolated from citrus fruits increase TrkB and BDNF expression in mice [67] and promoted hippocampal neurogenesis in a rat model of depression [68].

Phytochemicals directly prevent the formation of disease-specific protein aggregates

Accumulation of senile plaques and neurofibrillary tangle is the major histopathological finding in AD, and that of Lewy bodies and neurites in PD. Abnormal-mutated and -modified protein is aggregated through oligomerization and polymerization, changes in the secondary structure (β-sheet formation), formation of insoluble aggregate structure and dysfunction of the proteolytic pathways. Recently, the process, rather than the end product, is considered to decide the neurotoxicity, and the therapeutic strategy should mainly aim to deplete toxic oligomers and fibrils, and aggregation intermediates.

Phytochemicals were found to inhibit the synthesis of amyloidogenic monomers and formation of oligomeric and fibrillar aggregates, promote nontoxic aggregate formation, and activate the proteolytic systems to ameliorate neuronal dysfunction caused by Aβ (Figure 4) [69,70]. Amyloidogenic Aβ peptides with 40–42 amino acids are produced by sequential cleavage of APP by β-secretase (BACE1) and γ-secretase. Tannic acid (a polyphenol), ferulic acid, polymethoxyflavones (nobiletin, tangeretin, sinensetin), genistein and galangin have been shown to inhibit β-secretase and improve behavioral impairment in animal models of AD [71,72]. Luteolin, icariin, rutin, quercetin, EGCG, resveratrol, curcumin and 7,8-dihydroxyflavine reduced the expression of β-secretase. EGCG, oleuropein, genistein and curcumin promoted APP cleavage by α-secretase into nontoxic N-terminal product named as soluble APPα and C-terminal fragment α [73].

Curcumin, myricetin and rosmarinic acid produced soluble Aβ oligomer and profibrillar species and decreased the toxic oligomers and fibrils [74,75]. EGCG was found to interact with the hydrophobic sequences of amino acids 14–24 and 27–37 of insoluble Aβ, break β-sheet motif, prevent formation of fibrils and convert large mature Aβ and αSyn fibrils into nontoxic, smaller, amorphous protein aggregate [76]. Luteolin, myricetin and honokiol (the Magnolia-derived lignan) bound to the hydrophobic region of the amyloid pentamer and inhibited Aβ aggregation [77]. Theaflavins stimulated the assembly of Aβ and αSyn into nontoxic spherical aggregates that were incompetent in seeding amyloid formation, and remodeled Aβ fibrils into nontoxic aggregates [78].

Tau is a normal, unfolded, highly soluble protein, and the imbalance between kinases and phosphatases produces hyperphosphorylated tau. Tau hyperphosphorylation changes the conformation into aggregate-prone species: conversion of the monomer to oligomer and aggregation into pair helical filaments, resulting in production of neurofibrillary tangles. The immediate tau oligomer is a most toxic form and causes synaptic impairment in AD. EGCG, resveratrol, curcumin, caffeic acid and altenusin inhibited tau hyperphosphorylation [79,80]. EGCG prevented aggregation of tau into toxic oligomers [81], and anthraquinones (emodin, daunorubicin) inhibited tau aggregation and dissolved paired helical filaments in vitro and in cells [82].

Aggregation of presynaptic αSyn has been proposed as a pathogenic factor in ‘α-synucleinopathies’ including PD. αSyn aggregation and fibrillization are induced by the increase in, missense mutations (A30P, A53T, E46K), oxidative stress, phosphorylation, metal ions (Fe³⁺, Cu²⁺) and interaction with phospholipid membrane. As in the case with Aβ and tau, intermediate formation of the toxic oligomers is most critical for the αSyn neurotoxicity. EGCG, baicalein, myricetin, kaempferol, theaflavins, curcumin, nordihydroguaiaretic acid, rosmarinic acid and ferulic acid inhibited αSyn oligomer formation and assembly into aggregates [83,84]. EGCG converted mature αSyn fibrils into nontoxic, smaller, amorphous aggregates [85]. Curcumin was reported to bind to preformed toxic oligomer and fibrils, and alter the hydrophobic surface into more soluble species [86].

Protein aggregation is caused also by dysfunction of the cellular proteolytic systems, the UPS and the ALP, as discussed in Box 3. The UPS targets the unfolded ubiquitinated protein and the dysfunction results in accumulation of misfolded protein in PD, but the role of the USP in the pathogenesis has not been fully established. The ALP targets disease-specific abnormal protein aggregates, including Aβ, αSyn, N-terminal fragments of mutant Huntingtin in Huntington's disease and mutant TDP43 in amyotrophic lateral sclerosis (aggrephagy) and damaged mitochondria (mitophagy) [87]. Autophagy activation has been shown to remove aggregate-prone proteins, regulate axon homeostasis and neurogenesis, and modify the pathogenesis of PD, AD and other neurodegenerative disorders [88].

The mTOR/ULK1 is a major negative regulator of autophagy, and the Beclin-1 (ATG6) is a positive regulator. Phytochemicals induce autophagy and show neuroprotection through mTOR-dependent or -independent mechanisms. In mouse models of PD, resveratrol prevented MPTP-induced neurotoxicity via autophagic degradation of αSyn and efflux, which was mediated by Sirt-1-dependent deacetylation of LC3 (MAP1LC3A) and its translocation from nucleus to cytoplasm [89]. Curcumin protected PC12 cells against A53T αSyn-induced cytotoxicity by downregulation of mTOR/p70S6K (p70-S6 kinase) signaling and activation of autophagy [90]. In APP/PS1 double transgenic mice, curcumin activated autophagy and protected neurons via downregulation of PI3K/Akt/mTOR signaling pathway [91]. Oleuropein aglycone induced autophagy by activation of AMPK/mTOR signal pathway, decreased Aβ aggregation and ameliorated cognition impairment [92]. Resveratrol, curcumin and EGCG inhibited AMPK and initiated mTOR-mediated autophagic degradation in cellular models of PD and AD [93,94].

Conclusion

Phytochemicals protect neuron by targeting multiple pathogenic factors of neurodegenerative disorders. Epidemiological studies have presented that dietary habits promote health and longevity, preserve cognition and motor function, and prevent depressive disorders. Intervention with food-derived compounds has been proposed as the therapeutic strategy for age-dependent neurodegeneration. However, the beneficial effects of phytochemicals in the clinical studies have not been fully confirmed. Bioavailability of the food-derived compounds is limited by the rapid metabolism, insufficient permeability across the BBB, and decreased bioavailability and stability in the brain [95]. Lipophilic flavonoids, alkaloids and terpenes can be transferred into the brain across the BBB and have high bioavailability and good affinity to receptors in the brain [96]. Development of new neuroprotective compounds is expected among these phytochemicals and the derivatives. In order to increase bioavailability and pharmacological formulation, several delivery systems have been proposed, including nanoparticles, liposomes, complexes with phospholipids and amphiphilic polymers and conjugation with amino acids and glycosides [97].

In cellular models, phytochemicals suppress or promote the mitochondrial permeabilization and apoptosis dependently on the concentrations, suggesting that ‘therapeutic window’ of food-derived compounds is too narrow to present reproducible results in clinical trials. The effects of diet and food-derived compounds should vary also among individuals with different genetic background, metabolic profile, age and environmental exposure. These facts suggest that the results obtained by epidemiological study should be carefully evaluated. Highly sensitive analytical methods for pharmacokinetic study are required to identify the potent bioactive compounds and determine the effective concentration in the targeted brain regions. In addition, the quantitative evaluation of the clinical benefits and effects on disease progression has not been established, and more sensitive in vivo quantitative methods of the survival or death and function of distinct neurons are still required.

BDNF and GDNF have been applied to patients with PD, depressive disorders and cognition decline, and the BBB-permeable phytochemicals might be useful as a surrogate delivery tool by stimulating the de novo synthesis of NTFs in the brain. BDNF and GDNF have been reported to be selectively induced by flavonoids and nonflavonoids in glial cells. It may be relevant with the results that flavonoids improve memory, cognition and depression, whereas resveratrol and curcumin ameliorate neuronal dysfunction and promote survival in animal models. Induction of distinct NTF family should be systematically investigated to establish the structure linking to increase the specified NTF synthesis.

Recently, new neuroprotective multitargeted MAO-B inhibitors have been derived from the basic structure of phytochemicals. Pyrazoline analogs of MAO inhibitors were synthesized from sasamol, curcumin and flavones [98,99]. Coumarin is found in many plants and characterized by 1,2-benzopyrone or benzopyran-2-one groups, and various drugs, inducing warfarin, acenovoumarol and phenprocoumarin have been developed. Dual acetylcholinesterase-MAO-B inhibitors, such as tacrine-coumarin hybrids, were reported as new drug candidates for AD [100]. Tacrine was the first AChE inhibitor on the market and coumarins are inhibitors of MAO and AChE activity. AChE inhibitors not only increase cholinergic transmission, but also interfere with the synthesis, deposition and aggregation of Aβ. Homoisoflavonoid Mannich base derivatives were found to have high affinity toward Aβ aggregates and MAO-B inhibitory activities, and multifunctional agents with AChE inhibition, including tacrine derivatives, have been developed [101,102]. By in silico screening for AChE inhibitors, plant benzyl alkaloids (berberastine, berberine, yohimbine, sanguinarine) and naringenin were confirmed to inhibit AChE, suggesting finding of novel anti-AD drugs [103].

The antidepressant and anticancer activities of phytochemicals have been intensively investigated, but cannot be discussed in details in this review. Depressive disorders are caused not only by the deregulation of monoamine neurotransmitter systems, but also by impaired development and function of neural architecture by the interaction between genetic and environmental factors [104].

Diet, curcumin, ferulic acid, hypericum and resveratrol presented the preventive and therapeutic activities in depressive disorders by induction of BDNF expression and promotion of adult neurogenesis in the hippocampus [105]. Phytochemicals have inhibited or promoted mitochondrial apoptosis signaling to show neuroprotective or anticancer activities through regulation of mitochondrial membrane permeability [106]. Our recently reported analytical method for the apoptosis signaling at the mitochondrial membrane [9] can be applied to screen the structure required for the inhibitory or promoting potency of phytochemicals. These prominent functions of phytochemicals should be further studied for development of novel series of antidepressants and anxiolytics and also anticancer therapeutic agents.

Future perspective

Phytochemicals and the derivatives are applied as preventive and therapeutic compounds in patients with neuropsychiatric diseases, including AD, PD and other neurodegenerative disorders, and depression. Using more advanced brain image analyses, the effects on function of specified neurons in the brain regions can be followed quantitatively in vivo. More sensitive and exact analyses of phytochemical metabolites supply the on-time information of the distribution, metabolism and bioactivity of phytochemicals in the brain. These results are applied to develop new neuroprotective compounds with more stable bioavailability and higher neuroprotective potency by induction of in situ biosynthesis of NTFs, Bcl-2 protein family and antioxidant molecules. Molecular mechanisms underlying antiamyloidogenic effects of phytochemicals are clarified in more detail, and phytochemical derivatives are applied to prevent formation of toxic Aβ and αSyn oligomers in the brain of patients with AD and PD. Instead of once proposed antibody therapy, phytochemicals are applied to covert mature protein aggregates into soluble nontoxic forms. From the BBB-permeable phytochemicals, new multifunctional compounds will be synthesized with inhibitory potency to MAO and AChE in order to increase monoamine transmitters and acetylcholine in addition to the neuroprotective activity for the therapy of AD and PD.