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Alzheimer’s disease (AD) is the most common form of dementia in the older people and 6th leading cause of death in the United States. Deposition of amyloid-beta (Aβ) plaques, hyperphosphorylation of microtubule associated protein tau (MAPT), neuroinflammation and cholinergic neuron loss are the major hallmarks of AD. Deposition of Aβ peptides, which takes place years before the clinical onset of the disease can trigger hyperphophorylation of tau proteins and neuroinflammation, and the latter is thought to be primarily involved in neuronal and synaptic damage seen in AD. To date, four cholinesterase inhibitors or ChEI (tacrine, rivastigmine, donepezil and galantamine) and a partial NMDA receptor antagonist (memantine) are the only approved treatment options for AD. However, these drugs fail to completely cure the disease, which warrants a search for newer class of targets that would eventually lead to effective drugs for the treatment of AD. In addition to selected pharmacological agents, botanical and medicinal plant extracts are also being investigated. Apart from its culinary use, garlic (Allium sativum) is being used to treat several ailments like cancer and diabetes. Herein we have discussed the effects of a specific ‘Aged Garlic Extract’ (AGE) and one of its active ingredients, S-allyl-L-cysteine (SAC) in restricting several pathological cascades related to the synaptic degeneration and neuroinflammatory pathways associated with AD. Thus, based on the reported positive preliminary results reviewed herein, further research is required to develop the full potential of AGE and/or SAC into an effective preventative strategy for AD.
Alzheimer’s disease (AD) is the most common form of dementia affecting elderly populations all over the globe. Over 5.4 million people in the United States are diagnosed with AD and this number will triple by mid- century if definite curative or restrictive therapies are not developed. Neuropathologically, deposition of amyloid β (Aβ) peptides in the brain parenchyma, hyperphosphorylation of microtubule associated protein tau (MAPT) and loss of cholinergic neurons comprise the major hallmarks of AD. It is believed that deposited Aβ peptides trigger a series of inflammatory reactions including activation of microglia, generation of reactive oxygen species (ROS) and cytochemokines result in widespread neuronal loss . To date, four cholinesterase inhibitors (ChEI; tacrine, rivastigmine, donepezil and galantamine) and a partial NMDA receptor antagonist (memantine) are the only treatment options for AD. Although these drugs can provide some symptomatic relief, they fail to cure or restrict the disease progression. In this context, clinical trials with new drugs aiming to decrease Aβ production have been conducted. Unfortunately, some of these trials were halted because of serious adverse effects including cancer . Moreover, these drugs primarily target a single pathological pathway such as Aβ precursor protein (APP) processing. It is indicated that deposition of Aβ peptides as diffuse plaques appear much earlier in the disease process and it is possible that people may remain cognitively intact for decades. Neuroinflammation may be a key player responsible for the clinical onset of AD. Since AD has multifactorial etiopathogenesis, such as neuritic plaques, neurofibirillary tangles, severe neuroinflammation, glutamate induced excitotoxicity, defective Aβ breakdown, we hypothesize that an agent effective in modulating multiple pathological pathways could be an appropriate therapeutic agent in preventing and restricting AD progression. Although sequence of these events is poorly understood, they result in widespread neuronal and synaptic loss.
In addition to pharmacological agents, botanical and medicinal plant extracts are also being investigated. Some of these studies are based on dietary and epidemiological studies, including incidence and prevalence of AD in different cultures and countries. For example, a large epidemiological study had depicted a significant (~4.4-fold) less incidence of AD in Indian elderly when compared with a reference American populace. Another epidemiological study did not observe any association between AD and APOE ε4 in an elderly cohort in Nigeria. However, the latter was strongly associated with AD in African American populations. These findings indicate a strong environmental contribution in the development of AD, which may include dietary factors. In this context, Mediterranean diets are believed to protect against the development of AD. Resveratrol (a polyphenol present in fruits and red wine) increases cerebral blood flow in young adults . Curcumin (a polyphenol present in turmeric and curry powder) was shown to have protective or curative roles in AD models . However, curcumin failed to exert significant beneficial effects in clinical settings, which can be attributed to its poor bioavailability and rapid bio-transformation. In this context, we have observed increased bioavailability and significant neuroprotective properties of a nanoformulation of curcumin (NanoCurc™) in vivo as well as in neuronal cells . We studied further to evaluate potential beneficial roles of other naturally occurring compounds in the context of neurodegenerative disorders, such as AD, and observed significant neuroprotective and synaptopreservative properties of garlic derived compounds in animal and cell culture models . In this review, we will discuss ‘pleiotropic’ properties of garlic components in modifying several pathological cascades involved in AD.
Apart from culinary purposes, garlic (Allium sativum) is being used to treat different ailments such as cardiovascular , gastrointestinal , hematological  and as cancer . Recent studies have demonstrated beneficial effects of “Aged Garlic Extract” (AGE) and one of its active ingredients S-Allyl-L-Cysteine (SAC) in AD models [12, 17]. In these studies AGE and SAC treatments not only decreased Aβ loads in APP-transgenic (Tg) mice, but also ameliorated tau pathology and increased levels of synaptic protein markers versus vehicle treated mice. Previous studies also demonstrated potent antioxidant action of AGE .
Aβ peptide is a generated from APP by sequential enzymatic cleavage of β- and γ secretases. APP, which is encoded by a long gene localized in human chromosome 21, is a large (695–770 amino acids) glycosylated transmembrane protein. Apart from mammalian APP, two other non-mammalian homologous proteins were also identified which are appl (present in Drosophila melanogaster) and apl-1 (present in the worm Caenorhabditis elegans) [19–20]. APP is first cleaved by β secretase (also known as BACE-1) to produce sAPPβ (596 amino acids for APP 695 isoform) and a C-terminal 99 amino acid containing fragment (C99). In the final step of processing, C99 is cleaved by γ secretase to produce Aβ peptides, containing 39–43 amino acids residue . The Aβ peptide with 40 amino acids (Aβ40) is abundantly produced in this process and considered as less pathogenic. However, the Aβ peptide with 42 amino acids residue (Aβ42), although produced in lower quantities, is fibrilar in nature and can potentially form aggregates [23–24]. In addition to aggregated Aβ, soluble oligomeric Aβ species are neurotoxic, and the brain load of the latter was found to be positively correlated with the clinical symptoms of AD [25–26]. It is believed that Aβ deposition can also trigger hyperphosphorylation of tau and neuroinflammation including generation of ROS.
MAPT or Tau is a microtubular protein found in neurons and coded by a gene located at chromosome 17 . One of the primary functions of tau is to provide structural stability of microtubule by interacting with tubulin, another structural protein of the cell . Tau is a phosphoprotein, and hyperphosphorylation can prevent binding of tau with tubulin, hence destabilize axonal structures. Phosphorylation at Ser and/or Thr residues at different positions (such as Ser 262, Thr 231, Ser 235) of tau protein is considered as the major forms of phosphorylation and observed in neurodegenerative disorders including AD . Several protein kinases including glycogen synthase kinase-3 (GSK-3) and cyclin dependent kinase-5 (cdk-5) can phosphorylate tau protein . Postmortem AD brain revealed 3-4-fold more hyperphosphorylation of tau protein versus post mortem non-AD brain tissue . To date, it is not completely clear whether or not deposition of Aβ causes tau hyperphosphorylation. Intracerebral infusion of Aβ peptides in Tg mice models induced tauopathies . Further, intracerebral infusion of deposited Aβ peptides collected from aged APP-Tg mice to young tau-Tg mice resulted in an increase in tau pathology in the brain of the injected mice . In a triple transgenic mouse model (APP, presenilin-1 and tau), Aβ deposition was observed before the appearance of tangle pathology .
Beyond AD and in a clinical study, we also observed increased levels of CSF-phosphotau in normal pressure hydrocephalus (NPH) patients suffering for more than one year . Cardinal pathology of NPH involves disruption of CSF hemodynamics, which may result in increased Aβ deposition . However, tau pathology was not observed in the postmortem brain samples of patients suffered in a rare disease, hereditary cerebral hemorrhage with amyloidosis of Dutch type (HCHWA-Dutch), which also showed a considerable amount of Aβ burden . Moreover, a growing body of evidence suggests a link between hyperphosphorylation of tau in diabetes. Brain tissues of streptozotocin induced diabetic rats’ revealed decreased levels of insulin in the brain due to altered transport of insulin across the blood brain barrier (BBB) . Further, GSK-3β, a prime enzyme responsible for pathological phosphorylation of tau can be inactivated by insulin . Thus apart from AD, other disorders such as NPH, diabetes can induce hyperphosphorylation of tau.
As stated earlier, neuroinflammation is believed to play a central role in the clinical onset of AD. This is consistent with the observations that deposited Aβ can activate microglia, which produces cytochemokines (such as TNFα, ILs) and result in neuroinflammation. Further, ROS, nuclear factor kappa beta (NFκB), peroxisome proliferators-activated receptor-γ (PPARγ), apolipoprotein E (APOE) are also thought to play important roles in neuroinflammation associated with AD (for review see ). Neuroinflammation can be an important cause for widespread neuronal damage observed in AD, especially cholinergic neurons in basal forebrain are more susceptible for degeneration .
Garlic consists of several sulfur containing ingredients and alliin (S-allyl cysteine sulfoxide) is the major one. Alliin undergoes several chemical transformations to generate a series of other sulfur compounds such as allyl thiosulfinate, allicin, allyl methyl thiosulfinate, allyl propenyl thiosulfinate, diallyl disulfide, and allyl methyl sulfide . SAC is an important component of garlic which is produced by hydrolysis from γ-glutamyl-S-allyl cysteine . Some sulfur containing garlic compounds are sparely water soluble and upon ingestion can also cause adverse effects in humans. AGE is prepared by soaking garlic in ethanol-water mixture for 20 months, which removes irritant compounds from garlic and solubilize some of the insoluble compounds . SAC is one of the active ingredients of AGE. Previous report has proposed greater safety and efficacy of AGE than raw garlic as a therapeutic agent .
AGE and SAC were observed to possess potential anti-amyloidogenic properties both in vitro and in vivo studies. Previous research has demonstrated protection of cellular structures by AGE from Aβ- mediated damage [43–44]. Mechanistically, SAC was shown to have Aβ disaggregation property in vitro . In an AD animal model, four months of independent treatments of AGE and SAC resulted in a significant decrease of both Aβ load and numbers of Aβ plaques in the brain of APP-Tg mice (Tg-2576) versus non-treated Tg mice controls . In addition to levels of Aβ peptides, brain lysate of the animals from the above study was analyzed in Western immunoblotting to measure intracellular APP. Notably, we observed a significant decrease in the levels of intracellular APP in AGE-treated APP-Tg mice versus untreated Tg mice (Ray and Lahiri unpublished). The exact mechanism of AGE and SAC in modulating APP levels or processing is still not properly elucidated and could be mediated through NFκB (discussed later).
Apart from that, it was also observed that SAC can prevent Aβ-mediated hippocampal neurodegeneration by attenuating endoplasmic reticulum (ER) stress . Another important aspect of AGE and SAC’s mechanism is their ability in inhibiting the activation of caspase 3. Deposited Aβ can increase the level of activated caspase 3 , which was also observed in APP-Tg mice brain versus wild type mice. It was also observed that activation of caspase 3 can lead to decreased synaptic function and postsynaptic density . We have observed significant increase in the levels of pre-synaptic proteins SNAP25 and synaptophysin in brains of APP-Tg mice treated with either AGE or SAC versus non-treated APP-Tg mice . Further, we have observed decreased levels of cleaved caspase 3, which indicates inhibition of the caspase 3 activity in AGE-treated APP-Tg mice brain samples versus untreated Tg mice (data not shown).
Based on a variety of current research results, a strong positive correlation between diabetes mellitus and AD has been proposed. Insulin in the brain can display growth factor like functions . Further, insulin increases the expression of choline acetyl transferase (ChAT) in cholinergic neurons of basal forebrain . In addition, insulin degrading enzyme (IDE), which catalyzes insulin also takes part in breaking down of Aβ peptides in the brain . Hence diabetes mellitus, characterized by reduced production or peripheral resistance of insulin may lead to the development of AD. Interestingly, garlic can increase insulin secretion from pancreatic β cells, i.e. considered as an insulin secretagogue . Secretagogues, which are medicines that stimulate the beta cell to secrete insulin, include the sulfonylureas and glinides. In experimental settings one of the garlic derived compounds S-Allyl-L-cysteine sulfoxide (SACS) was shown to ameliorate symptoms of diabetic rats, and this action of SACS was comparable to glibenclamide, a known anti-diabetic agent . Another important aspect of AGE’s and SAC’s beneficial roles in Aβ-related pathology is by augmenting cardiovascular health. Epidemiological and clinical studies have documented that cardiovascular lesions are common in AD and are also potential risk factors [55–56]. Hypercholesterolemia, the most important causative factor for cardiovascular pathology, was shown to enhance brain Aβ accumulation . Refolo et al. demonstrated an alteration in APP processing towards amyloidogenic pathway by cholesterol in Tg mice model . Further, radio imaging study has documented that higher serum cholesterol level is associated with increases frequency of APOE ε4 alleles, one of the well known risk factors for AD . Interestingly, garlic and garlic derived compounds were shown to have potent antilipidemic properties in term of inhibiting cholesterol synthesis [60–62]. Several mechanisms of garlic and its components’ lipid lowering properties were proposed including inhibition of the rate limiting enzyme of cholesterol biosynthesis, HMGCoA-reductase .
The summary of potential effects of AGE and SAC in ameliorating Aβ-related pathology is illustrated in Fig. (2).
In the molecular sequel related to the pathogenesis of AD, hyperphosphorylation of tau proteins is believed to follow Aβ deposition and to form neurofibrillary tangles. Certain garlic compounds effects’ in ameliorating tau pathology in preclinical settings are encouraging. Working with APP-Tg mice, Chauhan observed a decreased level of tau phosphorylation in APP-Tg mice by independent treatments of AGE, SAC and diallyl disulfide (DADS), another compound present in garlic. Mechanistically, the decreased level of tau phosphorylation was most likely due to decreasing the activity of GSK-3β by garlic compounds . No change in the levels of Cdk5 was observed in the treated animals versus controls. As discussed, deposition of Aβ peptides can induce hyperphosphorylation of tau protein, AGE or SAC’s anti-amyloid properties can also be responsible for preventing hyperphosphorylation of tau. Interestingly, insulin secretagogue action of garlic compounds can increase the brain levels of insulin and insulin like growth factor (IGF), which can decrease brain Aβ burden and inhibit the activation of GSK-3β and can potentially prevent tau phosphorylation. Further, studies revealed an increased level of tau phosphorylation both in cortex and hippocampus of rats that were fed with high cholesterol containing diet for a prolog period of time (6 months) . In AD patients, increased in the levels of CSF-phosphotau is considered as one of the diagnostic criteria . It was observed that 14-weeks treatment with an HMGCoA- reductase inhibitor (simvastatin) decreased the levels of CSF-phosphotau in hyperlipidemic patients with dementia , and this can be attributed as an indirect evidence that hyperlipidemia can potentially cause hyper-phosphorylation of tau proteins. Due to antilipidemic properties, garlic compounds can play an important role in ameliorating tauopathies. In addition, several studies have shown that inflammation can induce tau pathology. Induction of microglial activation by lipopolysaccharide (LPS) was shown to increase tauopathies in tau-transgenic mice . Activated microglia produces interleukin-1 (IL-1), which was observed to participate in tau pathology . Moreover, activation of microglia can occur by secreted APP as well . Taken together, due to their anti-inflammatory (discussed later), ROS-scavenging and APP lowering properties, garlic compounds can ameliorate neuroinflammation, which in turn, can be effective in preventing excessive phosphorylation of tau.
Sporadic AD is generally a disease of the old age. Mitochondrial DNA is damaged in aging, which is not only associated with low ATP production but also with higher production of ROS, including highly charged free radicals . In AD, deposited Aβ can activate microglia and activated microglia generates ROS, which play an important role in damaging neurons . Apart from ROS, activated microglia can give rise to a number of pro-inflammatory cytochemokines like interleukins (ILs) and TNFα, leading to further neuronal loss . Because of the anti-amyloid properties, AGE and other garlic derived compounds like SAC can be helpful in ameliorating neuroinflammation. From a therapeutic point of view, since Aβ deposition precedes the onset of clinical dementia and the exact timing and sequence are unknown, lowering only Aβ loads may not be sufficient in halting or treating AD. In this context, ROS-scavenging property of AGE and SAC would be beneficial. Notably, we have observed a significant protection to neuronally differentiated rat PC12 cells by AGE and SAC from ROS (H2O2)-mediated insults .
Mechanistically, it is suggested that garlic compounds can modulate intracellular levels of glutathione (GSH), a key enzyme responsible for cellular protection against ROS. Upon interaction with ROS, GSH become converted to oxidized glutathione (GSSG) by the enzymatic action of glutathione peroxidase . The ratio of GSH to GSSG is an important parameter to determine the redox state of the cells; an increased ratio of GSH:GSSG denotes a reduced intracellular environment and the ratio decreases in oxidative stressed condition, which was observed in some specific regions of postmortem AD brain . AGE was observed to increase the levels of intracellular GSH . Further, in a rat model, AGE treatment was detected to preserve the levels of glutathione peroxidase and glutathione reductase, and the latter is involved in conversion of GSSG to GSH . Similar results were observed in SAC treatment in a cell culture-based study . Further, independent treatments of AGE and SAC to APP-Tg mice revealed a significant decrease in IL-1β immunoreactive microglia numbers compared to untreated APP-Tg mice .
One of the important mediators of inflammation in AD is activated NFκB, which is responsible for the production of various proinflammatory cytochemokines . In steady state, NFκB stays within cytosol of the cells and stays bound to an inhibitor protein IkB . NFκB activations requires phosphorylation of IkB and once IkB is phosphorylated, NFκB translocates to the nucleus and binds with promoter regions of several pro-inflammatory cytokines genes . One of the stimulatory factors for NFκB activation is Aβ peptide, which was observed to bind with a death receptor (75 kDa-neurotrophin receptor) resulting in NFκB activation . In addition to Aβ peptides, generated ROS from Aβ-microglia interaction can also stimulate NFκB activation . Promoter mapping of different genes shows the presence of NFκB binding sites within APP, presenilin-1 and BACE-1 genes, indicating up-regulation of these proteins following activation of NFκB . In this context, prevention of NFκB activation is considered as a rational strategy to ameliorate inflammation related to AD. Curcumin, one of the potential therapeutic candidates in AD and cancer, was shown to suppress the activation of NFκB . Suppression of NFκB activation was also observed by SAC in cultured cells when they were separately treated with ROS and TNF-α [83–84].
Peroxisome proliferators-activated receptors (PPAR) are a class of ligand-activated transcription factor and plays an important role in regulation of inflammation . PPAR has three distinct subtypes α, β and γ; out of those PPAR-γ is involved in adipocyte differentiation and also a target for anti-diabetic drugs like pioglitazone . It was shown that activation of PPAR-γ in microglia and macrophages is involved in alleviating inflammation by decreasing the production of pro-inflammatory cytochemokines [87–88]. Further, activation of PPAR-γ is involved in Aβ clearance . Regarding garlic compounds, diallyl disulfide (DADS), a component of AGE was shown to increase the expression of PPAR-γ in cell culture, hence can act as an anti-inflammatory agent [90–91].
Neuronal destruction and synaptic loss are the endpoints of neuroinflammation seen in AD, and cholinergic neurons in the basal forebrain are most vulnerable by this process . Hence, facilitating choline uptake by cholinergic neurons or preserving the rate limiting enzyme for choline synthesis, choline acetyl transferase (ChAT) can be considered as a possible intervention approach. Working with cholinergically differentiated human neuroblastoma (SK-N-SH) cells, we have observed significant ‘procholinergic’ properties of AGE in terms of increasing high affinity choline uptake (HACU) and ChAT activity when the cells were co-treated with AGE and ROS (H2O2)(Ray and Lahiri, unpublished data). Similar ‘procholinergic’ effects were observed in SAC and ROS cotreated cholinergic human SK-N-SH cells (data not shown).
Development of novel drugs to treat AD is one of the priorities in current biomedical research as the number of AD patients and societal costs are steadily increasing. Current FDA-approved drugs for the treatment of AD fail to completely cure the disease. Several new drugs had been tried in clinical trials but most of them fail to demonstrate a definite curative or disease restrictive effects in those trials. In our view, the reasons for not achieving desirable effects with the new drugs can be due to the fact that these drugs primarily target a single molecular pathway such as APP processing. As we reviewed here, clinical manifestation of AD may not solely depend on deposited Aβ but hyper-phosphorylation of tau, and severe neuroinflammation can also play vital roles. Based on the aforementioned discussions, we support the proposal of the necessity of agents with ‘pleiotropic’ properties in the treatment of AD . In fact, some pleiotropic properties have been observed in recent studies with ChEI (rivastigmine and phenserine) and memantine. Apart from their primary mode of actions, these drugs have been reported to display APP modulating and neuropreserving properties [94–96]. Rivastigmine, which increases acetylcholine levels by inhibiting acetylcholine esterase and butyrylcholine esterase was also observed to increase the levels of presynaptic proteins SNAP25 and synaptophysin in rat embryonic primary neurons .In this context, garlic compounds AGE and SAC can have significant effects in lowering brain amyloid load, reducing hyperphosphorylation of tau proteins and ameliorating neuroinflammation by modulating several interconnected pathways, however, how much these effects really translate into clinical trials remain to be seem.. In any case, the overall results of such intervention would have far reaching implications. Taken together, mechanistically, AGE and SAC can have great potential for an effective use in the prevention and treatment of AD. Particularly, as a pure synthesizable chemical compound, SAC can emerge as a potential therapeutic agent in preventing and treating neurodegenerative disorders, such as AD. In order to fully understand their efficacy, further research is warranted in larger clinical settings.
We thank Jason Bailey and Justin Long, and grant supports from the National Institutes of Health (AG18379 and AG18884) to DKL.