Preventive and Therapeutic Strategies in Alzheimer's Disease: Focus on Oxidative Stress, Redox Metals, and Ferroptosis

Abstract

Significance: Alzheimer's disease (AD) is the most common cause of dementia in the elderly. AD is currently ranked as the sixth leading cause of death, but some sources put it as third, after heart disease and cancer. Currently, there are no effective therapeutic approaches to treat or slow the progression of chronic neurodegeneration. In addition to the accumulation of amyloid-β (Aβ) and tau, AD patients show progressive neuronal loss and neuronal death, also high oxidative stress that correlates with abnormal levels or overload of brain metals.

Recent Advances: Several promising compounds targeting oxidative stress, redox metals, and neuronal death are under preclinical or clinical evaluation as an alternative or complementary therapeutic strategy in mild cognitive impairment and AD. Here, we present a general analysis and overview, discuss limitations, and suggest potential directions for these treatments for AD and related dementia.

Critical Issues: Most of the disease-modifying therapeutic strategies for AD under evaluation in clinical trials have focused on components of the amyloid cascade, including antibodies to reduce levels of Aβ and tau, as well as inhibitors of secretases. Unfortunately, several of the amyloid-focused therapeutics have failed the clinical outcomes or presented side effects, and numerous clinical trials of compounds have been halted, reducing realistic options for the development of effective AD treatments.

Future Directions: The focus of research on AD and related dementias is shifting to alternative or innovative areas, such as ApoE, lipids, synapses, oxidative stress, cell death mechanisms, neuroimmunology, and neuroinflammation, as well as brain metabolism and bioenergetics.

Introduction

Alzheimer's disease (AD) is a chronic degenerative brain disease and the most common cause of dementia in the elderly (sporadic late-onset AD). AD stands as the sixth leading cause of death in the United States and is currently incurable with no effective therapeutic approaches to treat, reverse, or slow down its progression (9, 102). The real cause of AD is still a controversy, but old age is the most significant risk factor. Etiology of AD is a complex combination of behavioral, genetic, and environmental risk factors that derive in pathogenic mechanisms, most notably the abnormal accumulation of amyloid-β (Aβ), hyperphosphorylated tau, neuroinflammation, progressive synaptic loss, and neuronal death. Consequently, therapeutic strategies have focused on the key components of the amyloid cascade: Aβ and tau. Small drugs and immunotherapies have been designed to inhibit Aβ production or aggregation, clearance or neutralization of neurotoxic Aβ oligomers/plaques, blocking extracellular spread and toxicity of tau (67).

In almost three decades of research, only five prescription drugs have been approved by the Food and Drug Administration (FDA) for AD, and none are based on amyloid or tau (Table 1) (2a). Although none of these medications cures or stops the disease, they are prescribed to alleviate dementia symptomatology, including memory loss and confusion. Patients diagnosed with mild to moderate AD receive galantamine, rivastigmine, and donepezil, whereas memantine and memantine/donepezil are prescribed to treat moderate to severe AD.

Table 1.

Food and Drug Administration Approved Medications for Alzheimer's Disease^a

Drug	Mechanism of action	Dosage	Side effects
Donepezil	Cholinesterase inhibitor	Oral 5–10 mg/day, maximum 23 mg/day	Nausea, vomiting, diarrhea, muscle cramps, fatigue, weight loss.
Rivastigmine	Cholinesterase inhibitor	Oral 3–12 mg/day	Nausea, vomiting, diarrhea, weight loss, indigestion, muscle weakness.
		Patch 4.6–13.3 mg/day
Galantamine	Cholinesterase inhibitor	Oral 8–24 mg/day	Nausea, vomiting, diarrhea, loss of appetite, dizziness, headache.
Memantine	NMDA antagonist	Oral 5–28 mg/day	Dizziness, headache, diarrhea, constipation, confusion.
Memantine+Donepezil	NMDA antagonist and cholinesterase inhibitor	Variable (7/10, 14/10, 21/10 or 28/10 mg) depending on whether patients are stabilized on memantine and/or donepezil.	Nausea, vomiting, diarrhea, headache, dizziness, loss of appetite.

^aRef. (2a).

NMDA, N-methyl-d-aspartate.

Unfortunately, more than 200 phase II/III clinical trials assessing more than 100 different drugs have dramatically failed, with the cost of several billion dollars and decades of research (67, 115). Some methodological factors in the design of AD clinical trials have been correlated with the unsuccessful outcomes rather than the effectiveness of the drug itself, including inadequate dosing, recruitment/selection of patients, inconsistency in cognitive scoring procedures, inappropriate time of intervention, and assessment of target engagement, among others (67, 115). This raises the question as to whether current therapies are targeting the wrong pathological components or whether a multitarget approach is needed, for effective disease modification rather than symptom remission. Herein, we review therapeutic approaches for AD and related dementia that identify alternative targets and brain mechanisms beyond the centralized Aβ and tau strategies. Some of them are in early or conceptual stages, tested on in vitro or animal models, and others are already under scrutiny in clinical trials.

Alterations of Redox Homeostasis

The course of AD is accompanied by a progressive alteration or detriment of brain metabolism. Patients with AD present alterations of the neuroendocrine system, glucose metabolism, antioxidants, neuroinflammation, and reactive oxygen species (ROS), affecting cognitive functions, behavior, and functionality of the brain. Overall, this state is commonly known as oxidative stress, characterized by an impaired response to oxidant or electrophile stress (Fig. 1). Oxidative stress affects many metabolic pathways in the brain; the interventions that counteract oxidative damage include antioxidants, polyphenols, glucose metabolism, diet, and lifestyle (18).

FIG. 1.

Imaging of ROS in neurons. Live cell imaging of N27 rat dopaminergic neurons treated with 250 μM of ammonium iron (III) citrate for 3 h. (A) Bright-field imaging. (B) DCFDA fluorescence as an indicator of ROS in cells. DCFDA, 2′,7′-dichlorofluorescin diacetate; ROS, reactive oxygen species. Color images are available online.

Antioxidants

Vitamin E (α-tocopherol) has been proposed as a neuroprotective agent and an antioxidant. Supplementation of vitamin E in AD models reduced deficits of learning and memory, attenuation of oxidative stress, decreased Aβ deposits, and improved cognition (75). Trials with AD patients receiving doses of 800, 1000, or 2000 IU/day of vitamin E for 6–48 months showed a slow functional decline in comparison with placebo, lower oxidative stress but no effects over the progression from mild cognitive impairment (MCI) to AD (51, 75). This antioxidant is also supplemented in combination with selenium or memantine, but with no significant changes in dementia progression, suggesting that vitamin E is beneficial only in mild-to-moderate AD to reduce functional decline (47, 97).

The most extensively consumed antioxidant is vitamin C (ascorbic acid). Ascorbic acid supplementation showed neuroprotective properties, reducing free radicals and neuroinflammation, also iron chelation and Aβ reduction (118). Ascorbate participates in neuronal maturation and differentiation, myelin formation, modulation of neurotransmission, and redox homeostasis (95). APP/PSEN1 mice with vitamin C deficiency showed increased levels of oxidative stress (malondialdehyde, protein carbonyls, F2-isoprostanes) in the brain cortex and decreased glutathione (GSH) compared with wild-type controls and also increased levels of Aβ (38). In a randomized phase I clinical trial with 78 subjects (NCT00117403), supplements were administered for 16 weeks, 800 IU/day of vitamin E + 500 mg/day of vitamin C + 900 mg/day of α-lipoic acid or 400 mg/day CoQ; the compounds did not affect cerebrospinal fluid (CSF) AD biomarkers (Aβ or tau) but caused reduction of oxidative stress. A longitudinal study of 600 elderly patients reported that around 8% of the subjects used vitamins C and/or E (normal dose or high dose), but the vitamins showed no correlation to reduce the time to develop dementia (52).

Vitamin B supplementation in elderly patients may reduce cognitive decline, by lowering levels of serum homocysteine, which has been linked as a potential risk factor for cognitive impairment. Clinical trials in phase III (NCT00056225) evaluating the efficacy of high-dose vitamin B12 (1 mg/day), B6 (25 mg/day), and B9 (folic acid, 5 mg/day) with AD patients reported reduction in homocysteine levels, but no significant improvement in cognition (6, 188). Some adverse effects of depression were observed in patients taking vitamin B. Vitamin supplementation can be included in nutritional formulations enriched in folate, α-tocopherol, B12, S-adenosyl methionine, N-acetyl cysteine, and acetyl-l-carnitine; in a phase II trial, the formulation was administered for 3–6 months to 106 individuals with AD (NCT01320527). The evaluation indicated that the formulation helped to maintain or improve cognitive performance and mood behavior (142). Vitamins B1/B6/B9/B12 modify brain metabolism, oxidative stress, inflammation, and cognition in AD. A 3-month pilot study of vitamin supplementation reduced levels of oxidized proteins (carbonyl groups) (145), whereas folic acid (1.25 mg/day for 6 months) reduced Aβ and inflammation biomarkers (TNFα, IL6) (23). Remarkably, a high B-vitamin dose for 2 years (folic acid 0.8 mg, vitamin B6 20 mg, vitamin B12 0.5 mg) slowed brain shrinkage and atrophy (45).

Serum levels of carotenoids in AD patients indicated that β-carotene was significantly lower in the demented group, whereas α-carotene was similar in comparison with controls (88). This deficiency could be related to a dietary deficiency, since AD patients usually have a weight and body mass index lower than controls. Low levels of β-carotene correlate with higher levels of CSF Aβ and tau and increased oxidative stress (165). The oxidative damage is related to low levels of blood antioxidants (uric acid, vitamins, and carotenes), since none of the major antioxidant enzymes is significantly decreased in AD (150). Remarkably, institutionalized AD patients receiving nutritional supplements showed reduced morbidity and mortality (65).

Caffeine has antioxidant properties, and its consumption reduced Aβ in an AD mouse model. The administration of 0.5 and 30 mg/day of caffeine was shown to have important effects in the sporadic AD-like rabbit model with a cholesterol-enriched diet, including reduction of cholesterol-induced Aβ levels and p-tau, control oxidative stress, and normalize levels of adenosine A(1) receptors (136). Lifelong consumption of caffeinated drinks (coffee and tea) has been linked with the prevention of cognitive decline and reducing the risk of neurodegeneration, but the recommended intake should be around 200–400 mg (2.5–5 cups of coffee) daily (122). Other benefits of caffeine could be linked to increasing alertness, concentration, improved mood, reduced depression, and even potentiation of the effect of other regular drugs such as analgesics. Caffeine trials have determined a lack of association with the prevention of AD and related dementia, but others showed the potential benefits of coffee, tea, and caffeine consumption (131). New studies testing the potential uses of caffeinated products for symptomatology control or prevention of AD will continue over the next few years.

Production of melatonin (N-acetyl-5-metoxytryptamine) is reduced with aging, suggesting that this decline is related to AD progression. Its mechanism of action is by stimulation of nonamyloidogenic processing of AβPP, activation of α-secretase, and downregulation of β- and γ-secretases (157). The neuroprotective effect of melatonin is an antioxidant, free radical scavenger, and mitochondrial protectant, stimulating the synthesis of antioxidant enzymes (SOD, GPx, and glutathione reductase) and production of glutathione, modulating aggregation of Aβ and tau, and regulating tau phosphorylation (10, 130). APP/PS1 mice supplemented with melatonin for 12 months showed a significant reduction of plasma levels and Aβ deposits (126). Clinical trials using melatonin (50–100 mg/day) for 10 days to 24 weeks showed safety, but no improvement of cognitive abilities in AD patients only improved sleep quality (178).

Among the herbal extract used for cognitive disorders and AD, Ginkgo biloba is one of the most popular. Standardized G. biloba extract is a supplement with antioxidant properties, improving memory and cognitive functions (161). In cultured neurons, Neuro2A overexpressing AβPP G. biloba extract was added at 100 μg/mL for up to 10 h, observing a reduction of ROS and malondialdehyde, enhancement antioxidant enzymes, and reduction of Aβ (24). The standardized extract of G. biloba was administered to rats, observing an increase in catalase and superoxide dismutase (SOD) activities in the hippocampus, striatum, and substantia nigra, and reduced lipid peroxidation, reducing overall oxidative damage (16). At least 21 clinical trials have been performed for G. biloba, indicating potential benefits in cognitive functions but with inconsistencies among the trials and with mild adverse events (186). A most recent trial with standardized G. biloba extract (EGb 761) tested its effectiveness for treating behavioral and psychological symptoms of dementia; after 22–24 weeks with a daily dose of 240 mg, the patients showed better results in symptom scores except for delusions, hallucinations, and elation/euphoria (148). The efficacy of G. biloba in AD remains controversial but it could be beneficial to control symptoms.

Polyphenols

Epigallocatechin-3-gallate (EGCG or Sunphenon EGCg) is the key bioactive polyphenolic flavonoid from green tea, with antioxidant, anti-inflammatory, and neuroprotective effects. The APP/PS1 mice treated with EGCG and/or ferulic acid (30 mg/kg) showed reversed cognitive impairment and reduced Aβ, promoting nonamyloidogenic AβPP processing, reducing neuroinflammation and oxidative stress (119). EGCG treatment decreased p-tau in rat primary cortical neurons, this independent of Nrf2 activation but with enhanced autophagy (26). Preclinical and translational studies of EGCG have shown anti-inflammatory and neuroprotective effects against neuronal damage and brain edema, but there are reports of a negative association between green tea and the prevalence of MCI (20). The benefits of EGCG seem to be related to improving mood and work performance, modulation of cerebral blood flow, and reduced stress. A phase III trial is testing the safety and tolerability of Sunphenon EGCg (high-purity EGCg, NCT00951834) with a dosage of 200–800 mg, but no results are published.

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a polyphenol with antioxidant properties found in red grapes (wine), blueberries, peanuts, soybeans, pomegranates, and dark chocolate. The mechanisms attributed to resveratrol are extensive, from cell signaling in cytokines, caspases, metalloproteins, inflammation, and glucose metabolism, among others. The utility of resveratrol for treating oxidative stress in neurodegeneration was tested in mice that were fed with 300 mg/kg resveratrol for 45 days, observing reduced Aβ plaque formation (48% reduction in the cortex, 89% reduction in the striatum, and 90% reduction in the hypothalamus), but glutathione declined by 21% and cysteine increased by 54% (90). A phase II trial with 119 participants analyzed a 500 mg/day of resveratrol; the supplement was safe and tolerated (with minor side effects); and after 1 year, the CSF Aβ and plasma Aβ showed a decline. Interestingly, the group that received resveratrol showed increased brain volume loss (169). Resveratrol has been extensively tested with 244 trials; in 2019, there were 27 ongoing clinical trials (phase III for AD and phase IV for MCI) (160).

Curcumin is a polyphenol from turmeric herb. Its properties include inhibition of Aβ aggregation, reduction of p-tau, copper-binding, reduction of cholesterol, microglia modulation, and antioxidant and anti-inflammatory effects (141, 167). In vitro and animal studies with curcumin determined its utility as an antioxidant and anti-inflammatory agent, but clinical trials showed limited effects due to its low solubility and bioavailability (69). The elderly receiving 1500 mg/day of curcumin for 12 months showed no significant differences between treated and placebo, as measured by cognitive tests (139). At least six clinical trials have used curcumin (alone or combined), reaching phase II.

Glucose metabolism

The brain requires a high amount of energy in the form of ATP, but in MCI and AD, glucose metabolism is significantly impaired contributing to oxidative damage (18). The therapeutic strategy has been to repurpose antidiabetics such as intranasal insulin, pioglitazone, rosiglitazone, metformin, sitagliptin, and liraglutide.

Intranasal insulin (commercial names: Detemir, Levemir, Humulin, Novolin) is currently in phase II/III for AD and phase II for MCI. A pilot study monitored the effects of 8 weeks of intranasal insulin (4 × 40 IU/day), indicating enhanced mood, improving memory, and the absence of systemic side effects (12). A phase II trial (NCT00438568) with 104 participants tested 20–40 IU of insulin for 4 months, indicating improved memory or preserved general cognition; CSF biomarkers did not change, but in an exploratory analysis, the ratio of tau/Aβ changed in insulin-treated patients (31). The response to insulin seems to differ by gender and ApoE genotype; both men and women showed cognitive improvement with low-dose insulin, but only men responded positively to high-dose insulin. Remarkably, men with ApoE4⁻ improved but women with ApoE4⁻ did not, whereas all ApoE4⁺ remained cognitively stable (28). Pioglitazone (commercial names: AD4833, Actos, Glustin, Piozone) is an insulin sensitizer or PPARγ agonist. Phase II trial NCT00982202 with 29 nondiabetic AD subjects was shown to be safe and well tolerated with a dose of 45 mg/day (58, 64). A phase III trial is ongoing to confirm observations of the potential use of pioglitazone for MCI and AD. Rosiglitazone (commercial name: Avandia) is another antidiabetic compound or PPARγ agonist that reached phase III until its discontinuation by GlaxoSmithKline due to no significant improvement and side effects. Two phase III studies evaluated its efficacy and safety (2–8 mg), indicating no statistical differences in cognition or global function between groups, with adverse events of edema in 14%–19% of patients (NCT00428090 and NCT00550420) (66, 79). Metformin is a common drug prescribed for type 2 diabetes. The antioxidant properties of the drug were probed in neuronal PC12 cells and primary hippocampal neurons dosed with H₂O₂, observing a reduction of cell death, ROS, and mitochondrial damage (192). A pilot study with 80 patients with MCI tested 1000 mg metformin twice per day for 12 months, showing differences in ADAS-Cog but no significant differences in memory, glucose uptake PET, or plasma Aβ (106). A crossover study with 20 nondiabetic subjects not only explored an 8-week dosage of metformin, showing safety and tolerability, but also improved executive functioning, learning/memory, and attention (96). Sitagliptin is a dipeptidyl peptidase (DPP4) inhibitor, with antioxidant, anti-inflammatory, and antiapoptotic properties (182). The APP/PS1 mice treated with 20 mg/kg of sitagliptin for 8 weeks showed reduced Aβ deposition and protected cognitive function (43). A pilot study with 205 elderly diabetic patients with or without cognitive impairment showed improvement of cognitive function after a 6-month treatment with sitagliptin, comparable to metformin treatment (83). The glucagon-like peptide receptor stimulating drug Liraglutide (commercial names: Victoza) works by modulating endoplasmic reticulum stress response and stimulation of autophagy (129). An intervention study with AD patients treated for 6 months with liraglutide showed no effects on Aβ deposition (48). In another trial, 38 AD patients received a 26-week dose of liraglutide. The treatment prevented cognitive impairment but with no effects on Aβ deposition or cognition (63). There is an active phase II trial of liraglutide (NCT01843075) with 204 patients that is monitoring cerebral glucose metabolic rate and Aβ and tau by MRI.

Lifestyle interventions in AD

Nonpharmacological therapies or lifestyle interventions aim at preventing or slowing AD progression and reducing symptomatology, proposed as alternatives to pharmacological therapies (Fig. 2).

FIG. 2.

Lifestyle nonpharmacological interventions for AD. AD, Alzheimer's disease. Color images are available online.

The Mediterranean diet has been correlated with a lower risk of chronic diseases, cardiovascular disease, cancer, and lower mortality. A community-based study monitored 2258 individuals for 4 years, and the subjects following a Mediterranean diet showed a lower risk for AD (149). The Mediterranean diet includes a variety of fresh fruits and vegetables, olive oil, fish, and moderate wine intake that contribute essential vitamins, minerals, polyphenols, and unsaturated fatty acids that may reduce oxidative stress and inflammation (116). A cohort of 82 elderly patients was examined: The group following the Mediterranean diet showed better learning and memory performance and even neuroimaging indicated that dentate gyri were larger in comparison with the control group (89). Interestingly, a change from a traditional Japanese diet to a Western diet increased the prevalence of AD, increasing rates from 1% in 1985 to 7% in 2008 (40) and reaching 11.3% in 2012 (128). The Western diet has been linked to a higher risk of cognitive decline and dementia: This diet is high in red meat, processed meat, processed foods, saturated fat, and fried foods. This dietary pattern showed a high correlation with neurodegeneration, including elevated amyloid levels, neuritic plaques, and small vessel disease (5, 49). Caloric restriction is defined as a dietary regime with a strong limitation on calorie intake without facing a lack of nutrients or malnutrition. A reduction in calories is linked with an extended lifespan, slowdown of aging, improved memory, and reduction of AD progression (170). Intermittent fasting is an eating pattern in which individuals go over extended periods (usually 16–48 h) with little or no food intake followed by a period of normal food intake (113). Animal models under intermittent fasting demonstrated benefits in age-related chronic diseases, such as diabetes, cardiovascular, cancer, and neurological disorders including AD. This, by activation of adaptive cellular stress signaling pathways, enhances mitochondrial metabolism, promoting DNA repair and autophagy (113). An AD rat model under intermittent fasting (3 h per day of food consumption) showed elevated fat oxidation as an energy source, decreased serum glucose levels, lowered cortisol levels, and overall preserved spatial memory function compared with normal diet (156). Physical exercise has many health benefits and may reduce the risk of developing chronic diseases, but there is no conclusive evidence to confirm that physical exercise prevents MCI or AD. Nevertheless, cumulative evidence from observational studies suggests the benefits of exercise for the brain, including slowdown of cognitive decline and fewer Aβ plaque and tau tangles (121a). Trials with MCI or AD patients demonstrated that physical activity may have a positive impact on the development of the disease, but until now there is inconclusive evidence that exercise improves cognition in AD (19). Sleep changes correlate with AD pathology (64a). Alterations in quality sleep have an inverse relationship with cognitive impairment, and it may be an early indicator of AD and Aβ/tau deposition (105). In particular, chronic sleep deprivation increases Aβ plaques and may promote tau accumulation (80).

Biometals in the AD Brain

Iron and copper are essential metals required for normal brain function: They serve mainly as co-factors in the synthesis of neurotransmitters, oxygen transportation, synapses, and ATP production. The course of AD seems to be correlated with a marked dyshomeostasis or overload of metals, causing oxidative stress (Figs. 3 and 4). Amyloid plaques can deposit or accumulate Fe and Cu, and this phenomenon is more evident when compared with healthy age-matched individuals (Fig. 4) (162). Therapeutic strategies to deal with abnormalities of metals in the brain have been proposed and tested in AD models, at both preclinical and clinical levels. These are described as metal-protein-attenuating compounds or as metal chelators.

FIG. 3.

Biometals in the brain. Human brain affected by AD suffers from altered homeostasis of essential biometals such as iron, copper, zinc, and calcium, affecting the overall redox state of the neurons. Color images are available online.

FIG. 4.

Histochemical location of redox-active iron in AD tissue sections. (A) Tissue section from AD brain. (B) Control. Arrowheads indicate neurofibrillary tangle and arrows indicate amyloid senile plaques. Scale bar = 200 μm. From Smith et al. (162).

Iron dyshomeostasis

The iron chelator deferoxamine or desferrioxamine was tested in AD patients (125 mg twice daily for up to 24 months), showing a significant reduction in progression, but no improvement in cognition or memory and presenting side effects such as appetite and weight loss (32). Intranasal deferoxamine in mice and rat models showed inhibition of Aβ aggregation and decreased memory loss (54, 76). Deferoxamine decreases protein oxidation and promotes the expression of the insulin receptor in the brain (53). The APP/PS1 mice treated with deferoxamine showed a reduction of Aβ and neuronal death accompanied by microglial activation (191).

Deferasirox and deferiprone have been used for iron chelation therapy in neurodegeneration. Deferasirox administered for 4 months to aged rats caused a reduction in iron accumulation and expression of TfR1 and ferritin, also reducing Aβ, oxidative stress, and inflammation biomarkers (11). Treatment with 10–50 mg/kg/day of deferiprone in rabbits caused a reduction in Aβ, BACE1, and p-tau, but no reduction in ROS (137). Deferiprone is currently under clinical trial (phase II, NCT03234686) with 171 subjects with prodromal AD or mild AD receiving 15 mg/kg to test the effect in cognitive decline. Similarly, deferiprone followed a phase II trial (10–15 mg/kg, for 6 months), showing to be well tolerated and reducing iron accumulation, but with no significance in the improvement of motor scores or cognitive function (110).

Clioquinol (iodochlorhydroxyquin or PBT1) has been proposed as an AD treatment. Its activity is through disruption of the interactions between Aβ and metal ions (copper and zinc) and, consequently, reducing Aβ plaques and oxidative stress. Preclinical studies with transgenic AD mice showed a 49% reduction in Aβ deposition after a 9-week course of clioquinol treatment and benefits in memory (25, 73). A phase II trial with 36 subjects (125–375 mg twice daily, 36 weeks) showed tolerability, reduction in Aβ, no effect in cognition, and no effect in plasma copper levels (143). Since clioquinol showed no significant effect in cognition, the phase III trial was terminated due to the presence of toxic contaminants and side effects (147).

PBT2 is an 8-hydroxyquinoline analog with activity as a translocator of copper and zinc and reducing Aβ. The preclinical analysis of PBT2 (30 mg/kg/day) restored dendritic spine density and reduced 13% Aβ (3, 35). A phase II trial with 78 patients (50–250 mg/day) revealed safety, reduction of Aβ, and improvement in cognition (98). A follow-up phase II trial with 40 subjects over 12-month treatment (250 mg/day) confirmed safety and tolerability, but with no significant difference in Aβ (174).

Copper dislocation

Lipophilic copper-containing complexes have been tested for antibacterial, antimycotic, or anticancer properties, but copper bis(thiosemicarbazonate) complex (Cu-GTSM) was explored as a neuroprotective agent. In vitro analysis revealed that Cu-GTSM promoted neurogenesis and neurite elongation (14), and a dose-dependent reduction of Aβ and p-tau, with an increase of intracellular copper (34, 44). A similar compound Cu-ATSM [diacetylbis(N(4)-methylthiosemicarbazonato) copper(II)] showed anti-inflammatory effects on microglia and astrocytes (27), but with limited effectiveness in AD mice (2). Cu(II)-orotate-dihydrate was tested in mild AD patients during a phase II trial. After 12-month administration of 8 mg, this compound reduced Aβ by 30%, but without effect on tau or cognition (92, 93).

Aggregation of Aβ is influenced by Fe and Cu. The metal-chelating tripeptide GHK (glycyl-l-histidyl-l-lysine) is a copper-binding ligand with antiaging properties (134). GHK interacts with Aβ and sequesters Cu²⁺ to prevent the formation of toxic aggregates and ROS (140). In fact, the Aβ aggregates contain copper and these are destabilized with chelators. We tested bathocuproinedisulfonic acid (BCDS) and bicinchoninic acid (BCA) chelators with amyloid plaque cores. The presence of both Cu chelators caused disassembly of high-order amyloid structures, obtaining mainly small and fibrillar aggregates (Fig. 5). BCDS binds Cu⁺, whereas BCA interacts with Cu²⁺; the fact that both compounds showed activity indicates that copper is present in both oxidation states within amyloid plaques. Interruption of Aβ–Cu interactions is an alternative approach to avoid or reduce the formation of neurotoxic protein aggregates. For example, APP/PS1 mice treated with BCDS showed nonamyloidogenic processing of AβPP and reduced Aβ deposition (179). A pilot study with 34 AD subjects testing the copper-chelating agent d-penicillamine showed that a 6-month treatment reduced oxidative stress but with no differences in cognitive decline (164). Triethylenetetramine (TETA or trientine) was approved for the treatment of Wilson's disease and diabetes: This compound is a Cu(II) chelator with potential therapeutic uses in AD. In APP/PS1 mice, a 3-month treatment of TETA reduced Aβ deposition and synapse loss (29, 175). In the same direction, tetrathiomolybdate [(NH₄)₂MoS₄] is a copper-chelating agent approved for Wilson's disease. This compound reduced inflammation biomarkers and increased SOD activity (146). In Tg2576 mice, the treatment with tetrathiomolybdate reduced copper in the brain and reduced Aβ deposition; here, the compound was proposed as prevention and not a treatment for AD (138). Similarly, APP/PS1 mice receiving tetrathiomolybdate showed a lower formation of Aβ plaques (179).

FIG. 5.

Amyloid plaque cores treated with Cu chelators. Electron microscopy imaging of (A) amyloid-β plaque without Cu chelators, (B) amyloid-β plaques with bathocuproine disulfonic acid, (C) amyloid-β plaques with bicinchoninic acid.

Zinc deficiency, calcium, and lithium

A marked zinc deficiency is linked with neurodegeneration, presumably as a dietary deficiency. The imbalance of zinc in the brain could be related to the interaction of Zn²⁺ with Aβ aggregates (17), but contradictory results indicate that zinc modulates the nucleation of Aβ retarding its aggregation (1). Zinc supplementation was tested in AD subjects with contradictory results. Compounds such as ZnS0₄, zinc bis-dl-hydrogen aspartate, zinc methionine, reaZin (zinthionein), zinc oxide, and zinc gluconate showed some signs of improvement in memory and cognitive outcomes, but in other cases they lacked significant benefits [reviewed in Adlard and Bush (4)].

Calcium suffers age-related dysregulation, playing a role in AD progression; it does this by causing synaptic deficits, and aggregation of Aβ and tau (168). Several compounds targeting calcium channels or proteins related to calcium dysregulation are tested as a therapeutic approach for AD (168). Memantine is an antagonist of glutamatergic NMDA receptors inhibiting Ca²⁺ influx, and it is one of the few FDA approved drugs for AD (Table 1). Another target for calcium dysregulation is the ionotropic transmembrane receptor AMPA. The antagonists of the AMPA receptor include LY451395, LY450108, and S18986. A clinical trial of LY450108 and LY451395 showed that single and multiple doses (1 and 5 mg) were safe and well tolerated (86). A clinical trial with LY451395 (0.2 mg for 28 days, 1.0 mg thereafter for 8-weeks) showed no significant changes in cognition, and the majority of patients showed mild adverse effects (22). Aged mice dosed with S18986 (0.03 and 0.1 mg/kg) showed a restoration of memory impairment. The utility of S18986 was compared with memantine to treat age-related cognitive dysfunction in mild AD (171). Nimodipine (or Nimotop) is a blocker of L-type VGCC calcium channels, targeting calcium dyschondrosteosis in dementia. The usefulness of nimodipine is not clear but sometimes it is prescribed for cognitive impairment and dementia (103). Clinical trials of nimodipine showed benefit when administered 90 mg/day (12 weeks) by improving cognitive function. A phase I trial showed no benefits or effects on secondary outcomes (CSF Aβ, tau, and p-tau) after an 8-week treatment of nimodipine (153). Nilvadipine (or Nilvad, Nivadil, ARC029) is a blocker of L-type VGCC calcium channels, initially prescribed for hypertension. Aged hTau mice treated with nilvadipine presented reduced inflammatory responses and significantly improved spatial memory (120). An initial clinical trial showed that nilvadipine reduced Aβ, reduced inflammation, and improved cerebral blood flow. A phase III clinical trial tested the effectiveness of nilvadipine in slowing cognitive decline (NCT02017340). Here, 511 subjects received 8 mg/day for 78 weeks, indicating that the compound was safe and well tolerated, but unfortunately without benefits in cognitive decline (99). ST101 (or ZSET1446) modulates T-type voltage-gated calcium channels. This compound induces AβPP processing in the nonamyloidogenic pathway, improves memory, and reduces Aβ in transgenic mice and nonhuman primates (71). ST101 was tested in a phase II trial with 210 AD patients dosed with 10, 60, or 120 mg for 12 weeks; the results indicated a dose-response in cognitive measures, especially in patients treated with donepezil (62). Hyperforin is an acylphloroglucinol isolated from Hypericum perforatum (St John's Wort), being an agonist of TRPC6 (transient receptor potential cation channel subfamily C member 6) with antioxidant and anti-inflammatory properties. This compound decreased Aβ in the hippocampus of a rat AD model, preventing neurotoxicity and reducing ROS (37). Hyperforin also showed dose-dependent neuroprotective effects against Aβ and H₂O₂ (87). Another TRPC6 activator is NSN21778, which works as a positive modulator of the neuronal-store-operated Ca²⁺ influx. This pathway is compromised in AD, and treatment with NSN21778 was able to rescue hippocampal long-term potentiation impairment in an AD mouse model (189).

Cytosolic calcium is regulated by pumping Ca²⁺ into the endoplasmic reticulum by sarco ER Ca²⁺-ATPase (SERCA), which also interacts with presenilins altering Aβ production (70). Thapsigargin is an SERCA inhibitor that causes downregulation of γ-secretase presenilin-2 but not presenilin-1, but thapsigargin also increases oxidative stress and induces apoptosis in vitro (33, 144). Dantrolene is an antagonist of ryanodine receptors that release Ca²⁺ from the sarco/endoplasmic reticulum. This compound showed neuroprotective properties in AD animal models (101). Long-term treatment of dantrolene on 3 × Tg-AD mice (5 mg/kg for 6 months) decreased the accumulation of Aβ and p-tau in the hippocampus, but with no changes in motor functions or cognition (183). Carvedilol (or Coreg, Artist, Aucardic, Dilatrend, Kredex) is a nonselective α/β-adrenergic receptor antagonist and α-adrenergic receptor blocker, currently prescribed for high blood pressure and cardiovascular problems. Treatment with carvedilol increased basal synaptic transmission in the TgCRND8 mice model, also enhancing neuronal plasticity and suppression of neuronal hyperexcitability (8). A phase IV trial with 29 participants is testing the effect of 25 mg/day dose of carvedilol on AD patients (NCT01354444). The trial was completed in January 2017, but no reports have been published. Xestospongin C is as an antagonist of the calcium channel activated by inositol triphosphate. When tested in APP/PS1 mice, intracerebroventricular injection of 5 μmol modulated intracellular Ca²⁺, improved cognition, reduced Aβ plaques, and reduced neuronal apoptosis (180).

Lithium is recognized as a neuroprotective and neurotrophic compound and is mainly prescribed to treat bipolar disorder. A study of the Danish population reported a nonlinear association of lithium consumption in drinking water with the diagnosis of dementia. Here, the authors found that elderly individuals (median age 80.3 years) with a higher low-term lithium exposure had a lower incidence of dementia (91). These conclusions are controversial, since other factors such as age, lifestyle, and health care were not associated. Another population study reported an inverse relationship between lithium in drinking water and AD (50). A most recent study did not find any significant benefit of groundwater lithium exposure in mental illnesses (132). This can be explained, because doses of therapeutic lithium are orders of magnitude higher than its concentration in drinking water. Lithium formulations (lithium carbonate, lithium salicylate, lithium salicylate-proline) have shown efficacy in reducing AD. In APP/PS1 mice, these compounds served as prophylactic to prevent cognitive decline, memory decline, and irritability when administered for 4 months (77). SK-N-SH neurons were treated with LiCl; the transcriptomic analysis revealed that the core AD pathways did not suffer significant changes, but they affected noncoding nucleolar RNA and microRNAs connected to the AβPP pathway (107). However, mixed results of therapeutic lithium, micro-dose, and low-dose lithium treatments (300–600 mg) seem to improve symptoms in AD subjects, in particular, by stabilizing cognitive impairment and reducing agitation/aggression.

Neuronal Death Through Ferroptosis

Oxidative stress and altered metals have been implicated in activating cell death. Examination of AD brains has revealed a notable reduction in the brain volume and overall cell number in comparison with healthy individuals. Nevertheless, the specific mechanisms that trigger neuronal death in AD still remain undefined. Recently, a novel form of regulated cell death mediated by iron was described and termed ferroptosis, which is morphologically, biochemically, and genetically distinctive from apoptosis and necrosis (59, 79a, 184). Ferroptosis has similar characteristics as oxytosis, a non-excitotoxic pathway for glutamate-induced cell death previously described (146a, 150a, 166a). Oxytosis is characterized by glutathione depletion, lipoxygenase activation, ROS accumulation, and calcium influx. Ferroptosis results from dysfunction of redox homeostasis, ROS overproduction, and lipid peroxidation. Neurodegeneration is closely correlated with high oxidative stress and abnormal levels or overload of redox-active and nonredox metals, consequently promoting Aβ/tau aggregation, mitochondrial dysfunction, inflammation responses, and synaptic failure. Misregulated ferroptosis pathways have been speculated to trigger neurodegeneration, but the cellular mechanisms are not completely understood. Different small drugs have been designed as ferroptosis inductors or ferroptosis inhibitors. These compounds target four main pathways: system x_c⁻, glutathione metabolism, lipid peroxidation, and iron metabolism (Fig. 6). Erastin, erastin2, IKE, sorafenib, sulfasalazine, and glutamate serve as inhibitors of system x_c⁻ (SLC73A2/SLC7A11), which operates as glutamate/cystine transport and regulation of intracellular glutathione precursors. The compound l-buthionine-(S,R)-sulfoximine (BSO) inhibits the enzyme γ-glutamylcysteine synthetase, which catalyzes an ATP-dependent condensation of cysteine and glutamate, leading to glutathione depletion. The selenoprotein glutathione peroxidase 4 (GPX4) has been identified as a critical component of ferroptosis, because GPX4 functions as phospholipid hydroperoxidase reducing lipid peroxidation. The compounds (1S,3R)-RSL3, ML-162, and ML-210 bind to GPX4, serving as potent inhibitors; whereas FIN56 reduces GPX4 expression and also interferes with the mevalonate pathway (cholesterol synthesis). An excessive amount of free iron (ferric Fe³⁺/ferrous Fe²⁺) may trigger ferroptosis. The labile pool of Fe is redox active and generates an excessive amount of hydroxyl radicals that cause lipid peroxidation. Ferroptosis is a topic that is actively evolving, and novel ferroptosis inductors have been designed or discovered with potential therapeutic uses.

FIG. 6.

Overview of ferroptotic pathways. Diagram indicating the main pathways in ferroptosis, cystine-glutamate exchange transporter, glutathione metabolism, iron metabolism, and mevalonate. Ferroptosis induces erastin; sulfasalazine inhibits system X_c⁻; BSO inhibits production of GSH; Fin56 and RSL3 inhibit GPX4; and Fe²⁺ promotes overproduction of ROS and lipid ROS. BSO, l-buthionine-(S,R)-sulfoximine; GPX4, glutathione peroxidase 4; GSH, glutathione. Color images are available online.

Ferroptosis inhibitors may prevent cell death by restoring cellular functions, being ROS scavengers or metal chelators. For example, liproxstatin-1, ferrostatin-1, and its analogs interfere with peroxyl radicals, and the activity of GPX4 is potentiated by compounds SRS16-86 and SRS11-92. The redox-active Fe can form complexes with deferasirox, deferiprone, deferoxamine mesylate, or ciclopirox to reduce oxidative stress that is mediated by ROS. The first recognized antiferroptotic gene is the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2), now known as ferroptosis suppressor protein 1 (FSP1) (41). This protein confers protection against ferroptosis mediated by GPX4 deletion, and it achieves this by catalyzing regeneration of CoQ₁₀ using NAD(PH)H and, consequently, trapping lipid peroxyl radicals.

Some ferroptosis-like biochemical and morphological features have been observed in AD in clinical, in vivo and in vitro studies, including GSH depletion (108), marked lipid peroxidation (15, 61, 127, 166), mitochondrial dysfunction (56, 60, 173, 177), GPX4 downregulation (78, 187), and overall altered redox homeostasis. Ferroptosis presents biochemical and morphological features that are different from apoptosis and necrosis (Table 2 and Fig. 7), but since ferroptosis is a newly described and accepted mechanism of programmed cell death, new molecular pathways, proteins, and genes are continuously described. The activation of ferroptosis has been described as ferrosenescence and correlated to iron dyshomeostasis observed in aging and neurodegenerative disorders (152). Ferroptosis can be prevented through selenium donors, lipoxygenase inhibitors, iron chelators, and ferroptosis inhibitors. The use of ferroptosis inhibitors is an alternative to prevent or treat iron-dependent disorders in neurodegenerative diseases, delay or prevent neuronal failure, block lipid peroxidation, reduce oxidative stress, and reduce cell death. The ferroptosis inhibitors function by targeting key molecules in the main ferroptotic pathways (Fig. 6).

Table 2.

Ferroptosis in Neurodegeneration

Morphology changes	Biochemical changes	Inductors	Inhibitors
Rounding up and detachment (cultured cells) Lack of membrane rupture and no blebbing Small mitochondria and changes in its morphology Normal size of nucleus and no chromatin condensation	Iron overload Overproduction of ROS Inhibition system Xc− Glutathione depletion Lipid peroxidation Inflammatory markers	Erastin Erastin2 Sorafebib Sulfasalazine Glutamate l-Buthionine-sulfoximine 1S,3R-RSL3 ML-162, ML-210 FIN56 Free iron (III)/iron (II) Artemisin, Artesunate	Liproxsatatin-1 Ferrostatin-1 UAMC-3203 SRS16, SRS11-92 Deferasinox Deferiprone Deferoxamine Ciclopirox CoQ10 Selenium Glutathione

Adapted from Refs. (59) and (184).

ROS, reactive oxygen species.

FIG. 7.

Ferroptotic neurons. Live cell imaging of SH5Y5Y neuronal cells treated with 100 mM of ferroptosis inductors for 24 h. (A) Control. (B) Buthionine sulfoximine. (C) Sulfasalazine.

Redox-active iron

Iron chelators such as deferasirox, deferiprone, and deferoxamine have shown potential to inhibit ferroptosis (39, 76). The relationship of these compounds with AD is discussed in the Alterations of Redox Homeostasis section. Ciclopirox possesses iron-binding properties. This compound showed neuroprotective properties and significantly reduced the age-dependent acceleration of mortality rate in Caenorhabditis elegans (104). The mechanism of ciclopirox and deferoxamine is a G1/S cell cycle blocker. Low-affinity iron chelators are proposed as an alternative to reduce the disruption of iron metabolism in AD. Examples of these natural iron chelators are curcumin, kolaviron, procyanidins, baicalein, tetramethylpyrazine, ferulic acid, polyphenols, catechins, epigallocatechin-3-gallate, phytate, epimedium, astragalus, kudzu root (Radix puerariae), and neem tree (Azadirachta indica) (152). The copper chelator Cu(II)-ATSM inhibits ferroptosis, by delaying the progression of degeneration caused by erastin and RSL3. The efficacy of Cu(II)-ATSM was similar to ferroptosis inhibitor liproxstatin-1 (163). Similar to iron chelators, copper-binding agents have potential as ferroptosis inhibitors in neurodegeneration.

Glutathione peroxidase

Ferrostatin-1 is a potent ferroptosis inhibitor: It is a radical scavenger by blocking erastin-induced ROS production and lipid peroxidation (39). This compound is one of the first ferroptosis inhibitors identified: Its activity was confirmed in cancer models and also by inhibition of glutamate-induced cell death in organotypic rat brain slices (39). Analogs of ferrostatin-1, such as ferrostatin-1 dyine and UAMC-3203, are new therapeutic compounds with the potential for enhanced stability and solubility. The use of ferrostatin and liproxstatin-1 helped to prevent mitochondrial dysfunction and neuronal death in mouse hippocampal neurons dosed with erastin (123).

Glutathione depletion in ferroptosis is caused by the disruption of GPX4. Consequently, the redox system fails, and ROS accumulates causing protein and lipid oxidation. The compound RSL3 inhibits GPX4 when RSL3 is added to neuronal HT22 cells and mouse fibroblasts; it causes dose-dependent mitochondrial damage and lipid peroxidation. Ferroptotic response in HT22 neurons was reversed with the use of deferoxamine, ferrostatin-1 and liproxstatin-1, and mitochondrial ROS scavenger MitoQ or by genetic modification of BID receptor (85). GPX4 depletion in neurons caused neurodegeneration in mice. The inactivation of this enzyme could be prevented with α-tocopherol, lipoxygenase inhibitors or through siRNA-mediated silencing of AIF (151). Another important characteristic of GPX4 is that it is a selenoprotein. Site-directed mutation in GPX4 of the selenocysteine residue activated ferroptosis (82). Selenium is essential for a healthy brain: It is inversely associated with DNA hypomethylation, protects against DNA damage, and is used by neuronal proteins such as GPX4 (152). Elevated blood levels of iron and copper correlate with a selenium decline and cognitive impairment in AD (172). Selenium-based supplements are alternative interventions for ferroptosis and neurodegeneration (152). Ebselen is a lipid-soluble antioxidant with GPX-like activity. In AD models, Ebselen showed inhibition of oxidative stress, reducing Aβ and BACE; it also reduced p-tau and improved postsynaptic density. Further, the spatial and memory test of 3 × Tg mice treated with Ebselen showed significant improvement (185). In a mice model of sporadic AD, Ebselen treatment (1–10 mg/kg) helped to reverse memory impairment, and it reduced hippocampal oxidative stress and apoptosis (111). Se compounds have been proposed as supplements for neurodegeneration, maintaining functional levels of selenoproteins in the brain. Additional compounds with potential therapeutic applications are selenoneine, organo-Se-compounds- (PhSe)₂, sodium selenite, sodium selenate, selenomethionine, Selol, and selenoglutathione (42).

Lipid peroxidation in ferroptosis

The lipid content in the human brain is extremely high, being 36%–40% in gray matter, 49%–66% in white matter, and 78%–81% in myelin (125). Under oxidative stress conditions, the brain lipids may suffer a series of complex modifications that will have an impact on normal neuronal and brain functions (Fig. 8). Abnormally elevated levels of lipid peroxidation have been observed in the brain and body fluids of AD patients (15, 133). The formation of oxidized lipids is mediated by acyl-CoA synthetase long-chain family member 4 (ACSL4), lysophosphatidylcholine acyltransferase 3 (LPCAT3), and lipoxygenases. Thiazolidinediones are inhibitors of ACSL4. Currently, there are three compounds of this family approved for treatment in humans (pioglitazone, rosiglitazone, and lobeglitazone); at this time, none of these treatments focuses on neurodegenerative diseases. Interestingly, the ApoE gene polymorphism influences the efficacy of thiazolidinediones. In a clinical trial with 2381 subjects, rosiglitazone significantly decreased cognitive scores in ApoE4⁻ patients, but it increased in ApoE⁺ carriers (81).

FIG. 8.

Lipid peroxidation in microglia. Live cell imaging of BV2 mouse microglia treated with 250 μM of ammonium iron (III) citrate for 3 h. (A) Bright-field imaging. (B) DPPP fluorescence as an indicator of lipid peroxidation in cells. Color images are available online.

Lipoxygenases (LOX) catalyzes the deoxygenation of polyunsaturated fatty acids (PUFA) into lipids. In particular, 12/15-lipoxygenase is upregulated in the AD brain and linked to the oxidation of phospholipids. Since PUFA are prone to oxidative damage through both enzymatic and nonenzymatic processes, overproduction of lipid peroxides can be ameliorated through inhibition of free-radicals ROS or inhibition of lipoxygenases. Small molecules such as the flavonoid baicalein can inhibit LOX with therapeutic potential by reducing oxidative stress, as an anti-inflammatory and neuroprotectant (100). The APP/PS1 mice fed with baicalein for 2 months showed inhibition of 12/15-LOX, reduced BACE1 activity, and decreased Aβ and p-tau (74). There were similar observations in an AD rat model, where baicalein improved the behavioral test and promoted overexpression of antioxidant proteins, stress response, phospholipid metabolism, and antiapoptosis in the hippocampus (181). Nordihydroguaiaretic acid (NGDA) is another LOX inhibitor; this phenolic compound is obtained from the leaves of the evergreen desert shrub Larrea tridentata (Creosote bush). When NGDA was administered to a transgenic Drosophila model of AD for 30 days at 20–80 μM, it promoted reduced oxidative stress, prevented memory loss, and increased the life span of flies, but without effect on Aβ (158). The NGDA showed neuroprotective properties in rat hippocampal neurons dosed with Aβ. Moreover, the treatment suppressed the accumulation of ROS induced by iron (68). Zileuton inhibits LOX5; in the 3 × Tg mice model of AD, a 3-month treatment of zileuton reduced γ-secretase, Aβ, and tau (36). In addition, there was a decrease in brain damage and a reduction of inflammatory cytokine expression (TNFα, IFNγ, IL1β, IL6, CXCL1, CCL3, and CCL5) (159).

The substrates of LOX are PUFA, such as omega-3 and omega-6. Another therapeutic strategy consists of dietary supplements to replace those that have suffered peroxidation. Long-chain PUFA from omega-3, -6, or -9 series are administered as supplements. The beneficial effects of omega-3 acids of fish oil, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in AD stand as antioxidants, anti-inflammatory, antiapoptotic, and neurotrophic, also improving cognitive function (7). In vitro and in vivo neuronal models revealed a reduced production of lipid peroxides and ROS, also a reduced number of apoptotic cells after omega-3 supplementation (176). In vitro neurons showed neuroprotective effects of DHA, because oxidized lipids may cause a shift from nonamyloidogenic to amyloidogenic AβPP processing (72). In a clinical trial with 40 AD subjects, a daily dose of 1.7 g DHA and 0.6 g EPA for 6 months showed no significant effects in oxidative stress and inflammation biomarkers in comparison with controls (55). Additional trials focusing on supplements and interventions of the Mediterranean diet are running; the results will provide new information on the therapeutic use of omega acids to manage AD. However, the epidemiological and nutritional studies of DHA/EPA in AD are still controversial and with no reliable conclusions.

CoQ10 plays a critical role in the ferroptosis pathway as a potent antioxidant ROS scavenger preventing or reducing lipid peroxidation (Fig. 6). CoQ10 supplementation was tested in old mice, using a dose of 0.72–2.81 mg/g for 15 weeks. CoQ10 treatment improved spatial learning and decreased oxidative damage, even delaying senescence (154). CoQ10 alone (123 mg/kg/day) or in combination with α-tocopherol (vitamin E, 200 mg/kg/day) improved brain function in old mice, suggesting a synergistic effect of these compounds in brain metabolism (114). A combination of CoQ10+α-tocopherol ameliorated age-related impairment and overall protein oxidation; these two supplements showed better performance when administered together (155). Tg19959 mice treated with CoQ10 showed decreased levels of Aβ in the hippocampus and cortex, also a reduction in oxidative stress biomarkers (46). A phase I clinical trial with 75 subjects (60–85 years old) evaluated vitamin E (800 IU), vitamin C (200 mg), alpha-lipoic acid (200 mg), and CoQ10 (400 mg) administered three times per day for 4 months (NCT00117403). The results indicated no changes in AD biomarkers (Aβ, tau, and p-tau), but a 19% reduction of oxidative stress (biomarker F2-isoprostane) (57). Mechanistically, the FSP1 function as an oxidoreductase that uses CoQ10 as a lipophilic radical-trapping antioxidant that stops the lipid peroxidation reactions (13). In conjunction, FSP1+CoQ10 and glutathione+GPX4 serve as a ferroptosis-resistance antioxidant mechanism in the cells.

GSH is a major antioxidant that counteracts oxidative stress. The levels of GSH suffer a significant reduction in the hippocampus and cortex in MCI and AD (109). Glutathione supplementation has been proposed as a therapeutic strategy for MCI and AD. Elevation of GSH is possible through supplementation with the precursors N-acetyl-cysteine and γ-glutamylcysteine ethyl ester, which are the substrates for GSH synthesis (135). Intranasal administration of GSH (100–200 mg, 3 times per day for 3 months) followed a phase IId clinical trial with 45 patients with Parkinson's, indicating improvements in symptomatology but no significance in outcome measures (117). The use of GSH supplementation in clinical trials with MCI or AD patients has not been evaluated yet (109).

Ferroptosis and senescence in AD

The mechanisms, cellular pathways, and biomolecules involved in ferroptosis are still partially described, but there is an overlap with other mechanisms of cell arrest and programmed death. Cellular senescence is a potential contributor to the accumulation of iron in AD and related dementia; astrocytes, microglia, and even neurons display senescent features (112). The iron content is increased during senescence, and this phenomenon can be induced by a low dose of H₂O₂, causing increased oxidative stress and cellular dysfunction (94). Nonferritin-bound iron showed a marked dysregulation, cytosolic accumulation, and induced both a senescent phenotype and ferroptosis in induced pluripotent human fibroblast and differentiated neurons (30). Transcriptomics of AD transgenic mouse revealed an expression pattern consistent with senescence, including stress response, aberrant cell cycle, and β-galactosidase activity; similar observations were confirmed in postmortem human AD brain (121). Analysis of the brain of AD patients and AD mice exhibited senescence-like phenotype in oligodendrocyte progenitor cells, with overexpression of p21/CDKN1A, p16/INK4/CDKN2A, and detection of senescence-associated β-galactosidase (190). A potential treatment for senescence is the FDA-approved senolytic cocktail of dasatinib-quercetin, which reduces inflammation and ameliorates cognitive deficits. Overall, senolytics promote clearance of senescent cells: This therapy may help to remove cells with altered or irreversible cell cycle arrest that once accumulated in the tissues could trigger inflammation and oxidative stress. Currently, a phase II clinical trial is testing senolytic therapy for modulating the progression of AD (NCT04063124). Around 40 patients >65 years old are receiving dasatinib (D) and quercetin (Q) (D + Q). These compounds are administered intermittently for 2 days on/14 days off for 12 weeks (6 cycles), and the dosage of D + Q was not disclosed. The trials will be completed at the end of 2020, with assessments of CSF biomarkers (Aβ, tau, IL6, p16) and cognitive tests.

Conclusions

Due to recent failures of some amyloid-focused therapeutic compounds evaluated in several clinical trials, researchers, public health/funding agencies, and pharmaceutical companies are seeking AD drugs targeting other mechanisms than Aβ, tau, and secretases. AD is a complex and mostly multifactorial chronic condition; it is extremely complicated that a single drug or intervention can avoid, reduce, or reverse the disease. Here, we presented and discussed potential therapeutic compounds and interventions that focused on re-establishing redox homeostasis, biometal alterations, and neuronal failure in Alzheimer's. Some of these strategies may be beneficial in maintaining the mental function, management of symptoms, or slowdown the progression of neurodegeneration.

Abbreviations Used

Aβ

amyloid-β

AβPP

amyloid-β precursor protein

ACSL4

acyl-CoA synthetase long-chain family member 4

AD

Alzheimer's disease

AIF

apoptosis-inducing factor

AIFM2

apoptosis-inducing factor mitochondria-associated 2

BACE

β-secretase

BCA

bicinchoninic acid

BCDS

bathocuproinedisulfonic acid

BSO

l-buthionine-(S,R)-sulfoximine

CSF

cerebrospinal fluid

Cu-GTSM

copper bis(thiosemicarbazonate) complex

DCFDA

2′,7′-dichlorofluorescin diacetate

DHA

docosahexaenoic acid

EGCG

epigallocatechin-3-gallate

EPA

eicosapentaenoic acid

FDA

Food and Drug Administration

FSP1

ferroptosis suppressor protein 1

GHK

glycyl-l-histidyl-l-lysine

GPX4

glutathione peroxidase 4

GSH

glutathione

IKE

imidazole ketone erastin

LOX

lipoxygenases

LPCAT3

lysophosphatidylcholine acyltransferase 3

NGDA

nordihydroguaiaretic acid

NMDA

N-methyl-d-aspartate

MCI

mild cognitive impairment

PUFA

polyunsaturated fatty acids

p-tau

hyperphosphorylated tau

ROS

reactive oxygen Species

SERCA

sarco ER Ca²⁺-ATPase

SOD

superoxide dismutase

TETA

triethylenetetramine

TRPC6

transient receptor potential cation channel subfamily C member 6

Funding Information

This research project was possible by the generous support from Alzheimer's Association (AARFD-17-529742), The Lowe Foundation, Semmes Foundation, and NIH R01AG066749.