Methylxanthines And Neurodegenerative Diseases: An Update Part 2

For the efficiency of caffeine, its distribution to and in the brain is highly important. It is important to mention that ATP-binding cassette (ABC) transporters in the brain can be affected and therefore might have an impact on methylxanthine distribution in the brain under pathological conditions [38]. 

ATP (adenosine triphosphate) is an important substance in cells. It is the main form of energy reserve in cells. However, in addition to playing the role of energy reserve in cells, ATP also plays an important role in memory.

There are many ATP binding boxes on the neuronal membrane, which can be linked to nerve conduction, synaptic plasticity, and memory formation by regulating ion channels, receptors, and signal transduction pathways.

Studies have shown that the regulation of ATP binding boxes can affect the formation of long-term potentiation (LTP) and long-term depression (LTD). LTP and LTD are two important forms of synaptic plasticity in the brain. LTP strengthens the connection between neuronal synapses, thereby strengthening the information transmission between neurons; while LTD weakens the synaptic connection and regulates the signal transmission between neurons, thus affecting cognition and learning memory.

In addition to its role in neurons, ATP also plays an important role in glial cells. Glial cells and neurons together constitute the nervous system. They have irreplaceable roles in synapse formation, neuronal metabolism, and regulating signal transmission between neurons. ATP can participate in memory formation in glial cells by regulating the concentration of calcium ions, promoting the health of neuronal synapses, and maintaining the metabolic state of neurons.

Therefore, we can see that the relationship between ATP binding box and memory is inseparable. Good ATP binding box regulation can promote synaptic plasticity, and strengthen memory formation and cognitive ability. Maintaining a healthy lifestyle, including a regular biological clock, proper exercise, and diet, can also maintain the health of neurons and glial cells by regulating the role of the ATP binding box. Therefore, we should focus on maintaining good health to promote the improvement of our own memory ability and cognitive level. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because Cistanche can also regulate the balance of neurotransmitters, such as increasing the levels of acetylcholine and growth factors, which are very important for memory and learning. In addition, Cistanche can also improve blood flow and promote oxygen delivery, which can ensure that the brain obtains sufficient nutrition and energy, thereby improving brain vitality and endurance.

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In addition, it has been shown that caffeine can inhibit ABCC4 and ABCC5 with affinities of similar magnitude as for the adenosine receptor and thereby might diminish its efflux from the brain [39]. 

The anti-inflammatory and antioxidant potential of caffeine was analyzed by Badshah and colleagues in an animal study using lipopolysaccharide (LPS)-induced oxidative stress and neuroinflammation. 

LPS was identified as a Toll-like receptor 4 (TLR-4) ligand, primarily expressed in the central nervous system. Activation of TLR-4 leads to the production of proinflammatory cytokines, key mediators of the neuro-inflammatory process [40]. 

The authors reported upregulated expression levels of nuclear factor erythroid-2-related factor 2 and enzyme hemeoxygenase 1, two endogenous antioxidant regulators, and significantly downregulated expression levels of toll-like receptor 4, phosphor-nuclear factor kappa B and phosphor-c-jun n-terminal kinase in the LPS-injected mouse model treated with 30 mg/kg/day caffeine intraperitoneally for four weeks. 

Accordingly, this study was able to show-by using Western blot, immunofluorescence studies, and biochemical assays- that caffeine prevents LPS-induced oxidative stress in the mouse brain and suppresses inflammatory mediators simultaneously [41]. 

The same authors showed in a second study using in vitro (HT-22 and BV-2 cells) and in vivo (B57BL/6N mice) models, reducing effects of caffeine regarding reactive oxygen species, lipid peroxidation, and inflammatory mediators. 

Cadmium-induced cognitive deficits, neuronal loss, or synaptic dysfunction were rescued by caffeine in their mouse model. On a molecular level, the neuroprotective effects of caffeine are exerted via the regulation of nuclear factor-2 erythroid-2 and nuclear factorκB [42]. Moreover, neuroinflammation and Aβ may cause neuronal death by activating the complement system. 

The potential impact of complement- and C-reactive protein (CRP)- mediated neuronal death in AD has already been discussed in previous literature. Fischer and colleagues reported increased levels of complement component 1q (C1q) in the frontal cortex of patients suffering from AD [43]. 

In human AD/stroke patients, a co-localization of monomeric CRP (mCRP) with Aβ plaques, tau-like fibrils, and insulin receptor substrate 1 (IRS-1)/Phospho-Tau positive neurons was reported, and the authors suggested mCRP may be responsible for promoting dementia after ischemia [44]. 

Importantly, it has been shown that coffee consumption can reduce CRP levels and therefore reduce inflammation, which has been analyzed in a dose-response meta-analysis including eleven studies and 61,047 participants. Analysis of the three studies with the largest sample sizes shows a statistically significant association between coffee and CRP levels. 

However, by combining all studies, no significant associations were found in a dose-response meta-analysis, indicating a further need to investigate these inconsistent associations [24]. In line with the reported slightly anti-inflammatory effects of coffee, Rodas and colleagues showed in their recent study that caffeine intake is a negative predictor of CRP. 

They analyzed the effect of regular caffeine intake in 244 healthy participants aged 18–55 years [25]. Recently, another additional mechanism by which caffeine might exert its potential therapeutic benefits in AD has been suggested by Zhao and colleagues [45]. In the presence of caffeine, Wnt signaling was found to be upregulated. 

Caffeine can bind to and inhibit the activity of notum, a hydrolase that inhibits Wnt signaling via enzymatic delipidation of Wnt ligands. Restoration of Wnt signaling is discussed to be beneficial in AD pathologies since this pathway is essential for neuronal differentiation, development, adult neurogenesis, and neural stem cell maintenance. 

Interestingly, only caffeine, but not its demethylated metabolites paraxanthine, theobromine, or theophylline exerts this inhibitory property. Using structure analysis, the authors were able to show that caffeine binds at the enzymatic pocket and overlaps the position of notum´s natural substrate palmitoleic lipid [45]. 

The relatively low inhibitory potency of caffeine (IC50 19 µM) indicates that in adults consuming moderate amounts of three or four cups of coffee per day, resulting in plasma concentrations of about 15 µM [46], physiological notum inhibition seems unlikely. However, in the case of using caffeine as a therapeutic medicine, plasma levels of 150 µM were obtained [47]. 

Moreover, it is discussed that caffeine in physiological concentrations mediates its neuroprotective effects via its inhibitory interactions with adenosine receptors and thereby increases the concentration of adenosine, known to have neuromodulatory properties. 

Nabbi-Schroeter and colleagues investigated the influence of long-term caffeine consumption (30 mg/kg/day for 84 days in drinking water) on the adenosine A1 receptor (A1AR) using small animal positron emission tomography and the highly selective radioligand [18F]CPFPX in the brain of Sprague-Dawley rats. 

The selected dose of caffeine in this study corresponds to a human consumption of four to five cups of coffee per day, which is a common daily intake in industrialized countries. 

The authors reported no long-persistent upregulation of functionally available A1ARs in vivo under their experimental conditions of chronic caffeine intake and based on this, they excluded this mechanism as a molecular basis for mediating the neuroprotective effects reported for this methylated xanthine [48]. Regarding the A1AR, Mendiola-Precoma, and colleagues reported in their study-examining the effects of a cholesterol-enriched diet on cognitive processes in a rat model-that the caffeine metabolite theobromine was able to restore A1 receptor levels, which were reduced as a consequence of the diet. 

Moreover, theobromine showed prophylactic neuroprotection against damage to cognitive functions and levels of Aβ when 30 mg/L were added to drinking water. The authors suggest that the reducing Aβ effect could be mediated by the antioxidant and anti-inflammatory capacities of theobromine via decreased SOD1 and NFκB levels [49]. 

Moreover, theobromine isonucleotide analogs were synthesized and evaluated as active compounds in the inhibition of cholinesterases, since their low cytotoxicity makes them interesting as therapeutic molecules for AD [50]. 

Recently, Ciaramelli and colleagues reported theobromine and catechins as the chemical components of cocoa that hinder Aβ peptide aggregation and toxicity. 

They analyzed the contents of Lavado cocoa concerning their anti-amyloidogenic properties using NMR spectroscopy, preparative reversed-phase chromatography, atomic force microscopy, and biochemical and cell assays in a human neuroblastoma SH-SY5Y cell line, which is commonly used to elucidate underlying mechanisms in respect to AD [51]. 

Recently, methylxanthines were reported to influence the expression of genes linked to pathways involved in processes like oxidative stress, lipid homeostasis, signal transduction, transcriptional regulation, and neuronal function, all known to be influenced in the pathophysiology of AD, by Janitschke and colleagues. 

In their profiling study on a human neuroblastoma cell line, they found that caffeine shows different or inverse effects on gene regulation compared to the other analyzed methylxanthines theobromine, theophylline, pentoxifylline, and propentofylline [52]. 

In line with the outcome of the above-mentioned studies, summarized in Figure 1 and Table 2, significant differences between the individual methylxanthines were detected, further complicating a prediction of neuroprotective effects of other methylxanthines by referring to caffeine.

3. Parkinson's Disease

3.1. Epidemiological and Clinical Studies

Parkinson´s disease (PD) is one of the most common neurodegenerative diseases characterized by a loss of dopaminergic neurons in the substantia nigra and accumulation of misfolded α-synuclein protein aggregates in Lewy bodies. 

Leading symptoms of PD are bradykinesia or akinesia with rest tremor, postural instability, or rigor. Additionally, patients suffering from PD show a broad range of non-motor symptoms differing in early and late-stage symptoms, e.g., obstipation and dementia. Despite an unknown etiology of PD, many risk factors such as age, male gender, environmental factors, intestinal inflammation, and genetics are described [53,54]. 

Besides the known risk factors it is discussed that nutrition may be associated with increased (dairy products) or decreased (phytochemicals, Omega-3 fatty acids, tea) risk or progression in PD [55]. Multiple epidemiological studies could already demonstrate an inverse association between coffee drinking and PD and thereby suggest a link to caffeine, and consequently methylxanthines, as A2A receptor antagonists [56–58]. 

Recent studies have confirmed this association (for an overview see Table 3). Bakshi et al. found a significantly lower caffeine intake, determined through a questionnaire, in idiopathic PD patients compared to a control group from the Harvard Biomarkers Study cohort with 369 cases of PD and 197 healthy controls [59]. In a study from Fujimaki et al., the authors investigated serum from 108 patients with PD by liquid chromatography-mass spectrometry (LC-MS) compared to 31 age-matched healthy control participants and found significantly lowered serum levels of not only caffeine but also its downstream products like theophylline, theobromine and paraxanthine [60]. 

Crotty et al. demonstrated similar results in a recent metabolomics study showing a significantly lower plasma and CSF concentration of caffeine (71% lower) and its degradation products, e.g., paraxanthine and theophylline (57%, 56% lower), in PD patients versus control group measured via LC-MS. 

Subgroup analysis of carriers of the LRRK2 mutation, a mutation in the leucine-rich repeat kinase 2 gene (known for an increased risk for sporadic PD) [61], revealed an even greater decrease of plasma caffeine level by 76% for PD LRRK2 mutation carriers compared to healthy LRRK2 carriers [62]. 

Ohmichi et al. found similar results for the methylxanthine analog theophylline and confirmed a significantly lower plasma concentration in patients with PD versus an age-matched control group [63]. 

A recent meta-analysis by Hong et al., which included 13 studies in total, showed that for healthy individuals, caffeine consumption results in a significantly lower risk of developing PD-specific symptoms (HR = 0.797, 95% CI: 0.748–0.849, p < 0.001, I2: ~ 15.41%, 9/13 studies). Furthermore, a significant deceleration of PD progression among early-stage PD individuals with higher caffeine consumption was found (HR = 0.834, 95% CI = 0.707–0.984, p = 0.03, I2: ~39.7%, 4/13 studies) [64]. 

Another interesting recent study, carried out by Maclagan et al., used a computational approach to rank 620 drugs with the ability to inhibit α-synuclein aggregation. 

They examined associations between the top 15 drugs in a case-control validation study by using health administrative databases. Their logistic regression models found that patients exposed to methylxanthines, especially the synthetic pentoxifylline (PTX) and the naturally occurring theophylline, were associated with decreased odds of occurrence of PD. 

Additionally longer durations of PTX administration revealed a trend towards dose-response [65]. One modified methylxanthine, istradefylline (ISD), has been used to decrease daily "off episodes", a state where symptoms recur when the effect of L-DOPA weakens, in PD patients. ISD was approved first in Japan in May 2013 and by the FDA in the US in 2019 [66]. 

A meta-analysis including six studies revealed that treatment with ISD 40 mg/day decreased the duration of "off episodes" and enhanced the motor symptoms of PD patients. Similar effects could be found for 20 mg/day. ISD treatment revealed no significant effect on adverse effects. In summary, the meta-analysis showed that ISD 20mg and 40mg both improved the unified Parkinson's disease rating scale III (UPDRS) [67].

3.2. Animal Studies/Molecular Pathways

Older studies have already demonstrated the neuroprotective effects of caffeine in PD mouse models [68]. In line with these previous encouraging results, Luan et al. demonstrated that chronic caffeine treatment reduced the effect of intra-striatal injected human A53T α-Synuclein fibrils in mice [69]. Caffeine was administered seven days before the injection and applied for 120 days at concentrations of ~0.4–2 mg/L. 

They found significantly decreased inclusion of α-Synuclein in the striatum of the caffeine-treated mice. Furthermore, apoptosis, microglial activation, and astrogliosis were significantly reduced [69]. 

A similar study used 52 rats with a control group, a PD model group-generated by injecting 1.5 mg/kg rotenone intraperitoneally (i.p.) for 45 days-and two caffeine groups. One of the caffeine groups was injected with 30 mg/kg caffeine i.p. in addition to the administered rotenone for 45 days, whereas the other group was treated with caffeine after the induction of PD via rotenone. 

The study revealed that co-treatment and post-treatment with caffeine in the intoxicated rats enhanced the dysfunction of rotenone-induced motor symptoms by recovering dopamine levels in the midbrain and striatum. In addition, caffeine improved the antioxidant effect by reducing rotenone-induced lipid peroxidation and superoxide dismutase activity in the striatum and midbrain. 

Furthermore, the data revealed reduced rotenone-induced TNF-α activity in the caffeine groups indicating an anti-inflammatory effect of caffeine. Caffeine protection and treatment restored open field test parameters, forelimb hanging test, and traction test to near control group values. 

Histopathological investigation revealed a degeneration of neurons and the presence of Lewy bodies in the rotenone-induced PD group, which was prevented in both caffeine groups with a more prominent effect in the caffeine-protected group [70]. Pardo et al. demonstrated in another animal study that the naturally occurring methylxanthine theophylline can reverse motor symptoms in rats. 

In this study theophylline reversed locomotion, catalepsy, and tremulous jaw movement (resembling parkinsonian tremor) induced by pimozide, a D2 dopamine antagonist [71]. 

In conclusion, these studies show a link between caffeine and other methylxanthines acting as A2A receptor antagonists with a decreased risk for PD and modification of progression, indicating new adenosine receptor ligands might be encouraging targets in treating PD. In a study by Rohilla et al., newly synthesized xanthine derivates as selective AR antagonists were analyzed. 

Evaluation of the antiparkinsonian effect in the study was carried out by inducing catatonia in rats with perphenazine. Most xanthines significantly lowered the catatonic score compared to the control. 

The most potent antiparkinsonian effect was found for the methylxanthine RB-531 (8-[3-(3-Chloropropxy)]-1,3-dipropyl-7- methylxanthine), showing a similar response as the standard treatment L-DOPA [72]. The abovementioned studies and their outcomes are summarized in Figure 2 and Table 4.

4. Multiple Sclerosis

4.1. Epidemiological and Clinical Studies

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) where the immune system impairs the myelin sheath that covers nerve fibers. 

Thus, communication between the CNS and the peripheral nervous system (PNS) is damaged resulting in a potentially disabling neurologic disease. The underlying cause of MS is unclear, however, epidemiological studies have shown that low serum levels of Vitamin D, smoking, and Epstein-Barr-Virus infections play a role in the development of the disease [73,74]. 

Unlike in PD or AD, a recent study from Lu et al. revealed no association between coffee consumption and the risk of MS [75] (see Table 5). Table 5. Summary of recent clinical studies investigating the relationship between methylxanthines and multiple sclerosis (MS). n: sample size.

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