Methylxanthines And Neurodegenerative Diseases: An Update Part 1

Abstract: 

Methylxanthines (MTX) are purine-derived xanthine derivatives. Whereas naturally occurring methylxanthines like caffeine, theophylline, or theobromine are widely consumed in food, several synthetic but also non-synthetic methylxanthines are used as pharmaceuticals, in particular in treating airway constrictions. Besides the well-established bronchoprotective effects, methylxanthines are also known to have anti-inflammatory and anti-oxidative properties, mediate changes in lipid homeostasis, and have neuroprotective effects. 

As the aging population intensifies, more and more people are beginning to pay attention to how to protect their health. Among them, antioxidants have been proven to be an effective way to help people slow down the aging process and improve memory.

Antioxidants refer to substances that can resist free radical oxidation in the body. Free radicals are highly active chemical molecules produced by metabolism in the body or external environmental factors. When free radicals accumulate to a certain extent, they will attack body cells and cause damage to the body's defenses, which is also one of the main causes of human aging. Antioxidants can neutralize and remove free radicals, thereby slowing down the degree of cell damage.

Some studies have shown that antioxidants can also improve people's memory. Memory is the brain's ability to process and store information. With age and changes in physical condition, memory will gradually decline. However, a variety of antioxidants such as vitamin C, vitamin E, flavonoids, etc. have been shown to protect and improve brain function. These substances can promote the growth and development of brain cells, reduce the degree of damage to brain cells, and strengthen the connection between brain cells. In addition, antioxidants can help maintain the integrity of neuronal cell membranes, thereby improving thinking and cognitive abilities.

In short, antioxidants are a way to benefit human health and memory. Through proper diet and lifestyle changes, we can take in more antioxidants, thereby strengthening the body's defenses and maintaining an excellent state of health and memory. It can be seen that we need to improve memory, and Cistanche can significantly improve memory because it has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidative and inflammatory responses in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

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Known molecular mechanisms include adenosine receptor antagonism, phosphodiesterase inhibition, effects on the cholinergic system, Wnt signaling, histone deacetylase activation, and gene regulation. 

By affecting several pathways associated with neurodegenerative diseases via different pleiotropic mechanisms and due to its moderate side effects, the intake of methylxanthines has been suggested to be an interesting approach to dealing with neurodegeneration. Especially in the past years, the impact of methylxanthines in neurodegenerative diseases has been extensively studied and several new aspects have been elucidated. 

In this review we summarize the findings of methylxanthines linked to Alzheimer´s disease, Parkinson's disease, and Multiple Sclerosis since 2017, focusing on epidemiological and clinical studies and addressing the underlying molecular mechanisms in cell culture experiments and animal studies to assess the neuroprotective potential of methylxanthines in these diseases.

Keywords: methylxanthines; caffeine; theobromine; theophylline; pentoxifylline; propentofylline; istradefylline; Alzheimer´s disease; Parkinson´s disease; Multiple Sclerosis.

1. Introduction

This review summarizes the latest findings on the relationship between methylxanthines and neurodegenerative diseases, especially Alzheimer's disease (AD), Parkinson´s disease (PD), and Multiple Sclerosis (MS). 

These multifactorial disorders share several common histopathological hallmarks and mechanisms like neuronal cell loss linked to gliosis, misfolding and accumulation of proteins, oxidative stress, and neuroinflammation. 

In the case of AD, disease progression is strongly associated with Aβ peptide generation and aggregation, associated with pathological extracellular and intracellular filamentous deposits, hyperphosphorylated tau proteins, neuroinflammation, and synaptic loss. 

PD is characterized by dopaminergic neurodegeneration and accumulation of α-synuclein in Lewy bodies. Concerning MS, neuroinflammatory processes leading to the demyelination of neurons are the focus of the disease. 

Numerous studies have been performed to address the question of whether xanthine derivatives like caffeine and theobromine have beneficial properties concerning the characteristic histopathological changes that occur in the above-mentioned diseases and cognitive decline. 

Although the outcome of the clinical studies, especially for AD, was heterogeneous [1], some mechanisms were identified, showing how methylated xanthine derivatives could protect against neuronal damage.

In general, in physiological concentrations, achieved for example by coffee consumption or by intake of methylxanthine-containing beverages, methylxanthines act as antagonists of the adenosine receptor (AR), histone deacetylase activator, or antioxidant. 

Affecting these pathways, xanthine derivatives can modulate molecular mechanisms associated with neurodegenerative diseases like accumulation of misfolded proteins, oxidative stress, and neuroinflammation. Interestingly, in particular, the beneficial role of adenosine receptor antagonists in the treatment of neurodegenerative diseases has become more and more apparent in the last years [2–5]. 

In supraphysiological concentrations, which can be reached for example, by intake of methylxanthines as pharmaceuticals, additional mechanisms like inhibition of phosphodiesterases and high-affinity ATP-dependent cyclic nucleotide transporters are reported, also contributing to neuroprotective properties of methylxanthines [6]. 

Besides the abovementioned properties of xanthine derivatives, further mechanisms were suggested or excluded in the last years and are summarized in detail below together with new clinical trials addressing these issues (also see figures and tables).

2. Alzheimer's Disease

2.1. Epidemiological and Clinical Studies

In the past four years, several new clinical and epidemiological studies were published examining the influence of caffeine or other methylxanthines on cognitive performance concerning AD, see Table 1. 

Jee Wook Kim and colleagues analyzed the relationship between lifetime coffee intake and AD biomarkers in the human brain using neuroimaging techniques like positron emission tomography (PET), magnetic resonance imaging scans, and clinical assessment. The study included 411 participants with an inclusion criterion for patients already suffering from mild cognitive impairment (MCI) but without a diagnosis of dementia. 

Study participants were divided into high and low/no coffee consumers: 269 participants, consuming no coffee or <2 cups/day and 142 participants stated to drink two or more than two cups per day, reflecting higher coffee intake. The authors reported that a higher lifetime coffee intake was significantly associated with a reduced pathological cerebral amyloid deposition and could therefore be linked to a lowered risk for AD or cognitive decline even after controlling for potential cofounders. 

Besides the association of coffee intake with reduced amyloid deposition, no correlation between coffee intake and AD-associated glucose hypometabolism, atrophy or cortical thickness, and cerebral white matter hyperintensities were detected [7]. This study is in line with older results obtained by transgenic mouse models or animals revealing a decreased Aβ concentration [8,9]. 

Interestingly, this study also revealed that the effect was more pronounced in long-term coffee drinkers suggesting that the positive effects of higher coffee intake against Aβ pathology involve more chronic effects which are associated with a regular long-term exposure rather than a short-term effect of caffeine. These results might help to explain why some previous studies including participants with shorter caffeine exposure showed weak or no correlation of caffeine with cognitive decline or amyloid burden, emphasizing that not only the caffeine concentration but also the duration of exposure plays a critical role for the beneficial effects of methylxanthines in AD. 

In this context, a new meta-analysis of eight observational prospective studies from Larsson and Orsini should be noticed. No statistically significant association between coffee consumption and risk of dementia was detected by the authors. The 95% confidence interval of dementia for one cup of coffee/day was 0.92, two cups/day 0.90, three cups/day 0.93, four cups/day 1.01, and five cups/day 1.11. 

In line with the authors, it has to be mentioned that especially over a long time, coffee consumption may vary and is hard to be exactly estimated. Moreover, three studies showing the effects of coffee consumption on AD risk were not included because they used different quantitative categories of coffee consumption [10–13]. 

Another interesting aspect is revealed by a recent study from Iranpour and colleagues analyzing data from the National Health and Nutrition Examination Survey (NHANES) including 1440 participants older than 60 years [14]. 

By applying different cognitive tests, the authors reported a weak positive relation between high caffeine intake with cognitive function. Importantly the correlation was stronger amongst males than females emphasizing a potential need for subgroup-analysis besides combining studies in meta-analysis to obtain large but probably even more heterogeneous cohorts. 

In summary, concerning dose dependency, a correlation between coffee consumption and AD risk is still controversial and can currently not be answered without uncertainty in the absence of further studies.

2.1.1. Are the Positive Effects Due to Caffeine or Other Compounds in Coffee?

Besides caffeine, coffee contains several biological compounds known to be biologically active-such as phenylindanes or molecules produced during roasting of coffee beans [15,16]. 

Therefore, it is difficult to answer the question of whether the potential positive observed effects are due to caffeine or other substances. Addressing exactly this important question, Xue Dong and colleagues analyzed in their study the association of coffee, caffeinated coffee, decaffeinated coffee, and caffeine intake from coffee with cognitive performance. 

CERAD (Consortium to Establish a Registry for Alzheimer´s Disease) test and DSST (Digit Symbol Substitution Test) were performed with over 2500 participants from the NHANES aged 60 years or older without any diagnosis of AD. 

Significant associations with cognitive performance were reported for coffee, caffeinated coffee, and caffeine from coffee, but not for decaffeinated coffee [17], underlining the important role of methylxanthine caffeine in coffee. 

However, as older studies could also show beneficial effects of other compounds in coffee-besides caffeine-this result should emphasize the impact of caffeine but does not rule out that other substances might contribute, especially in combination with caffeine, to a protective effect as well [18,19].

2.1.2. The Effect of Other Methylxanthines on Alzheimer's Disease

Besides caffeine, several other methylxanthines are known that have potential protective effects concerning their molecular mechanism. However, very little is known about their potential protective function concerning AD in clinical studies or data obtained from human samples. 

De Leeuw investigated the association of different nutritional biomarkers with clinical progression in patients with cognitive decline. Theobromine was revealed to be increased in a subgroup of patients with lower Aβ42 levels but also higher total and phosphorylated tau levels. In addition, high theobromine levels were found to be associated with cognitive decline in mild cognitive impairment (MCI) patients [20]. 

At first glance, these results might point towards a negative impact of methylxanthines in disease progression. However, it has to be taken into consideration, that caffeine is metabolized in the liver to theobromine. 

A faster or altered metabolization rate of caffeine might increase theobromine but in a decreased caffeine level. Theobromine compared to caffeine has been shown to have a lower anti-amyloidogenic potential [21]. 

Therefore, the association of increased theobromine with cognitive decline might be influenced by the faster degradation of caffeine resulting in lower caffeine levels. 

However, as caffeine is not studied in this paper this potential explanation is speculative. Nevertheless, in line with this argumentation, a recent article from Mullins and colleagues discussed whether genome-wide single nucleotide polymorphism (SNP) data can help to predict the individual response to dietary interventions. 

The CYP1A2 gene encoding cytochrome P450 1A2 is responsible for approximately 95% of caffeine metabolization, and thirteen SNPs are associated with this gene. One of these SNPs (rs762551) influences the sensitivity for caffeine and how fast it gets metabolized [22]. 

Unfortunately, it is unknown whether this SNP is associated with AD, but undoubtedly-this SNP in the CYP1A2 gene having an impact on caffeine intake might explain the heterogeneous results of clinical studies and should be taken into consideration for further clinical trials, since SNP data are nowadays quickly and inexpensively acquired. 

Therefore, in clinical studies, CYP1A2 variants have to be considered as an additional important factor highly affecting the pharmacokinetics of methylxanthines. 

Importantly, the pharmacokinetics of methylxanthines can be further influenced by lifestyle or dietary habits. For example, caffeine consumption combined with smoking cessation has been reported to be associated with more than two times increased caffeine plasma levels that could even induce caffeine toxicity symptoms [23]. 

In summary, the lack of knowledge of the methylxanthine plasma levels or the lifestyle habits that interfere with the pharmacokinetics of methylxanthines, and the additional analysis of CYP1A2 variants, makes it hard to estimate the potency of methylxanthines in treating or preventing neurodegenerative diseases and should be considered as a caveat in interpreting previous studies.

Moreover, synthetic methylxanthines are an interesting substance class that might be suitable to treat or prevent AD, because of their capability to act as phosphodiesterase inhibitors. 

A recent systematic review of clinical trials, epidemiology, and meta-analyses reported propentofylline as the only phosphodiesterase inhibitor having completed efficacy testing clinical trials showing improvement of cognition and dementia severity in mild-to-moderate AD patients. 

Propentofylline was found to be the most effective inhibitor out of phosphodiesterase-inhibiting xanthine derivatives by inhibiting several phosphodiesterase isoforms, especially phosphodiesterase 2 and 4. 

The authors therefore suggest a co-treatment of propentofylline with the phosphodiesterase 5 inhibitor sildenafil for the prevention or treatment of AD [6]. Based on these data further clinical trials analyzing the effect of this synthetic methylated xanthine on AD symptoms and severity should be performed. 

It has to be considered that clinical studies could have limitations like unrepresentative sample populations, implications of confounds, or personal motivation of the participants for self-reporting their coffee consumption. Other interesting points are the individual caffeine sensitivity and the above-mentioned pharmacokinetic of methylxanthines.

2.2. Animal Studies/Molecular Pathways

Concerning the uptake of caffeine, an animal study by Liang Jin and colleagues shows that intestinal permeability and oral absorption of caffeine were not affected in a mouse model of familial AD. They reported that plasma caffeine concentration as well as total brain exposure did not differ between wildtype and APP/PS1 mice after oral administration. 

APP/PS1 mice contain human transgenes for both APP bearing the Swedish mutation and PSEN1 containing the L166P mutation, therefore representing a transgenic mouse model that overproduces Aβ and that is often used to study the neuropathologic mechanisms of AD as well as the therapeutic effects of drugs on AD [26]. 

In line with this, the authors demonstrated in a previous study, that the abundance of intestinal and hepatic Cyp1a2 was not different in APP/PS1 mice in comparison to wildtype mice [27]. These data suggest caffeine is a potential therapy for AD since its uptake is not impaired in patients suffering from this neurodegenerative disease. 

Another animal study, performed by Zappettini and colleagues, used a mouse model of AD-like Tau pathology (THY-Tau22 transgenic mice) for investigation of the long-term consequences of early life exposure to caffeine during pregnancy. 

Their findings suggest that Tau pathology-related pathological traits appear earlier in the offspring of caffeine-exposed mice and therefore suggest caffeine exposure during pregnancy as a risk factor for early-onset AD-like pathology [28]. 

In addition to caffeine, the neuroprotective effects of its metabolite theobromine were also analyzed in animal studies. Yoneda and colleagues reported that orally administered theobromine (0.05% for 30 days) is detectable in plasma and cerebral cortex in wild-type mice. (The utilized mouse strain here was C57BL/6NCr mice, the nomenclature of these mice is due solely to their origin, since after establishing the substrain C57BL/6 at The Jackson Laboratory the sublines C57BL/6N and C57BL/6J were separated and Cr stands for Charles River because this company acquired their breeding colonies in 1974 [29]). 

It can act as a phosphodiesterase inhibitor in the brain and enhances cAMP/CREB/BDNF pathways in a way that supports cell survival and neuronal functions. Moreover, the theobromine-fed mice showed better performances on a three-lever motor learning task [30]. These findings suggest a beneficial influence of cacao products on learning and memory. 

In the case of the adenosine A2 receptor (A2AR) antagonist istradefylline, a pharmaceutic that is approved for PD in Japan (for detailed information see later), an animal study performed by Orr and colleagues investigated the ability of this reagent to enhance cognitive functions in aging mice with AD-like amyloid plaque pathology. 

The authors reported increased spatial memory and habituation in APP transgenic mice treated with low doses of istradefylline (≤10 mg/kg/day) and underline the importance of further investigations of this adenosine receptor antagonist as a potential therapeutic approach for AD or other neurodegenerative diseases besides PD [31]. 

In this context, a recent in vitro study from Franco and colleagues suggests that antagonists of A2AR affect the function of N-methyl D-aspartate ionotropic glutamate receptors (NMDAR) since A2AR activation leads to higher NMDAR functionality in neurons [32].

In the past years, several experimental studies were performed to elucidate the molecular mechanisms that underlie the observed beneficial effects of methylxanthines like caffeine in AD. 

Gastaldo and colleagues examined in their study if food ingredients (among others caffeine) can affect Aβ peptide aggregation in AD through an indirect,membrane-mediated pathway since membranes are known to play a crucial role in the early stages of peptide aggregation. 

The authors used synthetic brain membranes to analyze the influence of caffeine on the size and volume fraction of aggregates consisting of cross-β sheets of the membrane active fragment Aβ25-35. 

Caffeine was reported to spontaneously partition into the membranes in the first 150 ns of the molecular dynamics simulation and was found to mainly position in the head-tail interface of the membranes-and some also temporarily inside the hydrophobic core. The Aβ25-35 peptides were found via microscopy to form pronounced amyloid fibrils, located on the top of the membranes in the presence of caffeine. 

Moreover, using X-ray diffraction they found that caffeine leads to membrane thickening and a decrease in membrane fluidity, and the presence of the Aβ25-35 peptides an increase in local membrane curvature, which is likely induced by the formation of extracellular Aβ aggregates and fibrils. Additionally, they found in their UV-visible spectroscopy studies using thioflavin T, a significant increase in the fluorescent signal of β-sheets at 420 nm after the addition of caffeine [33]. 

Similar results regarding the influence of caffeine on membranes were obtained in one of the authors' earlier studies [34]. Concerning caffeine and Aβ peptides, Gupta and colleagues reported as a result of their molecular dynamics simulations a disorganization of cross-β structures of Aβ17-42 fibrils in the presence of caffeine. This destabilization effect might further inhibit the formation of aggregates [35]. 

Concerning Aβ homeostasis, Janitschke, and colleagues examined the effects of the methylxanthines caffeine, theobromine, theophylline, pentoxifylline, and propentofylline in human neuroblastoma cells. 

They concluded that the analyzed xanthine derivatives reduce the levels of Aβ via pleiotropic mechanisms by shifting the processing of the amyloid precursor protein from the amyloidogenic to the non-amyloidogenic pathway via influencing protein stabilities and gene expressions as well as affecting the involved secretases directly. 

Moreover, these methylxanthines decrease oxidative stress, levels of cholesterol, and aggregation of Aβ1-42 in SH-SY5Y neuroblastoma cells [21]. Another pathway, by which caffeine could mediate its pharmacological activity, is the cholinergic system. 

In this context, Fabiani and colleagues used single-channel recordings and fluorescent measurements to examine the influence of caffeine on the nicotinic acetylcholine receptor (AChR). 

Their results show that caffeine acts as a partial agonist and an ion channel blocker at neuronal α7 and muscle nicotinic receptors (AChR) at different concentrations and suggest this methylxanthine as a multitarget-directed drug for the treatment of AD [36]. 

Interestingly, Kumar and colleagues screened more than 600 molecules of natural origin for their ability to modulate acetylcholine metabolism. They found that caffeine has a comparable AChE inhibitory potential to donepezil, a commonly used drug to treat mild to moderate dementia. 

Moreover, caffeine shows no neurotoxicity in primary (E18) hippocampal neurons but significantly improves neuronal survival and protects from neurodegeneration [37].

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