Unique Role of Caffeine Compared to Other Methylxanthines (Theobromine, Theophylline, Pentoxifylline, Propentofylline) in Regulation of AD Relevant Genes in Neuroblastoma SH-SY5Y Wild Type Cells

Methylxanthines are a group of substances derived from the purine base xanthine with a methyl group at the nitrogen on position 3 and different residues at the nitrogen on position 1 and 7. They are widely consumed in nutrition and used as pharmaceuticals. Here we investigate the transcriptional regulation of 83 genes linked to Alzheimer’s disease in the presence of five methylxanthines, including the most prominent naturally occurring methylxanthines—caffeine, theophylline and theobromine—and the synthetic methylxanthines pentoxifylline and propentofylline. Methylxanthine-regulated genes were found in pathways involved in processes including oxidative stress, lipid homeostasis, signal transduction, transcriptional regulation, as well as pathways involved in neuronal function. Interestingly, multivariate analysis revealed different or inverse effects on gene regulation for caffeine compared to the other methylxanthines, which was further substantiated by multiple comparison analysis, pointing out a distinct role for caffeine in gene regulation. Our results not only underline the beneficial effects of methylxanthines in the regulation of genes in neuroblastoma wild-type cells linked to neurodegenerative diseases in general, but also demonstrate that individual methylxanthines like caffeine mediate unique or inverse expression patterns. This suggests that the replacement of single methylxanthines by others could result in unexpected effects, which could not be anticipated by the comparison to other substances in this substance class.


gene gene name reference oxidative stress
CAT Catalase -depletion of cellular hydrogen peroxide -deficiency or malfunction of catalase can be linked to degenerative diseases [1] -there is a broad agreement in the role of oxidative stress in the development of cardiovascular injuries [2] -oxidative stress is reported as important pathophysiological change in COPD [3] GPX1 Glutathione peroxidase 1 -detoxification of H2O2 -GPX shows an age related reduced activity and are decreased in plasma samples of AD patients [4,5] -modulation of GPx-3 activity is associated with altered risk of cardio-renal disease [6] -a study reported a reduced activity of GPx in COPD cases compared to healthy controls [7] GPX3 Glutathione peroxidase 3

GPX7
Glutathione peroxidase 7 NQO1 NAD(P)H dehydrogenase, quinone 1 -polymorphism in NQO1 as a risk factor for sporadic AD [8,9] -gender-dependent (women) association with coronary artery disease and NQO1 polymorphism was found [10] NUDT15 Nudix type motif 15 -Methylation levels of NUDT15 were altered in mild cognitive impairment (MCI) patients [11] PARK7 Parkinsonism associated deglycase -up-regulated DJ-1 expression related to oxidative stress and AD [12] -PARK7 protein found to be regulating vascular contractility and blood pressure, thus being a risk factor for cardiovascular diseases [13] -Protein levels of PARK7 were decreased in the lungs of COPD patients [ [45] -a strong correlation between XPA expression and fibrous plaques was observed in NSTEMI patients [46] -genes which demonstrated a high module membership and gene significance for clinical indices of COPD severity included XPA (& ERCC5) [47] lipid & energy metabolism

ABCA1
ATP-binding cassette, subfamily A, member 1 -ABCA1 has a regulatory role in brain amyloidogenesis and amyloid clearance [48] -an ABCA1 gene variation was reported to be associated with the risk of a heart disease during pravastatin treatment [49] -ABCA1 is discussed as therapeutic target for therapy of cardiovascular diseases (cholesterol is major cardiovascular disease risk factor) [50] -as reviewed in [51] ATP-binding cassette transporters appear to have a crucial and protective role in respiratory diseases

ACAT1
Acetyl-CoA acetyltransferase 1 -cholesteryl ester levels were correlated with Aβ generation and blocking ACAT activity diminished Aβ generation [52,53] -polymorphism in the ACAT1-gene is linked with a higher risk of coronary artery disease (CAD) [54] -expression of ACAT1 seems to be induced by oxidative stress [55] APOA1 Apolipoprotein A-I -the HDL-cholesterol transporter ApoA1 inhibits amyloid-beta aggregation [56] -Abeta production is influenced by cholesterol levels [57] -The ratio of ApoB and ApoA1 is suggested as marker for atherosclerotic risk in prevention of coronary artery disease [58] -ApoA1 is suggested to be beneficial in the prevention and the treatment of lung diseases [59] -overexpression of ApoA1 suppressed the generation of ROS in lungs of cigarette smoke exposed mice [60] HMGCR

ERN1
Endoplasmatic reticulum to nucleus signaling 1 -polymorphism in the ERN1 promoter are discussed as risk factor for the development of AD [80] -Evidence arise that ERN1 plays an important role in cardiovascular disease [81] CLU Clusterin -CLU is a cellular biosensor of oxidative load alterations and is linked to different pathological conditions like atherosclerosis [82] -in regard to AD clusterin was identified as Aβ toxicity mediator [83] AASS Aminoadipate-semialdeh yde synthase -AD is associated with protein carbonylation [84] -hyperexpression of this gene in cardiac tissue is discussed as protection against the harmful action of ROS [15]

signal transduction & gene expression
PRKCA Protein kinase C, alpha -direct activation of the alpha secretase mediated cleavage of APP; gain-of-function-mutations in PKCalpha may promote synaptic defects [85,86] -Protein kinase C activation has shown a link to cardiovascular disease states as reviewed in [87] PRKCD Protein kinase C, delta -PKCd-/-mice showed an attenuation of pulmonary fibrosis via inhibition of the NF-KB [88] PRKCE Protein kinase C, epsilon -direct activation of the alpha secretase mediated cleavage of APP [85] -PRKCE showed a strong association with forced one second expiratory volume and the FEV1/FVC ratio [89] PRKCG Protein kinase C, gamma -decreased PKCG level in AD fibroblasts [90] PRKCQ Protein kinase C, theta -PRKCQ was reported to influence the permeability of the blood-brain barrier via tight junction protein disruption [91] -PRKCQ is an important intermediate in signaling pathways for a robust lung inflammatory response [92] PRKCZ Protein kinase C, zeta -PRKCZ may contribute to the development of cardiomyopathy [93] -Patients with AECOPD showed a dynamic gene expression in an up-up pattern [94] ALS2 alsin Rho guanine nucleotide exchange factor -Alsin has neuroprotective activity against ALS-related insults [95] -ALS2-KO neurons are reported to be predisposed to oxidative stress [96] GNAO1 G protein subunit alpha o1 -It is discussed that PS1 can interact and regulate G protein activity [97] -Aβ can influence function of G-proteins and its receptors directly or indirectly [98] -G proteins are related to cardiovascular diseases [99] -first signal transduction cascade in response to oxidative stress includes G proteins

EP300
E1A (adenovirus early region 1A) binding protein p300 -Ep300 is dysregulated in AD brains compared to healthy controls [100] -alterations of histone acetylation on PS1-and BACE1 promoters were described to be mediated by EP300 [101] -EP300 was linked to the development of recurrent cardiovascular events [102] HDAC1 Histone deacetylase 1 -in AD patients the level of HDAC1 is significantly attenuated [103] -the HDAC activity reflects the severity of COPD [104] -HDACs are regulators that are playing a role in certain cellular processes e.g. apoptosis, fibrosis, oxidative stress and inflammation [105]

CDKL1
Cyclindependent kinase-like 1 -Cdkl1 is a kinase involved in the pathologic phosphorylation of tau, a neuropathological feature of AD [106] GAP43 Growth associated protein 43 -increased GAP43 mRNA and protein level were reported in AD brains [107,108] GSK3a Glycogen synthase kinase 3 alpha -GSK3 is a key kinase contributing to abnormal tau-phosphorylation causing neurofibrillary tangles in AD [109,110] -GSK3 is discussed as potential therapeutic target for myocardial diseases [111] -GSK3b mediates ROS-induced glucocorticoid unresponsiveness in COPD [112]

APBA1
APP binding, family A, member 1 -APBA proteins fine-tune APP trafficking and processing [113,114] -regulation of APP processing [115] -interaction of APP with APP-BP1 is required for activation of DNA synthesis and apoptosis in neurons [116] -APP and its processing products are reported to be present in artherosclerotic plaques, which are part of the pathomechanism of cardiovascular diseases [117,118] -circulating Aβ40 levels were predictive of cardiovascular mortality in patients with coronary heart disease (CHD) as reported in a retrospective cohort study [119] -serum levels of Aβ40, Aβ42 and total Aβ are significantly increased in patients with chronic obstructive pulmonary disease compared to healthy controls [ -Notch is suggested to be involved in the aetiology of different cardiovascular diseases and discussed as therapeutic target [128] -the -secretase is discussed to mediate the oxidative stress-induced increase of the beta-secretase enzyme [129] APH1b aph1 homolog B, gamma secretase subunit CASP3 caspase 3 in AD cases there is a high degree of co-localization of activated caspases and neurofibrillary tangles / senile plaques (see also CASP4) CTSB cathepsin B -Cathepsin was described to play a role in APP metabolism [130] -in AD impairments in the cathepsin family have been observed [131] -Plasma cathepsins are potential biomarkers for cardiovascular disease in humans [132] -Cathepsin L is discussed to degrade an alternative APP c-terminal fragment [133] CTSL cathepsin L PSENEN presenilin enhancer, gamma secretase subunit -encodes a component of the gamma secretase complex [126] (see also APH1a and APH1b)

LRP1
low density lipoprotein receptor-related protein 1 -LRP is associated with the transport, the production and the clearance of Abeta [139] -Inactivation of LRP1 leads to a higher risk of atherosclerosis and could result in aneurysm formation [140] MMP2 matrix metallopeptidase 2 -Aβ-Degradation [141] -functional polymorphisms of MMPs were related to arterial stiffness and CAD [142] -MMP members are up-regulated during acute or chronic phase of lung diseases [143] PKP4 plakophilin 4 -interaction with presenilin 1 [144] PRNP prion protein -Significantly altered levels of PRNP mRNA levels in ventricular autopsy samples from children and young adults which were exposed to high concentrations of air pollutants indicating increased myocardial inflammation [145] -Significant association with COPD was found only in smokers [146] SNCA synuclein, alpha -up-regulated expression of SNCA in AD subjects than in control subjects [147] -link between alpha-synuclein levels in the blood and insulin resistance in SNCA knock-out mice; SNCA association to diabetes indirectly links it to CVD as diabetes is a risk factor [148] SNCB synuclein, beta -levels of beta-synuclein were decreased in AD [149] CASP4 Caspase 4 -in AD cases there is a high degree of co-localization of activated caspases and neurofibrillary tangles / senile plaques [150,151] -anti-apoptotic approaches seem to be an effective therapeutic strategy for cardiovascular diseases [152] -in the pathogenesis of COPD apoptosis of structural lung cells might possibly be an important event [153] IL1A Interleukin 1, alpha -Altered expression is observed in AD [154,155] -IL-1 cytokines and the inflammasome are linked to the pathogenesis of cardiovascular diseases [156] -IL-1 alpha is secreted via alveolar macrophages and leads to subsequent lung inflammation [157] SH2D3C/CH AT

SH2 domain containing 3C
-SH2D3C is reported to be overexpressed in Aß-enriched fractions [158] -expression of SH2D3C was found to correlate negatively with smoking, a risk factor for COPD, in a transcriptomic study in lymphocytes [159] neuronal genes

MAP2
Microtubule-associated protein 2 -Map2 is negative correlated to the pathomechanism of AD [160] -As a part of the MAPK-Cascade Map2 is involved in pulmonary cell adhesion and cell motility [161] MAPT Microtubule-associated protein tau -hyperphosphorylated tau  neurofibrillary tangle [162,163] -top gene identified in COPD patients vs control group in a sherlock analysis [164] PLAU Plasminogen activator, urokinase -Polymorphism in PLAU gene could increase the risk of AD [165] -Gene variations of the PLAU gene were significantly associated with an increased risk of MI [166] -via activation of TGF-beta1 inhibition of SERPINE2, PLAU could be linked to COPD [167] PLAT Plasminogen activator, tissue -t-PA is associated with Abeta degradation [168] -t-PA protein level were significantly lower for healthy control patients than CAD patients [169] ACHE Acetylcholinesterase -decreased AChE activity in AD brains [170,171] -Treatment with acetylcholinesterase inhibitors was linked to a reduced risk of acute coronary syndrome in patients with dementia [172] -Alterations in AChE activities can be associated with development and severity of COPD [173] -AChE was reported to regulate lipid metabolism in the brain via transcriptional influence on lipid-related genes [174] BDNF Brain-derived neurotrophic factor -declined BDNF level in AD leads to poorly differentiated neurons, synapse loss and cognitive dysfunction [175][176][177] -A direct interplay of oxidative stress indicators and the levels of BNDF is suggested in the brain [178] -BDNF is indicated as important biomarker associated with parameters of COPD severity [179] -reduced plasma BDNF concentration is suggested to be linked to coronary heart disease pathogenesis [180] 1