Table 2.

In vitro and in vivo studies summary.

Study	In-Vivo/In Vitro Study	Mechanism of Neuroprotective Effect	Study Methodology	Main Outcomes
[49] Gastaldo et al., 2020	In Vitro	Effecting membrane	➢Studied interaction of resveratrol, caffeine, carotene, and epigallocatechin gallate (EGCG) on Aβ peptide aggregate by using synthetic membranes that contained cross-sheets of Aβ 25–35 ➢The effect on the size and volume fraction of Aβ fragments noted using microscopy, x-ray diffraction, UV-vis spectroscopy, and molecular dynamic simulations	➢caffeine was membrane-active and simultaneously partitioned into the synthetic membrane, where caffeine caused membrane thickening ➢caffeine attracted water and promoted the expulsion of plaques from the membrane leading to more pronounced amyloid fibrils ➢caffeine by causing early expulsion of peptides prevents crosslinking with neighboring monomers and reduces peptide aggregation
[50] Janitschke et al., 2019	In Vitro	Altering APP processing	➢human neuroblastoma (SH-SY5Y WT and SH-SY5Y APP695) was incubated in MTX (0.1 nmol per 1 g protein) 30 min before α and β-secretase activity measurements using WB	➢caffeine decreased total secreted Aβ levels by 15.5% through elevation of non-amyloidogenic α-secretase APP processing ➢caffeine reduced ROS, cholesterol levels, and Aβ aggregation
[51] Arendash et al., 2006	In Vivo	Altering APP processing	➢studied the effect of chronic caffeine administration (1.5 mg/day in drinking water, 4–9 months of age) on transgenic APPswedish mice (Tg) ➢last 8 weeks of the study, the mice were subjected to behavioral assessment ➢rodent’s brain subjected to post-mortem WB analysis to measure soluble/insoluble Aβ levels, PCS1, BACE and adenosine levels, and adenosine receptor density	➢Tg mice with chronic caffeine administration performed significantly better than control Tg mice across multiple cognitive domains ➢caffeine treated Tg mice had lower hippocampal Aβ levels, reduced presenilin 1 (PS1) and β-secretase (BACE1) levels, restored brain adenosine levels, and unchanged A1 and A2A receptor density compared to control Tg mice
	In Vitro		➢Studied effect of caffeine (0–10 μM) on Aβ production in vivo in APPswedish mice	➢APPswedish mice and N2a neuronal cultures showed a caffeine concentration-dependent decrease in Aβ1–40 and Aβ1–42 production
[52] Arendash et al., 2009	In Vivo	Altering APP processing	➢effect of caffeine administration (0.3 mg/mL) on aged transgenic APPswedish mice (18–19 months) showing impaired working memory ➢after 4–5 weeks of caffeine treatment, the mice were subjected to behavioral testing ➢post-mortem tissue was subjected to immunohistochemistry, Aβ ELISA, pcRaf-1, and PKA analysis	➢caffeine administration on aging Tg mice showed markedly improved working memory and overall cognition than Tg control mice (p < 0.05) ➢caffeinated Tg mice had lower Aβ deposition and lowered soluble Aβ levels than Tg control ➢mechanistically the neuroprotective effect of caffeine involves BACE1 suppression in Tg caffeinated through cRaf-1/NFηB pathway and PKA
			➢9-month-old Tg mice were gavage with caffeine (1.5 mg/twice daily for 2 weeks), ➢sacrificed and subjected to pcRaf-1 and PKA analysis
			➢5.5-month WT mice were put on caffeine (0.3 mg/L) ➢age of 15–16 months was subjected to 6-week behavioral screening
	In Vitro		➢effect of concentration-dependent caffeine administration (0–20 μM for 1h) or time-dependent caffeine administration (20 μM for 0–180 min) on APPswedish mice N2a neuronal cultures	➢there was reduce glycogen synthases kinase 3 levels in N2a cells
[53] Cao et al., 2009	In Vivo	Altering APP processing	➢acute (1.5 mg caffeine IP or gavage) and chronic caffeine administration (2× daily 1.5 mg caffeine gavage for 7 days) on Aβ levels of (Tg) and (NT)	➢acute and chronic caffeine administration in Tg mice led to reduced Aβ levels in brain interstitial fluid and plasma
			➢microdialysis of living rodent hippocampus to study the effect of acute caffeine administration on interstitial fluid Aβ levels	➢plasma Aβ or caffeine levels did not correlate with brain Aβ levels or cognitive performance
[54] Zappettini et al., 2019	In Vivo	Altering excitation and inhibition	➢investigated the long-term effect of early-life exposure to caffeine in THY-Tau22 transgenic mice ➢caffeine dose of 3 g/L was given to parental THY-Tau22 Mice and WT mice, starting 2 weeks before mating and continued to postnatal day 15 ➢then learning of offspring Tg and WT mice was accessed at 8 and 12 months ➢offspring Tg and WT mice were subjected to in vivo electrophysiology examination of hippocampal neuronal activity and post-mortem biochemical analysis	➢in vitro electrophysiology assessment showed that early life caffeine exposure altered glutamatergic and GABAergic circuits ➢complex non-linear Tau-age-caffeine interaction rather than the predicted simple caffeine-induced aging-like increase in glutamatergic and GABAergic drives
[55] Mancini et al., 2018	In Vitro	Altering protein aggregation	➢studied ability of caffeine, chlorogenic acid, quinic acid, caffeic acid, quercetin, and phenylindole at 25 mM to inhibit fibrilization of Aβ and Tau ➢using (ThT) and (ThS) fluorescence assay	➢caffeine on its own could not interfere with Aβ, Tau aggregation, and Aβ oligomerization
[56] Laurent et al., 2014	In Vivo	Altering protein aggregation	➢effect of chronic caffeine intake (0.3 g/L drinking water) on THY-Tau22 mouse ➢rodent subjected to cognitive test, biochemical analysis, mRNA extraction, and caffeine metabolite sampling	➢chronic caffeine Tg mice performed significantly better than control Tg mice ➢Caffeinated Tg mice had significantly lower Tau phosphor-isotopes, pro-inflammatory and oxidative stress markers than Tg control mice
[57] Alzoubi et al., 2018	In Vivo	Antioxidant properties	➢caffeine (0.3 g/mL added to drinking water) to reduce the cognitive decline caused by increased oxidative stress due to administration of L-methionine (1.7 g/kg/day orally) for a treatment period of 4 weeks ➢cognition and hippocampal tissue antioxidant markers were assessed	➢L-methionine administration caused (short and long) term memory impairment while caffeine negated that effect ➢L-methionine administration caused reduced catalyze and GPx enzyme activities; reduced GSH, GSSG ratio compared to controls, while caffeine administration normalized these effects
[58] Moy et al., 2013	In Vivo	Effect on BNDF levels	➢effect of caffeine on rats placed on a high-fat diet ➢Rodents’ hippocampus was subjected to microdialysis and then spontaneous alternating testing to test the working memory of rodents. ➢Post mortem the rodent brains were subjected to histology, WB, and enzyme-linked immunosorbent assay (BNDF quantification)	➢caffeine treatment was sufficient to prevent high-fat diet weight gain and high-fat diet memory impairment ➢caffeine diet prevented reduction in BNDF induced by a high-fat diet and allowed maintenance of synaptic plasticity
[59] Han et al., 2013	In Vivo	Effect on BNDF levels	➢effect of caffeine 0.75 mg/day or 1.5 mg/day on saline vehicle treatment for 8 weeks on the expression of BNDF and TrKB receptors in Tg mice ➢rodents subjected to a Morris water maze test on and WB	➢caffeine administration Tg mice significantly improved cognitive performance compared to control Tg mice ➢dose-response increase of hippocampal BNDF and TrKB expression in caffeinated Tg mice
[60] Zhao et al., 2017	In Vivo	AR antagonist properties	➢administration of 3 g/L caffeine in drinking water or A2AR KO mouse model can increase cognitive impairment by reducing Tau-hyperphosphorylation induced by TBI mouse model ➢cognition was assessed using the Morris water maze test (day 7 and week 4 post-treatment) ➢post-mortem (immunohistochemistry, Golgi staining, Western blotting were also performed	➢post-TBI mechanism of A2AR activation that triggers hyperphosphorylation of Tau, causing memory impairment may be normalized by chronic caffeine administration
[61] Bortolotto et al., 2015	In Vivo	AR antagonist properties	➢effects of acute of caffeine (10 mg/kg), ZM241385 (10 μg/kg,), DPCPX (0.5 mg/kg), dipyridamole (5 mg/kg), ELINA (100 μg/kg,) on scopolamine (200 μM) induced memory loss in adult WT Zebrafish ➢subjected to behavioral tests such as inhibitory avoidance task, exploratory assessment, and social interaction test	➢caffeine pre-treatment prevented scopolamine-induced amnesia
[62] Li et al., 2015	In Vitro	AR antagonist properties	➢caffeine (200 μM), selective AR-1a,2a,2b,3r antagonists, A3R gene knockout treatment can reduce AβPP and LDL internalization, therefore reducing Aβ generation in rat embryonic primary cerebral cortical neurons and human blastoma SH-SY5Y ➢the cells were examined by Western blotting, surface immunostaining, RT-PCR, Lactate dehydrogenase	➢caffeine, A3R antagonist, A3R gene knockout showed a concentration-dependent reduction in LDL internalization, suppression of LDL-induced Aβ generation, suppressed AβPP internalization
[63] Espinosa et al., 2013	In Vivo	AR antagonist properties	➢effect of caffeine consumption (30 μm plasma) in adult Wistar rats with sporadic AD (induced by STZ, 5 μL) ➢rodents were subjected to cognitive tests, immunohistochemistry, immunoblotting, and quantitative-PCR	➢caffeine can prevent STZ-induced memory decline while simultaneously controlling the hippocampal A2AR population
[64] Dall’Igna et al., 2007	In-Vivo	AR antagonist properties	➢CF1 adult mice with cognitive decline induced by Aβ injection (3 nmol)and treated them with caffeine or selective A2AR treatment (acute, subchronic, prolonged, or combined) ➢Subjected to inhibitory avoidance and spontaneous alteration cognitive tests	➢prolonged caffeine or selective A2AR treatment using: (subchronic, chronic, and combined prolonged) protocols showed a protective effect against cognitive decline
[65] Soliman et al., 2017	In Vitro	Effect on endolysosomes dysfunction	➢SH-SY5Y with HIV-1 Tat (200 μM) for 2 days in the presence/absence of caffeine (200 μM) ➢quantified Aβ levels, vacuolar-ATPase, and phosphorylated Tau protein levels	➢caffeine was able to prevent HIV-1 Tat induced increase in Aβ and Tau levels and prevent a decrease in vacuolar-ATPase
[66] Mohamed et al., 2013	In Vitro In Silico	Acetylcholinesterase inhibition	➢effect of xanthine (caffeine, pentoxifylline, propentofylline) on the inhibition of AChE through in vitro and molecular modeling studies	➢caffeine was a weak AChE inhibitor
[67] Pohanka et al., 2013	In vitro In silico	Acetylcholinesterase inhibition	➢used standard Elman test and in silico examinations to determine whether caffeine could inhibit human BChE and ACh	➢caffeine is a strong non-competitive inhibitor of AChE and a weak non-competitive inhibitor of BChE
[68] Cao et al., 2011	In Vivo	Effect on granulocyte-colony stimulating factor, IL-6, and IL-10	➢examined the effects of decaffeinated coffee, caffeinated coffee (1.5 mg caffeine), and pure caffeine (1.5 mg caffeine) on plasma cytokines measured in 8-month-old Tg and NTg mice ➢chronic effects of decaffeinated coffee, caffeinated coffee (0.75 mg caffeine), saline, and pure caffeine (0.75 mg caffeine) administered twice weekly through gavage on plasma cytokines of 10-month-old Tg and NTg ➢Rodents were subjected to cognitive interference task, and blood samples were analyzed using Luminex assay and ELISA	➢acute treatment, plasma levels of GCSF, IL-6, IL-10 was elevated for Tg and NTg mice treated with caffeinated coffee only ➢chronic experiment, it was identified that both caffeinated coffee and caffeine treatment allowed better preservation of working memory compared to NTg controls and higher plasma GCSF levels correlated with better cognition
[69] Qosa et al., 2012	In Vivo In Vitro	Effect on Aβ-clearance	➢treated WT mice with caffeine/rifampicin (20 mg/kg intraperitoneally, 2-week caffeine, and 3-week rifampicin) and subjected them to a brain efflux index study	➢brains of mice treated with caffeine showed significantly higher Aβ clearance across BBB compared to controls
			➢treated mouse bEnd3 with 50 μM of caffeine/rifampicin and analyzed them using PT-PCR and WB	➢in vitro caffeine treatment significantly upregulates the expression of P-GP-mechanism that increases Aβ across BBB
[70] Shukitt-hale et al., 2013	In Vivo	Nill	➢WT mice were subjected for 8 weeks to varying diets of coffee extract (0%, 0.165%, 0.275%, 0.55%, 0.825%) in study 1 ➢study 2 WT mice were subjected to coffee (0.387%, 0.55%) and (0.0181%, 0.0258%) for 8 weeks ➢then the rodents were subjected to psychomotor and cognitive testing ➢the brain and serum concentrations of caffeine and hydroxycinnamic acid metabolites were also recorded	➢pure caffeine did not fully account for the protective effect of coffee

Study did not find a neuroprotective caffeine link: ; study found a neuroprotective caffeine link: .