Significance Coffee consumption is linked with reduced risk of Parkinson’s disease (PD), and caffeine is generally believed to be the protective agent. However, several lines of evidence suggest the presence of additional compound(s) in coffee that can be protective as well. Here we show that eicosanoyl-5-hydroxytryptamide, which we purified from coffee as an agent that leads to enhanced enzymatic activity of the specific phosphatase PP2A that dephosphorylates the pathogenic protein α-synuclein, works in synergy with caffeine in protecting against mouse models of PD and Dementia with Lewy bodies. The mechanism of this synergy is also through enhancing PP2A, which is dysregulated in the brains of individuals with these α-synucleinopathies.

Abstract Hyperphosphorylated α-synuclein in Lewy bodies and Lewy neurites is a characteristic neuropathological feature of Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB). The catalytic subunit of the specific phosphatase, protein phosphatase 2A (PP2A) that dephosphorylates α-synuclein, is hypomethylated in these brains, thereby impeding the assembly of the active trimeric holoenzyme and reducing phosphatase activity. This phosphatase deficiency contributes to the accumulation of hyperphosphorylated α-synuclein, which tends to fibrillize more than unmodified α-synuclein. Eicosanoyl-5-hydroxytryptamide (EHT), a fatty acid derivative of serotonin found in coffee, inhibits the PP2A methylesterase so as to maintain PP2A in a highly active methylated state and mitigates the phenotype of α-synuclein transgenic (SynTg) mice. Considering epidemiologic and experimental evidence suggesting protective effects of caffeine in PD, we sought, in the present study, to test whether there is synergy between EHT and caffeine in models of α-synucleinopathy. Coadministration of these two compounds orally for 6 mo at doses that were individually ineffective in SynTg mice and in a striatal α-synuclein preformed fibril inoculation model resulted in reduced accumulation of phosphorylated α-synuclein, preserved neuronal integrity and function, diminished neuroinflammation, and improved behavioral performance. These indices were associated with increased levels of methylated PP2A in brain tissue. A similar profile of greater PP2A methylation and cytoprotection was found in SH-SY5Y cells cotreated with EHT and caffeine, but not with each compound alone. These findings suggest that these two components of coffee have synergistic effects in protecting the brain against α-synuclein−mediated toxicity through maintenance of PP2A in an active state.

Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are characterized by intraneuronal aggregates of α-synuclein in Lewy bodies and Lewy neurites (1). This normally soluble intrinsically disordered protein misfolds and forms amyloid fibrils in neuropathological hallmark inclusions. The initial oligomerization and eventual fibrillization are believed to be critical steps leading to neuronal dysfunction and death (2). Aggregates of α-synuclein can also propagate across neurons to synaptically connected brain regions (3), a phenomenon that explains the progressive nature of these disorders, their prodromal phase, and the emergence of additional clinical manifestations over time (4). Postmortem brain studies also show that α-synuclein in these aggregates is characteristically hyperphosphorylated at serine 129, and antibodies directed at phospho-Ser129-α-synuclein (p-α-Syn) are commonly used to detect these inclusions (5⇓–7). Hyperphosphorylated α-synuclein is also a consistent feature of aggregates in transgenic rodent and Drosophila models expressing human α-synuclein (8⇓⇓⇓–12) as well as aggregates deposited in remote brain regions following intracerebral inoculation of α-synuclein preformed fibrils (α-Syn PFF) (3). Additionally, α-synuclein phosphorylation at serine 129 accelerates its oligomerization and fibrillization in vitro (5). Accordingly, this posttranslational modification is of both pathogenetic and therapeutic interest in α-synucleinopathies.

While α-synuclein can be phosphorylated at serine 129 by multiple kinases (13⇓–15), its dephosphorylation is carried out by a specific isoform of protein phosphatase 2A (PP2A) (8). This heterotrimeric holoenzyme is the primary serine/threonine phosphatase in the brain and consists of a structural A subunit, a catalytic C subunit, and one of multiple regulatory B subunits that determine substrate specificity (16⇓–18). The isoform that dephosphorylates α-synuclein contains the B55α subunit, and its assembly and activity are highly regulated by reversible carboxyl methylation of Leu309 in the catalytic C subunit (8, 16⇓–18). PP2A carboxyl methylation is regulated by the opposing activities of a PP2A-specific leucine carboxyl methyltransferase (LCMT-1) and a PP2A-specific methylesterase (PME-1) (19⇓–21). Interestingly, the levels of these methylation-regulating enzymes are perturbed in PD and DLB brains, with down-regulation of LCMT-1 and up-regulation of PME-1. This profile is associated with reduced relative levels of methylated PP2A (methyl-PP2A), which is the enzymatically more active form of the phosphatase (22). Furthermore, treating α-synuclein transgenic mice (SynTg) with eicosanoyl-5-hydroxytryptamide (EHT), an inhibitor of the PME-1 methylesterase activity, increases both brain PP2A methylation and phosphatase activity, reduces the accumulation of phosphorylated α-synuclein aggregates, improves neuronal integrity, represses the neuroinflammatory response to the transgene, and preserves behavioral phenotype (8, 23).

It is of interest to note that we identified EHT initially in a series of extractions and purifications as a component of coffee with PME-1 methylesterase inhibitory activity in vitro (8). Subsequent studies showed that EHT also exhibits antioxidant and antiinflammatory activities (23). Structurally, EHT is unrelated to caffeine (CAF). It is a serotonin derivative wherein the C20 saturated fatty acid, eicosanoic acid, is linked to the serotonin amino group. Considering the epidemiological data linking coffee consumption with reduced risk of developing PD (24⇓⇓–27), the identification and characterization of the properties of EHT provide a mechanistic insight into the beneficial effects of this widely consumed beverage. Prior studies about the protective potential of coffee in PD have focused largely on CAF, because epidemiological data are consistent with CAF as a major source of neuroprotective activity (25, 27). In addition, CAF has been shown to protect mice against the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and this effect has been attributed to CAF acting as an adenosine A 2A receptor antagonist (28). A recent study also reported protection against mutant α-synuclein fibril injections in the striatum (29). However, among patients with early PD, the amount of CAF consumption does not impact the rate of progression of the disease (30), and decaffeinated coffee has been found to be protective in Drosophila models of PD (31), raising some question about the protective effect of only CAF among the numerous other compounds in coffee.

The present study was carried out to test the potential synergy between EHT and CAF in protecting against α-synuclein−mediated pathology in two mouse models, transgenic mice expressing human α-synuclein panneuronally driven by the Thy1 promoter (SynTg) (32), and unilateral α-Syn PFF inoculation of the striatum (3). The potential synergy between these coffee components was also tested in cultured cells.

Discussion The present findings demonstrate that subtherapeutic doses of CAF and EHT, two unrelated compounds found in coffee, can work in synergy to effect biochemical and molecular changes in the mouse brain leading to protection in two models of α-synucleinopathy. This is reflected in better behavioral performance of both SynTg mice and α-Syn PFF-inoculated mice treated chronically with a combination of these compounds for 6 mo, but not if each is given separately. In addition to preserved neuronal integrity and function, and dampened inflammatory response to α-synuclein, PP2A methylation is modulated by this cotreatment in a manner that favors enhanced phosphatase activity. This is associated with reduced accumulation of p-α-Syn. Similar biochemical changes occur in a cellular model challenged with α-Syn PFF and treated with the combination leading to increased PP2A methylation, reduced p-α-Syn levels, and cytoprotection. These observations collectively suggest that CAF and EHT may function through a common mechanism. We purified EHT from coffee in an analytical assay set up to identify specifically compounds that inhibit the PP2A methylesterase PME-1 (8). EHT administered alone to SynTg mice for 9 mo also modulated PP2A methylation in brain tissue lysates in a dose-dependent manner in favor of the enzymatically active form and reduced the accumulation of p-α-Syn. This was more evident at a dose 10 times higher (120 mg/kg/d) than the dose used in the present study (8). CAF, on the other hand, is an adenosine A2a receptor antagonist, a property that is believed to mediate its protective function in the MPTP model of DA neuron degeneration (28). In addition, deleting the A2a receptor gene in mice has been shown to protect against dopaminergic neuron degeneration induced by human α-synuclein transgene containing two pathogenic mutations, A53T and A30P (44). Moreover, CAF was recently reported to protect against A53T mutant α-synuclein fibril injections in the striatum (29). By blocking A2a receptor signaling, CAF may enhance PP2A methylation through preventing cAMP-dependent protein kinase A/glycogen synthase kinase 3β-mediated activation of PME-1 (45⇓–47). The combined effect of EHT and CAF is greater PP2A activity than either compound could achieve alone. The latter could underlie the synergy with EHT observed in the present study. Thus, these results suggest that chronic consumption of coffee and, therefore, chronic coingestion of both EHT and CAF have added benefits in α-synucleinopathy−prone brains. Additionally, the increase in methyl-PP2A with these treatments associated with neuroprotection, along with hypomethylation of PP2A in PD and DLB brains (22), suggests a pathogenetic significance of this phosphatase in these disorders. The protection in the α-Syn PFF model with CAF and EHT coadministration provides some insight into the role of α-synuclein phosphorylation in nucleation and propagation of pathologic aggregates. This treatment was initiated upon weaning of WT mice, and α-Syn PFF inoculation occurred 6 wk later. Thus, the reduced phosphorylation level of α-synuclein in the brain appears to have decreased the ability of exogenous fibrils to nucleate endogenous α-synuclein at the site of striatal injection, leading to reduced propagation of aggregates to the substantia nigra pars compacta and the contralateral striatum. This may suggest that pharmacological interventions leading to reduced phosphorylation of α-synuclein may retard nucleation and propagation of pathology and slow down disease progression. The present observations also have implications for the epidemiological data linking coffee consumption with reduced risk of PD (48⇓–50) and attribution of this protection to CAF (24, 25). However, coffee is a complex chemical mixture containing more than a thousand different compounds (51). Thus, it is not unlikely that other components of coffee play a beneficial role as well. Factors that are not accounted for in epidemiological studies include the method of preparation of coffee. Different components of coffee are present in the final consumed product, depending on conditions of coffee growth and harvesting, methods of roasting and brewing, whether the coffee is filtered or not, and what kind of filter is used. EHT is a principal constituent of coffee wax that coats the coffee bean (52). The levels of EHT and closely related N-alkanoyl-5-hydoxytryptamides are appreciable in unfiltered coffee, whereas some preparation methods remove lipids such as EHT (53). In addition to deficiencies in PP2A methylation in α-synucleinopathies (PD and DLB) (22), similar PP2A dysregulation is found in postmortem brains from patients with tauopathies such as Alzheimer’s disease (AD) and Progressive Supranuclear Palsy (PSP) (54, 55). Our present results, therefore, raise the possibility of similar synergy between EHT and CAF in both classes of chronic neurodegenerative diseases. Several epidemiologic studies have shown that greater consumption of coffee or CAF is associated with reduced risk of AD (56), and dietary supplementation with EHT is efficacious in two rodent models of AD: mice exposed to oligomeric beta-amyloid injections into the hippocampus (57) and rats injected in the lateral ventricle with viral vector expressing the endogenous PP2A inhibitor SET that exhibit tau hyperphosphorylation, elevated levels of cytoplasmic amyloid-β protein, and cognitive impairment (58). Additionally, in AβPPsw+PS1 transgenic mice, administration of caffeinated coffee increased plasma levels of cytokines, while neither CAF alone nor decaffeinated coffee had this effect, raising the likelihood that CAF synergizes with another component of coffee (59). Based on our present findings, we submit that at least one of the synergizing components is EHT. Coffee is not the only botanical extract that contains agents that act to inhibit PME-1-mediated PP2A methylation. As we begin to unravel the polypharmacology of the micronutrients in commonly consumed botanical extracts such as coffee, it seems likely that it will be possible to optimize their composition to enhance efficacy so as to provide widely available, inexpensive, and effective therapeutics for the prevention and treatment of neurodegenerative diseases such as PD, DLB, PSP, and AD.

Materials and Methods Detailed information on the experimental methods and materials used can be found in SI Appendix. All animal procedures were approved by the Rutgers Robert Wood Johnson Medical School Institutional Animal Care and Use Committee. SynTg on BDF1 background overexpressing human WT α-synuclein under the control of the Thy1 promoter were used as a model of diffuse α-synucleinopathy to study the effects of EHT and CAF treatments. The α-Syn PFF inoculation model was prepared using C57BL/6J mice (3). To prepare α-Syn PFF, plasmid pT7-7 encoding mouse α-synuclein cDNA was used to transform Escherichia coli BL21(DE3) strain (60, 61). EHT was synthesized at Signum Biosciences. Striatal DA content was measured using HPLC-MS. TH immunoreactive nigral neurons were counted using an automated, unbiased, context-intelligent neural network algorithm developed by Fimmic Oy.

Acknowledgments We thank Eliezer Masliah (then at the University of California, San Diego) for providing SynTg mice, and acknowledge technical assistance from Gina M. Moriarty in Jean Baum’s laboratory (Rutgers University) with purifying recombinant mouse α-synuclein from plasmid pT7-7 originally obtained from Peter Lansbury. Technical assistance from Chelsea Bautista and Katherine Skibiel is also acknowledged. This study was supported by National Institutes of Health (NIH) Grant AT006868 (to M.M.M. and J.B.S.). Analyses conducted by the Rutgers Robert Wood Johnson Medical School Biological Mass Spectrometry Facility were supported by NIH Grant P30NS046593. E.J. is supported by NIH Grants NS070898 and NS095003 and the State of New Jersey. M.M.M. is the William Dow Lovett Professor of Neurology and is supported by the Michael J. Fox Foundation for Parkinson’s Research, the American Parkinson Disease Association, the New Jersey Health Foundation/Nicholson Foundation, and NIH Grants NS073994, NS096032, and NS101134. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Footnotes Author contributions: R.Y., E.J., J.B.S., and M.M.M. designed research; R.Y., J.Z., H.-J.P., E.S.P., S.O., H.Z., and M.V. performed research; R.Y., J.Z., H.-J.P., E.S.P., S.O., H.Z., E.J., M.V., J.B.S., and M.M.M. analyzed data; and R.Y., E.J., J.B.S., and M.M.M. wrote the paper.

Conflict of interest statement: J.B.S. has a financial interest in Signum Biosciences, which is developing PP2A phosphatase enhancing agents. M.V. is employed by Signum Biosciences. J.B.S. and M.M.M. are inventors of a patent application relevant to this work.

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