Introduction
Largely because of significant increases in life span, Alzheimer's disease (AD)
3The abbreviations used are:
AD
Alzheimer's disease
Aβ
amyloid β-protein
APP
amyloid β-protein precursor
sAPP-α
soluble APP-α
EGCG
(−)-epigallocatechin-3-gallate
FA
ferulic acid
ADAM10
a disintegrin and metalloproteinase domain-containing protein 10
APP/PS1
the mutant human APP and presenilin 1
APP/PS1 mice
APP/PS1 transgenic mouse model of cerebral amyloidosis
RAWM
radial arm water maze
ANOVA
analysis of variance
RSC
retrosplenial cortex
EC
entorhinal cortex
H
hippocampus
ROI
regions of interest
CAA
cerebral amyloid angiopathy
sAPP-β
soluble APP-β
β-CTF/C99
β-C-terminal APP fragment
P-β-CTF/P-C99
phospho-β-CTF
BACE1
β-site APP-cleaving enzyme 1
pADAM10
precursor ADAM10
mADAM10
mature (active) ADAM10
TNF-α
tumor necrosis factor-α
IL-1β
interleukin-1β
SOD1
superoxide dismutase 1
GPx1
GSH peroxidase 1
QPCR
quantitative real-time PCR
GFAP
glial fibrillary acidic protein
Iba1
ionized calcium-binding adapter molecule 1
IR
immunoreactivity
LD
50lethal dose for 50% survival.
has become a worldwide public health concern. AD is a fatal neurodegenerative illness that likely begins with brain changes 20 years or more prior to clinical symptoms. AD is characterized by progressive decline in memory and other cognitive functions, eventually leading to dementia and death. Neuropathological hallmarks of AD include extracellular senile plaques and intracellular neurofibrillary tangles, accompanied by neuroinflammation, synaptic toxicity, and neuron loss (
1Alzheimer's disease: genes, proteins, and therapy.
). Amyloid β-protein (Aβ), a peptide derived from amyloid β-protein precursor (APP), has garnered major interest as a therapeutic target for AD. Two enzymes, the β- and γ-secretases, cleave APP into smaller amyloidogenic peptides: Aβ(1–40) and Aβ(1–42), which can form both oligomeric and multimeric aggregates (
2- De Strooper B.
- Saftig P.
- Craessaerts K.
- Vanderstichele H.
- Guhde G.
- Annaert W.
- Von Figura K.
- Van Leuven F.
Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein.
3Cellular mechanisms of β-amyloid production and secretion.
,
4- Vassar R.
- Bennett B.D.
- Babu-Khan S.
- Kahn S.
- Mendiaz E.A.
- Denis P.
- Teplow D.B.
- Ross S.
- Amarante P.
- Loeloff R.
- Luo Y.
- Fisher S.
- Fuller J.
- Edenson S.
- Lile J.
- et al.
β-Secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE.
5- Kimberly W.T.
- LaVoie M.J.
- Ostaszewski B.L.
- Ye W.
- Wolfe M.S.
- Selkoe D.J.
γ-Secretase is a membrane protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2.
). Once cleaved from APP, Aβ enters into systemic equilibrium between soluble and deposited forms (
6- DeMattos R.B.
- Bales K.R.
- Cummins D.J.
- Paul S.M.
- Holtzman D.M.
Brain to plasma amyloid-β efflux: a measure of brain amyloid burden in a mouse model of Alzheimer's disease.
). Conversely, nonamyloidogenic α-secretase cleavage of APP precludes Aβ formation and produces soluble APP-α (sAPP-α) (
3Cellular mechanisms of β-amyloid production and secretion.
,
7- Postina R.
- Schroeder A.
- Dewachter I.
- Bohl J.
- Schmitt U.
- Kojro E.
- Prinzen C.
- Endres K.
- Hiemke C.
- Blessing M.
- Flamez P.
- Dequenne A.
- Godaux E.
- van Leuven F.
- Fahrenholz F.
A disintegrin-metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer disease mouse model.
). Because the nonamyloidogenic pathway is the homeostatic default, this results in constitutively low Aβ levels.
Despite intense investigation, a synthetic drug that is both safe and effective has not yet been developed for AD. This inconvenient truth has led to the consideration of alternative strategies, including naturally occurring dietary compounds with therapeutic potential (so-called “nutraceuticals”). Another important matter is a recent mandate to develop combination therapy for AD. Because nutraceuticals are generally safe and well-tolerated, these agents are more amenable to combination than designer drugs. In this report, we explore combination therapy with two promising nutraceuticals that have complementary anti-amyloidogenic properties: (−)-epigallocatechin-3-gallate (EGCG, an α-secretase enhancer) (
8- Rezai-Zadeh K.
- Shytle D.
- Sun N.
- Mori T.
- Hou H.
- Jeanniton D.
- Ehrhart J.
- Townsend K.
- Zeng J.
- Morgan D.
- Hardy J.
- Town T.
- Tan J.
Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice.
,
9- Obregon D.F.
- Rezai-Zadeh K.
- Bai Y.
- Sun N.
- Hou H.
- Ehrhart J.
- Zeng J.
- Mori T.
- Arendash G.W.
- Shytle D.
- Town T.
- Tan J.
ADAM10 activation is required for green tea (−)-epigallocatechin-3-gallate–induced α-secretase cleavage of amyloid precursor protein.
) and ferulic acid (FA, a β-secretase modulator) (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
).
EGCG is a polyphenol catechin (the ester of epigallocatechin and gallic acid) (
11- Legeay S.
- Rodier M.
- Fillon L.
- Faure S.
- Clere N.
Epigallocatechin gallate: a review of its beneficial properties to prevent metabolic syndrome.
) that is found in high abundance in tea leaves, including green tea, and in carob flour (
i.e. Ceratonia siliqua) at lesser abundance. Trace amounts are found in fruits (
e.g. apple and cranberries) and nuts (
e.g. pecans and hazelnuts) (
12- Harnly J.M.
- Doherty R.F.
- Beecher G.R.
- Holden J.M.
- Haytowitz D.B.
- Bhagwat S.
- Gebhardt S.
Flavonoid content of U.S. fruits, vegetables, and nuts.
) (
http://www.ars.usda.gov/nutrientdata).
4Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site.
EGCG has garnered attention as a medicinal agent to improve cognition, reduce inflammation, and even to treat cancer. Beneficial effects are often attributed to anti-oxidant, metal-chelating, anti-inflammatory, anti-carcinogenic, and anti-apoptotic properties (
13- Singh N.A.
- Mandal A.K.
- Khan Z.A.
Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).
). Substantial quantities pass from the small to the large intestine, where EGCG undergoes degradation by gut flora and absorption. EGCG can be reabsorbed from the intestine through enterohepatic re-circulation (
14- Lee M.J.
- Maliakal P.
- Chen L.
- Meng X.
- Bondoc F.Y.
- Prabhu S.
- Lambert G.
- Mohr S.
- Yang C.S.
Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability.
,
15Degradation and metabolism of catechin, epigallocatechin-3-gallate (EGCG), and related compounds by the intestinal microbiota in the pig cecum model.
). It has been shown that EGCG crosses the blood–brain barrier after systemic administration (
16- Lin L.C.
- Wang M.N.
- Tseng T.Y.
- Sung J.S.
- Tsai T.H.
Pharmacokinetics of (−)-epigallocatechin-3-gallate in conscious and freely moving rats and its brain regional distribution.
), and we were the first to show that EGCG enhances APP cleavage to sAPP-α and reduces Aβ abundance both in neuron-like cells and in Tg2576 mouse brains (
8- Rezai-Zadeh K.
- Shytle D.
- Sun N.
- Mori T.
- Hou H.
- Jeanniton D.
- Ehrhart J.
- Townsend K.
- Zeng J.
- Morgan D.
- Hardy J.
- Town T.
- Tan J.
Green tea epigallocatechin-3-gallate (EGCG) modulates amyloid precursor protein cleavage and reduces cerebral amyloidosis in Alzheimer transgenic mice.
). A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) activation is mechanistically responsible for EGCG promotion of nonamyloidogenic α-secretase APP cleavage (
9- Obregon D.F.
- Rezai-Zadeh K.
- Bai Y.
- Sun N.
- Hou H.
- Ehrhart J.
- Zeng J.
- Mori T.
- Arendash G.W.
- Shytle D.
- Town T.
- Tan J.
ADAM10 activation is required for green tea (−)-epigallocatechin-3-gallate–induced α-secretase cleavage of amyloid precursor protein.
).
Like EGCG, FA is also a plant-derived compound. Seed plants and leaves (
e.g. rice, wheat, and oats), vegetables (
e.g. tomatoes and carrots), and fruits (
e.g. pineapples and oranges) are the main sources of dietary FA. The compound has both anti-inflammatory and anti-oxidant properties (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
,
17- Kanski J.
- Aksenova M.
- Stoyanova A.
- Butterfield D.A.
Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies.
,
18- Srinivasan M.
- Sudheer A.R.
- Menon V.P.
Ferulic acid: therapeutic potential through its antioxidant property.
), and we have previously shown that FA is a β-secretase modulator that inhibits amyloidogenic APP cleavage. We further reported that FA reverses cognitive/behavioral deficits and mitigates AD-like pathology after 6 months of oral treatment in a transgenic mouse model of cerebral amyloidosis, and it alters amyloidogenic β-secretase APP cleavage in mutant APP-overexpressing neuron-like cells (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
). Because FA has a relatively low molecular weight, the compound is freely cell-permeable and bioavailable. FA is absorbed in free form via stomach mucosa and is then transported into the hepatic portal vein where it is metabolized in the liver. Of note, oral FA can be recovered in rat plasma after only 5 min, when the ratio of free FA to total FA is markedly increased, but it rapidly gives rise to conjugated FA. Both free and conjugated FA are distributed via the systemic circulation into peripheral tissues (
18- Srinivasan M.
- Sudheer A.R.
- Menon V.P.
Ferulic acid: therapeutic potential through its antioxidant property.
,
19- Zhao Z.
- Egashira Y.
- Sanada H.
Ferulic acid is quickly absorbed from rat stomach as the free form and then conjugated mainly in liver.
20Chemistry, natural sources, dietary intake and pharmacokinetic properties of ferulic acid: a review.
). Whereas FA is negatively charged at physiological pH due to a hydroxyl moiety (
21- Sultana R.
- Ravagna A.
- Mohmmad-Abdul H.
- Calabrese V.
- Butterfield D.A.
Ferulic acid ethyl ester protects neurons against amyloid β-peptide(1–42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity.
), the compound has been shown to cross the blood–brain barrier in rodents after peripheral administration (
22- Qin J.
- Chen D.
- Lu W.
- Xu H.
- Yan C.
- Hu H.
- Chen B.
- Qiao M.
- Zhao X.
Preparation, characterization, and evaluation of liposomal ferulic acid in vitro and in vivo.
,
23- Wu K.
- Wang Z.Z.
- Liu D.
- Qi X.R.
Pharmacokinetics, brain distribution, release and blood–brain barrier transport of Shunaoxin pills.
).
Given that both compounds share anti-inflammatory and anti-oxidant properties and have complementary modes of action on APP cleavage, we tested whether combination therapy with EGCG and FA (each at 30 mg/kg) ameliorated learning and memory changes, cerebral amyloidosis, AD-like pathology, and amyloidogenic APP processing in the mutant human APP and presenilin 1 (APP/PS1) transgenic mouse model of cerebral amyloidosis (APP/PS1 mice). We orally administered EGCG/FA (alone or in combination) or vehicle to APP/PS1 mice once daily for 3 months (beginning at 12 months of age) and evaluated animals at 15 months old. Here, our pre-clinical results show that combination therapy with EGCG plus FA has significant advantages over single treatment with either compound.
Discussion
A key drug development challenge is delivering an agent with few side effects that not only treats symptoms but actually modifies disease. Here, we report that combination therapy with EGCG and FA completely reverses transgene-associated behavioral deficits in the APP/PS1 transgenic mouse model (see
Fig. 1). In concert with learning and memory improvement, pathological findings demonstrate that combined therapy posts additional benefits over single treatments to attenuate parenchymal and vascular β-amyloidosis (see
Figure 2,
Figure 3,
Figure 4). In this regard, clinicopathological studies have investigated the relationship between cerebral amyloid pathology and cognitive deficits in human AD (
31- Sabbagh M.N.
- Cooper K.
- DeLange J.
- Stoehr J.D.
- Thind K.
- Lahti T.
- Reisberg B.
- Sue L.
- Vedders L.
- Fleming S.R.
- Beach T.G.
Functional, global and cognitive decline correlates to accumulation of Alzheimer's pathology in MCI and AD.
,
32- Robinson J.L.
- Geser F.
- Corrada M.M.
- Berlau D.J.
- Arnold S.E.
- Lee V.M.
- Kawas C.H.
- Trojanowski J.Q.
Neocortical and hippocampal amyloid-β and tau measures associate with dementia in the oldest-old.
). Similar to our results, numerous reports have linked reversal of transgene-associated behavioral deficits to attenuated cerebral amyloid pathology in mouse models (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
,
26- Town T.
- Laouar Y.
- Pittenger C.
- Mori T.
- Szekely C.A.
- Tan J.
- Duman R.S.
- Flavell R.A.
Blocking TGF-β-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology.
,
27- Mori T.
- Rezai-Zadeh K.
- Koyama N.
- Arendash G.W.
- Yamaguchi H.
- Kakuda N.
- Horikoshi-Sakuraba Y.
- Tan J.
- Town T.
Tannic acid is a natural β-secretase inhibitor that prevents cognitive impairment and mitigates Alzheimer-like pathology in transgenic mice.
28- Mori T.
- Koyama N.
- Segawa T.
- Maeda M.
- Maruyama N.
- Kinoshita N.
- Hou H.
- Tan J.
- Town T.
Methylene blue modulates β-secretase, reverses cerebral amyloidosis, and improves cognition in transgenic mice.
,
33- Schenk D.
- Barbour R.
- Dunn W.
- Gordon G.
- Grajeda H.
- Guido T.
- Hu K.
- Huang J.
- Johnson-Wood K.
- Khan K.
- Kholodenko D.
- Lee M.
- Liao Z.
- Lieberburg I.
- Motter R.
- et al.
Immunization with amyloid-β attenuates Alzheimer-disease-like pathology in the PDAPP mouse.
,
34- Kotilinek L.A.
- Bacskai B.
- Westerman M.
- Kawarabayashi T.
- Younkin L.
- Hyman B.T.
- Younkin S.
- Ashe K.H.
Reversible memory loss in a mouse transgenic model of Alzheimer's disease.
,
35- Hartman R.E.
- Izumi Y.
- Bales K.R.
- Paul S.M.
- Wozniak D.F.
- Holtzman D.M.
Treatment with an amyloid-β antibody ameliorates plaque load, learning deficits, and hippocampal long-term potentiation in a mouse model of Alzheimer's disease.
,
36- Mouri A.
- Noda Y.
- Hara H.
- Mizoguchi H.
- Tabira T.
- Nabeshima T.
Oral vaccination with a viral vector containing Aβ cDNA attenuates age-related Aβ accumulation and memory deficits without causing inflammation in a mouse Alzheimer model.
), although others have shown that cognitive function and cerebral amyloidosis can be uncoupled (
37- Holcomb L.A.
- Gordon M.N.
- Jantzen P.
- Hsiao K.
- Duff K.
- Morgan D.
Behavioral changes in transgenic mice expressing both amyloid precursor protein and presenilin-1 mutations: lack of association with amyloid deposits.
,
38- Westerman M.A.
- Cooper-Blacketer D.
- Mariash A.
- Kotilinek L.
- Kawarabayashi T.
- Younkin L.H.
- Carlson G.A.
- Younkin S.G.
- Ashe K.H.
The relationship between Aβ and memory in the Tg2576 mouse model of Alzheimer's disease.
). In humans, the elderly with preserved cognitive function reportedly have cerebral amyloid pathology comparable with prodromal or frank AD at autopsy (
39- Price J.L.
- McKeel Jr., D.W.
- Buckles V.D.
- Roe C.M.
- Xiong C.
- Grundman M.
- Hansen L.A.
- Petersen R.C.
- Parisi J.E.
- Dickson D.W.
- Smith C.D.
- Davis D.G.
- Schmitt F.A.
- Markesbery W.R.
- Kaye J.
- et al.
Neuropathology of nondemented aging: presumptive evidence for preclinical Alzheimer disease.
,
40- Mufson E.J.
- Malek-Ahmadi M.
- Perez S.E.
- Chen K.
Braak staging, plaque pathology, and APOE status in elderly persons without cognitive impairment.
). One explanation provided for the discordancy between senile plaques and cognitive impairment in the human AD literature is that soluble, oligomeric forms of Aβ are the true neurotoxic species (
41- Walsh D.M.
- Klyubin I.
- Fadeeva J.V.
- Cullen W.K.
- Anwyl R.
- Wolfe M.S.
- Rowan M.J.
- Selkoe D.J.
Naturally secreted oligomers of amyloid β protein potently inhibit hippocampal long-term potentiation in vivo.
42- Cleary J.P.
- Walsh D.M.
- Hofmeister J.J.
- Shankar G.M.
- Kuskowski M.A.
- Selkoe D.J.
- Ashe K.H.
Natural oligomers of the amyloid-β protein specifically disrupt cognitive function.
,
43Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid β-peptide.
44- Shankar G.M.
- Li S.
- Mehta T.H.
- Garcia-Munoz A.
- Shepardson N.E.
- Smith I.
- Brett F.M.
- Farrell M.A.
- Rowan M.J.
- Lemere C.A.
- Regan C.M.
- Walsh D.M.
- Sabatini B.L.
- Selkoe D.J.
Amyloid-β protein dimers isolated directly from Alzheimer's brains impair synaptic plasticity and memory.
). In support of this hypothesis, others have demonstrated that behavioral deficit occurs in parallel with elevated Aβ oligomers in rodent models (
34- Kotilinek L.A.
- Bacskai B.
- Westerman M.
- Kawarabayashi T.
- Younkin L.
- Hyman B.T.
- Younkin S.
- Ashe K.H.
Reversible memory loss in a mouse transgenic model of Alzheimer's disease.
,
38- Westerman M.A.
- Cooper-Blacketer D.
- Mariash A.
- Kotilinek L.
- Kawarabayashi T.
- Younkin L.H.
- Carlson G.A.
- Younkin S.G.
- Ashe K.H.
The relationship between Aβ and memory in the Tg2576 mouse model of Alzheimer's disease.
). Interestingly, we show that combination treatment significantly decreases Aβ oligomers (see
Fig. 5), which inversely correlates with cognitive function (see
Fig. 1).
Sequential APP endoproteolysis by the secretases generates amyloidogenic C99 and sAPP-β followed by Aβ, whereas the nonamyloidogenic pathway generates sAPP-α and C83. Our data show significantly increased sAPP-α in brain homogenates from APP/PS1 mice treated with EGCG alone or in combination with FA, whereas sAPP-β, C99, and P-C99 production are reduced, providing evidence that APP cleavage is shifted toward the nonamyloidogenic pathway. Soluble APP-α has beneficial properties, including neurotrophism and neuroprotection (
45- Mattson M.P.
- Cheng B.
- Culwell A.R.
- Esch F.S.
- Lieberburg I.
- Rydel R.E.
Evidence for excitoprotective and intraneuronal calcium-regulating roles for secreted forms of the β-amyloid precursor protein.
,
46Cellular actions of β-amyloid precursor protein and its soluble and fibrillogenic derivatives.
), and even enhancing long-term potentiation (
47- Ishida A.
- Furukawa K.
- Keller J.N.
- Mattson M.P.
Secreted form of β-amyloid precursor protein shifts the frequency dependency for induction of LTD, and enhances LTP in hippocampal slices.
). Interestingly, treatment with EGCG alone not only increases nonamyloidogenic processing but also reduces amyloidogenic APP processing (see
Fig. 5). Combined treatment significantly enhances protein expression of α-secretase candidate ADAM10, whereas BACE1 protein abundance is significantly reduced, even by EGCG treatment alone. This latter result is interesting, because a previous study showed that EGCG can act as a β-secretase modulator in a cell-free system (
48- Jeon S.Y.
- Bae K.
- Seong Y.H.
- Song K.S.
Green tea catechins as a BACE1 (β-secretase) inhibitor.
). Nonetheless, combination therapy further significantly decreases BACE1 protein expression compared with EGCG alone (see
Fig. 6). This agrees with our prior report that sAPP-α can indirectly modulate β-secretase activity and Aβ generation through a negative feedback loop (
49- Obregon D.
- Hou H.
- Deng J.
- Giunta B.
- Tian J.
- Darlington D.
- Shahaduzzaman M.
- Zhu Y.
- Mori T.
- Mattson M.P.
- Tan J.
Soluble amyloid precursor protein-α modulates β-secretase activity and amyloid-β generation.
), and others have shown that sAPP-α can autoregulate APP cleavage (
50- Kaden D.
- Munter L.M.
- Joshi M.
- Treiber C.
- Weise C.
- Bethge T.
- Voigt P.
- Schaefer M.
- Beyermann M.
- Reif B.
- Multhaup G.
Homophilic interactions of the amyloid precursor protein (APP) ectodomain are regulated by the loop region and affect β-secretase cleavage of APP.
,
51- Young-Pearse T.L.
- Chen A.C.
- Chang R.
- Marquez C.
- Selkoe D.J.
Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin β1.
52- Gralle M.
- Botelho M.G.
- Wouters F.S.
Neuroprotective secreted amyloid precursor protein acts by disrupting amyloid precursor protein dimers.
). Hence, EGCG can inhibit Aβ generation both by directly enhancing α-secretase and by indirectly modulating β-secretase activity. Collectively, our data show that combination therapy further polarizes APP cleavage toward nonamyloidogenic α-secretase cleavage
versus either single treatment.
In the AD brain, reactive microglia and astrocytes co-exist in both temporal and spatial proximity to β-amyloid deposits, and glial activation leads to neuroinflammation that can cause bystander neuronal injury (
53- Akiyama H.
- Barger S.
- Barnum S.
- Bradt B.
- Bauer J.
- Cole G.M.
- Cooper N.R.
- Eikelenboom P.
- Emmerling M.
- Fiebich B.L.
- Finch C.E.
- Frautschy S.
- Griffin W.S.
- Hampel H.
- Hull M.
- et al.
Inflammation and Alzheimer's disease.
,
54- Heneka M.T.
- Carson M.J.
- El Khoury J.
- Landreth G.E.
- Brosseron F.
- Feinstein D.L.
- Jacobs A.H.
- Wyss-Coray T.
- Vitorica J.
- Ransohoff R.M.
- Herrup K.
- Frautschy S.A.
- Finsen B.
- Brown G.C.
- Verkhratsky A.
- et al.
Neuroinflammation in Alzheimer's disease.
55- Andreasson K.I.
- Bachstetter A.D.
- Colonna M.
- Ginhoux F.
- Holmes C.
- Lamb B.
- Landreth G.
- Lee D.C.
- Low D.
- Lynch M.A.
- Monsonego A.
- O'Banion M.K.
- Pekny M.
- Puschmann T.
- Russek-Blum N.
- et al.
Targeting innate immunity for neurodegenerative disorders of the central nervous system.
). Both EGCG and FA have additional beneficial properties, including reducing inflammation (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
,
13- Singh N.A.
- Mandal A.K.
- Khan Z.A.
Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).
,
18- Srinivasan M.
- Sudheer A.R.
- Menon V.P.
Ferulic acid: therapeutic potential through its antioxidant property.
). Strikingly, in fact, combination therapy confers the greatest additional benefit over single treatments on neuroinflammation. When considering neuroinflammatory biomarkers, results show that combined treatment dampens plaque-associated microgliosis and astrocytosis and completely blocks brain mRNA expression of TNF-α and IL-1β to baseline levels (see
Figure 7,
Figure 8,
Figure 9). Given the recent intense focus on inflammatory mechanisms in AD (
54- Heneka M.T.
- Carson M.J.
- El Khoury J.
- Landreth G.E.
- Brosseron F.
- Feinstein D.L.
- Jacobs A.H.
- Wyss-Coray T.
- Vitorica J.
- Ransohoff R.M.
- Herrup K.
- Frautschy S.A.
- Finsen B.
- Brown G.C.
- Verkhratsky A.
- et al.
Neuroinflammation in Alzheimer's disease.
,
55- Andreasson K.I.
- Bachstetter A.D.
- Colonna M.
- Ginhoux F.
- Holmes C.
- Lamb B.
- Landreth G.
- Lee D.C.
- Low D.
- Lynch M.A.
- Monsonego A.
- O'Banion M.K.
- Pekny M.
- Puschmann T.
- Russek-Blum N.
- et al.
Targeting innate immunity for neurodegenerative disorders of the central nervous system.
), it remains possible that mitigated neuroinflammation after combination therapy plays a dominant role to reduce AD-like pathology and improve cognition in APP/PS1 mice.
Reactive oxygen species are produced during host defense against pathogens. However, when activated inappropriately or overproduced, oxidative substances can damage DNA, proteins, and lipids, leading to cellular injury and death (
56- Valko M.
- Leibfritz D.
- Moncol J.
- Cronin M.T.
- Mazur M.
- Telser J.
Free radicals and antioxidants in normal physiological functions and human disease.
). Oxidative damage has been linked to AD pathogenesis (
57- Yan S.D.
- Chen X.
- Fu J.
- Chen M.
- Zhu H.
- Roher A.
- Slattery T.
- Zhao L.
- Nagashima M.
- Morser J.
- Migheli A.
- Nawroth P.
- Stern D.
- Schmidt A.M.
RAGE and amyloid-β peptide neurotoxicity in Alzheimer's disease.
,
58Oxidative stress hypothesis in Alzheimer's disease.
), and both EGCG and FA have anti-oxidant and free radical scavenging properties (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
,
11- Legeay S.
- Rodier M.
- Fillon L.
- Faure S.
- Clere N.
Epigallocatechin gallate: a review of its beneficial properties to prevent metabolic syndrome.
,
13- Singh N.A.
- Mandal A.K.
- Khan Z.A.
Potential neuroprotective properties of epigallocatechin-3-gallate (EGCG).
,
17- Kanski J.
- Aksenova M.
- Stoyanova A.
- Butterfield D.A.
Ferulic acid antioxidant protection against hydroxyl and peroxyl radical oxidation in synaptosomal and neuronal cell culture systems in vitro: structure-activity studies.
). Therefore, we explored whether the compounds modulated oxidative stress in APP/PS1 mice. Results show that combined treatment with EGCG and FA completely reduces cerebral mRNA expression of SOD1 and GPx1 to baseline levels in APP/PS1 mice, whereas EGCG or FA single treatment has only modest effects. We actually observe the greatest reductions in SOD1 or GPx1 proteins with combination therapy (see
Fig. 9).
Synaptic loss is a cardinal feature of the AD brain, and the role of Aβ in synapse loss and dysfunction is well-documented. Importantly, synaptic plasticity is considered to form the cellular basis for learning and memory (
59Alzheimer's disease is a synaptic failure.
,
60Alzheimer's disease: synaptic dysfunction and Aβ.
61- Klyubin I.
- Cullen W.K.
- Hu N.W.
- Rowan M.J.
Alzheimer's disease Aβ assemblies mediating rapid disruption of synaptic plasticity and memory.
). Dysregulated plasticity leads to synaptic loss, and decreased synapse distribution is a durable predictor of disease progression and behavioral decline (
59Alzheimer's disease is a synaptic failure.
). Synaptophysin is a pre-synaptic marker that is most commonly used as a surrogate for synaptic density (
62- Valtorta F.
- Pennuto M.
- Bonanomi D.
- Benfenati F.
Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis?.
). In this regard, we quantified synaptophysin IR in hippocampal CA1 and EC regions of APP/PS1 mice and found significant increases in each APP/PS1 treatment group, with the greatest benefit after combined treatment (see
Fig. 10).
Even more than with single treatment approaches, safety is a key issue with combination therapy, and adverse effects must be taken into account. Importantly, single or dual treatment with EGCG and FA for 3 months is well-tolerated, and we did not observe any adverse events in any of the mice included in our study. Based on the chemical database for EGCG by Registry of Toxic Effects of Chemical Substances, the acute oral lethal dose for 50% survival (LD
50) is as high as 2,710 mg/kg in mice. Likewise, FA has a high acute oral LD
50 of 2,370 mg/kg in mice (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
), with a human equivalent dose of 2.43 mg/kg for EGCG or FA, which equates to a 146 mg dose for a 60 kg person (
63- Reagan-Shaw S.
- Nihal M.
- Ahmad N.
Dose translation from animal to human studies revisited.
). Moreover, the tolerable daily intake in a human can be extrapolated from rodent LD
50 threshold data (
64Reference dose (RfD): description and use in health risk assessments.
). Assuming the body surface area calculation factor for interspecies variation (
63- Reagan-Shaw S.
- Nihal M.
- Ahmad N.
Dose translation from animal to human studies revisited.
), the tolerable human daily intake for EGCG is equivalent to 13.18 g, whereas FA is 11.53 g (both assume 60 kg human weight). Thus, the doses (30 mg/kg/day for a mouse: 146 mg for a human) used in this study are quite low, and such advantageous pharmacotoxicity profiles reinforce the notion that combined EGCG/FA administration is safe. Nonetheless, it remains possible that increased ADAM10 and/or decreased BACE1 activity might also affect processing of other substrates, leading to unwanted side effects. Hence, further translational research is warranted to investigate whether long-term EGCG and FA therapy is safe in humans.
With respect to the pharmacokinetics of EGCG in humans, the compound is absorbed at <5% abundance and reaches peak plasma concentration time (
tmax) at 2 h after per os administration (
65- Williamson G.
- Dionisi F.
- Renouf M.
Flavanols from green tea and phenolic acids from coffee: critical quantitative evaluation of the pharmacokinetic data in humans after consumption of single doses of beverages.
,
66- Manach C.
- Williamson G.
- Morand C.
- Scalbert A.
- Rémésy C.
Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies.
). Green tea catechins are primarily absorbed in the intestine (
i.e. jejunum and ileum) by paracellular diffusion through epithelial cells (
67- Moore R.J.
- Jackson K.G.
- Minihane A.M.
Green tea (Camellia sinensis) catechins and vascular function.
). Once absorbed, free EGCG is mainly detected in plasma (>75%) (
14- Lee M.J.
- Maliakal P.
- Chen L.
- Meng X.
- Bondoc F.Y.
- Prabhu S.
- Lambert G.
- Mohr S.
- Yang C.S.
Pharmacokinetics of tea catechins after ingestion of green tea and (−)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability.
,
68- Ullmann U.
- Haller J.
- Decourt J.P.
- Girault N.
- Girault J.
- Richard-Caudron A.S.
- Pineau B.
- Weber P.
A single ascending dose study of epigallocatechin gallate in healthy volunteers.
) and has a half-life (
t½) of ∼3 h (
65- Williamson G.
- Dionisi F.
- Renouf M.
Flavanols from green tea and phenolic acids from coffee: critical quantitative evaluation of the pharmacokinetic data in humans after consumption of single doses of beverages.
,
66- Manach C.
- Williamson G.
- Morand C.
- Scalbert A.
- Rémésy C.
Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies.
). Similar to EGCG, FA is absorbed from the stomach, jejunum, and ileum. Free FA can be found in human plasma just 10 min after oral administration of sodium ferulate (
69- Yang C.
- Tian Y.
- Zhang Z.
- Xu F.
- Chen Y.
High-performance liquid chromatography-electrospray ionization mass spectrometry determination of sodium ferulate in human plasma.
). Plasma FA concentrations reach
tmax at 24 min following oral administration, with a
t½ of 42 min (
69- Yang C.
- Tian Y.
- Zhang Z.
- Xu F.
- Chen Y.
High-performance liquid chromatography-electrospray ionization mass spectrometry determination of sodium ferulate in human plasma.
). Free FA and its glucuronic conjugate are detected in the plasma, and both free FA and its glycine conjugate are found in urine after ingestion of FA containing wheat bran (
70- Kern S.M.
- Bennett R.N.
- Mellon F.A.
- Kroon P.A.
- Garcia-Conesa M.T.
Absorption of hydroxycinnamates in humans after high-bran cereal consumption.
). The urinary excretion of FA in humans plateaus between 7 and 9 h after ingestion (
71- Bourne L.C.
- Rice-Evans C.
Bioavailability of ferulic acid.
).
An important question is whether EGCG and FA can cross the blood–brain barrier. Using LC coupled with tandem MS electrospray ionization, EGCG was detected in the rodent brain after systemic administration (
16- Lin L.C.
- Wang M.N.
- Tseng T.Y.
- Sung J.S.
- Tsai T.H.
Pharmacokinetics of (−)-epigallocatechin-3-gallate in conscious and freely moving rats and its brain regional distribution.
). With respect to FA, standard HPLC approaches have detected the compound in the brain after peripheral administration (
22- Qin J.
- Chen D.
- Lu W.
- Xu H.
- Yan C.
- Hu H.
- Chen B.
- Qiao M.
- Zhao X.
Preparation, characterization, and evaluation of liposomal ferulic acid in vitro and in vivo.
,
23- Wu K.
- Wang Z.Z.
- Liu D.
- Qi X.R.
Pharmacokinetics, brain distribution, release and blood–brain barrier transport of Shunaoxin pills.
). This result has been validated using a more advanced technique: ultra-performance LC coupled to tandem MS (
72- Gasperotti M.
- Passamonti S.
- Tramer F.
- Masuero D.
- Guella G.
- Mattivi F.
- Vrhovsek U.
Fate of microbial metabolites of dietary polyphenols in rats: Is the brain their target destination?.
).
The vast majority (> 95%) of AD cases are “sporadic” and are characterized by onset at 65 years or later, whereas less than 5% of cases are early-onset autosomal dominant AD. To date, a series of mutations in three different genes (
i.e. APP,
PS1, and
PS2) cause autosomal dominant AD (
73The genetics of Alzheimer's disease.
). These mutations have been used with varying success to study basic and translational aspects of AD. One of the most widely used tools is transgenic mice expressing one or more of these mutant human transgenes. In this study, we utilized APP/PS1 mice harboring human
APP and
PS1 mutations. It is important to note that, although APP/PS1 mice demonstrate some of the AD-relevant phenotypes, including cerebral amyloidosis, neuroinflammation, and cognitive disturbance, they do not recapitulate all AD features and are therefore not a complete model of human AD.
In conclusion, we report that administering 3-month combination therapy with EGCG and FA to the aged APP/PS1 transgenic mouse model of cerebral amyloidosis confers additional benefits over single treatments on improving behavioral deficits, ameliorating cerebral amyloidosis, and reducing Aβ generation. Furthermore, combined treatment potently stabilizes neuroinflammation, alleviates oxidative stress, and mitigates synaptotoxicity. We propose long-term combination therapy with EGCG and FA as an effective disease-modifying therapy for AD.
Experimental procedures
Ethics statement
All experiments were performed in accordance with the guidelines of the National Institutes of Health, and all animal studies were approved by the Saitama Medical University Institutional Animal Care and Use Committee. Animals were humanely cared for during all experiments, and all efforts were made to minimize suffering.
Mice
Male B6.Cg-Tg(APP
Swe, PSEN1dE9)85Dbo/Mmjax mice (bearing “Swedish”
APPK595N/M596L and
PS1 exon 9-deleted mutant human transgenes) on the congenic C57BL/6J background (designated APP/PS1 mice) (
74- Jankowsky J.L.
- Slunt H.H.
- Gonzales V.
- Jenkins N.A.
- Copeland N.G.
- Borchelt D.R.
APP processing and amyloid deposition in mice haplo-insufficient for presenilin 1.
) and female C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME). For colony maintenance, male APP/PS1 mice on the congenic C57BL/6J background were bred with female C57BL/6J mice to yield APP/PS1 and WT offspring, so all experimental APP/PS1 mice and WT littermates in this study are on the same C57BL/6J genetic background.
EGCG and FA were obtained from Sigma. Thirty mg of each reagent was resuspended in 25 μl of 100% DMSO and then diluted with distilled water to a final concentration of 0.2% DMSO. All reagents were freshly prepared daily prior to each treatment. We randomly assigned APP/PS1 mice to four treatment groups (n = 8 per condition; four males and four females): EGCG (APP/PS1-EGCG); FA (APP/PS1-FA); EGCG + FA (APP/PS1-EGCG/FA); or vehicle (distilled water containing DMSO at a final concentration of 0.2%; APP/PS1-V). Additionally, WT littermates received the same four treatments (n = 8 per group; four males and four females) as follows: WT-EGCG; WT-FA; WT-EGCG/FA mice; or vehicle (WT-V). To determine whether treatment prevented versus reversed kinetics of cerebral amyloid accumulation, we included eight untreated APP/PS1 mice at 12 months of age (APP/PS1–12M mice, four males and four females) for analysis of β-amyloid pathology. After baseline behavioral assessment just before dosing (at 12 months of age), animals were gavaged with EGCG, FA, EGCG plus FA (all at 30 mg/kg), or vehicle once daily for 3 months. Mice were housed in a specific pathogen-free barrier facility under a 12/12-h light/dark cycle with ad libitum access to food and water.
Behavioral analyses
To assess novel object recognition and retention, animals were habituated in a cage for 4 h, and two objects of different shapes were concurrently provided for 10 min. The number of times that the animal explored the familiar object (defined as number of instances where an animal directed its nose 2 cm or less from the object) was counted for the initial 5 min of exposure (training phase). To test memory retention on the following day, one of the familiar objects was replaced with a different shaped novel object, and explorations were recorded for 5 min (retention test). The recognition index, taken as a measurement of episodic memory, is reported as frequency (%) of explorations of the novel
versus familiar objects (
75- De Rosa R.
- Garcia A.A.
- Braschi C.
- Capsoni S.
- Maffei L.
- Berardi N.
- Cattaneo A.
Intranasal administration of nerve growth factor (NGF) rescues recognition memory deficits in AD11 anti-NGF transgenic mice.
).
To measure exploratory activity and spatial working memory, animals were individually placed in one arm of a radially symmetric Y-maze made of opaque gray acrylic (arms, 40 cm long and 4 cm wide; walls, 30 cm tall), and the sequence of arm entries and total number of entries were counted over a period of 8 min, beginning when the animal first entered the central area. Percent alternation was defined as entries into sequentially different arms on consecutive occasions using the following formula: % alternation = number of alternations/(number of total arm entries − 2) × 100% (
24- Arendash G.W.
- King D.L.
- Gordon M.N.
- Morgan D.
- Hatcher J.M.
- Hope C.E.
- Diamond D.M.
Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes.
).
To assess spatial reference learning and memory, the RAWM test was conducted over 2 days and consisted of triangular wedges in a circular pool (80 cm diameter) configured to form swim lanes that enclosed a central open space. Mice naïve to the task were placed in the pool and allowed to search for the platform for 60 s. Animals were dropped into a random start arm and allowed to swim until they located and climbed onto the platform (goal) over a period of 60 s. Latency to locate the platform and errors were recorded. Each mouse was given a total of 15 trials. On day 1, the goal alternated between visible and hidden, although on day 2, the goal was always hidden. All data were organized as individual blocks of three trials each. The goal arms remained in the same location for both days, whereas the start arm was randomly altered. All trials were done at the same time of day (±1 h), during the animals’ light phase. To avoid interference with behavioral testing, each treatment was carried out 1 h after concluding behavioral testing (
76- Alamed J.
- Wilcock D.M.
- Diamond D.M.
- Gordon M.N.
- Morgan D.
Two-day radial-arm water maze learning and memory task; robust resolution of amyloid-related memory deficits in transgenic mice.
).
Brain tissue preparation
At 15 months of age, animals were anesthetized with sodium pentobarbital (50 mg/kg) and euthanized by transcardial perfusion with ice-cold physiological saline containing heparin (10 units/ml). Brains were isolated and quartered (sagittally at the level of the longitudinal fissure of the cerebrum and then coronally at the level of the anterior commissure). Left anterior hemispheres were weighed and snap-frozen at −80 °C for Western blotting. Right anterior hemispheres were weighed and immersed in RNA stabilization solution (RNAlater®, Applied Biosystems, Foster City, CA) and then snap-frozen at −80 °C for QPCR analysis. Left posterior hemispheres were immersed in 4% paraformaldehyde at 4 °C overnight and routinely processed in paraffin. Right posterior hemispheres were weighed and snap-frozen at −80 °C for ELISA.
Immunohistochemistry
Five coronal paraffin sections (per set) were cut with a 100-μm interval and 5-μm thickness spanning bregma −2.92 to −3.64 mm (
77- Franklin K.B.J.
- Paxinos G.
The Mouse Brain in Stereotaxic Coordinates.
). Three sets of five sections were prepared for analyses of β-amyloid plaques, astrocytosis, and microgliosis. An additional set of five sections was used for analysis of synaptophysin IR. Primary antibodies were as follows: biotinylated anti-Aβ(17–24) monoclonal (1:200 dilution, 4G8; Covance Research Products, Emeryville, CA); anti-GFAP polyclonal (1:500 dilution, Dako, Carpinteria, CA); C-terminal anti-Iba1 polyclonal (1:500 dilution, FUJIFILM Wako Pure Chemical, Osaka, Japan); and C-terminal anti-synaptophysin monoclonal (1:20 dilution, DAK-SYNAP; Dako) antibodies. Immunohistochemistry was performed using a Vectastain ABC
Elite kit (Vector Laboratories, Burlingame, CA) coupled with the diaminobenzidine reaction, except that the biotinylated secondary antibody step was omitted for the biotinylated Aβ(17–24) mAb.
Image analysis
Images were acquired and quantified using SimplePCI software (Hamamatsu Photonics, Shizuoka, Japan). Images of five 5-μm sections through each anatomic ROI (
i.e. RSC, EC, and H) were captured based on anatomical criteria (
77- Franklin K.B.J.
- Paxinos G.
The Mouse Brain in Stereotaxic Coordinates.
), and we set a threshold optical density that discriminated staining from background. Selection bias was controlled for by analyzing each ROI in its entirety. For Aβ, GFAP, and Iba1 burden analyses, data were reported as the percentage of positive pixels captured divided by the full area captured. For conventional Aβ burden analysis, the Aβ(17–24) mAb was used, and its epitope lies within amino acids 18–22 (VFFAE).
For β-amyloid plaque morphometric analysis, diameters (maximum lengths) of β-amyloid plaques were measured, and three mutually exclusive plaque size categories (<25, 25–50, or >50 μm) were blindly tabulated. For quantitative analysis of CAA, numbers of Aβ antibody-positive cerebral vessels were blindly counted in each ROI. To evaluate synaptophysin IR, images of five 5-μm sections through hippocampal CA1 and EC were blindly captured based on anatomical criteria (
77- Franklin K.B.J.
- Paxinos G.
The Mouse Brain in Stereotaxic Coordinates.
) and converted to grayscale. The average optical density of positive signals from each image was quantified as a relative number from zero (white) to 255 (black) and expressed as mean intensity of synaptophysin IR.
ELISA
Brain Aβ(1–40) and Aβ(1–42) species were detected by a three-step extraction protocol with modifications (
78- Kawarabayashi T.
- Younkin L.H.
- Saido T.C.
- Shoji M.
- Ashe K.H.
- Younkin S.G.
Age-dependent changes in brain, CSF, and plasma amyloid (β) protein in the Tg2576 transgenic mouse model of Alzheimer's disease.
). Brains were homogenized using TissueLyser LT (Qiagen, Valencia, CA) in Tris-buffered saline (TBS: 25 m
m Tris-HCl, pH 7.4, 150 m
m NaCl) containing protease inhibitor mixture (Sigma) and centrifuged at 18,800 ×
g for 60 min at 4 °C, and supernatants were collected (representing the TBS-soluble fraction). Resulting pellets were treated with TNE buffer (10 m
m Tris-HCl, 1% Nonidet P-40, 1 m
m EDTA, and 150 m
m NaCl) with protease inhibitors and homogenized using TissueLyser LT. Homogenates were then centrifuged at 18,800 ×
g for 30 min at 4 °C, and supernatants were collected (representing the detergent-soluble fraction). Remaining pellets were treated with 5
m guanidine HCl and dissolved by occasional mixing on ice for 30 min and centrifuged at 18,800 ×
g for 30 min at 4 °C. Supernatants were then collected; this is taken as the guanidine HCl–soluble fraction. Aβ(1–40) and Aβ(1–42) species were separately quantified in each sample in duplicate by sandwich ELISA (IBL, Gunma, Japan) (
79- Horikoshi Y.
- Sakaguchi G.
- Becker A.G.
- Gray A.J.
- Duff K.
- Aisen P.S.
- Yamaguchi H.
- Maeda M.
- Kinoshita N.
- Matsuoka Y.
Development of Aβ terminal end-specific antibodies and sensitive ELISA for Aβ variant.
). Aβ oligomers were quantified in the TNE-soluble fraction in duplicate by human amyloid β oligomers (82E1-specific) assay kit (IBL) (
80- Xia W.
- Yang T.
- Shankar G.
- Smith I.M.
- Shen Y.
- Walsh D.M.
- Selkoe D.J.
A specific enzyme-linked immunosorbent assay for measuring β-amyloid protein oligomers in human plasma and brain tissue of patients with Alzheimer disease.
). All samples fell within the linear range of the standard curve.
Western blotting
Brain homogenates were lysed in TBS containing protease inhibitor mixture (Sigma) followed by TNE buffer, and aliquots corresponding to 10 μg of total protein (equally loaded per lane) were electrophoretically separated for each mouse brain using Tris glycine gels. Electrophoresed proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked for 1 h at ambient temperature. Membranes were then hybridized for 1 h at ambient temperature with primary antibodies as follows: N-terminal anti-APP polyclonal (1:2,000 dilution, IBL); C-terminal anti-presenilin 1 (PS1) monoclonal (1:500 dilution, PS1-loop, Merck Millipore, Darmstadt, Germany); C-terminal anti-sAPP-α monoclonal (1:300 dilution, 2B3; IBL); C-terminal anti-sAPPβ-sw monoclonal (1:100 dilution, 6A1; IBL), N-terminal anti-Aβ(1–16) monoclonal (1:500 dilution, 82E1; IBL); C-terminal anti-BACE1 polyclonal (1:400 dilution, IBL); C-terminal anti-ADAM10 polyclonal (1:1,500 dilution, Cell Signaling Technology, Danvers, MA); anti-Cu/Zn SOD polyclonal (1:3,000 dilution, Enzo Life Sciences, Farmingdale, NY); anti-GPx1 polyclonal (1:4,000 dilution, Boster Biological Technology, Pleasanton, CA); and anti-β-actin monoclonal (1:5,000 dilution, 13E5; Cell Signaling Technology; as a loading control). Membranes were then rinsed and incubated for 1 h at ambient temperature with appropriate horseradish peroxidase-conjugated secondary antibodies (1:50,000 dilution for both primary monoclonal and polyclonal antibodies). After additional rinsing, membranes were incubated for 5 min at ambient temperature with enhanced chemiluminescence substrate (SuperSignal West Dura Extended Duration Substrate, ThermoFisher Scientific, Waltham, MA), exposed to film, and developed. Western blottings were done for each brain (n = 8 mice per group), and quantitative data were averaged.
QPCR
We quantified TNF-α, IL-1β, SOD1, GPx1, and β-actin mRNA levels by QPCR analyses (
10- Mori T.
- Koyama N.
- Guillot-Sestier M.V.
- Tan J.
- Town T.
Ferulic acid is a nutraceutical β-secretase modulator that improves behavioral impairment and Alzheimer-like pathology in transgenic mice.
,
26- Town T.
- Laouar Y.
- Pittenger C.
- Mori T.
- Szekely C.A.
- Tan J.
- Duman R.S.
- Flavell R.A.
Blocking TGF-β-Smad2/3 innate immune signaling mitigates Alzheimer-like pathology.
,
27- Mori T.
- Rezai-Zadeh K.
- Koyama N.
- Arendash G.W.
- Yamaguchi H.
- Kakuda N.
- Horikoshi-Sakuraba Y.
- Tan J.
- Town T.
Tannic acid is a natural β-secretase inhibitor that prevents cognitive impairment and mitigates Alzheimer-like pathology in transgenic mice.
). Total RNA was extracted using the RNeasy mini kit (Qiagen), and first-strand cDNA synthesis was carried out using the QuantiTect reverse transcription kit (Qiagen) in accordance with the manufacturer's instructions. We diluted cDNA 1:1 in H
2O and carried out QPCR for all genes of interest using cDNA-specific TaqMan primer/probe sets (TaqMan Gene Expression Assays, Applied Biosystems) on an ABI 7500 Fast real-time PCR instrument (Applied Biosystems). Each 20-μl reaction mixture contained 2 μl of cDNA with 1 μl of TaqMan Gene Expression Assay reagent, 10 μl of TaqMan Fast Universal PCR Master Mix (Applied Biosystems), and 7 μl of H
2O. Thermocycler conditions consisted of 95 °C for 15 s, followed by 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Mouse TaqMan probe/primer sets were as follows: TNF-α (number Mm00443258_m1); IL-1β (number Mm00434228_m1); SOD1 (number Mm01700393_g1); GPx1 (number Mm00656767_g1); and β-actin (number Mm00607939_s1; used as an internal reference control) (Applied Biosystems). Samples that were not subjected to reverse transcription were run in parallel as negative controls to rule out genomic DNA contamination, and a “no template control” was also included for each primer set. The cycle threshold number (
CT) method (
81- Monney L.
- Sabatos C.A.
- Gaglia J.L.
- Ryu A.
- Waldner H.
- Chernova T.
- Manning S.
- Greenfield E.A.
- Coyle A.J.
- Sobel R.A.
- Freeman G.J.
- Kuchroo V.K.
Th1-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease.
) was used to determine relative amounts of initial target cDNA in each sample. Results were expressed relative to vehicle control WT mice.
Statistical analysis
Data are presented as means with associated standard deviations. A hierarchical analysis strategy was used in which the first step was an overall ANOVA (repeated measures was used for behavioral data) to assess significance of main effects and interactive terms. If significant, post hoc testing was done with Tukey's HSD or Dunnett's T3 methods, where appropriateness was determined based on Levene's test for equality of the variance. In instances of multiple mean comparisons, one-way ANOVA was used, followed by post hoc comparison of the means using Bonferroni's or Dunnett's T3 methods (depending on Levene's test for equality of the variance). The α levels were set at 0.05 for all analyses. All analyses were performed using the Statistical Package for the Social Sciences, release 23.0 (IBM SPSS, Armonk, NY).
Article info
Publication history
Published online: December 18, 2018
Accepted:
December 13,
2018
Received:
June 2,
2018
Edited by Paul E. Fraser
Footnotes
This work was supported, in whole or in part, by National Institutes of Health Grants 2R01NS076794-06A1, 1RF1AG053982-01A1, 5P01AG052350-03 and 5R21AG053884-02 (to T. T.), the Japan Society for the Promotion of Science KAKENHI JP26430058 (to T. M.), an Alzheimer's Association Sex and Gender in Alzheimer's disease grant (SAGA; to T. T.), Cure Alzheimer's Fund (to T. T.), Coins for Alzheimer's Research Trust (to T. T.), and generous start-up funds from the Zilkha Neurogenetic Institute of the Keck School of Medicine at the University of Southern California (to T. T.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
This article contains Tables S1–S15.
Copyright
© 2019 Mori et al.