Activation of peroxisome proliferator-activated receptor α stimulates ADAM10-mediated proteolysis of APP Grant T. Corbetta,b, Frank J. Gonzalezc, and Kalipada Pahanb,d,1 aGraduate Program in Neuroscience, Department of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612; bDepartment of Neurological Sciences, Rush University Medical Center, Chicago, IL 60612; cLaboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and dDivision of Research and Development, Jesse Brown Veterans Affairs Medical Center, Chicago, IL 60612 Edited by Thomas C. Südhof, Stanford University School of Medicine, Stanford, CA, and approved May 21, 2015 (received for review March 10, 2015) Amyloid precursor protein (APP) derivative β-amyloid (Aβ) plays an and catabolism. Although the hippocampus does not metabolize important role in the pathogenesis of Alzheimer’s disease (AD). fat, recently we have demonstrated that PPARα is constitutively Sequential proteolysis of APP by β-secretase and γ-secretase gen- expressed in the hippocampus and hippocampal neurons (11). erates Aβ. Conversely, the α-secretase “a disintegrin and metal- Here, we describe that activation of PPARα induces the expression ” β of ADAM10 and subsequent α-secretase proteolysis of APP. Fur- loproteinase 10 (ADAM10) cleaves APP within the eventual A −/− β thermore, 5XFAD mice null for PPARα (5X/α ) exhibited exac- sequence and precludes A generation. Therefore, up-regulation β of ADAM10 represents a plausible therapeutic strategy to combat erbated brain A load relative to traditional 5XFAD mice. These β results highlight the importance of PPARα in reducing endoge- overproduction of neurotoxic A . Peroxisome proliferator-acti- β vated receptor α (PPARα) is a transcription factor that regulates nous A production by shifting APP processing toward the α-secretase pathway. genes involved in fatty acid metabolism. Here, we determined that Adam10 the promoter harbors PPAR response elements; that knock- Results down of PPARα, but not PPARβ or PPARγ, decreases the expression PPARα Modulates ADAM10 Expression. To determine the roles of of Adam10; and that lentiviral overexpression of PPARα restored Ppara−/− PPAR family members in the expression of APP-relevant sec- ADAM10 expression in neurons. Gemfibrozil, an agonist retases, expression of the α-secretases ADAM9, ADAM10, and of PPARα, induced the recruitment of PPARα:retinoid x receptor α, β γ γ α α Adam10 ADAM17; the -secretase BACE1; and the -secretase catalytic but not PPAR coactivator 1 (PGC1 ), to the promoter in component presenilin-1 were measured in the hippocampus and − − wild-type mouse hippocampal neurons and shifted APP processing frontal cortex of transgenic mice null for PPARα (Ppara / ) and toward the α-secretase, as determined by augmented soluble APPα β −/− −/− PPAR (Pparb ) and in wild-type (WT) neurons transduced and decreased Aβ production. Accordingly, Ppara mice displayed with PPARγ shRNA (PpargKD) (Fig. S1A), as PPARγ-null β β elevated SDS-stable, endogenous A and A 1–42 relative to wild-type mutations are embryonically lethal (12). Expression of Adam10 −/− littermates, whereas 5XFAD mice null for PPARα (5X/α ) exhibited (Fig. 1A), but not Adam9 (Fig. S1B), Adam17 (Fig. 1B), Bace1 NEUROSCIENCE greater cerebral Aβ load relative to 5XFAD littermates. These results (Fig. 1C), and Psen1 (Fig. 1D), mRNA was significantly reduced α identify PPAR as an important factor regulating neuronal ADAM10 in the hippocampus [F(2,6) = 18.480; P = 0.003] and frontal cortex −/− −/− expression and, thus, α-secretase proteolysis of APP. [F(2,6) = 20.302; P = 0.002] of 4-mo-old Ppara , but not Pparb , animals relative to WT controls or in WT neurons silenced for PPARalpha | ADAM10 | APP | Alzheimer’s disease PPARγ relative to empty vector-transduced neurons. Next, we de- termined the subcellular expression of ADAM10 and ADAM17. ’ After removal of the prodomain, enzymatically mature ADAMs are lzheimer s disease (AD) is the most prevalent neurodegen- transported to the cell membrane. Accordingly, mature ADAM10 Aerative disease. Although the precise physiologic changes that (mA10) and ADAM17 (mA17) were enriched in membrane frac- trigger development of AD remain unknown, abnormal metabo- tions prepared from hippocampi extracted from 4-mo-old WT mice lism of the type 1 transmembrane amyloid precursor protein (APP) (Fig. 1E). Nondenaturing solubilization of the membrane pellet β β into amyloid- (A ) plays a causative role in AD (1). Sequential (or the hippocampus as a whole) in 1% CHAPS buffer greatly proteolytic processing of APP by the aspartic proteases β-secretase γ 1(BACE1)and -secretase (reviewed in ref. 2) at ectodomain and Significance intramembrane sites, respectively, generates pathogenic Aβ frag- ments between residues 36 and 43. Conversely, juxtamembrane β β cleavage of APP between K16/L17 residues by α-secretase pre- Although -amyloid (A ) peptides participate in the patho- β genesis of Alzheimer’s disease (AD), the mechanisms that regu- cludes A generation and results in clearance of APP (3). β Several proteases have been suggested as AD-relevant α-secre- late A production are poorly understood. Here, we demonstrate tases, many of which belong to the “a disintegrin and metallopro- that activation of the nuclear receptor peroxisome proliferator- + teinase” (ADAM) Zn2 sheddase family (reviewed in ref. 4) and activated receptor α (PPARα) upregulates transcription of the “a include ADAM9, ADAM10, and ADAM17. However, ADAM10 disintegrin and metalloproteinase” 10 (Adam10) gene and shifts has emerged as the constitutive and inducible APP α-secretase APP processing toward the α-secretase pathway. These findings in neurons (5). Of note, ADAM9 and ADAM17 do not recover suggest PPARα could be a therapeutic target for reducing Aβ α-secretase proteolysis of APP in the absence of ADAM10 (5), burden in AD. neuron-specific overexpression of ADAM10 decreases Aβ load in a mouse model of AD (6), and impaired ADAM10 trafficking to the Author contributions: G.T.C. and K.P. designed research; G.T.C. performed research; F.J.G. synapse generates a model of sporadic AD (7). Similarly, human contributed new reagents/analytic tools; G.T.C. and K.P. analyzed data; and G.T.C., F.J.G., studies have observed deficits in ADAM10 expression (8), trafficking and K.P. wrote the paper. (9), and activity (10) in AD. Therefore, dysregulation of ADAM10 The authors declare no conflict of interest. may play a significant role in the establishment of Aβ pathology. This article is a PNAS Direct Submission. However, little is known about the genetic regulation of ADAM10. 1To whom correspondence should be addressed. Email: [email protected]. Peroxisome proliferator-activated receptor (PPAR)-α is a tran- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. scription factor that regulates genes involved in fatty acid transport 1073/pnas.1504890112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1504890112 PNAS | July 7, 2015 | vol. 112 | no. 27 | 8445–8450 Downloaded by guest on October 1, 2021 significantly induced surface immunoreactivity of ADAM10 [Fig. 2 C and D; F(1,76) = 4.231; P = 0.043], but not APP [Fig. 2 D and E; F(1,76) = 0.079; P = 0.779], in fully differentiated (as determined by MAP-2 immunolabeling in adjacent cultures), unpermeabilized Fig. 1. PPARα deficiency results in impaired ADAM10 expression. (A–D) Quan- titative PCR results of Adam10 (A), Adam17 (B), Bace1 (C), and Psen1 (D)mRNA −− −− expression in the hippocampus and cortex of 4-mo-old WT Ppara / and Pparb / animals or in WT neurons knocked down for PPARγ (PpargKD). Values are cor- rected for Gapdh and are expressed as percentage of WT. (E) Subcellular and detergent-soluble (1% CHAPS) expression of precursor (pA10) and mature (mA10) ADAM10 and COOH terminus cleaved ADAM10 (A10CTF) or precursor (pA17) and mature (mA17) ADAM17 in hippocampi from 4-mo-old WT animals. (F–J)Repre- sentative immunoblots (F) and quantification of pA10 (G), mA10 (H), A10CTF, pA17 (I), mA17 (J), and A17CTF membrane expression in the hippocampus and cortex of 4-mo-old WT, Ppara−/− and Pparb−/− animals or in 18DIV PpargKD neurons. (K and L) Representative immunoblots of detergent-soluble BACE1 (K), N-terminal and C-terminal presenilin-1 (PS1NT and PS1CT, respectively) (L) expression in the hippocampus of 4-mo-old Ppara WT (+/+) and null (−/−) animals. Values are corrected for α-tubulin, indicate the mean ± SEM relative to WT, and represent n = 3 or 4 for each genotype. *P < 0.05 and **P < 0.01 using one-way ANOVA. Ctx, cortex; Hpc, hippocampus; KD, knockdown; Neu, neurons; OD, relative optical density. ●, nonspecific band. increased the extraction of a truncated, transmembrane C-terminal ADAM10 fragment (A10CTF) (13). Using a subcellular pre- fractionation protocol, we found that membrane expression of precursor A10 (pA10) (Fig. 1 F and G), mA10 (Fig. 1 F and H), and A10CTF (Fig. 1F and Fig. S1C), but not pA17 (Fig. 1 F and I), mA17 (Fig. 1 F and J), and A17CTF (Fig. 1F and Fig. S1D), was significantly impaired in the hippocampus [pA10, F(2,6) = 19.418 (P = 0.002); mA10, F(2,6) = 14.707 (P = 0.005); A10CTF, F(2,6) = 15.690 (P = 0.004)] and frontal cortex [pA10, F(2,6) = 19.519 (P = 0.002); mA10, F = 34.704 (P = 0.001); A10CTF, F = 12.369 (2,6) − − − − (2,6) (P = 0.007)] of 4-mo-old Ppara / , but not Pparb / , animals relative to WT controls or in WT neurons silenced for PPARγ relative to α−/− empty vector-transduced neurons. In contrast, Ppar mice did α not differ significantly from WT mice in terms of hippocampal, Fig. 2. PPAR agonists induce the expression of ADAM10 in primary hip- pocampal neurons. (A and B) Representative immunoblots (A) and quanti- CHAPS-soluble BACE1 (Fig. 1K) or presenilin-1 (PS1) (Fig. 1L) fication (B) of pA10, mA10, and A10CTF membrane expression in 18DIV expression.