Activation of Peroxisome Proliferator-Activated Receptor Α Stimulates ADAM10-Mediated Proteolysis of APP

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 determined 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 fragments 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 corrected 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 prefractionation 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 hippocampal 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. These results suggest that transcription and subcellular neurons after 24 h treatment with DMSO or agonists of PPARα (WY14643), localization of ADAM10 is impaired in animals null for PPARα. β δ γ – α PPAR / (GW501516), or PPAR (GW1929). (C E) Representative immunocy- Next, we examined whether PPAR agonists could augment tochemical images (C) and quantification of ADAM10 (D) and APP (E)sur- ADAM10 levels. Using serially isolated neurons [18 d in vitro face expression in live-stained, unpermeabilized WT and Ppara−/− neurons (DIV)], astrocytes (14DIV), and microglia (16DIV) from the treated with DMSO or 25 μM Gem for 24 h. (F–J) Representative immuno- same E18 WT hippocampi, an enrichment of membrane-bound blots (F) and quantification of pA10 (G), mA10 (H), A10CTF (I), and APP − − ADAM10 in neurons and astrocytes was observed (Fig. S1A), (J) membrane expression in WT and Ppara / neurons treated with DMSO, suggesting these cells as primary sources of brain ADAM10. 25 μM Gem, or 0.2 μM9-cis-retinoic acid (9cRA) for 24 h. (K) Representative Because it was suggested that neuronal activity regulates Aβ immunoblots of membrane-bound ADAM10 expression in hippocampi from generation (14) and a majority of the brain PPARα is local- 2-mo-old Ppara WT (+/+), heterozygous (+/−), and null (−/−) mice. (L and M) ized to neurons (15), we determined the relationship between Representative immunoblots (L) and quantification (M) of pA10 and mA10 α membrane expression in WT neurons transduced with GFP alone and in PPARs and ADAM10 in neurons. Agonists specific for PPAR −/− α (WY14643), but not PPARβ/δ (GW501516) or PPARγ (GW1929), Ppara neurons transduced with GFP alone or with flPPAR .(N) Quantifi- cation of surface ADAM10 expression in live-stained, unpermeabilized, significantly elevated membrane-bound pA10 [F(6,14) = 56.240; P < −/− = < = 18DIV WT neurons transduced with GFP alone and in Ppara neurons 0.001], mA10 [F(6,14) 21.088; P 0.001], and A10CTF [F(6,14) α < transduced with GFP alone or with flPPAR . The corresponding immunocy- 17.949; P 0.001] expression in hippocampal neurons (Fig. 2 A tochemical images are presented in Fig. S2D. All values are corrected for β-or and B). Gemfibrozil (Gem), a known PPARα agonist, activated α-tubulin, indicate the mean ± SEM relative to control, and represent n = 3 PPAR-driven luciferase activity in a dose-dependent manner in or 4 for each treatment with the exception of E, F,andN,inwhichn ≥ 20. *P < hippocampal neurons, with maximal transcriptional activation 0.05 and **P < 0.01, using two-way (B, D, E, M) or one-way (N) ANOVA. (Scale achieved at 25 μM(Fig. S1B). In line with this finding, Gem bar, 5 μm.) n.s., not statistically significant; OD, relative optical density.

8446 | www.pnas.org/cgi/doi/10.1073/pnas.1504890112 Corbett et al. Downloaded by guest on October 1, 2021 − − WT, but not Ppara / , hippocampal neurons. There was also a main effect of Ppara genotype on ADAM10 [Fig. 2D; F(1,76) = 21.242; P < 0.001], but not APP [Fig. 2E; F(1,76) = 2.444; P = 0.070], surface expression. Interestingly, ADAM10 and APP puncta were 1.78 times more likely to colocalize in WT neurons treated with Gem (r2 = 0.742) relative to DMSO (r2 = 0.416) (Fig. S1C). To − − support these findings, WT and Ppara / hippocampal neurons were treated with Gem or 9-cis-retinoic acid (9cRA), an agonist of the PPARα allosteric heterodimer partner retinoid x receptor (RXR)(16).ThemaineffectsofGemand9cRA treatments and − − Ppara / genotype on expression of membrane-bound pA10 (Fig. 2G) [treatment, F(2,12) = 13.940 (P = 0.001); genotype, F(1,12) = 116.353 (P < 0.001)], mA10 (Fig. 2H) [treatment, F(2,12) = 19.312 (P < 0.001); genotype, F(1,12) = 217.105 (P < 0.001)], and A10CTF (Fig. 2I) [treatment, F(2,12) = 3.974 (P = 0.047); genotype, F(1,12) = 26.746 (P < 0.001)], but not pA17 and mA17 or cellular APP (Fig. − − 2J) expression in 18DIV WT, and not Ppara / , hippocampal neurons, highlight the importance of PPARα in Gem- and 9cRA- induced ADAM10 expression. − − We next asked whether recovery of PPARα in Ppara / tissues − − restored ADAM10 expression. Ppara / mice were backcrossed + + with WT C57BL/6J mice to create cogenic Ppara WT (Ppara / ), + − − − + − heterozygous (Ppara / ), and null (Ppara / )animals.Ppara / mice expressed roughly half and twice as much membrane-bound + + − − ADAM10 in the hippocampus relative to Ppara / and Ppara / mice, respectively (Fig. 2K), suggesting a gene–dose effect of PPARα on ADAM10 expression. To support these findings, −/− transduction of 18DIV Ppara hippocampal neurons with lenti- Fig. 3. Gem augments the recruitment of PPARα to the Adam10 pro- viruses coexpressing GFP and WT, full-length PPARα (flPPARα) moter. (A) Cartoon depicting alignment of PPRE located upstream of the significantly restored membrane-bound pA10 [F(2,6) = 5.448; P = transcription start site in ADAM10 promoter sequences (GXPs). (B–E) Global 0.045] and mA10 [F(2,12) = 33.550; P = 0.001] to levels comparable PPAR/RXR (B and C) and PPARγ (D and E) response element nucleotide with WT neurons expressing GFP-fused empty vector alone (Fig. 2 probability distributions and consensus frequencies represented by sequence − − L and M). In addition, introduction of flPPARα to 18DIV Ppara / logos (D) and index vectors (consensus index vector; E), respectively. (F) Quan- hippocampal neurons resulted in significant recovery of surface titative PCR results for PPREs 203.1 (1), 203.2 (2), and 207.1 (3) after chromatin −/− immunoprecipitation with antibodies against PPARα,RXRα,CBP,p300,PGC1α, ADAM10 immunoreactivity relative to Ppara neurons trans- − − = = NcoR1, and RNA Pol II (x-axis, left to right) from 18DIV WT and Ppara / neurons

fected with GFP alone [Fig. 2N and Fig. S2D; F(2,57) 7.549; P NEUROSCIENCE α treated with DMSO or 25 μMGemfor2h.(G) Cartoon depicting DNA binding 0.003]. These results suggest that overexpression of PPAR is α −/− of the PPAR transcriptional complex to the Adam10 promoter in the absence necessary and sufficient to recover ADAM10 levels in Ppara or presence of Gem. All values are corrected for input DNA and are relative to hippocampal neurons. IgG, indicate the mean ± SEM relative to control, and represent n = 4foreach treatment. *P < 0.05 and **P < 0.01 using Student’s t test. bp, base pairs; ChIP, Gem Induces the Recruitment of PPARα to the Adam10 Promoter. chromatin immunoprecipitation; IP, immunoprecipitating antibody. Five experimentally verified Adam10 promoter variants were analyzed for PPRE with higher-than-chance consensus index vectors, using MatInspector (17). A number of PPREs are lo- PPARα Modulates APP Processing. We devised an in vitro assay to cated upstream of the transcription start site (Fig. 3A), and the reliably detect murine APP fragments and circumvent the need PPRE nucleotide distributions varied depending on whether the for transgenic overexpression of nonendogenous APP (18) or its sequences are responsive to PPAR/RXR heterodimers (Fig. 3 B rate-limiting secretases. Murine soluble APPα (sAPPα; ∼110 kDa) β ∼ and C) or PPARγ (Fig. 3 D and E). We then extracted the full and monomeric A ( 4 kDa) were immunoprecipitated from promoter sequences with ElDorado and designed flanking pri- conditioned media harvested from WT 18DIV hippocampal mers (Table S1) upstream and downstream of all seven PPRE. neurons with antibody M3.2 (Fig. 4A, first panel), the murine α analog to human-specific 6E10 (Fig. 4A, third panel). Oligomeric Gem significantly induced the promoter enrichment of PPAR β [203.1, t = 6.981 (P = 0.005); 203.2, t = 9.014 (P = 0.002)] A was not enriched with antibody A11 (Fig. 4A, second panel), (6) (6) likely because murine Aβ lacks the propensity to rapidly self- and its heterodimer RXRα [203.1, t(6) = 10.281 (P = 0.001); 203.2, t = 12.336 (P < 0.001)], cAMP response element- associate, especially under cell culture conditions. M3.2 pre- (6) cipitation of sAPPα and Aβ from neuronal media was confirmed binding protein (CBP) [203.1, t = 3.787 (P = 0.024); 203.2, t = (6) (6) by precipitation of human APP fragments from the hippocampus P = t = P = 4.033 ( 0.019)], and p300 [203.1, (6) 6.213 ( 0.009); of 6-mo-old 5XFAD mice, using antibody 6E10 before or after 203.2, t(6) = 3.989 (P = 0.023)], and the RNA Pol II [203.1, t(6) = < = = samples were immunodepleted with an antibody specific for the 11.453 (P 0.001); 203.2, t(6) 8.890 (P 0.002)], at two direct N terminus of all APP species (antibody 22C11) (Fig. 4A,third repeat 1 PPRE located in one Adam10 promoter variant in −/− panel). Precipitated proteins were detected (immunoblot) with anti- 18DIV WT, but not Ppara , hippocampal neurons relative to bodies against M3.2 (first panel), D54D2 (second panel), or 6E10 α DMSO (Fig. 3 F,1 and F,2). PGC1 was not involved, and pro- (third panel). Please see Table S2 for antibodies and concentrations. moter occupancy of transcriptional corepressor NcoR1 was sig- Using this assay, we found that Gem or 9cRA significantly = = = nificantly abrogated [203.1, t(6) 3.648 (P 0.031); 203.2, t(6) elevated sAPPα (Fig. 4 B and D)[F = 8.400; P = 0.005] and = (2,12) 6.703 (P 0.006)] in the presence of Gem relative to DMSO reduced Aβ (Fig. 4 B and E)[F(2,12) = 4.594; P = 0.033] and (Fig. 3 F,1 and F,2). As expected, Gem did not induce the re- sAPPβ (Fig. 4 B and C)[F = 3.940; P = 0.048] release into (2,12) − − cruitment of any of these factors to a likely PPARγ response el- culture media in WT, but not Ppara / , neurons. Accordingly, ement (Fig. 3F,3), indicating specificity of binding. Amplification Gem and 9cRA treatments reduced the production of cell-bound of the other direct repeat 1 PPRE did not occur, possibly because β-secretase-cleaved APP C-terminal fragments (CTFβ; Fig. 4 B of the absence of these promoter variants in neurons. and F)[F(2,12) = 4.265; P = 0.040] and increased the production

Corbett et al. PNAS | July 7, 2015 | vol. 112 | no. 27 | 8447 Downloaded by guest on October 1, 2021 C-terminal presenilin-1 (PS1CT) (Fig. 5 A and I), and BACE1 (Fig. 5 A and J) did not differ between genotypes at either age. Next, as antibody M3.2 nonselectively recognizes both 40(1–40) and 42(1–42) Aβ peptides, we used highly sensitive, murine- specific sandwich ELISAs to quantify Aβ1–42 species in serially isolated aqueous (TBS) and detergent-soluble (TBS-Tx) hippocampal fractions (Fig. 5K). Relative to their age-matched − − WT controls, Ppara / mice expressed significantly higher levels of aqueous (Left) and detergent-soluble (Right)Aβ1–42 at 6 mo of age [TBS, t(6) = 2.854 (P = 0.029); TBS-Tx, t(6) = 6.815 (P < 0.001)] and 18 mo of age [TBS, t(6) = 5.337 (P = 0.002); TBS-Tx, t(6) = 6.742 (P = 0.001)] (Fig. 5M). Taken together, these findings describe that PPARα plays a vital role in the regulation of Aβ generation.

Ablation of PPARα Propagates Cerebral Aβ Load and Augments Lethality in 5XFAD Mice. To examine the effect of deleting endogenous PPARα on the production of transgenically derived Aβ,we − − crossed Ppara / mice with 5XFAD mice overexpressing familial AD-linked human APP and presenelin 1 (PSEN1) mutations − − (19) to create bigenic 5XFAD mice null for Pparα (5X/α / ). As expected, the Ppara disruption did not alter insertion or expression of the 5XFAD transgenes, and vice versa (Fig. S3A). − − − − Six-month-old WT, Ppara / , 5XFAD, and 5X/α / mice did not α β Fig. 4. Activation of PPAR attenuates endogenous A generation. (A) Rep- differ significantly with respect to wet brain or gross body weight resentative immunoblots of APP species IP from 18DIV neuronal culture (Fig. S3 B and C), and we did not observe any overt phenotypic media using antibodies M3.2 and A11 or from the hippocampal TBS fraction (TBS Frac) of 6-mo-old 5XFAD mice with antibody 6E10. (B–G) Representative differences, including diet, fecal boli, social interaction, and ag- β α β itation across genotypes at this age. immunoblots (B) and quantification of murine sAPP (C), sAPP (D), and A β (E) immunoprecipitated from conditioned media, and β-secretase cleaved We monitored brain A load in two cohorts of 5XFAD and α−/− β APP C-terminal fragments (CTFβ)(F) and α-secretase cleaved APP C-terminal 5X/ mice by colabeling coronal sections for the -pleated- fragments (CTFα)(G) immunoprecipitated from lysates harvested from WT sheet marker thioflavin-S (Thio-S) and the N-terminal Aβ anti- − − and Ppara / neurons treated with DMSO, 25 μM GEM, or 0.2 μM all-trans body 6E10 (Fig. 6A). As expected, robust Thio-S and Aβ fluo- retinoic acid (9cRA) for 24 h. All values are corrected for IgG, indicate the rescence was observed throughout the cortex and hippocampus − − mean ± SEM relative to control, and represent n = 3 or 4 for each treatment. in 5- to 6-mo-old 5XFAD (Fig. 6A,1) and 5X/α / (Fig. 6A,2) − − *P < 0.05 and **P < 0.01, using two-way ANOVA. IB, immunoblot; Beads, mice and in 10- to 12-mo-old 5XFAD (Fig. 6A,3) and 5X/α / −/− protein G beads alone; IgGHC/LC, IgG heavy chain and light chain; Media, (Fig. 6A,4) mice, but not 5- to 6- or 10- to 12-mo-old Ppara unconditioned media; OD, relative optical density. ●, nonspecific band. mice (Fig. S4B). Quantification of Thio-S staining area revealed significantly more amyloid deposition in the cortex and hippo- − − campus of 5X/α / mice relative to age-matched 5XFAD mice at of CTFα (Fig. 4 B and G)[F = 14.735; P = 0.001] in WT, but − − (2,12) 5–6 mo of age [cortex, t = −4.012 (P = 0.001); hippocampus, not Ppara / , neurons. Importantly, there was also a strong main (25) − − t = −4.769 (P < 0.001)] and 10–12 mo of age [cortex, t = Ppara / α F = P < (25) (35) effect of genotype on baseline sAPP [ (1,12) 81.114; −7.603 (P < 0.001); hippocampus, t = −5.768 (P < 0.001)] 0.001], sAPPβ [F = 124.822; P < 0.001], Aβ [F = 212.166; (35) (1,12) (1,12) (Fig. 6B). Further characterization of Thio-S staining revealed a P < 0.001], CTFβ [F = 125.371; P < 0.001], and CTFα [F = (1,12) (1,12) significantly greater number of plaques in the hippocampus of 128.399; P < 0.001], but not APP, expression (Fig. 4 C–G). − − 5X/α / mice relative to age-matched 5XFAD mice at 5–6moof To validate these in vitro findings, 6- and 18-mo-old WT and −/− age [t(25) = −3.524; P = 0.001] and in the hippocampus and Ppara mice were subjected to subcellular prefractionation – = − = (lysate) or solubilized in Tris-buffered saline (TBS) for immu- cortex at 10 12 mo of age [cortex, t(35) 2.591 (P 0.014); hippocampus, t = −4.534 (P < 0.001)], and larger plaque size noblotting (Fig. 5A). Again, we found that membrane expression (35) − − in the hippocampus and cortex of 5X/α / mice relative to age- of pA10 (Fig. 5 A and B), mA10 (Fig. 5 A and C), and A10CTF – = − = (Fig. 5 A and D) was significantly impaired in the hippocampus matched 5XFAD mice at 5 6 mo of age [cortex, t(25) 3.207 (P 0.004); hippocampus, t(25) = −6.006 (P < 0.001)] and in the cortex at of 6-mo-old [pA10, t(6) = 9.084 (P < 0.001); mA10, t(6) = 13.834 10–12 mo of age [t(35) = −3.136 (P = 0.003)] (Fig. S4 C and D). (P < 0.001); A10CTF, t(6) = 3.808 (P = 0.009)] and 18-mo-old [pA10, t = 3.726 (P = 0.010); mA10, t = 6.892 (P < 0.001); Next, we prepared serially isolated aqueous (TBS) and de- (6) −(6)− A10CTF, t = 2.771 (P = 0.032)] Ppara / mice relative to age- tergent-soluble (TBS-Tx) fractions from hippocampi from 2- (6) α−/− – matched WT mice. Interestingly, significantly elevated and re- and 6-mo-old 5XFAD (5X), 5X/ , and WT mice (Fig. 6 A C). α−/− duced expression of sAPPα (Fig. 5 A and K) and Aβ (Fig. 5 A and Relative to 5XFAD littermates, 5X/ mice expressed signifi- β = L), respectively, was observed in the TBS fraction in 6-mo-old cantly more aqueous, low-n, SDS-stable A at 2 mo of age [t(6) α = < β = − = 4.826; P = 0.003] and 6 mo of age [t(6) = 4.394; P = 0.020] (Fig. 6 [sAPP , t(6) 9.307 (P 0.001); A , t(6) 2.572 (P 0.042)] −/− and 18-mo-old [sAPPα, t = 10.707 (P < 0.001); Aβ, t = A and B). In agreement with this, 5X/α mice expressed sig- (6)− − (6) −2.832 (P = 0.030)] Ppara / mice relative to age-matched WT nificantly higher levels of aqueous (Left) and detergent-soluble β = = mice. We also observed significantly reduced CTFα (Fig. 5 A and (Right)A 1–42 at 2 mo of age [TBS, t(8) 4.211 (P 0.003); TBS- = < = G) and elevated CTFβ (Fig. 5 A and F) and sAPPβ (Fig. 5 A and Tx, t(8) 4.975 (P 0.001)] and 6 mo of age [TBS, t(8) 4.446 = = < H) levels in hippocampal lysates from 6-mo-old [CTFα, t = (P 0.002); TBS-Tx, t(8) 8.491 (P 0.001)] relative to age- (6) – 3.449 (P = 0.014); CTFβ, t(6) = −7.840 (P < 0.001); sAPPβ, t(6) = matched 5X mice. Furthermore, Kaplan Meier estimates (Fig. − = α = = 6F) revealed significantly different survival distributions for both 4.213 (P 0.006)] and 18-mo-old [CTF , t(6) 4.716 (P 0.003); − − CTFβ, t = −20.374 (P < 0.001); sAPPβ, t = −4.282 (P = 5X (16.2 ± 1.22 mo) and 5X/α / (13.9 ± 1.283 mo) mice relative (6) − − (6) − − 0.005)] Ppara / mice relative to age-matched WT mice. Im- to WT (22.0 ± 0.685 mo) and Pparα / (21.846 ± 0.862 mo) mice 2 −/− munoblotting for CTFα with antibody C1/6.1 was performed in [χ(3) = 27.688; P < 0.001] and revealed that 5X/α mice dis- lysates immunodepleted with M3.2. Conversely, and in agree- played significantly shorter lifespans relative to their 5X lit- 2 ment with previous data, expression of APP (Fig. 5 A and E), termates[(χ(2) = 13.955; P = 0.001].

8448 | www.pnas.org/cgi/doi/10.1073/pnas.1504890112 Corbett et al. Downloaded by guest on October 1, 2021 − − γ-secretase catalytic protease (PSEN1) in Pparα / brains. Second, − − lentiviral delivery of WT, full-length PPARα to Ppara / neurons was necessary and sufficient to restore ADAM10 expression. Third, WY14643, a specific agonist of PPARα, but neither the PPARβ/ δ-specific agonist GW501516 nor the PPARγ-specific agonist GW1929, increased expression of ADAM10 in hippocampal NEUROSCIENCE − − Fig. 5. APP processing is shifted away from the α-secretase pathway in Ppara / mice. (A–L) Representative immunoblots (A) and quantification of precursor ADAM10 (pA10) (B), mature ADAM10 (mA10) (C), COOH terminus-cleaved ADAM10 (A10CTF) (D), APP (E), β-secretase-cleaved APP C-terminal fragment (CTFβ)(F), α-secretase-cleaved APP CTFα (G), β-secretase-cleaved soluble APP (sAPPβ)(H), C-terminal-cleaved PS1 (PS1CT) (I), BACE1 (J), and immunoprecipitated, α-secretase-cleaved soluble APP (sAPPα)(K)andAβ (L) expression in hip- − − pocampi from 6- and 18-mo-old WT and Ppara / mice. (M) ELISA quantification

of murine Aβ1–42 expression in serially extracted TBS (Left)andTBS+1% Triton X-100 (TBS-Tx; Right) fractions from 6- and 18-mo-old WT and Pparα−/− hippocampi. All values are corrected for α-tubulin or IgG, indicate the mean ± SEM relative to WT, and represent n = 4 for each genotype and age, except for (M),

where n = 5. *P < 0.05 and **P < 0.01, using Student’s t test. IgGHC/LC,IgG heavy chain and light chain; OD, optical density. ●, nonspecific band.

Fig. 6. Disruption of PPARα exacerbates cerebral Aβ load and decreases lifespan in 5XFAD mice. (A) Representative images of coronal sections from Discussion − − 5- to 6-mo-old 5XFAD (A1), 5- to 6-mo-old 5X/Pparα / (A2), 10- to 12-mo-old One of the pathologic hallmarks of AD is the presence of ex- α−/− β 5XFAD (A3), and 10- to 12-mo-old 5X/Ppar (A4) mice colabeled with thio- tracellular amyloid plaques containing A peptides, which orig- flavin-S (Thio-S, green) and 6E10 (red). (B) Quantification of Thio-S-positive inate from the amyloidogenic proteolytic processing of APP, area as a percentage of total area in the cortex (CTX) and hippocampus (HPC) through the sequential action of β- and γ-secretases. In contrast, of 5XFAD and 5X/Ppara−/− mice. All values represent the mean ± SEM and APP can also be cleaved by a nonamyloidogenic pathway by represent n = 14, 13, 19, and 18 for 5- to 6-mo-old 5XFAD, 5- to 6-mo-old − − − − α-secretase, precluding the formation of Aβ peptides. Accord- 5X/Ppara / , 10- to 12-mo-old 5XFAD, and 10- to 12-mo-old 5X/Ppara / mice, ingly, mutations in the α-secretase ADAM10 prodomain are respectively. (C and D) Representative immunoblots (C) and quantification of β 6E10-detected Aβ (D) expression in TBS fractions from 2- and 6-mo-old WT, associated with exacerbated A generation and AD susceptibility − − − − (20). Therefore, understanding the mechanistic regulation of 5XFAD (5X), and 5X/Pparα / (5X/α / ) hippocampi. Values are corrected for ADAM10 is an important area of research. This study reveals a α-tubulin and indicate the mean ± SEM relative to WT. (E) ELISA quantification of human Aβ1–42 in serially extracted TBS (Top) and TBS+1% Triton X-100 (TBS- pathway for the up-regulation of ADAM10 involving the lipid- − − Tx; Bottom) fractions from 5XFAD and 5X/α / hippocampi. (F)Kaplan–Meier lowering transcription factor PPARα. Our conclusion is based on −/− −/− survival analysis of WT (n = 15), Pparα (n = 13), 5XFAD (n = 19), and 5X/α the following: First, we demonstrated that brains and neurons = + α β δ γ (n 16) animals across 24 mo. Plus ( ) symbols represent censored cases. All null for PPAR ,butnotPPAR/ or PPAR , were deficient for values represent the mean ± SEM and represent n = 4(C and D)or5(E)for membrane-bound, catalytic ADAM10. In contrast, we failed to each genotype and age. *P < 0.05, **P < 0.01, and ***P < 0.001, using Stu- observe any deficits in ADAM17, an enzyme with potential APP dent’s t test, and #P < 0.01, using Breslow Generalized Wilcoxon χ2.(Scalebar, α-secretase functional redundancy, the β-secretase BACE1, and the 500 μm.) IB, immunoblot; OD, optical density. ●, nonspecific band.

Corbett et al. PNAS | July 7, 2015 | vol. 112 | no. 27 | 8449 Downloaded by guest on October 1, 2021 neurons. Similarly, Gem, a lipid-lowering drug approved by the transcriptional induction of ADAM10. Therefore, the outcome of US Food and Drug Administration and an agonist of PPARα, this investigation highlights undiscovered properties of PPARα, also elevated the expression of ADAM10 in hippocampal neu- describes a mechanism for reducing Aβ generation, and fuels in- −/− rons isolated from WT, but not Ppara , mice. Fourth, Gem terest in understanding the link between APP processing and lipid α α treatment led to the recruitment of PPAR and RXR to two metabolism pathways. direct repeat 1 PPREs in the Adam10 promoter. This inducible transcriptional mechanism also involved the histone acetyl- Methods and Materials α transferases CBP and p300. Although PGC1 has been identi- Animals. Mice were maintained and experiments conducted in accordance fied as an important transcriptional coactivator being involved in with National Institute of Health guidelines and were approved by the Rush diverse cellular functions including metabolism of lipids and University Medical Center Institutional Animal Care and Use Committee. carbohydrates, mitochondrial biogenesis and function, and cho- − − C57BL/6J, Ppara / , and 5XFAD (19) mice were obtained from Jackson. lesterol homeostasis (21), we did not observe recruitment of − − − − Ppara / and Pparβ/δ / mice (27) were maintained homozygous for the PGC1α to the Adam10 promoter upon Gem treatment, indicating the specificity of this process. Fifth, earlier studies have mutations unless otherwise indicated. shown a role of RXR (22) and retinoic acid receptor (23) in the induction of ADAM10 in neuronal cell lines. We found that Immunocytochemistry and Quantification. Primary hippocampal neurons were − − RXR agonists were unable to up-regulate ADAM10 in Ppara / double-immunostained and quantified according to Glynn and McAllister neurons, indicating an obligatory role of PPARα in this process. (28), with modification (SI Materials and Methods). Can PPARα modulate APP processing and, therefore, generation of Aβ? This is an important question depending on the fact Subcellular Prefractionation, Immunoblotting, Densitometry, and ELISA. Sub- that ADAM10 favors the nonamyloidogenic pathway. We found cellular prefractionation of cultured neurons and brain tissue was per- evidence that APP processing was shifted toward the β-secretase- formed as described elsewhere (29), except the membrane pellet was − − · mediated pathway in the hippocampus of Ppara / mice. Crossing solubilized in 1% CHAPS buffer (30 mM Tris HClatpH7.5,150mMNaCl, − − + 5XFAD mice with Ppara / mice led to robust elevations in plaque 1% CHAPS) protease and phosphatase inhibitors (Sigma) instead of area, number, and size in the hippocampus and cortex of both PBS. For immunoblotting, densitometry and ELISA details, please see SI young and aged mice. Biochemically, we observed drastically ex- Materials and Methods. acerbated Aβ1–42 accumulation and aggregation in the hippocampus and cortex relative to traditional 5XFAD littermates. In Conditioned Media Preparation and Immunoprecipitation of APP Fragments. addition, 5XFAD mice null for PPARα displayed significantly Endogenous APP fragments were detected in conditioned neuronal media shorter life spans (by ∼2 mo) compared with their traditional according to Shankar et al. (30), with modification (SI Materials and Methods). 5XFAD littermates. Although there is no definitive correlation An extended section is provided in SI Materials and Methods. between Aβ and lifespan per se, there is evidence that increased Aβ burden induces early mortality in mice (24) and lower organisms ACKNOWLEDGMENTS. We thank Dustin Wakeman and Benjamin Hiller for (25). There is also evidence that elevated Aβ levels are associated their assistance with histological preparation. A portion of this work was completed while G.T.C. was supported by a National Institutes on Aging withreducedlifespaninthehumandisease(26). Predoctoral training Grant (5T32 AG000269). This work was supported by In summary, we describe that activation of PPARα induces grants from National Institutes of Health (AT6681 and NS083054) and a α-secretase proteolysis of APP in hippocampal neurons via merit award from Veteran Affairs (I01BX003033-01).

1. LaFerla FM, Green KN, Oddo S (2007) Intracellular amyloid-beta in Alzheimer’s dis- 17. Cartharius K, et al. (2005) MatInspector and beyond: Promoter analysis based on ease. Nat Rev Neurosci 8(7):499–509. transcription factor binding sites. Bioinformatics 21(13):2933–2942. 2. O’Brien RJ, Wong PC (2011) Amyloid precursor protein processing and Alzheimer’s 18. Podlisny MB, et al. (1995) Aggregation of secreted amyloid beta-protein into sodium disease. Annu Rev Neurosci 34:185–204. dodecyl sulfate-stable oligomers in cell culture. J Biol Chem 270(16):9564–9570. 3. Allinson TM, Parkin ET, Turner AJ, Hooper NM (2003) ADAMs family members as 19. Oakley H, et al. (2006) Intraneuronal beta-amyloid aggregates, neurodegeneration, – amyloid precursor protein alpha-secretases. J Neurosci Res 74(3):342 352. and neuron loss in transgenic mice with five familial Alzheimer’s disease mutations: 4. Seals DF, Courtneidge SA (2003) The ADAMs family of metalloproteases: Multidomain Potential factors in amyloid plaque formation. J Neurosci 26(40):10129–10140. proteins with multiple functions. Genes Dev 17(1):7–30. 20. Suh J, et al. (2013) ADAM10 missense mutations potentiate β-amyloid accumulation 5. Kuhn PH, et al. (2010) ADAM10 is the physiologically relevant, constitutive alpha- by impairing prodomain chaperone function. Neuron 80(2):385–401. secretase of the amyloid precursor protein in primary neurons. EMBO J 29(17): 21. Villena JA (2015) New insights into PGC-1 coactivators: Redefining their role in the 3020–3032. regulation of mitochondrial function and beyond. FEBS J 282(4):647–672. 6. Postina R, et al. (2004) A disintegrin-metalloproteinase prevents amyloid plaque 22. Prinzen C, Müller U, Endres K, Fahrenholz F, Postina R (2005) Genomic structure formation and hippocampal defects in an Alzheimer disease mouse model. J Clin Invest 113(10):1456–1464. and functional characterization of the human ADAM10 promoter. FASEB J 19(11): 7. Epis R, et al. (2010) Blocking ADAM10 synaptic trafficking generates a model of 1522–1524. sporadic Alzheimer’s disease. Brain 133(11):3323–3335. 23. Tippmann F, Hundt J, Schneider A, Endres K, Fahrenholz F (2009) Up-regulation of the 8. Colciaghi F, et al. (2004) Platelet APP, ADAM 10 and BACE alterations in the early alpha-secretase ADAM10 by retinoic acid receptors and acitretin. FASEB J 23(6): stages of Alzheimer disease. Neurology 62(3):498–501. 1643–1654. 9. Marcello E, et al. (2013) Endocytosis of synaptic ADAM10 in neuronal plasticity and 24. Héraud C, et al. (2014) Increased misfolding and truncation of tau in APP/PS1/tau Alzheimer’s disease. J Clin Invest 123(6):2523–2538. transgenic mice compared to mutant tau mice. Neurobiol Dis 62:100–112. 10. Colciaghi F, et al. (2002) [alpha]-Secretase ADAM10 as well as [alpha]APPs is reduced 25. Greeve I, et al. (2004) Age-dependent neurodegeneration and Alzheimer-amyloid in platelets and CSF of Alzheimer disease patients. Mol Med 8(2):67–74. plaque formation in transgenic Drosophila. J Neurosci 24(16):3899–3906. 11. Roy A, et al. (2013) Regulation of cyclic AMP response element binding and hippo- 26. Mayeux R, et al. (2003) Plasma A[beta]40 and A[beta]42 and Alzheimer’s disease: α campal plasticity-related genes by peroxisome proliferator-activated receptor . Cell Relation to age, mortality, and risk. Neurology 61(9):1185–1190. – Reports 4(4):724 737. 27. Peters JM, et al. (2000) Growth, adipose, brain, and skin alterations resulting 12. Barak Y, et al. (1999) PPAR gamma is required for placental, cardiac, and adipose from targeted disruption of the mouse peroxisome proliferator-activated receptor tissue development. Mol Cell 4(4):585–595. beta(delta). Mol Cell Biol 20(14):5119–5128. 13. Tousseyn T, et al. (2009) ADAM10, the rate-limiting protease of regulated intra- 28. Glynn MW, McAllister AK (2006) Immunocytochemistry and quantification of protein membrane proteolysis of Notch and other proteins, is processed by ADAMS-9, colocalization in cultured neurons. Nat Protoc 1(3):1287–1296. ADAMS-15, and the gamma-secretase. J Biol Chem 284(17):11738–11747. 29. Guillemin I, Becker M, Ociepka K, Friauf E, Nothwang HG (2005) A subcellular pre- 14. Kamenetz F, et al. (2003) APP processing and synaptic function. Neuron 37(6): 925–937. fractionation protocol for minute amounts of mammalian cell cultures and tissue. 15. Gofflot F, et al. (2007) Systematic gene expression mapping clusters nuclear receptors Proteomics 5(1):35–45. according to their function in the brain. Cell 131(2):405–418. 30.ShankarGM,WelzelAT,McDonaldJM,Selkoe DJ, Walsh DM (2011) Isolation of 16. Heyman RA, et al. (1992) 9-cis retinoic acid is a high affinity ligand for the retinoid X low-n amyloid β-protein oligomers from cultured cells, CSF, and brain. Methods receptor. Cell 68(2):397–406. Mol Biol 670:33–44.

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