Chronic Treatment with the γ-Secretase Inhibitor LY-411,575 Inhibits β-Amyloid Peptide Production and Alters Lymphopoiesis and Intestinal Cell Differentiation
2004; Elsevier BV; Volume: 279; Issue: 13 Linguagem: Inglês
10.1074/jbc.m311652200
ISSN1083-351X
AutoresGwendolyn T. Wong, Denise Manfra, Frederique M. Poulet, Qi Zhang, Hubert Josien, Thomas Bara, Laura Engstrom, Maria Pinzon-Ortiz, Jay S. Fine, Hu-Jung J. Lee, Lili Zhang, Guy A. Higgins, Eric M. Parker,
Tópico(s)Nuclear Receptors and Signaling
ResumoInhibition of γ-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic β-amyloid (Aβ) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other γ-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by γ-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of γ-secretase inhibition, the γ-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Aβ production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4-CD8-CD44+CD25+ precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak γ-secretase inhibitor. These studies show that inhibition of γ-secretase has the expected benefit of reducing Aβ in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing. Inhibition of γ-secretase, one of the enzymes responsible for the cleavage of the amyloid precursor protein (APP) to produce the pathogenic β-amyloid (Aβ) peptides, is an attractive approach to the treatment of Alzheimer disease. In addition to APP, however, several other γ-secretase substrates have been identified (e.g. Notch), and altered processing of these substrates by γ-secretase inhibitors could lead to unintended biological consequences. To study the in vivo consequences of γ-secretase inhibition, the γ-secretase inhibitor LY-411,575 was administered to C57BL/6 and TgCRND8 APP transgenic mice for 15 days. Although most tissues were unaffected, doses of LY-411,575 that inhibited Aβ production had marked effects on lymphocyte development and on the intestine. LY-411,575 decreased overall thymic cellularity and impaired intrathymic differentiation at the CD4-CD8-CD44+CD25+ precursor stage. No effects on peripheral T cell populations were noted following LY-411,575 treatment, but evidence for the altered maturation of peripheral B cells was observed. In the intestine, LY-411,575 treatment increased goblet cell number and drastically altered tissue morphology. These effects of LY-411,575 were not seen in mice that were administered LY-D, a diastereoisomer of LY-411,575, which is a very weak γ-secretase inhibitor. These studies show that inhibition of γ-secretase has the expected benefit of reducing Aβ in a murine model of Alzheimer disease but has potentially undesirable biological effects as well, most likely because of the inhibition of Notch processing. Alzheimer disease (AD) 1The abbreviations used are: AD, Alzheimer disease; APP, amyloid precursor protein; NICD, Notch intracellular domain; DN, double negative; Aβ, β-amyloid peptide; LY-D, inactive diastereoisomer of LY-411,575. is the third most common cause of death and the leading cause of dementia in the United States (1Ewbank D.C. Am. J. Public Health. 1999; 89: 90-92Crossref PubMed Scopus (66) Google Scholar). Although the exact cause of AD is still unknown, the etiology of the disease is almost certainly linked to several neuropathological hallmarks observed in the brains of AD victims, particularly extracellular neuritic amyloid plaques and intracellular neurofibrillary tangles (2Arnold S.E. Hyman B.T. Flory J. Damasio A.R. Van Hoesen G.W. Cereb. Cortex. 1991; 1: 103-116Crossref PubMed Scopus (1138) Google Scholar, 3Games D. Adams D. Alessandrini R. Barbour R. Berthelette P. Blackwell C. Carr T. Clemens J. Donaldson T. Gillespie F. Nature. 1995; 373: 523-527Crossref PubMed Scopus (2246) Google Scholar, 4Dickson D.W. Crystal H.A. Bevona C. Honer W. Vincent I. Davies P. Neurobiol. Aging. 1995; 16: 285-298Crossref PubMed Scopus (364) Google Scholar). Although both of these neuropathological lesions probably contribute to progressive neuronal cell death in AD, the proximal lesion appears to be the amyloid plaques and their principal component, the Aβ peptides. A large body of evidence strongly suggests that overproduction, aggregation, and/or plaque deposition of the Aβ peptides, particularly Aβ42, are central to the pathogenesis of AD (reviewed in Ref. 5Hardy J. Selkoe D.J. Science. 2002; 297: 353-356Crossref PubMed Scopus (11018) Google Scholar). In fact, two recent studies of patients immunized against the Aβ42 peptide have provided the first preliminary clinical evidence that Aβ does indeed contribute to the cognitive decline in AD patients (6Nicoll J.A. Wilkinson D. Holmes C. Steart P. Markham H. Weller R.O. Nat. Med. 2003; 9: 448-452Crossref PubMed Scopus (1294) Google Scholar, 7Hock C. Konietzko U. Streffer J.R. Tracy J. Signorell A. Muller-Tillmanns B. Lemke U. Henke K. Moritz E. Garcia E. Wollmer M.A. Umbricht D. de Quervain D.J. Hofmann M. Maddalena A. Papassotiropoulos A. Nitsch R.M. Neuron. 2003; 38: 547-554Abstract Full Text Full Text PDF PubMed Scopus (731) Google Scholar). The Aβ peptides are produced by the sequential proteolytic cleavage of the amyloid precursor protein (APP) by β- and γ-secretase. γ-Secretase is a complex composed of at least four proteins, namely presenilins (presenilin 1 or -2), nicastrin, PEN-2, and APH-1 (8De Strooper B. Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar). Presenilin 1 and -2 have been proposed to be the novel aspartyl proteases responsible for the catalytic activity of γ-secretase (9Wolfe M.S. Xia W. Ostaszewski B.L. Diehl T.S. Kimberly W.T. Selkoe D.J. Nature. 1999; 398: 513-517Crossref PubMed Scopus (1692) Google Scholar, 10Zhang Z. Nadeau P. Song W. Donoviel D. Yuan M. Bernstein A. Yankner B.A. Nat. Cell Biol. 2000; 2: 463-465Crossref PubMed Scopus (359) Google Scholar). Because of the essential role of γ-secretase in the generation of Aβ peptides, γ-secretase inhibitors may be useful in the treatment of AD. To date, several γ-secretase inhibitors have been identified that lower Aβ production in intact cells and cell-free systems (reviewed in Ref. 11Josien H. Curr. Opin. Drug Discovery Dev. 2002; 5: 513-525PubMed Google Scholar). In addition, two potent γ-secretase inhibitors, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine tert-butyl ester (DAPT) and LY-411,575, have also been shown to decrease Aβ production after administration to transgenic mice overexpressing human APP (12Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D. Hyslop P.A. Johnstone E.M. Li W.-Y. Little S.P. Mabry T.E. Miller F.D. Ni B. Nissen J.S. Porter W.J. Potts B.D. Reel J.K. Stephenson D. Su Y. Shipley L.A. Whitesitt C.A. Yin T. Audia J.E. J. Neurochem. 2001; 76: 173-181Crossref PubMed Scopus (797) Google Scholar, 13May P.C. Altstiel L. Bender M.H. Boggs L.N. Calligaro D.O. Fuson K.S. Gitter B.D. Hyslop P.A. Jordan W.H. Li W.Y. Mabry T.E. Mark R.J. Ni B. Nissen J.S. Porter W.J. Sorgen S.G. Su Y. Audia J.E. Dovey H.F. Games D. John V. Freedman S.B. Guido T. Johnson-Wood K.L. Khan K. Latimer L.H. Lieberburg I.M. Seubert P.A. Soriano F. Thorsett E.D. Schenk D.B. Abstracts of the Society for Neuroscience 31st Annual Meeting, San Diego, CA, November 10-15, 2001. 2001; : 681Google Scholar, 14Lanz T.A. Himes C.S. Pallante G. Adams L. Yamazaki S. Amore B. Merchant K.M. J. Pharmacol. Exp. Ther. 2003; 305: 864-871Crossref PubMed Scopus (144) Google Scholar). It is appreciated now that APP is not the only substrate for γ-secretase. Other proteins that have been shown to be substrates for γ-secretase cleavage include Notch (16De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1800) Google Scholar) and the Notch ligands Delta1 and Jagged2 (20Ikeuchi T. Sisodi S.S. J. Biol. Chem. 2003; 278: 7751-7754Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), ErbB4 (17Ni C.-Y. Murphy M.P. Golde T.E. Carpenter G. Science. 2001; 294: 2179-2181Crossref PubMed Scopus (756) Google Scholar), CD44 (18Lammich S. Okochi M. Takeda M. Kaether C. Capell A. Zimmer A.K. Edbauer D. Walter J. Steiner H. Haass C. J. Biol. Chem. 2002; 277: 44754-44759Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), and E-cadherin (19Marambaud P. Shioi J. Serban G. Georgakopoulos A. Sarner S. Nagy V. Baki L. Wen P. Efthimiopoulos S. Shao Z. Wisniewski T. Robakis N.K. EMBO J. 2002; 21: 1948-1956Crossref PubMed Scopus (624) Google Scholar). The cleavage of Notch by γ-secretase has been studied most extensively. Notch plays an evolutionarily conserved role in regulating cell growth and lineage specification particularly during embryonic development (21Greenwald I. Genes Dev. 1998; 12: 1751-1762Crossref PubMed Scopus (460) Google Scholar, 22Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4917) Google Scholar, 23Conlon R.A. Reaume A.G. Rossant J. Development. 1995; 121: 1533-1545Crossref PubMed Google Scholar). Notch is activated by several ligands (Delta, Jagged, and Serrate) and is then proteolytically processed by a series of ligand-dependent and -independent cleavages. γ-Secretase catalyzes the terminal cleavage event (S3 cleavage), which releases a fragment known as the Notch intracellular domain (NICD). The NICD fragment then translocates to the nucleus where it acts as a nuclear transcription factor (15Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1361) Google Scholar, 24Struhl G. Adachi A. Cell. 1998; 93: 649-660Abstract Full Text Full Text PDF PubMed Scopus (635) Google Scholar). As expected from its role in Notch S3 cleavage, γ-secretase inhibitors have been shown to block NICD production in vitro (25Lewis H.D. Perez Revuelta B.I. Nadin A. Neduvelil J.G. Harrison T. Pollack S.J. Shearman M.S. Biochemistry. 2003; 42: 7580-7586Crossref PubMed Scopus (70) Google Scholar). In vivo, Notch function appears to be critical for the proper differentiation of T and B lymphocytes (reviewed in Ref. 26Allman D. Punt J.A. Izon D.J. Aster J.C. Pear W.S. Cell. 2002; 109: S1-S11Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and γ-secretase inhibitors reduce the thymocyte number and block thymocyte differentiation at an early stage in fetal thymic organ cultures (27Hadland B.K. Manley N.R. Su D. Longmore G.D. Moore C.L. Wolfe M.S. Schroeter E.H. Kopan R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7481-7491Crossref PubMed Scopus (189) Google Scholar, 28Doerfler P. Shearman M.S. Perlmutter R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9312-9317Crossref PubMed Scopus (155) Google Scholar). These data suggest that in addition to their potential therapeutic benefit in AD, γ-secretase inhibitors may have liabilities because of inhibition of the proteolytic processing of Notch (and other signaling molecules) that are critical for a variety of cellular functions. Despite data clearly suggesting both the potential benefits and the liabilities of γ-secretase inhibition, there are no published reports identifying the in vivo biological consequences of γ-secretase inhibitors apart from the inhibition of APP processing and Aβ production. Such data are necessary to fully assess the strengths and liabilities of γ-secretase inhibition as an approach to the treatment of AD. In this study, chronic treatment of mice with the potent γ-secretase inhibitor LY-411,575 (13May P.C. Altstiel L. Bender M.H. Boggs L.N. Calligaro D.O. Fuson K.S. Gitter B.D. Hyslop P.A. Jordan W.H. Li W.Y. Mabry T.E. Mark R.J. Ni B. Nissen J.S. Porter W.J. Sorgen S.G. Su Y. Audia J.E. Dovey H.F. Games D. John V. Freedman S.B. Guido T. Johnson-Wood K.L. Khan K. Latimer L.H. Lieberburg I.M. Seubert P.A. Soriano F. Thorsett E.D. Schenk D.B. Abstracts of the Society for Neuroscience 31st Annual Meeting, San Diego, CA, November 10-15, 2001. 2001; : 681Google Scholar) was used to characterize the effects of γ-secretase inhibition on Aβ production and to identify additional physiological consequences of γ-secretase inhibition. A structurally related analogue that is a weak γ-secretase inhibitor was used to distinguish effects of LY-411,575 that are due to γ-secretase inhibition from the nonspecific effects of the compound. Animals and Dosing—LY-411,575 and a diastereoisomer hereafter referred to as LY-D (see Fig. 1) were synthesized according to Wu et al. (29Wu J. Tung J.S. Thorsett E.D. Pleiss M.A. Nissen J.S. Neitz J. Latimer L.H. John V. Freedman S. Patent Application WO-09828268. 1998; Google Scholar). During synthesis, racemic 5-amino-7-methyl-5,7-dihydro-6H-dibenz[b,d]azepin-6-one was coupled with tert-butoxycarbonyl-alanine, and the two diastereoisomers were separated by high pressure liquid chromatography. Each diastereoisomer was then deprotected with trifluoroacetic acid and subsequently condensed with the appropriate chiral 3,5-difluoromandelic acid to provide the expected products (see Fig. 1). The structures of LY-411,575 and LY-D were confirmed by nuclear magnetic resonance spectroscopy and tandem liquid chromatography-mass spectrometry. Both compounds were formulated as 10 mg/ml solutions in 50% polyethylene glycol, 30% propylene glycol, 10% ethanol and diluted in 0.4% methylcellulose for dosing. TgCRND8 mice overexpress human APP with both the Swedish and Indiana familial AD mutations under the control of the Syrian hamster prion promoter (30Chishti M.A. Yang D.S. Janus C. Phinney A.L. Horne P. Pearson J. Strome R. Zuker N. Loukides J. French J. Turner S. Lozza G. Grilli M. Kunicki S. Morissette C. Paquette J. Gervais F. Bergeron C. Fraser P.E. Carlson G.A. George-Hyslop P.S. Westaway D. J. Biol. Chem. 2001; 276: 21562-21570Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). TgCRND8 mice on a C57BL/6 × C3H/He mixed genetic background were weaned, genotyped, and single-housed by the age of 4-5 weeks. Six-week-old female TgCRND8 or male C57BL/6 mice (Charles River Laboratories) were dosed orally once/day for 5 or 15 days with 0.4% methylcellulose vehicle, 1-10 mg/kg LY-411,575, or 1-10 mg/kg LY-D. At the end of the dosing period, mice were sacrificed by CO2 asphyxiation, total blood was collected from the posterior vena cava in EDTA Microtainer® tubes (BD Biosciences), and plasma was isolated. Cortices were surgically isolated from 1 hemibrain/mouse. Each cortex was homogenized in sucrose, extracted with guanidine, and sonicated (31Sawamura N. Morishima-Kawashima M. Waki H Kobayashi K. Kuramochi T. Frosch M.P. Ding K. Ito M. Kim T.W. Tanzi R.E. Oyama F. Tabira T. Ando S. Ihara Y. J. Biol. Chem. 2000; 275: 27901-27908Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Protein concentration was determined by the BCA assay (Pierce). All in vivo studies were approved by the Animal Care and Use Committee of the Schering-Plough Research Institute. Cell Lines—HEK293 cells expressing human APP carrying both the Swedish and London mutations have been described previously (32Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar). Human Notch1 was cloned from human fetal brain cDNA (Clontech), and a truncated Notch1 construct lacking the extracellular EGF repeat domain was prepared (referred to as NΔE) (15Schroeter E.H. Kisslinger J.A. Kopan R. Nature. 1998; 393: 382-386Crossref PubMed Scopus (1361) Google Scholar). NΔE was also stably expressed in HEK293 cells. Assays for Aβ and NICD—Procedures for measuring γ-secretase activity in membranes prepared from HEK293 cells expressing APP have been described previously (32Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar). Intact HEK293 cells expressing either APP or NΔE were treated with various concentrations of LY-411,575 or LY-D for 4 h at 37 °C. In the case of cells expressing NΔE, cells were lysed, the cell lysates were separated on a 4-12% NuPAGE gel, and the processed NICD fragment was detected via Western blot with a cleavage site-specific antibody. The inhibition of NICD production was quantified by spot densitometric analysis using FluorChem (Alpha Innotech Corp.). In the case of cells expressing APP, the conditioned medium was collected, centrifuged at 10,000 × g for 5 min to remove cell debris, and stored at -20 °C prior to the determination of Aβ levels. Aβ40 and -42 produced in HEK293 membrane- and cell-based assays, as well as plasma Aβ40 and cortex Aβ40 from TgCRND8 mice, were analyzed without pretreatment using an electrochemiluminescence detection-based immunoassay (32Zhang L. Song L. Terracina G. Liu Y. Pramanik B. Parker E. Biochemistry. 2001; 40: 5049-5055Crossref PubMed Scopus (92) Google Scholar). Plasma Aβ42 was measured by enzyme-linked immunosorbent assay as described previously (33Zhang L. Song L. Parker E. J. Biol. Chem. 1999; 274: 8966-8972Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). A commercially available enzyme-linked immunosorbent assay kit (BioSource International) was used to measure cortex Aβ42 according to the manufacturer's instructions. Flow Cytometry—Single cell suspensions from the thymus and spleen were prepared from individual tissues in RPMI 1640 medium containing 10% fetal calf serum (Invitrogen) by passage through a 100-μm nylon cell strainer (Falcon-BD Biosciences). Peripheral blood was collected as described above, and erythrocytes were lysed (erythrocyte lysis buffer, Sigma). Cells from the thymus, spleen, or blood were enumerated by trypan blue exclusion and then incubated for 20 min at 4 °C in the dark with 5 μg/ml Fc block (Pharmingen) and 300 μg/ml purified mouse IgG in phosphate-buffered saline, 1% bovine serum albumin, 0.1% sodium azide to minimize nonspecific antibody binding. Fluorochrome-conjugated monoclonal antibodies to the following mouse surface markers were then added, and the incubation was continued for 20 min at 4 °C in the dark: CD4 (GK1.5), CD8α (53-6.7), CD44 (IM7), CD25 (PC61), CD3 (145-2C11), αβ-TcR (H57-597), γδ-TcR (GL3), B220 (RA3-6B2), and IgM (R6-60.2) (all obtained from Pharmingen). Matched fluorochrome-conjugated isotype control antibodies were used to determine nonspecific binding. To determine viability, samples were stained subsequently with 5 μg/ml propidium iodide (Calbiochem). Events were acquired on a FACSCalibur instrument (BD Biosciences Immunocytometry Systems) and analyzed with FlowJo software (Stanford University). The number of cells of each phenotype was determined by multiplying the percentage of cells in the population by the total number of thymus or spleen cells, as appropriate. Histology—At the end of the dosing period the brain, thymus, spleen, liver, kidney, heart, stomach, adrenal glands, small intestine, large intestine, and lungs were collected in 10% neutral-buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin for light microscopic examination. In addition, small and large intestine were stained with periodic acid-Schiff stain to highlight mucin-containing goblet cells. Comprehensive bone marrow smear cytologic examinations were also performed. LY-411,575 Is a Potent γ-Secretase Inhibitor in Vitro and in Vivo—As reported previously (13May P.C. Altstiel L. Bender M.H. Boggs L.N. Calligaro D.O. Fuson K.S. Gitter B.D. Hyslop P.A. Jordan W.H. Li W.Y. Mabry T.E. Mark R.J. Ni B. Nissen J.S. Porter W.J. Sorgen S.G. Su Y. Audia J.E. Dovey H.F. Games D. John V. Freedman S.B. Guido T. Johnson-Wood K.L. Khan K. Latimer L.H. Lieberburg I.M. Seubert P.A. Soriano F. Thorsett E.D. Schenk D.B. Abstracts of the Society for Neuroscience 31st Annual Meeting, San Diego, CA, November 10-15, 2001. 2001; : 681Google Scholar), LY-411,575 is a very potent γ-secretase inhibitor in vitro as assessed by inhibition of Aβ production (IC50, 0.078 and 0.082 nm in membrane- and cell-based γ-secretase assays, respectively (Fig. 1)). LY-411,575 is also a potent inhibitor of Notch S3 cleavage (Fig. 1, IC50, 0.39 nm). A diastereoisomer of LY-411,575, LY-D, was also synthesized (Fig. 1). Depending on the assay, LY-D is 180-2000-fold less potent than LY-411,575 as an inhibitor of γ-secretase (Fig. 1). In vivo studies demonstrated that both 1 and 10 mg/kg oral doses of LY-411,575 decreased brain and plasma Aβ40 and -42 robustly when chronically administered to TgCRND8 mice (Fig. 2). In contrast, the administration of the less active diastereoisomer LY-D to TgCRND8 mice had no effect on Aβ levels in either the brain or plasma (Fig. 2). The different in vivo activities of LY-411,575 and LY-D are not because of different pharmacokinetic properties, because the two compounds achieved similar plasma levels after oral administration (data not shown). These in vitro and in vivo data demonstrate that LY-411,575 and LY-D are excellent tools to distinguish the mechanism-based effects of γ-secretase inhibition from the compound-related effects that are not due to γ-secretase inhibition. The time course of the effects of LY-411,575 on plasma and brain Aβ40 levels is shown in Fig. 3. A single oral dose of 10 mg/kg LY-411,575 rapidly decreased plasma Aβ40 with the maximal effect being observed by 1 h after administration (Fig. 3A). The reduction of plasma Aβ was still evident 24 h after the administration of LY-411,575 but returned to base-line levels by 48 h after administration (Fig. 3A). LY-411,575 also substantially decreased guanidine-soluble brain Aβ levels, although the maximal effect was less pronounced, the time required to reach the maximal effect was longer, and the duration of the effect was longer than that seen for reduction of plasma Aβ (Fig. 3B). These data demonstrate that dosing of LY-411,575 once/day is sufficient to maintain continual inhibition of γ-secretase. General in Vivo Effects of Chronic γ-Secretase Inhibition—LY-411,575 and LY-D (1 and 10 mg/kg) were administered to TgCRND8 mice once/day for either 5 or 15 consecutive days. In other experiments, LY-411,575 (1, 3, and 10 mg/kg) was administered to C57BL/6 mice once/day for 15 consecutive days. Similar results were observed in all three experiments. Unless indicated otherwise, therefore, data are presented only for the experiment in which LY-411,575 and LY-D were administered to TgCRND8 mice for 15 days. Mice receiving 10 mg/kg LY-411,575 for 15 days lost weight (-2 g) compared with mice treated with vehicle (+1.4 g), 1 mg/kg LY-411,575 (+1.4 g), 1 mg/kg LY-D (+2 g), or 10 mg/kg LY-D (+1.5 g). In addition, 20% of the mice dosed with 1 mg/kg LY-411,575 and 40% of the mice dosed with 10 mg/kg LY-411,575 died by day 15 of dosing. In contrast, there was no mortality for mice dosed with vehicle or with either dose of LY-D. Histopathological analysis demonstrated that neither LY-411,575 nor LY-D had any effect on the majority of the tissues examined, including the brain, liver, kidney, lungs, heart, adrenal glands, and stomach. Bone marrow smears also revealed no abnormalities (data not shown). In contrast, effects of LY-411,575 were observed in the thymus, spleen, and intestine. These observations are discussed individually below. Chronic γ-Secretase Inhibition Alters Lymphocyte Development—Chronic treatment of TgCRND8 mice with LY-411,575 induced a marked atrophy of the cortical zone of the thymus (Fig. 4, compare A with B). This effect was dose-dependent and was consistent with the dose-dependent reduction in the absolute thymocyte cell number caused by LY-411,575 (Fig. 5A). In contrast, LY-D had no effect on thymus histology (Fig. 4, compare A with C) or the absolute number of thymocytes (Fig. 5A). Flow cytometric analysis of intrathymic populations revealed that the number and proportion of thymocytes defined by CD4/CD8 expression was altered by treatment with LY-411,575 but not by treatment with LY-D. Consistent with the overall decrease in thymocyte number (Fig. 5A), the absolute number of CD4+CD8+ double positive, CD4-CD8- double negative (DN), CD4+ single positive, and CD8+ single positive cells also decreased in a dose-dependent manner (Fig. 5B). However, the proportion of DN and single positive cells increased, whereas the proportion of double positive cells decreased (Fig. 5C).Fig. 5Effect of LY-411,575 treatment on intrathymic populations. LY-411,575 (1 and 10 mg/kg), LY-D (1 and 10 mg/kg), or vehicle were orally administered once/day to TgCRND8 mice for 15 consecutive days, and various thymocyte populations were analyzed by flow cytometry as described under “Experimental Procedures.” A, the total number of cells in the thymus. B, the absolute number of cells in each of the four major populations defined by CD4 and -8. The inset illustrates the CD4+CD8+ double positive (DP) population on a separate scale. C, the percentage of total thymocytes in each of the four major populations defined by CD4 and -8. The inset depicts the CD4+CD8+ double positive population on a separate scale. D, evaluation of CD44/CD25 co-expression on intrathymic DN cells. The data shown are the mean ± S.E. (5-6 mice/group) and were analyzed by unpaired Student's t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001 all as compared with vehicle-treated animals). SP, single positive.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A detailed examination of the DN cell population demonstrated that LY-411,575 administration increased the relative percentage of the CD44+CD25- and CD44+CD25+ DN thymocytes while causing a commensurate decrease in the proportion of CD44-CD25+ and CD44-CD25- DN thymocytes (Fig. 5D) indicating a block at a very early stage of intrathymic T cell development. Once again, these effects were not observed in LY-D-treated animals (Fig. 5D). Interestingly, no differences in CD44 mean fluorescence intensity were observed across treatment groups implying that processing of this putative γ-secretase substrate was unaffected by LY-411,575 treatment. The percentage of thymocytes expressing intermediate or high levels of CD3/αβ-TcR was significantly reduced in accord with the inhibition of CD4/CD8 populations, although no consistent alterations in the percentage or number of γδ-TcR cells were noted. A slight increase in the proportion of thymic B220+ B cells was observed in LY-411,575-treated animals, although this did not reach statistical significance (data not shown). The effect of LY-411,575 treatment on peripheral leukocyte compartments was also examined. In contrast to the observations made in the thymus, neither LY-411,575 nor LY-D treatment significantly altered T cell populations in the peripheral blood or spleen (data not shown). However, LY-411,575 administration did cause a statistically significant elevation in the percentage and number of immature surface IgM- B220+ B cells and a corresponding reduction in the percentage and number of mature surface IgM+ B220+ B cells in the spleen (Fig. 6). These observations suggest that LY-411,575 treatment results in a developmental blockade of splenic B cell differentiation. Modest elevations in the number of immature B cells were observed in the blood as well. No remarkable findings on bone marrow cytology, myeloid populations in the blood, or complete blood count analysis were noted (data not shown). Chronic γ-Secretase Inhibition Increases Goblet Cell Number and Drastically Changes Tissue Architecture in the Gastrointestinal Tract—Treatment of TgCRND8 mice with 10 mg/kg LY-411,575 produced profound changes in the gastrointestinal tract. At this dose, LY-411,575 caused a significant increase in the number of mucin-containing goblet cells throughout the small and large intestine and an increased accumulation of mucin secreted into the intestinal lumen (Fig. 7, B, E, and H). In one mouse that was sacrificed in a moribund condition, LY-411,575 treatment (10 mg/kg) also caused epithelial erosion with underlying infiltration of inflammatory cells in the lamina propria (Fig. 7B, circled areas), the accumulation of necrotic cell debris, and dilation of crypts (Fig. 7B, arrowheads). In contrast, chronic treatment of TgCRND8 mice with 1 mg/kg LY-411,575 or with either dose of LY-D had no effect on the gastrointestinal tract (Fig. 7, C and F). Several recent reports (12Dovey H.F. John V. Anderson J.P. Chen L.Z. de Saint Andrieu P. Fang L.Y. Freedman S.B. Folmer B. Goldbach E. Holsztynska E.J. Hu K.L. Johnson-Wood K.L. Kennedy S.L. Kholodenko D. Knops J.E. Latimer L.H. Lee M. Liao Z. Lieberburg I.M. Motter R.N. Mutter L.C. Nietz J. Quinn K.P. Sacchi K.L. Seubert P.A. Shopp G.M. Thorsett E.D. Tung J.S. Wu J. Yang S. Yin C.T. Schenk D.B. May P.C. Altstiel L.D. Bender M.H. Boggs L.N. Britton T.C. Clemens J.C. Czilli D.L. Dieckman-McGinty D.K. Droste J.J. Fuson K.S. Gitter B.D.
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