Molecular Vision 2003; 9:179-83 © 2003 Molecular Vision Received 22 January 2003 | Accepted 30 April 2003 | Published 1 May 2003

β-Amyloid secretases and β-amyloid degrading enzyme expression in lens

Guanghui Li, Luigi Percontino, Qian Sun, A. Sami Qazi, Peter H. Frederikse

Department of Pharmacology & Physiology and the Integrative Neuroscience Program, UMDNJ-New Jersey Medical School, NJ

Purpose: β- and γ-Secretases are proteases involved in the processing of the Alzheimer precursor protein (AβPP) that releases the transmembrane β-amyloid fragment (Aβ), associated with age-dependent disease in lens and brain. γ-Secretase is a protein complex containing Presenilin and Nicastrin proteins, which also processes Notch and other receptors in- volved in the eye and lens development. Neprilysin (NEP), a major protease involved in degrading Aβ, acts with β- and γ- secretases to regulate steady-state levels of Aβ. Previously, we demonstrated AβPP and Presenilin expression and process- ing in the lens and demonstrated cell degeneration in classic Alzheimer disease (AD) transgenic and systemic oxidative stress animal models, suggesting that additional AβPP processing proteins are also present in the lens. Here we investigate lens expression of β-secretases, nicastrin and NEP proteins, and compare their protein distribution to Notch and Presenilin in lens. Methods: RT-PCR was used to analyze mRNA transcripts. Immunoblots and immunohistochemistry were used to exam- ine the protein expression and distribution of secretase and Aβ degrading proteins, as well as Presenilin and Notch pro- teins in mouse lenses. Results: β-Acting cleaving enzymes, BACE (BACE1) and BACE2, Nicastrin, Presenilins, Notch and NEP are expressed in the lens. In situ examination of protein distribution in lens indicates expression of each of these proteins is upregulated in peripheral elongating fiber cells at the lens equatorial margin and overlaps with Notch and Presenilin proteins, and also with the distribution of AβPP and Aβ proteins demonstrated in a previous study. Neprilysin exon 1-4 splicing, previously described as diagnostic for neuronal expression, also occurs in lens. Conclusions: BACE, BACE2, Nicastrin and NEP are expressed primarily in elongating peripheral fiber cells, overlap- ping with Notch, Presenilin, and AβPP protein distribution in lens, consistent with their role in regulating Notch and AβPP ectodomain shedding in lens. Lens expression of β- and γ-secretases together with NEP suggests these proteins may also regulate Aβ turnover in the lens. The presence of Aβ processing and degrading proteases in lens provides further evidence that Alzheimer-related cell biology is fundamentally involved in lens development, and provides additional evidence that mechanisms of Alzheimer pathophysiology can contribute to lens degeneration, suggesting further that therapeutics tar- geting Aβ proteases may be applicable to lens degenerative disease.

Alzheimer precursor protein (AβPP) is a transmembrane Previously, we demonstrated Presenilin 1 and Presenilin protein present in vesicle and cell surface membranes, that is 2 (PS 1 and 2) expression and processing in lens, similar to PS proteolytically processed, releasing β-amyloid (Aβ) peptides processing in neurons [5], and also demonstrated lens expres- [1,2]. AβPP has been linked with the major age-dependent sion of AβPP and Aβ [3,4]. PS proteins are multi-pass mem- diseases of brain and lens in Alzheimer transgenic mouse brane proteins in vesicle membranes and at the cell surface models [2-4]. γ-Secretase is a protein complex that includes that provide the proteolytic active site for the γ-secretase com- Presenilin and nicastrin proteins. γ-Secretase cleaves AβPP at plex [10-12]. In Alzheimer neurodegenerative disease, PS gene the C-terminus of the transmembrane Aβ peptide [5-12]. The mutations are the largest contributors to early-onset familial β-secretases include β-acting cleaving enzymes BACE forms of the disease [2]. PS proteins are obligately co-ex- (BACE1) and BACE2, which cleave AβPP at the N-terminal pressed with Notch proteins in Drosophila, C. elegans, and end of Aβ [13,14]. Aβ monomers or small oligomers are mammalian tissues [7,8,10]. In addition, the γ-secretase com- thought to contribute to age-dependent disease by producing plex requires nicastrin for proper activity [11,12]. γ-Secretase oxidative stress, in part due to metal binding [15]. In the lens, also processes a variety of other receptors including Notch Aβ is toxic to cultured lens cells and is also present in catarac- and the tyrosine kinase receptor ErbB4, also present in vesicle tous human lenses [4]. In contrast, the role of Aβ in normal membranes, in a process termed ectodomain shedding [9-12]. cell physiology is not well understood. Once released, the cytoplasmic AβPP, Notch, and ErbB4 do- mains travel to the cell nucleus to globally regulate gene ex- pression [7-9,16]. Although Alzheimer’s disease therapeutics Correspondence to: Peter H. Frederikse, Ph.D., UMDNJ-New Jer- involving γ-secretase protease inhibitors have much promise, sey Medical School, 185 South Orange Avenue, MSB H-645, New- the pleiotropic nature of ectodomain shedding now appears to ark, NJ, 07103 Phone: (973) 972-1686; FAX: (973) 972-7950; email: challenge the possible efficacy of γ-secretase inhibitors to regu- [email protected] late Aβ levels. 179 Molecular Vision 2003; 9:179-83 © 2003 Molecular Vision

The β-site AβPP cleaving enzymes, BACE and BACE2, Trizol reagent (Invitrogen, Carlsbad, CA). cDNA was produced are β-secretase glycoproteins that cleave AβPP at the N-ter- using random hexamer primers and reverse transcriptase minal end of Aβ [13,14,17]. This cleavage event is thought to (Invitrogen). The corresponding cDNAs were amplified in occur at the interior surface of vesicle membranes, or at the PCR reactions [3] using the following primers that correspond cell surface [1]. Like the AβPP gene, the BACE2 gene is lo- to sequences in neighboring exons: Mouse BACE: GTA TAG cated within the Down Syndrome (DS) critical region of hu- CGA GTG GTC GAT, TGT GCC CTA CAC CCA G; Rat man chromosome 21 and mouse chromosome 16 [17]. BACE BACE: ATT GGT GGT ATC GAC CAT TC, GCC TGT GGA protease inhibitors are also under investigation as potential β- TGA CTG TGA GA; BACE2: GAA GCA GTA ACT TCG amyloid disease therapeutic agents. CTG T, CAA GAC TAC CTC CGT TGG T; Neprilysin GCG Neprilysin (NEP) was identified as a primary Aβ degrad- GAG ATG TGC AAG TGG, ATC GGG AAC TGG TCT C; ing enzyme [18-21]. However, NEP was first identified some Nicastrin: CGA CTT ACG TTG TGC AG, TGG ATG TCT time ago as Common Acute Lymphoblastic Leukemia Anti- TTC CAG CG; Rat/Mouse Notch 1: CCT CTC CAC CAA gen 10 (CALLA10; CD10). Similar to Alzheimer’s disease TAC CTG, GAT GCC CTC GGA CCA ATC A. Nucleotide and cataract, Acute Lymphoblastic Leukemia (ALL) also oc- sequences were determined for all amplified products to con- curs at high rates in Down’s syndrome and, in addition, so- firm their identities. DNA contamination was also controlled matic trisomy 21 is also associated with ALL [3]. for in PCR reactions where RT was omitted in the preparation In the present study we demonstrate BACE and BACE2, of cDNAs, and no products were observed. Nicastrin, Notch, and NEP expression in mouse and rat lenses, Western blot and immunohistochemistry: Lenses were at the transcript and protein level. Further, each of these pro- placed in B-per extraction buffer (Pierce, Rockford, IL), and teins is expressed in differentiating mouse lens fibers that also briefly sonicated on ice. Proteins were resolved by SDS-PAGE, express Notch and AβPP in mammalian lenses, consistent with blotted onto filter paper, and probed with anti-BACE and anti- their required roles in AβPP and Notch ectodomain shedding. BACE2 antibodies, (Calbiochem, San Diego, CA and Santa Finally, we identified exon 1-4 Neprilysin splicing in lens that Cruz Biotechnology Santa Cruz, CA) to detect proteins on previously was considered diagnostic of its expression in neu- immunoblots [3,4]. Protein bands present on membrane fil- rons [19-21]. The present data demonstrate key AβPP and ters were visualized using VIP staining kits (Vector Labs, Notch regulatory proteases, and Aβ degrading enzymes in lens Burlingame, CA). that contribute to normal lens cell biology and possibly also For in situ immunohistochemical detection of protein in to lens Alzheimer pathophysiology. lenses [4], paraffin sections of whole mouse eyes were probed with anti-BACE, BACE2, Nicastrin, Notch or Presenilin anti- METHODS bodies (Santa Cruz Biotechnology), or with anti-NEP (Sigma, RT-PCR: C57/Bl6 mice and Sprague Dawley rats were used St. Louis, MO). Histological sections were deparaffinized in according to procedures prescribed by the US Public Health xylene, hydrated in graded alcohol washes, and then placed in Service Policy on humane care and use of laboratory animals. antigen unmasking solution (Vector labs) for 20 min at 60 °C. Total lens RNA was purified from mouse and rat lenses using Slides were first blocked in 2% horse serum (Vector labs) in PBS pH 7.4 for 2 h, and next incubated in antibodies diluted 1:200 in PBS with 2% horse serum. Immune complexes were visualized with fluor-conjugated secondary antibodies (Mo- lecular Probes, OR), and viewed by fluorescence microscopy. Photomicrographs were obtained with a CCD camera.

RESULTS The amplified cDNA products in Figure 1 demonstrate BACE and BACE2 expression in mouse and rat lenses. BACE and BACE2 transcripts are the spliced products of nine exon open reading frames, and BACE oligonucleotide primers are com- plimentary to sequences in neighboring exons. BACE and BACE2 amplified products contained the appropriate se- quences present in the Genbank database. We also examined the expression of nicastrin and neprilysin transcripts in rodent lenses. RT-PCR products cor- responding to NEP (Figure 1B) and Nicastrin (Figure 1C) were amplified from total rat and mouse lens RNA respectively. Figure 1. RT-PCR detection of BACE, BACE2, Neprilysin, and Neprilysin transcripts are alternatively spliced using one of Nicastrin in mouse and rat lens. A: BACE and BACE2 amplified cDNA products from mouse and rat lenses. B: Neprilysin transcripts three upstream non-coding exons. Previous investigators dem- in rat lens using oligonucleotide primers corresponding to exon 1 onstrated exon 1 to 4 splicing occurs only in neurons [19,21]. and exon 4. C: Nicastrin transcripts detected in mouse lens. D: Notch Here, we also identify exon 1-4 splicing in rat lenses, how- 1 transcripts in mouse and rat lens. ever exon 2-4 splicing was not detected in our experiments. 180 Molecular Vision 2003; 9:179-83 © 2003 Molecular Vision

This finding adds to the number of neuron specific gene splic- expression of Notch in rat and mouse lenses by RT-PCR (Fig- ing and gene products related to Alzheimer cell biology ob- ure 1D). Similar to previous studies in C. elegans, Drosophila, served in lens [3, and unpublished data]. and mammals, Notch is co-expressed in mouse lenses with In our previous study, we determined the expression and Presenilins and nicastrin. distribution of PS 1 and 2 in lens. Here, we determined lens We next examined BACE and BACE2 protein expres- sion in the lens with western blots. Figure 2 demonstrates the presence of BACE and BACE2 cross-reacting proteins of about 54 and 56 kDa on immunoblots. In addition, two higher MW cross-reacting protein bands were detected for both BACE and BACE2 proteins. These bands are consistent with glycosylation of these proteins in the lens, as demonstrated in other tissues [14]. To determine the spatial expression pattern for the pro- tein products of these genes in the lens we probed mouse eye sections with antibodies raised against Neprilysin (CD10), Nicastrin, BACE, BACE2, Notch 1, and also PS proteins. Fig- Figure 2. Western blot detection of BACE and BACE2 proteins in ure 3 demonstrates representative sections from three mice mouse lenses. The 56 kDa and 54 kDa bands correspond to BACE probed with these antibodies. No signal was obtained when and BACE2 proteins. The higher migrating bands are consistent with the primary antibody was omitted. Anti-Presenilin antibodies glycosylation of BACE proteins also occurring in lens. confirmed the localization pattern we demonstrated in our

Figure 3. Immunofluorescence detection of Neprilysin (CD10), Nicastrin, Notch receptor, BACE and BACE2 secretase proteins and Presenilin in mouse lenses. Lens protein distribution demonstrated for each of these gene products is greatest in peripheral nucleated fiber cells at the equatorial margin. This is consistent with the coordinated action of these proteins in Notch receptor and Alzheimer precursor protein (AβPP) protein processing. Neprilysin protein, a major β-amyloid (Aβ) degrading enzyme, is also greatest in peripheral fibers, consistent with expres- sion and proteolytic processing of AβPP, which releases the internal Aβ peptide. Brightfield photographs of the entire eye at 2x and 100x original magnifications are included. 181 Molecular Vision 2003; 9:179-83 © 2003 Molecular Vision earlier study [5]. The data demonstrate that the greatest ex- 4. Frederikse PH, Garland D, Zigler JS Jr, Piatigorsky J. Oxidative pression of β- and γ-secretase proteins, together with NEP, stress increases production of beta-amyloid precursor protein Notch receptor protein, and AβPP [3,4] occurs in peripheral and beta-amyloid (Abeta) in mammalian lenses, and Abeta has fibers that are in the process of cell differentiation and elonga- toxic effects on lens epithelial cells. J Biol Chem 1996; 271:10169-74. tion, and not in anterior epithelial cells. 5. Frederikse PH, Zigler JS Jr. Presenilin expression in the ocular lens. Curr Eye Res 1998; 17:947-52. DISCUSSION 6. Mitsiadis TA, Gayet O, Zhang N, Carroll P. Expression of Deltex1 The present study describes the expression of β- and γ- during mouse embryogenesis: comparison with Notch1, 2 and secretase and Aβ degrading proteins in lens. We determined 3 expression. Mech Dev 2001; 109:399-403. BACE, BACE2, Nicastrin, and NEP expression, and compared 7. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell their expression with PS and Notch 1 expression in mamma- fate control and signal integration in development. Science 1999; lian lenses. These proteins process Notch and AβPP proteins 284:770-6. that have key roles in fundamental aspects of lens develop- 8. Fortini ME. Notch and presenilin: a proteolytic mechanism emerges. Curr Opin Cell Biol 2001; 13:627-34. ment. Processed Notch intracellular domain (NICD) translo- 9. Lee HJ, Jung KM, Huang YZ, Bennett LB, Lee JS, Mei L, Kim cates to the nucleus to activate transcription of genes that regu- TW. Presenilin-dependent gamma-secretase-like intramembrane late cell generation, differentiation, and cell survival and it cleavage of ErbB4. J Biol Chem 2002; 277:6318-23. was recently demonstrated that the processed intracellular 10. Struhl G, Adachi A. Requirements for presenilin-dependent cleav- domain of AβPP (AID) binds Numb and Numb-like proteins age of notch and other transmembrane proteins. 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The print version of this article was created on 1 May 2003. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. 183