APPΔNL695 expression in murine tissue downregulates CNBP expression

Neuroscience Letters 482 (2010) 57–61

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APPNL695 expression in murine tissue downregulates CNBP expression Dana M. Niedowicz a,b , Tina L. Beckett b , Chris J. Holler a,b , Adam M. Weidner a,b , M. Paul Murphy a,b,c,∗ a

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, United States Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, United States c UK Center for Muscle Biology, University of Kentucky, Lexington, KY, United States b

a r t i c l e

i n f o

Article history: Received 30 April 2010 Received in revised form 18 June 2010 Accepted 2 July 2010 Keywords: Alzheimer’s disease Sporadic inclusion body myositis CNBP APP

a b s t r a c t The cellular nucleic acid binding protein (CNBP) is a ubiquitously expressed protein involved in regulation of transcription and translation. CNBP, and its encoding gene ZNF9, have been shown to be involved in type 2 myotonic dystrophy. Both Alzheimer’s disease (AD) and sporadic inclusion body myositis (sIBM) are age-related degenerative diseases associated with the accumulation of ␤-amyloid. Overexpression of amyloid precursor protein (APP) in mice has been used to generate models of both diseases. We show here that overexpression of APP in skeletal muscle from a mouse model of sIBM reduces the expression of CNBP significantly. We examined CNBP expression in a brain-specific APP-overexpressing strain, and a whole body APP knock-in strain, and found that there was a reduction in CNBP expression in tissue expressing APPSwe . We conclude that expression of APPSwe in murine tissue induces a decrease in CNBP expression. This effect does not appear to be due to alterations in CNBP transcription. APPSwe expression may provide a tool for the study of CNBP regulation and clues to the roles of both proteins in disease. © 2010 Elsevier Ireland Ltd. All rights reserved.

Alzheimer’s disease (AD) is an age-related neurodegenerative disease thought to occur as a result of ␤-amyloid (A␤) accumulation. A␤ is generated from the amyloid precursor protein (APP) through sequential proteolysis by the ␤- and ␥-secretases. A␤ readily oligomerizes to form multimers, fibrils, and plaques, the latter of which are deposited extracellularly in the brain. Due to the central role of A␤ in the disease process, AD models often use APP overexpression to recapitulate AD pathology in the brain. Sporadic inclusion body myositis (sIBM) is the most common age-related degenerative muscle disease. The symptoms include progressive muscle weakness and atrophy. sIBM has been associated with accumulation of A␤, though it is unclear whether it plays a significant causal role in the disease. In contrast to AD, A␤ is found in intracellular occlusions in the muscle of sIBM patients. APP is commonly overexpressed in the disease state [19,33]. Artificial APP overexpression does induce pathological changes resembling degenerative sIBM pathology in muscle and muscle cells [7,16,24,37]. The cellular nucleic acid binding protein (CNBP), encoded by the ZNF9 gene, is a ubiquitously expressed zinc finger protein that binds RNA and DNA [9,30,42]. While little is known about its endogenous targets, CNBP is thought to play a role in the regulation of transcription and translation [11,17,30,31]. A role for CNBP in vertebrate development has been described [1–3,9,10,12,14,21,22,34,39,40],

∗ Corresponding author at: 211 Sanders-Brown Center on Aging, 800 S. Limestone, Lexington, KY 40536, United States. Tel.: +1 859 257 1412x490; fax: +1 859 257 9479. E-mail address: [email protected] (M.P. Murphy). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.07.006

though its role in adult animals is less clear. A recent study suggests that CNBP may function as a nucleic acid chaperone [4]. A tetranucleotide expansion in ZNF9 causes type 2 myotonic dystrophy (DM2), which features cardiac arrhythmia, muscle wasting, and the development of neurofibrillary tangles in the brain that resemble that of AD [13,18,20,23]. A recent report indicates that ZNF9 haploinsufficiency in mice leads to a phenotype consistent with that of DM2, implying that the CNBP protein may play a functional role in muscle health [11]. Additionally, we have presented preliminary data suggesting that CNBP regulates ␤-secretase expression and that there are alterations in CNBP expression in disease, such as AD [26]. In this report, we show that overexpression of APP in the muscle of a murine sIBM model strikingly reduces expression of CNBP protein. Examination of brain and muscle from mouse strains that overexpress APP or a whole-body knock-in of APP leads us to the conclusion that expression of APPSwe reduces CNBP expression. We examined tissue from 4 mouse strains and their wild-type littermates: (1) A C57Bl/6 strain carrying a APPSwe (NL695) transgene under a creatine kinase promoter – the T7A6 mouse [37]. This sIBM model overexpresses APP 1.5- to 2-fold in muscle. Aged T7A6 mice (18–19 months) were fed either control (N = 19) or ketogenic (N = 15) diets (BioServ) for 2 months prior to euthanasia (N = 21 wild-type; 13 transgenic). We did not observe any effects of diet on CNBP expression (p = 0.363), so the data were pooled across genotype for both groups. (2) We crossed the sIBM strain to C3H mice and then subsequently crossed into a C57Bl/6/C3H hybrid (Charles River Laboratories), creating a hybrid sIBM strain (N = 3 wild-type; 3 transgenic). (3) A CD-1/129 strain with a whole-body knock-in

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of both APPSwe and the ␥-secretase subunit presenilin (PS1P264L ) under endogenous promoters (APP/PS1 mice; N = 3 wild-type; 3 knock-in) [32,35]. (4) A strain with tauP301L and APPSwe transgenes under a Thy1.2 promoter in a PS1M146V knock-in CD-1/129/C57Bl/6 hybrid background [29] (3xTG mice; N = 3 wild-type; 3 transgenic). 3xTG mice overexpress APP approximately 7-fold in brain. Mice were euthanized by CO2 asphyxiation, followed by decapitation. All animal work was conducted with prior IACUC approval and was performed in accordance with USDA and PHS guidelines. Chinese hamster ovary (CHO) cells were grown in Ham’s F12 media with 10% newborn calf serum and 1% penicillin/streptomycin (HyClone). Transfections were performed overnight with FuGene 6 liposomal transfection reagent (Roche) with either pAG3hyg-CNBP (CNBP cDNA was obtained from Dr. Irwin Flink (University of Arizona) [15]) or pAG3hyg-EGFP. Cells were lysed in 1% Triton X-100 in TBS (pH 8) with Complete Protease Inhibitor Cocktail (Roche). C57Bl6 skeletal muscle, cardiac muscle, and brain were homogenized in 1% Triton X-100 in TBS (pH 8) with the Complete Protease Inhibitor Cocktail. Following separation by SDSPAGE, proteins were transferred to PVDF and blocked with 5% nonfat dry milk. CNBP was detected using a rabbit polyclonal antiserum raised against the C-terminal 20 amino acids of human CNBP. This region has previously been used to generate a highly specific antiserum against CNBP that recognized both the human and mouse forms [38]. The antiserum was produced against the peptide coupled to KLH (Covance, PA). In some cases, the antiserum was pre-absorbed with 1 ␮g of the peptide immunogen. The T7A6 quadriceps were homogenized in RIPA buffer with a Complete Protease Inhibitor Cocktail (Amresco). The quadriceps and brain from the 3xTG and quadriceps from APP/PS1 mice were homogenized in 2% SDS with the Complete Protease Inhibitor Cocktail. The brains from APP/PS1 mice were homogenized in PBS with the Complete Protease Inhibitor Cocktail, and the pellet was subsequently extracted with 2% SDS. The proteins were separated by SDS-PAGE (either 10–20% Tris–HCl or 4–12% Bis–Tris) and were transferred to a nitrocellulose or PVDF membrane, blocked overnight with 1% BSA and 2% BlockAce and probed with our antiCNBP antiserum (1 h, room temperature; 1:1000 dilution in TBST with 5% nonfat dry milk), followed by a goat anti-rabbit IgG-HRP conjugate (1 h, room temperature; Rockland Immunochemicals). The membranes were then probed with an anti-GAPDH (Abcam) or anti-␤-actin (Sigma) antibody as a loading control. Triceps from T7A6 mice (N = 8 wild-type, 8 transgenic) were homogenized in TrizolTM (Invitrogen). Expression of CNBP was determined by two-step qRT-PCR, using iScript (BioRad) reverse transcription, followed by qPCR with PerfeCTa FastMixTM (Quanta). The geometric mean of the CT values for 18S rRNA, ␤-actin, and GAPDH was used as an internal control to calculate and compare relative expression (2−C T ). Gene specific primer sets were obtained from IDT. Densitometry was performed using Scion Image. ANOVA and multivariate analyses were performed using SPSS® for Windows. We developed an antiserum specific to the last 20 amino acids of the human and mouse CNBP protein (CYRCGESGHLARECTIEATA). This region is exceptionally well conserved and is 100% identical between humans and multiple other species, including but not limited to rodents, frogs and toads, and birds. In order to test the specificity of the antiserum, we transfected CHO cells with either pAG3-CNBP or pAG3-eGFP. CNBP was detected by Western blot with either pre-immune serum, antiserum, or antiserum preabsorbed with the peptide immunogen (Fig. 1A). The blot probed with pre-immune serum showed no detectable bands, while the blot probed with antiserum showed a single band running slightly above 20 kDa, approximately the predicted molecular weight of CNBP (19.6 kDa). This single band is more intense in the cells transfected with pAG3-CNBP and disappears when the antiserum is

Fig. 1. (A) Western blot of CHO cells transfected (0.1 ␮g) with either pAG3hyg-EGFP (−) or pAG3hyg-CNBP (+) overnight, probed with pre-immune serum, antiserum, or antiserum pre-absorbed with the peptide epitope of CNBP. The antiserum detects a single band running slightly above 20 kDa, which is augmented in cells overexpressing CNBP. This band was not detected by the pre-immune serum or following absorption with the CNBP peptide. (B) Western blot of mouse skeletal muscle, cardiac muscle, and brain proteins probed with the CNBP antiserum. A single band was detected, with more abundant expression in brain and cardiac muscle.

pre-absorbed with the immunogenic CNBP peptide. In an effort to determine tissue specificity and expression levels in vivo, we tested the antiserum on extracts of brain, cardiac muscle, and skeletal muscle from a C57Bl/6 mouse (Fig. 1B). While the antiserum is able to detect endogenous CNBP in all tissues tested, the strongest expression was detected in the brain. These data demonstrate that our antiserum can specifically detect the CNBP protein, both at endogenous and overexpressed levels, in cell culture and animal tissue. We next used this antiserum to examine CNBP protein expression in more detail. We extracted quadriceps from T7A6 sIBM mice and measured CNBP expression by Western blot (Fig. 2A). We observed a remarkable (∼90%; p < 0.0001; Fig. 2B) decrease in expression in the transgenic sIBM mice as compared with wild-type. We did not observe a change in brain (not shown), suggesting that the effect was due to expression of the transgene rather than an effect related to transgene insertion. In order to determine whether this change was due to transcriptional regulation, we measured

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Fig. 2. Quadriceps from T7A6 sIBM mice (IBM; N = 13) and their wild-type littermates (WT; N = 21) were homogenized, the proteins separated by SDS-PAGE and transferred to a nitrocellulose membrane. CNBP and GAPDH were visualized by Western Blot (A) and the resulting bands quantitated by densitometry using Scion Image. CNBP expression was standardized to GAPDH and subsequently standardized to WT mice (B). Though all animals tested were included in the densitometry analysis, we have shown only a representative Western Blot. APP overexpression in the muscle significantly reduces expression of CNBP (p < 0.0001). (C) Triceps from WT (N = 8) and sIBM (N = 8) mice were extracted in TrizolTM , and the RNA analyzed for CNBP mRNA expression using qRT-PCR. Expression was standardized to three housekeeping genes. There was no significant difference in CNBP expression between the WT and sIBM mice. Lysate from H4 cells was used as a positive control for expression.

CNBP mRNA expression in skeletal muscle from the T7A6 mice by qRT-PCR (Fig. 2C). There was no significant change in mRNA levels, suggesting that the observed affect is not due to APPinduced transcriptional regulation, but rather a translational or post-translational effect. In order to determine whether this change in CNBP expression was due to APP overexpression, we extracted protein from brain and quadriceps from several other mouse strains and compared them to their wild-type littermates (Fig. 3A). There was a reduction in CNBP expression in the quadriceps of the hybrid sIBM model that we generated in the lab (Fig. 3B), though this effect was less marked (∼13%). Like the T7A6 strain, these mice exhibit modest (1.3- to 1.5fold) overexpression of APPSwe in muscle (not shown). There was no effect in the brain (Fig. 3B). We also examined CNBP expression in the 3xTG [29] mice, which overexpress APPSwe specifically in the brain on a PS1 mutant knock-in background. There was no change in CNBP expression in muscle from the 3xTG mice, but a decrease in the brain was observed (Fig. 3B). CNBP expression was reduced in both brain and muscle from APP/PS1 knock-in mice (Fig. 3B), which harbor a whole-body knock-in of both APPSwe under its endogenous promoter [32]. Multivariate analysis of the standardized CNBP densitometry data revealed significant effects of strain, genotype, and a strain by genotype interaction (p ≤ 0.023). We observed a large reduction in the amount of CNBP protein expression in the skeletal muscle of the T7A6 mouse line, a proposed model of sIBM that overexpresses APPSwe in muscle. Reduced CNBP protein expression also occurs in the skeletal muscle of a hybrid line that also overexpresses APPSwe in skeletal muscle, the brain of the 3xTG mice, and in both brain and muscle of the wholebody knock-in APP/PS1 mice. Even with a limited sample size, it appears that the downregulation of CNBP expression occurs in tissue expressing APP containing the Swedish mutation (NL695), regardless of levels of APP expression or the presence of mutant PS1. The fact that the reduction in expression occurs across several different mouse strains suggests that the effect cannot be attributed solely to strain differences, although the magnitude of the reduction varies (90% in the T7A6 line, but only 13% in the hybrid derived from this line; Figs. 2B and 3B). The primary function of the APP holoprotein is unknown. APP is a type I transmembrane protein whose cleavage by the secretases yields both an extracellular, soluble APP (sAPP) and an intracellular C-terminal domain (AICD). Previous work suggests that sAPP has structural similarities to Kunitz-like serine protease inhibitors that

function in the regulation of thrombosis [41]. On the other hand, the AICD is theorized to translocate to the nucleus, where it possesses transcriptional regulatory ability in conjunction with other proteins [36], though its endogenous targets remain elusive. The mechanism of the APPSwe -mediated CNBP regulation is unknown, though it does not appear to be due to transcriptional regula-

Fig. 3. Brain and quadriceps from three mouse strains were homogenized and CNBP expression examined by Western Blot: (A) A T7A6 hybrid sIBM model, an APP/PS1 whole-body knock-in strain, and the 3xTG strain overexpressing APPSwe and tau in the brain in a PS1 knock-in. We examined both wild-type littermates (WT) and either transgenic (TG) or knock-in (KI) mice from each strain, where appropriate. (B) Densitometric analysis of CNBP expression, standardized first to ␤-actin (Brain) or GAPDH (Muscle) expression and then to the wild-type animals of each strain. CNBP expression is reduced in tissues expressing APPSwe .

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tion (Fig. 2C). The amount of CNBP knock-down does not seem to be dependent on the amount of APPSwe overexpression, however, since the strains with greater overexpression do not exhibit augmented reduction in CNBP. Additionally, the APP/PS1 knock-in strain, which does not overexpress APP, also exhibits a reduction in CNBP (13–30%; Fig. 3B) in both muscle and brain. These data imply that it is the presence of the Swedish mutation in APP rather than APP overexpression per se that causes the reduction and that this effect is via a translational or post-translational mechanism. Although no specific functional effect has been directly attributed to the Swedish mutation, it is possible that the preferential shift away from ␣-secretase and towards the ␤-secretase pathway has wide ranging consequences on cellular processes. For example, ␤secretase derived N-terminal fragments of APP may be important for activating caspases involved in neuronal pruning [28]. It is conceivable that these fragments have other functions. The consequences of CNBP knockdown in the muscle and brain of these mice are unclear. While there does not appear to be any gross abnormalities in the muscle, the T7A6 transgenic mice exhibit impaired motor function [8]. Whether this is due to A␤ accumulation or another factor of APP overexpression is not known. APP is often overexpressed in human sIBM cases [19,33], though A␤ accumulates as well [5,6]. It is unknown whether CNBP expression is altered in these cases, and this may be something to examine in the future. Overexpression of APPSwe in the brain of the 3xTG mice, as well as the knock-in APP/PS1 mice, induces substantial accumulation of A␤ [27,29,32]. In all cases, there is, at least, mild inflammation and some degeneration. Further studies will be needed to dissociate the role of these two processes on CNBP expression. Finally, it is possible that these mice could prove to be a useful tool in further study of CNBP. For example, a targeted replacement of CNBP in skeletal muscle or brain, followed by a gene expression or proteomic analysis could be used to identify unknown targets. Interestingly, the mutation used in these mouse strains (NL695) occurs naturally in many familial AD cases [25]. Given the data presented herein, tissue from these individuals may be a good resource in which to study CNBP. Acknowledgements Supported by NIH grants NS058382, AG005119, and RR020171. We would like to thank Dr. Frank LaFerla for the T7A6 mice, and Dr. Salvatore Oddo for the 3xTG mouse tissue. We would like to thank Dr. Christa M. Studzinski for the inception and preliminary data for the diet studies, and Marjorie Rochette for assistance in characterizing the CNBP antiserum. References [1] Y. Abe, W. Chen, W. Huang, M. Nishino, Y.P. Li, CNBP regulates forebrain formation at organogenesis stage in chick embryos, Dev. Biol. 295 (2006) 116–127. [2] P. Armas, T.H. Aguero, M. Borgognone, M.J. Aybar, N.B. Calcaterra, Dissecting CNBP, a zinc-finger protein required for neural crest development, in its structural and functional domains, J. Mol. Biol. 382 (2008) 1043–1056. [3] P Armas, S. Cachero, V.A. Lombardo, A. Weiner, M.L. Allende, N.B. Calcaterra, Zebrafish cellular nucleic acid-binding protein: gene structure and developmental behaviour, Gene 337 (2004) 151–161. [4] P. Armas, S. Nasif, N.B. Calcaterra, Cellular nucleic acid binding protein binds Grich single-stranded nucleic acids and may function as a nucleic acid chaperone, J. Cell. Biochem. 103 (2008) 1013–1036. [5] V. Askanas, W.K. Engel, Inclusion-body myositis: a myodegenerative conformational disorder associated with Abeta, protein misfolding, and proteasome inhibition, Neurology 66 (2006) S39–48. [6] V Askanas, W.K. Engel, R.B. Alvarez, Light and electron microscopic localization of beta-amyloid protein in muscle biopsies of patients with inclusion-body myositis, Am. J. Pathol. 141 (1992) 31–36. [7] V. Askanas, J. McFerrin, S. Baque, R.B. Alvarez, E. Sarkozi, W.K. Engel, Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle, Proc. Natl. Acad. Sci. U.S.A. 93 (1996) 1314–1319.

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