Transgenic expression of intraneuronal Aβ42 but not Aβ40 leads to cellular Aβ lesions, degeneration, and functional impairment without typical Alzheimer's disease pathology.

Neurobiology of Disease Transgenic Expression of Intraneuronal A ␤ 42 But Not A ␤ 40 Leads to Cellular A ␤ Lesions, Degeneration, and Functional Impairment without Typical Alzheimer’s Disease Pathology Dorothee Abramowski, 1 * Sabine Rabe, 1 * Ajeet Rijal Upadhaya, 2 Julia Reichwald, 1 Simone Danner, 1 Dieter Staab, 1 Estibaliz Capetillo-Zarate, 3 Haruyasu Yamaguchi, 4 Takaomi C. Saido, 5 Karl-Heinz Wiederhold, 1 Dietmar Rudolf Thal, 2 and Matthias Staufenbiel 1 1 Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland, 2 Institute of Pathology–Laboratory of Neuropathology, University of Ulm, 89081 Ulm, Germany, 3 Weill Medical College of Cornell University, New York, New York 10021, 4 Gunma University School of Health Sciences, Gunma 371-8514, Japan, and 5 Laboratory of Proteolytic Neuroscience, RIKEN Brain Science Institute, Saitama 351-0198, Japan An early role of amyloid- ␤ peptide (A ␤ ) aggregation in Alzheimer’s disease pathogenesis is well established. However, the contribution of intracellular or extracellular forms of A ␤ to the neurodegenerative process is a subject of considerable debate. We here describe transgenic mice expressing A ␤ 1–40 (APP47) and A ␤ 1–42 (APP48) with a cleaved signal sequence to insert both peptides during synthesis into the endoplasmic reticulum. Although lower in transgene mRNA, APP48 mice reach a higher brain A ␤ concentration. The reduced solubilityandincreasedaggregationofA ␤ 1–42 mayimpairitsdegradation.APP48micedevelopintracellularA ␤ lesionsindendritesand lysosomes. The hippocampal neuron number is reduced already at young age. The brain weight decreases during aging in conjunction with severe white matter atrophy. The mice show a motor impairment. Only very few A ␤ 1–40 lesions are found in APP47 mice. Neither APP47 nor APP48 nor the bigenic mice develop extracellular amyloid plaques. While intracellular membrane expression of A ␤ 1–42 in APP48micedoesnotleadtotheAD-typicallesions,A ␤ aggregatesdevelopwithincellsaccompaniedbyconsiderableneurodegeneration. Introduction Various lines of evidence point to a central role of the amyloid- ␤ peptide (A ␤ ) in the development of Alzheimer’s disease (AD) (for review, see Citron, 2010). Although the disorder is etiologically heterogeneous, aggregation of A ␤ appears as an early pathogenic event common to all forms of AD. Aggregated A ␤ shows no overt acute toxicity in vivo in accordance with the slow progression of this chronic neurodegenerative condition (Jack et al., 2010). In human brain, A ␤ deposits may persist for extended periods of time until clinical symptoms become evident. Amyloid plaque-forming ␤ -amyloid precursor protein (APP) transgenic mouse models of AD show correspondingly little neurodegeneration during their life span. A ␤ aggregates can affect neuronal processes at multiple levels, which may lead to a slow decompensation of functionally connected networks (Palop and Mucke, 2010). The molecular structure of the pathogenic species remains a matter of considerable debate. Both amyloid plaques, one of the pathological hallmarks of AD, as well as oligomeric forms of A ␤ have been implicated as pathogenic (Shankar et al., 2008; Nimmrich and Ebert, 2009). It also remains unclear to what extent intracellular and extracellular A ␤ aggregates contribute to pathogenesis (Gouras et al., 2010). Recently, transgenic mice have been described expressing either of the two major A ␤ isoforms, A ␤ 1–40 and A ␤ 1–42 , fused to the C terminus of the BRI protein (McGowan et al., 2005). Cleavage of the fusion proteins at a furin site leads to efficient secretion of A ␤ peptides. These animals demonstrated that A ␤ 1–42 but not A ␤ 1–40 is sufficient to promote A ␤ deposition in vivo. Overt toxicity, however, has not been found, suggesting that intracellular species might be responsible. To address this question, we have generated transgenic mice expressing intracellular A ␤ 1–40 and A ␤ 1–42 . The peptides are preceded by a cleaved N-terminal signal sequence to cotranslationally insert them into the endoplasmic reticulum. Both transgenic lines do not develop extracellular amyloid plaques, but A ␤ 42 mice (APP48) show intracellular A ␤ lesions. Additionally, hippocampal neurons and white matter are reduced along with a motor impairment indicating neurodegeneration in the absence of typical AD pathology. Materials and Methods Animal studies. A cDNA fragment encoding the rat preproenkephalin signal peptide (SPENK) was amplified from a rat brain cDNA library and ligated to cDNAs encoding human A ␤ 1–40 or A ␤ 1–42 , followed by a TAG stop codon. The resulting SPENK-A ␤ 40 or SPENK-A ␤ 42 cDNA was Received Sept. 8, 2011; accepted Dec. 2, 2011. Authorcontributions:D.A.,S.R.,J.R.,K.-H.W.,D.R.T.,andM.S.designedresearch;D.A.,S.R.,A.R.U.,J.R.,S.D.,D.S., andK.-H.W.performedresearch;E.C.-Z.,H.Y.,andT.C.S.contributedunpublishedreagents/analytictools;D.A.,S.R., A.R.U., J.R., S.D., D.S., K.-H.W., D.R.T., and M.S. analyzed data; D.R.T. and M.S. wrote the paper. This work was supported by Deutsche Forschungsgemeinschaft Grant TH624/6-1 and Alzheimer Forschung Initiative Grant 10810 (D.R.T.). We gratefully thank Irina Kosterin, Domenico Ammaturo, and Andre Schade for technical help. We also acknowledge Dr. Laura Jacobson for help with statistical analyses. The authors declare no competing financial interests. *D.A. and S.R. contributed equally to this work. Correspondence should be addressed to Dr. Matthias Staufenbiel, Novartis Pharma AG, Forum 1, Novartis Campus, CH-4056 Basel, Switzerland. E-mail: [email protected]. DOI:10.1523/JNEUROSCI.4586-11.2012 Copyright © 2012 the authors 0270-6474/12/321273-11$15.00/0 The Journal of Neuroscience, January 25, 2012 •32(4):1273–1283 • 1273 cloned into the pTSC21 plasmid for expression under the control of the mouse Thy-1 promoter. The same promoter was used to express APP with the KM670/671NL “Swedish” mutation in APP23 mice as described previously (Sturchler-Pierrat et al., 1997). The mice were on a C57BL/6 background and hemizygous for the transgene. They were killed, and tissues were prepared as described previously (Abramowski et al., 2008). All animal experiments were in compliance with protocols approved by the Swiss Animal Care and Use Committees. Biochemical assays. Brain samples were processed and analyzed for A ␤ peptides [immunoprecipitation of A ␤ and Western blotting or matrixassisted laser desorption ionization time-of-flight (MALDI-TOF), electrochemiluminescence-linked immunoassay (MSD 96-Well MultiArray Human (6E10) A ␤ 40 or A ␤ 42 Ultra-Sensitive kits; Meso Scale Discovery)] as described previously (Abramowski et al., 2008). Sequential A ␤ extraction and immunoprecipitation. For Triton X-100 (TX-100) (93418; Fluka/Sigma-Aldrich) extraction, forebrain homogenates were extracted with 1% TX-100 in TBS–Complete for 15 min on ice and ultracentrifuged (100,000 ⫻g, 4°C, 15 min), and the clear supernatants were immunoprecipitated as described below. The pellets were used for further SDS extraction. Subsequently, TX-100 pellets were extracted with SDS in TBS–Complete for 15 min at room temperature either with 1 or 2% SDS. The extracts with 1% SDS were diluted after extraction to 0.1% final SDS concentration with TBS–Complete. After ultracentrifugation (100,000 ⫻g, 4°C, 15 min), immunoprecipitation from the clear supernatant was done with 6E10 and the pellets were used for further formic acid extraction. Extracts with 2% SDS were ultracentrifuged (100,000 ⫻g, 20°C, 15 min), and the supernatants were subsequently diluted to a final 0.1% SDS concentration. These SDS extracts and formic acid-extracted pellets were immunoprecipitated with 4G8 (SIG-39200; Covance) only. SDS pellets were extracted with 70% formic acid for 15 min at room temperature, neutralized with 19 vol (v/v) 1 MTris-base/1% TX-100/Complete and ultracentrifuged (100,000 ⫻g, 4°C, 15 min). The clear supernatant was used for immunoprecipitation, and the pellet was discarded. A ␤ standards were prepared by spiking synthetic A ␤ peptides to nontransgenic forebrain extracts processed the same as described for the samples. All extracts were either immunoprecipitated with 6E10 (SIG-39300; Covance) bound to Dynabeads Protein G (100.03; Invitrogen) or 4G8 (SIG-39200; Covance) bound to Protein G-Sepharose 4 Fast Flow (170618-01; GE Healthcare Life Sciences) and incubated overnight at 4°C on end-over-end rotor. After incubation, the supernatants were removed, and the Dynabeads were washed with TBS–Complete/1% NP-40, then with 10 mMTris-HCl, pH 7.5, and finally with 1 mMTris-HCl, pH 7.5. Sepharose beads were washed once with 20 mMTris-HCl, pH 7.5. Samples were boiled with sample buffer for 5 min at 95°C and analyzed on Western blot. Sequential immunoprecipitation. For sequential immunoprecipitation, forebrain homogenates were extracted with 1% TX-100 as described above. To the extract, ␣ A ␤ (N3pE) antibody (18591; IBL)-bound Dynabeads Protein G were added and incubated overnight at 4°C on end-overend rotor. After incubation, the supernatants were transferred to fresh tubes and the extracts were immunoprecipitated a second time with 6E10 antibody bound to Dynabeads Protein G as described above. The beads from both immunoprecipitations were processed the same as described above. Western blot. For A ␤ peptide determination on Western blot, forebrain homogenates were separated on a 13% Tris-bicine gel with 8 Murea as described previously (Klafki et al., 1996; Staufenbiel and Paganetti, 2000). In this gel system, the different A ␤ peptides are well separated. Proteins were transferred to Immobilon-P membranes (Millipore). A ␤ peptides were heat fixed to the membrane by boiling for 3 min in PBS (P4417; Sigma-Aldrich). A ␤ peptides were detected with 6E10 (SIG39300; Covance) or N3pE A ␤ with ␣ A ␤ (N3pE) antibody (18591; IBL). Proteins were detected by visualizing chemiluminescence (ECL Advance or ECL Plus; GE Healthcare) on autoradiographic films (Hyperfilm ECL; GE Healthcare). In situ hybridization. The spatial distribution pattern of SPENK-A ␤ 40 or SPENK-A ␤ 42 transgene expression was determined by in situ hybridization (Sturchler-Pierrat et al., 1997) with a 33 P-labeled oligonucleotide (5⬘- CGCCCACCATGAGTCCAATGATTGCACCTTTGTTTGAACC-3⬘). The probe binds entirely within the A ␤ -coding part. It contains four mismatches compared with the mouse APP sequence and did not cross-react with mouse APP RNA. RNA quantification. Total RNA extraction, cDNA synthesis, and realtime PCR gene expression analysis and quantification were done as described by Reichwald et al. (2009). TaqMan Gene Expression Assays were ordered from Eurogentec (18s rRNA control kit FAM-TAMRA; RTCKFT-18s) or designed (SPENK40/42F1: CAG AGG AAG GAC CTC GAA GCT; SPENK40/42R1: AAC AAA GGT GCA ATC ATT GGA CT; MGB Taq40: FAM-TCG ACC TAG ACA ACA CC-MGBNFQ; MGB Taq42: FAM-TCG ACC TAC GCT ATG ACA-MGBNFQ). Real-time PCR quantifications were run in triplicate for each sample and the average determined. Mice were analyzed in groups of 10 per genotype. Neuropathology and immunocytochemistry. Tissue fixation, sectioning, and processing were done as described previously (Sturchler-Pierrat et al., 1997; Abramowski et al., 2008). Conventional silver staining for axonal neurofilaments was performed with the Bodian method. The Campbell–Switzer silver impregnation was used to stain fibrillar A ␤ with high sensitivity (Braak and Braak, 1991; Thal et al., 1999). Immunohistochemistry was performed for the detection and quantification of A ␤ pathology in APP48. Before the use of monoclonal mouse antibodies, 100- ␮ m-thick free-floating sections were incubated with goat anti-mouse IgG for blocking intrinsic mouse IgG (Thal et al., 2007). To detect A ␤ 1–42 -positive material, the sections were stained with monoclonal antibodies specifically detecting the C terminus of A ␤ 42 [MBC42 (Yamaguchi et al., 1998); 1/200; formic acid pretreatment; 24 h at 22°C] or with an antibody raised against A ␤ 17–24 (4G8; Covance; 1/5000; formic acid pretreatment; 24 h at 22°C), with an antibody directed against the N terminus of A ␤ 1–42 [A ␤ N1D (Saido et al., 1995); polyclonal rabbit; 1/100; formic acid and microwave pretreatment], and with anti-A ␤ N3pE (polyclonal rabbit; IBL; 1/100; formic acid and microwave pretreatment). To exclude tau and TDP43 pathology, an antibody against abnormal phosphorylated tau protein (AT-8; monoclonal mouse; Thermo Fisher Scientific; 1/1000; 24 h at 22°C) and an antibody against phosphorylated TDP43 (pTDP43: pS409/410-2; Cosmo Bio; 1/10,000; microwave pretreatment) were used. Astrocytes were labeled with an antibody directed against the glial fibrillary acidic protein (GFAP) (polyclonal rabbit; Dako; 1/1000; 24 h at 22°C), microglial cells with Ricinus communis agglutinin (RCA) (Vector Laboratories; 1/250; 24 h at 22°C). To test whether APP was present in A ␤ aggregates or in plaque-associated dystrophic neurites, antibodies directed against APP were used (22C11; monoclonal mouse; Millipore Bioscience Research Reagents; 1/75; 24 h at 22°C). To identify abnormalities in the neuronal network, sections of each mouse were stained with antibodies against 68 kDa subunits of neurofilament protein (NF-L; SPM 204; Zytomed; 1/100; microwave pretreatment; 24 h at 22°C) and synaptophysin (polyclonal rabbit; Dako; 1/1000; microwave pretreatment). The primary antibodies were detected with a biotinylated secondary antibody and the ABC complex (Biomeda), and visualized with diaminobenzidine-HCl (DAB) (Hsu et al., 1981). Sections were mounted in Eukitt (Kindler). Biotinylated RCA was detected with the ABC complex and visualized with DAB. For double immunofluorescence, 20- ␮ m-thick free-floating sections were incubated with rabbit A ␤ antiserum NT11 and monoclonal antibody (clone AP20; Millipore Bioscience Research Reagents) against microtubule-associated protein 2 (MAP2) as dendritic marker. Alternatively, CD45 monoclonal antibody MCA1031G (Serotec) was used to label microglia cells. Primary antibodies were detected with horseradish peroxidase (HRP)-labeled anti-rabbit IgG (Dako) and HRP-labeled antimouse IgG (Dako) secondary antibodies. Signal Amplification has been done by applying Cy3or FITC-conjugated Tyramide (NEL741; PerkinElmer). To determine the intracellular location of A ␤ -reactive structures, labeling with A ␤ antibody 4G8 was colocalized with antibody labeling of different compartmental markers: LAMP-1 (ab62562; Abcam) for late endosomes/lysosomes, EEA1 (ab2900; Abcam) for early endosomes, BiP (anti-KDEL; SPA-827; Stressgen) for post-endoplasmatic reticulum compartments, and TIA-1/TIAR(D-9) (sc-48371; Santa Cruz) for stress granules. A ␤ was detected with Cy2-labeled secondary antibodies, 1274 •J. Neurosci., January 25, 2012 •32(4):1273–1283 Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 whereas the compartmental markers were detected with Cy3-labeled secondary antibodies. In the event that single-label immunohistochemistry was performed on paraffin sections, a counterstaining with hematoxylin was applied. The immunostained sections were analyzed with a Leica DMLB fluorescence microscope (Leica). Quantification of A ␤ pathology in APP48 was performed in the area of the frontocentral neocortex. Stereology. Six APP48 and six wild-type mice, 18 months of age, were used for stereology. One hundred-micrometer-thick coronal sections were stained with the aldehyde fuchsin–Darrow red method exhibiting a Nissl-like staining pattern of the neurons and the pigment architecture for anatomical parcellation (Braak, 1974). The frontocentral cortex volume for stereology was defined as the volume of the subfields M2, M1, S1 starting at the level of the anterior commissure as described previously (Capetillo-Zarate et al., 2006). The CA1 volume was measured in serial 100- ␮ m-thick sections. Quantification of neurons was performed for the frontocentral cortex and the hippocampal sector CA1, separately, according to the principles of unbiased stereology (Schmitz and Hof, 2000). The number of specific types of A ␤ aggregates in the frontocentral neocortex was counted in accordance with the principles of unbiased stereology. The relative volume of the forebrain white matter was determined by measuring the area of the Luxol fast blue (LFB)-stained forebrain white matter and the total area of the forebrain in the same section. The percentage of the hemispheres covered by white matter was calculated as follows: Forebrain white matter volume (%) ⫽(forebrain area stained with LFB ⫻ 100)/total forebrain area. Electron microscopy and immuno-electron microscopy. One 100- ␮ m-thick vibratome section of the frontocentral cortex of six 18month-old wild-type and APP48/167 mice was stained with osmium tetroxide and flatembedded in Epon (Fluka). A second vibratome section was also stained with osmium tetroxide and then flat embedded in LR-WhiteResin (Hard-Grade Acrylic Resin; London Resin Company). A part of the frontocentral cortex covering all six cortical layers was dissected under microscopic control and pasted on Epon blocks with a drop of Epon. Ultrathin sections were cut at 70 nm. Epon sections were block stained with uranyl acetate and lead citrate, and viewed with a Philips EM400T 120 kV. LR-White sections were immunostained with anti-MAB42 antibodies and visualized with anti-mouse secondary antibodies (Aurion ImmunoGold Reagents & Accessories) labeled with 10 nm nanogold particles. Digital pictures were taken. Rotarod test. To measure motor coordination, 5to 7-month-old mice were placed on a horizontal cylinder (Ugo Basile; treadmill for mice Typ 7600) rotating at 13 rounds per minute. The time until the mice fell off the cylinder was measured. Three trials were performed on consecutive days. Trials were terminated after a maximum of 120 s. Statistical analyses. For statistical analysis, we used Student’s ttests (two-tailed) or ANOVA followed by Tukey’s test for pairwise comparison of all groups as indicated in the figure legends. Rotarod data were Figure 1. APP47 and APP48 transgenes and brain mRNA expression. A, Schematic representation of the APP47 and APP48 expression constructs. The box represents the translated sequence comprising the cleaved N-terminal signal sequence SPENK (signal peptide preproenkephalin) followed by A ␤ 1–40 or A ␤ 1–42 . The gray C-terminal end denotes the hydrophobic amino acid stretchofA ␤ ,whichis approximatelyone-halfof theAPPtransmembrane region(locatedin the luminalleaflet of themembrane bilayer). B,In situ hybridization to locate the transgene expression in 2-month-old APP47 and APP48 mouse brain. C, Relative transgenemRNAlevels inforebrainof femaleAPP47,APP48, and APP47⫻APP48 (47 ⫻48)miceas determinedbyquantitative PCR. Animals were 2 months of age. Differences between A ␤ 1–40 and A ␤ 1–42 mRNAs were statistically significant (Student’s t test, two-tailed, p⬍0.0001), whereas the same mRNAs did not differ significantly (p⬎0.1) between singleand doubletransgenic mice. Error bars indicate SEM. Figure 2. Characterization of human A ␤ 40 and A ␤ 42 peptides in brain. A, Western blot of forebrain homogenates from representative A ␤ -expressing mice at 2 months. Female mice are shown, but results were similar for males. Homogenates were dissolved in SDS-sample buffer and run on a SDS/urea gel to separate A ␤ 1–40 and A ␤ 1–42 . The gel was overloaded to improve detection. Synthetic A ␤ peptides spiked into a nontransgenic mouse brain homogenate are shown on the left. Note that A ␤ 1–40 andA ␤ 1–42 blotwithdifferentefficiencyandcannotbecompareddirectly.B,Formicacid-extractedtotalA ␤ wasquantifiedbyan electrochemoluminescence assay (MSD). Significant differences (Student’s ttest, two-tailed) are indicated by asterisks: ***p⬍ 0.001. One-way ANOVA and Tukey’s test showed a significant difference between A ␤ 40 and A ␤ 42 in APP47 versus APP48 mice (p⬍0.001) but not within the double-transgenic animals (47 ⫻48; Student’s ttest, two-tailed, p⫽0.83). Error bars indicate SEM.C,MALDIspectraofimmunoprecipitateswithA ␤ antibody6E10frombrainextractsofAPP47andAPP48miceshowedpeaks attheexpectedmolecularweightofA ␤ 1–40 andA ␤ 1–42 ,respectively.D,Forebrainhomogenatesof2-and21-month-oldAPP47, APP48,andAPP47⫻APP48(47 ⫻48)micewereimmunoprecipitatedwithA ␤ antibody6E10,separatedona SDS/ureagel,and detected by Western blotting using an N3pE (pyroglutamate; upper panel) or general (6E10; lower panel) A ␤ antibody. The two fastermigratingbandsareindicatedbyarrowheads.PyroglutamateA ␤ wasdetectedasaminorportionofthefastestbandinaged double-transgenic mice only. The migration positions of synthetic standards are indicated on the right. Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 J. Neurosci., January 25, 2012 •32(4):1273–1283 • 1275 analyzed by Mann–Whitney Utest followed by Tukey’s test; p⬍0.05 was considered significant for all tests; analyses were done with Systat for Windows 11 (Systat Software) or SPSS 16.0 (SPSS). Results Within APP, the N terminus of the A ␤ peptide is located in the lumen of the intracellular membrane systems, while its C terminus resides in the center of the transmembrane region (Kang et al., 1987). To insert human A ␤ 1–40 or A ␤ 1–42 in the same membrane orientation during translation at the endoplasmic reticulum, cDNA constructs were made encoding the rat preproenkephalin signal sequence (SPENK) in front of both peptides (Fig. 1A). In vitro translation of these constructs indicated signal sequence cleavage in the presence of microsomes accompanied by an association of the A ␤ peptides with the membrane vesicles (data not shown). Studies with transfected HEK cells had shown approximately equal amounts of A ␤ 1–40 remaining associated with cells and released into the culture medium. In contrast, A ␤ 1–42 largely remained cell associated as also noted by others (Maruyama et al., 1995) (our unpublished data). The murine Thy-1 promoter (Lu¨thi et al., 1997) was used to drive neuronal expression in brain. For both constructs, the lines with the highest brain A ␤ concentration were selected for further studies, APP47 (A ␤ 1–40 ) and APP48 (A ␤ 1–42 ). A ␤ expression in APP47 and APP48 mice The spatial transgene expression pattern in brain was analyzed by in situ hybridization (Fig. 1B). For both APP47 and APP48 mice, prominent labeling was found in cerebral cortex and hippocampus as expected for the Thy-1 promoter. Other regions including thalamus, cerebellum, and some subcortical nuclei also showed substantial expression. Relative transgene mRNA concentrations in forebrain of young (2month-old) APP47 and APP48 mice are shown in Figure 1C. They were approximately threefold higher for APP47 than for APP48. In double transgenic mice, the expression of both constructs remained unchanged indicating that coexpression did not result in detectable interference of the transgenes. Young mice were analyzed for A ␤ peptides using Western blotting of forebrain homogenates dissolved in SDS-sample buffer (Fig. 2A). In contrast to the corresponding mRNA, A ␤ 1–42 reached a considerably higher level than A ␤ 1–40 , in agreement with its reduced clearance following brain injection of synthetic peptides (Ji et al., 2001). Quantification of the A ␤ peptides after formic acid extraction indicated a ⬃10-fold higher steady-state concentration of A ␤ 42 than A ␤ 40 (Fig. 2B). Interestingly, in APP47 ⫻APP48 mice, A ␤ 42 was reduced by ⬃75% compared with single transgenic mice, while A ␤ 40 was elevated ⬃2.5-fold. The absolute concentrations of both peptides were very similar, suggestive of an interaction. The identity of the A ␤ peptides in APP47 and APP48 mice was confirmed by MALDI-TOF analysis following immunoprecipitation of SDS-dissolved forebrain extracts (Fig. 2C). The molecular weights determined for the A ␤ peaks in both transgenic lines were in agreement with the full-length A ␤ 1–40 and A ␤ 1–42 peptides, respectively. These data demonstrate the proper cleavage of the signal sequence. No other A ␤ peptides were detectable. SDS gels in addition showed one or two faster migrating bands, most notably in APP47 ⫻APP48 mice, which varied in amount but always remained minor forms (Fig. 2D). The upper band comigrated with A ␤ 2–42 and the lower one with A ␤ 4–40/42 and pyroglutamate A ␤ (N3pEA ␤ 3–40/42 ). To further characterize the lower band, A ␤ was immunoprecipitated from forebrain homogenates followed by Western blotting with an N3pEA ␤ antibody. Only Figure 3. Age-dependent changes and solubility characteristics of brain A ␤ in APP47 and APP48 mice. Formic acid-extracted total A ␤ 40 and A ␤ 42 in cerebral cortex was analyzed at the indicated ages using electrochemoluminescence assays. A, The A ␤ 42 concentration in APP48 brain significantly increased with age (Student’s ttest vs 1 month, two-tailed) as indicated by asterisks: *p⬍0.05,**p⬍0.01,***p⬍0.001.Linearregressionanalysis(regressionANOVAF (1,32) ⫽317.7,p⬍0.001)indicatedageas a strong and linear determinant of A ␤ 42 .B, The A ␤ 40 concentration in APP47 mouse brain remained unchanged (p⬎0.05, Student’s ttest, two-tailed, and linear regression). C, Compared with APP47, A ␤ 40 was considerably elevated in aged APP47 ⫻ APP48mice,whereasA ␤ 42 wasnotconsistentlydifferentfromAPP48(Student’sttestvssingle-transgenicmice,two-tailed).Error bars indicate SEM. D, Forebrain homogenates of 1.5and 21-month-old APP47, APP48, and APP47 ⫻APP48 (bi) mice were sequentiallyextracted with1%TritonX-100,2%SDS,and 70%formic acid.Extracts wereimmunoprecipitatedwithantibody4G8 and analyzed by Western blotting with antibody 6E10, both directed against A ␤ . A gel without urea was used, which does not separate A ␤ 1–40 and A ␤ 1–42 . The faster migrating band corresponds to the truncated A ␤ isoforms separated on SDS/urea gels (see above). This band is primarily found in the Triton and SDS extracts from young mice. An extract from a young APP transgenic mouse (APP23) is shown for comparison. Table 1. Average forebrain weight of APP47, APP48, and double-transgenic mice at 2 and 21 months of age Age group Wild type APP47 APP48 APP47 ⫻APP48 2 months Forebrain weight (mg) a 164 ⫾10 165 ⫾9 152 ⫾10 152 ⫾7 p(vs wild type)* NS 0.006 0.002 21 months Forebrain weight (mg) a 161 ⫾10 154 ⫾9 132 ⫾8 131 ⫾10 p(vs wild type)* NS ⬍0.0001 ⬍0.0001 a Shown are mean ⫾SD. *Student’s ttest, two-tailed. NS, Nonsignificant. 1276 •J. Neurosci., January 25, 2012 •32(4):1273–1283 Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 after prolonged exposure was a band detectable in 21-month-old APP47 ⫻ APP48 mouse brains but not young double transgenic or the single-transgenic brains. In contrast, A ␤ antibody 6E10 recognized the lower band to almost the same extent in young and old animals. Sequential immunoprecipitation with the N3pE followed by the 6E10 A ␤ antibodies confirmed that only a minor A ␤ fraction in this band contained pyroglutamate (data not shown). The main portion of the lowest band most likely corresponded to A ␤ 4–40/42 in agreement with the lack of detection by antibody ␤ 1 against the A ␤ 3–6 epitope (data not shown). Age-related increase of insoluble A ␤ 42 but not A ␤ 40 To estimate the overall changes with age, total A ␤ 42 in APP48 mice was analyzed after formic acid extraction of the cerebral cortex. An approximately threefold increase was found between 1 and 19 months of age (Fig. 3A). By contrast, A ␤ 40 in APP47 mice remained at a similar level during aging (Fig. 3B). In aged double-transgenic APP47 ⫻ APP48 mice, A ␤ 40 increased considerably compared with APP47 alone, while A ␤ 42 reached the same level as found in APP48 (Fig. 3C). These results and the discrepancy between mRNA and protein steady-state levels of A ␤ 40 and A ␤ 42 prompted us to analyze their solubility. Whereas Triton X-100 extraction almost completely solubilized A ␤ 40 from APP47 brains, A ␤ 42 in APP48 brain remained largely in the insoluble pellet after Triton X-100 and SDS extraction (data not shown). For more systematic analysis, forebrains from young andaged animals (1.5 and 21 months ofage) were sequentially extracted with Triton X-100, SDS, and formic acid and analyzed by Western blotting (Fig. 3D). Regardless of the age, the initial Triton X-100 treatment completely extracted A ␤ 40 from APP47 brain. In contrast, Triton X-100 hardly dissolved any A ␤ 42 either from young or aged APP48 brains. A ␤ 42 mostly distributed betweenthe SDS and formic acid extracts. This is consistent with the very low level of A ␤ 42 secretion from cells transfected with the same construct. Young APP47 ⫻APP48 mice showed increased A ␤ about equally distributed between the Triton X-100and SDS-soluble fractions. A ␤ in the SDSinsoluble material was hardly detectable. It increased considerably in this and the SDS fraction at old age, while the Triton X-100soluble A ␤ remained constant or decreased slightly. The complete extraction of the A ␤ peptides in every step required large buffer Figure 4. Macroscopic analysis of brains from APP48 mice. The forebrain of APP48 (B) mice was ⬃10% smaller than that of wild-type mice (A) as measured by the bi-hemispheric diameter, which is indicated as black bar below the brain section. The relativeforebrainwhite matter volume wasdecreasedin APP48 miceasseen morphologically inthecentral white matterandthe corpus callosum (B, stars) and as documented by quantitative assessment (Student’s ttest, two-tailed, **p⬍0.01; C). D,E, The spinalcord wasalso⬃10%smallerin APP48mice(E)thaninwild-typemice (D).However,morphologicalchangesandespecially white matter loss did not become obvious. Figure5. N3pEA ␤ staininginAPP47 and APP48 mice.Neocorticalsections from 18-month-oldAPP47(A–C) and APP48(D,E) mice were immunostained with antibodies specific for A ␤ 40 (A,D), A ␤ 42 (B,E), or N3pEA ␤ (C,F). As expected, APP47 brains were stained with A ␤ 40 but not A ␤ 42 antibodies, whereas APP48 reacted with A ␤ 42 but not A ␤ 40 antibodies. Pyroglutamate was found in neuropil A ␤ grains and few dendritic A ␤ treads but not in somatic A ␤ granules of APP48 but not in APP47 mice. Scale bar, 15 ␮ m. Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 J. Neurosci., January 25, 2012 •32(4):1273–1283 • 1277 volumes and 2% SDS/sonication in the second step. Smaller extraction volumes or less harsh SDS treatment reduced the A ␤ peptides in the corresponding fractions with a concomitant increase in the following extracts (data not shown). Independent of the extraction conditions, these data demonstrate an age-related increase of insoluble A ␤ 42 . Such an increase did not occur with A ␤ 40 alone but was detectable in the presence of A ␤ 42 (Fig. 3D). A ␤ from a young APP23 transgenic mouse (APP with Swedish mutation) analyzed in parallel distributed between all fractions. Distinct neuropathology after intracellular A ␤ 42 expression The forebrain weights of the different mouse lines were compared at 2 and 21 months of age. No significant difference from wild-type mice was found for APP47. However, APP48 mice showed a reduction in forebrain weight at 2 months, which became more pronounced with age. The same reduction was observed for doubletransgenic mice of the corresponding age groups (Table 1). Further investigation of APP48 mice showed a reduced bihemispheric diameter in forebrain compared with wild-type animals (Fig. 4A,B). Quantitative analysis demonstrated a ⬃37% reduction in white matter volume (Fig. 4C). A slightly reduced overall size (⬃10%) was observed for the spinal cord (Fig. 4D,E) without other obvious changes. Analyses of APP47 brains by A ␤ immunohistochemistrydemonstrated weak staining of A ␤ 40 granules in the soma of neurons (Fig. 5A–C). N3pEA ␤ (pyroglutamate-A ␤ ) was not detected. Amyloid plaques or other signs of histopathology were not found even at the oldest age analyzed (24 months). Immunohistochemistry did not detect amyloid plaques in APP48 mice even at the age of 24 months. Instead, three types of lesions were stained with different A ␤ antibodies (MBC42, 4G8, NT11, A ␤ N1D): (1) dendrites filled with A ␤ -positive material of thread-like appearance (dendritic A ␤ threads), (2) dot-like granules in the soma of nerve cells (somatic A ␤ granules), and (3) grain-like structures in the neuropil (A ␤ grains) (Figs. 5E,6A–G). The three types of A ␤ lesions were found throughout the entire gray matter of the CNS. Their distribution varied among different CNS regions with the most severe pathology in neocortical and allocortical areas (Table 2). A ␤ threads were less frequently found in the brainstem and cerebellum and were not seen in the spinal cord, whereas A ␤ granules were found in all these regions (Fig. 6I,J). Staining with an N3pEA ␤ antibody visualized A ␤ grains, few dendritic A ␤ threads, but no somatic A ␤ granules (Fig. 5E,F). A ␤ -positive lesions in APP48 mice were not stained with an A ␤ 40 antibody (Fig. 5D). Double-label immunohistochemistry corroborated the presence of A ␤ in MAP2-positive dendrites of APP48 mice (Fig. 6H). Campbell–Switzer silver staining indicated a fibrillar structure of dendritic A ␤ with the pattern of threads (Fig. 7A). Ultrastructurally, the dendritic A ␤ -positive material showed a fibril-like appearance (Fig. 7B,C). Axonal A ␤ was not observed. The intracellular location of somatic A ␤ granules was further analyzed by immuno-electron microscopy, which detected A ␤ - positive material in lysosomes of neurons (Fig. 7D,E). Neuropil grains were associated with CD45-positive microglial cells (Fig. Figure6. A ␤ lesionsinAPP48mice.A,B,Intheneocortex,A ␤ changesweremainlylocatedinlayersII,III,V,andVIin3-aswell as in 18-month-old animals. No amyloid plaques were visible. C, At 3 months of age, A ␤ antibodies predominantly stained grain-like structures, whereas thread-like material and somatic granules were less frequently found. D, In 18-month-old APP48 mice, thread-like lesions predominated, while grains and granules became less abundant (see Fig. 8 for quantification). E–G, Higher magnification of the three major A ␤ accumulations in APP48 mice: dendritic A ␤ threads (E) representing dendrites filled with A ␤ ; somatic A ␤ granules (F), which are dot-like A ␤ -positive structures within the perikaryon of neurons. They were distributedinthecellsoma;A ␤ grains(G)representingextraneuronalaccumulationsofA ␤ .H,Double-labelimmunofluorescence for A ␤ and MAP2 confirmed the dendritic localization of the A ␤ threads. These three A ␤ lesions are found throughout the gray matterofthe CNS, althoughA ␤ threadsweremissing in thespinalcord (I,J). I,At5 months ofage,A ␤ grainswerepredominant (arrows). Only few interneurons with somatic granules were observed (black arrowhead). Motor neurons were free of A ␤ (open arrowheads). J, At 19 months of age, somatic granules were also seen in motor neurons (arrowheads) and A ␤ grains were less abundant (arrows). 1278 •J. Neurosci., January 25, 2012 •32(4):1273–1283 Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 7F). Immuno-electron microscopy confirmed the microglial A ␤ inclusions and indicated a lysosomal association (Fig. 7G,H). We did not observe A ␤ in multivesicular bodies of APP48 mice but found minor staining in the endoplasmic reticulum (Fig. 7E, arrowheads). Double-labeling immunofluorescence showed colocalization of neuronal A ␤ granules and microglial A ␤ grains with BIP and LAMP-1 in agreement with a late endosomal/lysosomal localization of both structures (Fig. 8A–I). Dendritic A ␤ threads did not colocalize with BIP and LAMP-1, further indicating that these aggregates are different from A ␤ granules and grains (Fig. 8J–L). No colocalization was found with markers for early endosomes (EEA1) and stress granules [TIA/ TIAR(D-9)] (data not shown). Structural analysis in Epon-embedded sections by electron microscopy revealed few lysosomal, lipofuscin-like aggregates in the soma of neurons (Fig. 7I,J). Further structural changes, especially in dendrites, were neither detected at the ultrastructural (Fig. 7K) nor at the light-microscopic level. Immunohistochemisty did not show alterations of the dendritic tree or of axons in neurofilament-stained sections of 3and 18-month-old APP48 mice when comparing with age-matched wild-type animals. Dystrophic neurites were not observed in the APP staining. The cortical distribution of synaptophysin-positive material did not differ between APP48 and wild-type mice. Abnormally phosphorylated tau and pTDP-43 were not found in the brains of APP48 mice. There were no obvious differences in the distribution pattern of GFAP-positive astrocytes and RCA-positive microglial cells between wild-type and APP48 mice. APP47 ⫻ APP48 mice showed qualitatively similar alterations as APP48. Quantification of the three A ␤ lesions in the frontocentral cortex of APP48 mice (Fig. 9A–C) revealed an approximately Figure7. Immuno-electron-microscopiclocalizationofA ␤ lesionsandultrastructuralanalyzes.A,Campbell–Switzersilverstainingof dendritic A ␤ threads indicated the fibrillar nature of the aggregates (arrows). B, At the immuno-electron-microscopic level, a distinct number of dendrites (example marked by the dashed line) in the neuropil of the frontocentral neocortex contained A ␤ -positive material (arrows).NotethattheaxondidnotcontainA ␤ .C,Athighermagnification,theA ␤ -positivedendriticmaterialexhibitedafibrillarstructure (arrows)consistent withthe SDSresistance ofan A ␤ 1–42 subpopulation.D,Immuno-electron microscopyshowed aneuron withsomatic A ␤ granules. E, Higher magnification (area of the top two arrows in D) detected A ␤ within lysosomes (arrows) and more rarely in the endoplasmic reticulum (arrowheads). F, Double-label immunofluorescence indicated that A ␤ grains (labeled in green) were associated with CD45-positive microglial cells. G, Using immuno-electron microscopy, microglial cells were found, which exhibited lysosomal A ␤ 42 - reactive material. H, Higher magnification of the lysosomal region outlined by arrows. I–K, Epon-embedded tissue exhibited a better structuralresolutionthantheimmuno-electronmaterial.I,Neuronsdidnotshowobviousalterationsoftheirsubcellularorganization.J,No specific changes were found in lysosomes (arrow) at higher magnification (I, frame). K, Dendrites and synapses appeared normal. Mitochondria(arrowheads)within thedendritesand axonsshowedno obvious changes(arrowsindicate dendriticthreads). Table 2. Distribution of morphological changes in APP48 mice Brain regions Conventional histology A ␤ 42 staining Neocortex — A ␤ grains, A ␤ granules, A ␤ threads Allocortex (including hippocampus) Reduction of neurons in CA1 Few A ␤ grains, A ␤ granules, A ␤ threads Basal ganglia — A ␤ grains, A ␤ threads Thalamus — A ␤ grains, A ␤ granules, and few A ␤ threads Basal forebrain nuclei — A ␤ granules, single A ␤ threads, and A ␤ grains in aged animals Midbrain — A ␤ granules, single A ␤ threads, and A ␤ grains in aged animals Brainstem — A ␤ granules, single A ␤ threads, and A ␤ grains in aged animals Cerebellum: dentate nucleus — A ␤ granules, single A ␤ threads, and A ␤ grains in aged animals Cerebellum: granule cell layer — A ␤ grains Cerebellum: Purkinje cells — — Spinal cord Reduction of spinal cord diameter A ␤ grains, A ␤ granules Cerebral, cerebellar, and spinal white matter Reduction of cerebral white matter in aged animals — Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 J. Neurosci., January 25, 2012 •32(4):1273–1283 • 1279 threefold increase in the number of dendritic A ␤ threads between 3 and 18 month of age. In contrast, an age-dependent ⬃45–48% decrease of A ␤ granules and grains was found. The frontocentral neocortex did not show alterations in neuron number compared with wild-type mice (Fig. 9D). However, we found considerable neuron loss in the hippocampus at 3 and 18 months of age (Fig. 9E). Motor deficit in APP48 mice Compared with wild-type animals the APP47 genotype had no significant effect on body weight, while it was reduced in APP48 at 12 to 15 months (Table 3). Inspection over time showed no difference from wild type at 1 month but a body weight reduction from 2 months onward. An intermediate weight was found for double-transgenic APP47 ⫻APP48 mice. APP48 but not APP47 mice presented with minor motor anomalies at ⬃6 months, which increased with age, and occasionally paralysis developed above 18 months of age. No increase in spontaneous mortality was apparent in these mouse lines. To evaluate the apparent motor deficit quantitatively, 5to 7-month-old APP48 were analyzed in the Rotarod test compared with littermate controls (Fig. 10). During three consecutive trials done, APP48 mice fell off the rod much more quickly than the controls. These data indicate a considerable impairment in motor coordination in aged APP48 mice. Discussion In the present study, we describe transgenic mouse lines expressing A ␤ 1–40 (APP47) and A ␤ 1–42 (APP48) in neurons. The expression constructsencode a signalsequence toinsert both A ␤ peptides into the endoplasmic reticulum presumably with a similar orientation as after cleavage from APP. In contrast to regular cleavage of A ␤ from APP, which largely occurs in endosomes and is followed by rapid secretion (Selkoe et al., 1996), the A ␤ peptides are synthesized in the endoplasmic reticulum. Cell culture studies have shown substantial amounts of intracellular A ␤ and comparably little secretion in particular of A ␤ 1–42 (Maruyama et al., 1995) (our unpublished data). Brains from young APP48 mice contained considerably more A ␤ than the corresponding APP47 brains, while the inverse was true on the mRNA level. Different translational efficacies of these very similar constructs appear unlikely. Moreover, the mRNA levels remained unchanged in APP47 ⫻APP48 mice relative to the parent lines, whereas the amount of both A ␤ isoforms was considerably altered. This argues in favor of a differential posttranslational regulation of the A ␤ isoforms. A higher clearance of A ␤ 1–40 has been observed after brain injection (Ji et al., 2001), and this more soluble peptide may also undergo faster intracellular degradation. Accordingly, A ␤ 1–40 in APP47 mice remained at a similar level during aging, whereas A ␤ 1–42 showed a moderate elevation in APP48 mice. A ␤ 1–42 seems able to stabilize A ␤ 1–40 albeit at the expense of its own stability. In young APP47 ⫻APP48 mice, A ␤ 1–42 was decreased and more soluble while A ␤ 1–40 was increased compared with the parent lines. The relative ratio of both peptides may strongly influence their stability as indicated by a recent in vitro study (Kuperstein et al., 2010). During aging of APP47 ⫻ APP48 mice, A ␤ 1–42 increased moderately just compensating the decrease at young age compared with the single-transgenic mice (APP48). For A ␤ 1–40 , a very large increase was found in doublecompared with single-transgenic mice (APP47). Consistent with an intracellular interaction of both A ␤ peptides, no such effect on the steady-state levels was observed when both peptides were fused to the C terminus of the BRI protein and rapidly secreted after cleavage (Kim et al., 2007). However, intracellular A ␤ cannot be completely excluded in these mice as its analysis has not been a topic of the study. Nonetheless, secreted A ␤ 1–40 inhibited amyloid deposition in APP transgenic mice, which indicates an extracellular interaction affecting overall solubility of the A ␤ peptides. Among the lines, APP48 mice develop the more advanced pathology and show three types of A ␤ lesions. Neurons contain Figure 8. Localization of A ␤ lesions at intracellular membrane compartments by double immunolabeling. Double-label immunohistochemistryforBIP(A,D),amarkerofpost-endoplasmaticreticulumcompartments,andA ␤ (B,E)showedcolocalization of BIP and A ␤ (C,F)inA ␤ granules (A–C, arrow) and microglial A ␤ grains (D–F, arrow). The lysosomal marker LAMP-1 (G) demonstratedasimilarcolocalization(I)withA ␤ (H)ingranules(arrowhead)andgrains(arrow)asBIP,indicatingtheirlysosomal location. A ␤ labeling (K) of dendritic threads (J–L, arrows) did not colocalize (L) with the lysosomal marker LAMP-1 (J), further distinguishing A ␤ threads from A ␤ granules and grains. 1280 •J. Neurosci., January 25, 2012 •32(4):1273–1283 Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 A ␤ threads in dendrites and somatic A ␤ granules in lysosomes. Additionally, A ␤ grains are present in lysosomes of microglia cells. Dendritic A ␤ threads appear fibrillar at the electron-microscopic level and can be silver stained. They accumulate with age possibly because their fibrillar structure prevents efficient degradation. In contrast, A ␤ 1–42 found as granules in neuronal lysosomes neither appears fibrillar nor shows other evidence of accumulation, suggesting that it may be degraded. With progressing dendritic A ␤ aggregation, an increased number of assembly sites becomes available. These changes may lead to a shift of A ␤ toward dendritic threads and a reduced lysosomal transport resulting in the observed decrease of A ␤ granules with age. The detection of A ␤ in dendrites and lysosomes demonstrates that the peptide is transported within the neuron from the site of synthesis at the endoplasmic reticulum to other locations. The small A ␤ signal in the endoplasmic reticulum observed at the electron-microscopic level is in agreement with the synthesis of A ␤ at this location. We didnot detect A ␤ inmultivesicular bodies as described for APP transgenic mice and AD brain (Takahashi et al., 2002). APP47 mice only show somatic A ␤ granules consistent with a more rapid and complete degradation of A ␤ 1–40 . In APP48 brain, A ␤ is also found in microglial lysosomes even though the Thy-1 cassette drives expression in neurons (Calhoun et al., 1999). It may derive from A ␤ secretion known to occur to a certain extent in cell culture. Alternatively, microglial A ␤ may originate from degenerated neurons or neuronal processes, but the lack of PAS-positive lysosomal/lipofuscin-like material and the absence of phagosomes at the electron-microscopic level argue against strong phagocytosis. Interestingly, the very small amount of pyroglutamate A ␤ is mainly associated with microglial but not neuronal lysosomes, indicating that pyroglutamate-A ␤ (N3pEA ␤ ) formation is largely avoided when A ␤ is directly targeted for degradation. In agreement with a slow conversion of A ␤ 1–42 to pyroglutamate A ␤ , this isoform was also detected in dendritic threads. Intracellular A ␤ in APP47 and APP48 mice does not lead to amyloid plaque formation, although the total brain A ␤ concentrations are comparable with preplaque APP transgenic mice, which form plaques during aging (Abramowski et al., 2008). Intracellular membrane expression and aggregation of A ␤ as in APP48 is apparently not sufficient for plaque formation. This does not exclude that plaque development requires A ␤ generation and aggregation in a specific intracellular location, which is reached by APP or its C-terminal fragments but not by A ␤ as it lacks the trafficking signals. However, intraneuronal A ␤ accumulation in the absence of extracellular amyloid plaques has also been observed in transgenic mice expressing APP with the ADlinked E693⌬mutation (Tomiyama et al., 2010). In contrast, amyloid plaque formation has been observed in A ␤ 1–42 transgenic mice using the BRI protein as vehicle to secrete the A ␤ peptides (McGowan et al., 2005). Together, the studies favor the notion that amyloid plaques are formed after secretion of A ␤ . Single diffuse plaques have also been observed in A ␤ 3–42 transFigure 9. Quantification of A ␤ lesions and neuron numbers in APP48 mice. Dendritic A ␤ threads (A), somatic A ␤ granules (B), and microglia A ␤ grains (C) were quantified in the frontocentral neocortex of 3and 18-month-old APP48 mice. This analysis revealed an increase in the number of dendritic A ␤ threads with age but a decrease of somatic A ␤ granules and microglial A ␤ grains. Stereology was used to quantify neurons in the frontal cortex (D) of 18-month-old APP48 mice compared with wild-type littermate controls, which did not show a difference (p⫽0.613). The total number ofneuronswasreducedin hippocampus(E)atboth3 and18monthsofage.Significantdifferencesareindicated (Student’s ttest, two-tailed, *p⬍0.05). Figure 10. Motor impairment of APP48 mice. APP48 mice (open circles) and littermate controls (closed circles) at the age of 5–7 months were evaluated in the Rotarod test on 3 consecutivedays. Thetimeontherodisshownforeach individualanimal, andthemedian isindicated.Inallthreetests,APP48micestayedsignificantlylesslongontherodthanthe controls (Mann–Whitney Utest; trial 1, p⬍0.001; trial 2, p⬍0.0003; trial 3, p⬍ 0.0007). Table 3. Average body weight of APP47, APP48, and double-transgenic mice at 12–15 months of age Gender Body weight Wild type APP47 APP48 APP47 ⫻APP48 Females Body weight (g) a 39 ⫾538⫾523⫾330⫾5 p(vs wild type)* NS ⬍0.0001 ⬍0.0001 Males Body weight (g) a 45 ⫾10 41 ⫾630⫾436⫾5 p(vs wild type)* NS ⬍0.0001 ⬍0.005 a Shown are mean ⫾SD. *Student’s ttest, two-tailed. NS, Nonsignificant. Abramowski et al. •Neurodegeneration Induced by Intraneuronal A ␤ 42 J. Neurosci., January 25, 2012 •32(4):1273–1283 • 1281