Enhanced Neurofibrillary Degeneration in Transgenic Mice Expressing Mutant Tau and APP
10.1126/science.1058189
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

JNPL3 transgenic mice expressing a mutant tau protein, which develop neurofibrillary tangles and progressive motor disturbance, were crossed with Tg2576 transgenic mice expressing mutant β-amyloid precursor protein (APP), thus modulating the APP-Aβ (β-amyloid peptide) environment. The resulting double mutant (tau/APP) progeny and the Tg2576 parental strain developed Aβ deposits at the same age; however, relative to JNPL3 mice, the double mutants exhibited neurofibrillary tangle pathology that was substantially enhanced in the limbic system and olfactory cortex. These results indicate that either APP or Aβ influences the formation of neurofibrillary tangles. The interaction between Aβ and tau pathologies in these mice supports the hypothesis that a similar interaction occurs in Alzheimer's disease.

Bibliography

Lewis, J., Dickson, D. W., Lin, W.-L., Chisholm, L., Corral, A., Jones, G., Yen, S.-H., Sahara, N., Skipper, L., Yager, D., Eckman, C., Hardy, J., Hutton, M., & McGowan, E. (2001). Enhanced Neurofibrillary Degeneration in Transgenic Mice Expressing Mutant Tau and APP. Science, 293(5534), 1487–1491.

Authors 14
  1. Jada Lewis (first)
  2. Dennis W. Dickson (additional)
  3. Wen-Lang Lin (additional)
  4. Louise Chisholm (additional)
  5. Anthony Corral (additional)
  6. Graham Jones (additional)
  7. Shu-Hui Yen (additional)
  8. Naruhiko Sahara (additional)
  9. Lisa Skipper (additional)
  10. Debra Yager (additional)
  11. Chris Eckman (additional)
  12. John Hardy (additional)
  13. Mike Hutton (additional)
  14. Eileen McGowan (additional)
References 48 Referenced 1,209
  1. 10.1016/0006-291X(84)91209-9
  2. 10.1002/j.1460-2075.1985.tb04000.x
  3. 10.1073/pnas.85.12.4506
  4. 10.1016/0165-6147(91)90609-V
  5. 10.1097/00005072-199409000-00003
  6. 10.1097/00005072-199409000-00002
  7. 10.1038/349704a0
  8. 10.1038/375754a0
  9. 10.1126/science.7638622
  10. 10.1038/nm0896-864
  11. 10.1016/S0896-6273(00)80230-5
  12. 10.1038/nm0197-67
  13. 10.1002/ana.410430217
  14. 10.1038/373523a0
  15. 10.1126/science.274.5284.99
  16. 10.1038/383710a0
  17. 10.1016/S0896-6273(00)80974-5
  18. 10.1038/nm0198-097
  19. Lewis J., Hutton M., Neurosci. News 3, 63 (2000). / Neurosci. News by Lewis J. (2000)
  20. 10.1038/78078
  21. 10.1038/ng0892-345
  22. 10.1016/0304-3940(94)90463-4
  23. Double mutant mice were generated by crossing hemizygote Tg2576 × hemizygote JNPL3. Mice were maintained on a SW/DBA2/C57B6 background. Mice were genotyped for the tau transgene by polymerase chain reaction (PCR) between exons 1 and 5 of the human tau cDNA against an internal PS-2 control PCR. Mice were genotyped for the APP transgene by PCR between exon 15 of the human APP gene and the hamster prion protein promoter against an internal β-actin control PCR.
  24. For pathological studies immersion-fixed (4% paraformaldehyde or 10% formalin) paraffin-embedded brain sections (5 to 7 μm thick) were immunostained with an avidin biotin peroxidase (ABC) method (Vector Labs Burlingame CA) and the following tau antibodies: Ab39 (Yen 1:10); Alz50 (Davies 1:10); PHF1 (Davies 1:100); TG3 (Davies 1:10); CP3 (Davies 1:100); CP13 (Davies 1:100); PG5 (Davies 1:100); E-1 (Yen 1:1000); RT97 (Anderton 1:100); AT180 (Innogenetics 1:100); a polyclonal antibody to ubiquitin UH19 (Ksiezak-Reding 1:500); a polyclonal antibody to αB-crystallin (Novacastra 1:250); an antibody to mitotic phosphoepitopes MPM-2 (Accurate Chemicals & Scientific Products 1:200); glial fibrillary acidic protein (GFAP) mAb (Biogenex 1:100); and APP mAb 22C11 (Boehringer Mannheim 1:20). Double immunohistochemical methods were used to evaluate senile plaques in TAPP and Tg2576 mice. In some experiments sections that had been immunostained with peroxidase methods for dystrophic neurites were counterstained with thioflavin-S. In other experiments double staining was performed with species- and isotype-specific secondary antibodies applied as cocktails after incubating in cocktails containing two primary antibodies in various combinations. The secondary antibodies were labeled with alkaline phosphatase or horseradish peroxidase. The chromogens were developed sequentially. In all experiments the labeled secondary antibodies were reversed and formalin-fixed paraffin-embedded sections of human AD brain were run concurrently as positive controls. Paraffin-embedded sections were used for histochemical stains (hematoxylin and eosin) thioflavin-S Congo Red and Braak's modification of the Gallyas silver stain. NFTs were counted in Gallyas-stained sections of brain and spinal cord in eight neuroanatomic regions with an Olympus BX50 microscope using a 40× objective lens (×400). In each region counts were made in three nonoverlapping fields chosen to include the highest lesion density and the average NFT count was calculated. Amyloid plaques were counted in thioflavin-S–stained sections of brain in five neuroanatomic regions with an Olympus BH2 fluorescent microscope using a 10× objective lens (×100). A single low-power field was chosen from each region to include the maximum lesion density. Statistical comparisons for lesion density and of male-female differences in TAPP and JNPL3 were performed with the Student t test and Mann-Whitney rank sum using SigmaStat version 2; a significance level of P < 0.05 was used.
  25. The following antibodies were used to stain NFTs in TAPP mice. Conformational tau epitopes: Alz50 MC-1 and Ab39. Phosphorylated tau epitopes: RT97 (phospho-46) CP13 (phospho-202/205) CP3 (phospho-214) AT180 (phospho-231/235) TG3 (phospho-231/235) PHF-1 (phospho-396/404) and PG5 (phospho-409). Other antibodies: E1 (human-specific tau exon 1 polyclonal antibody) and MPM-2 (mitotic phosphoepitope).
  26. 10.1002/(SICI)1097-4547(19970415)48:2<128::AID-JNR5>3.0.CO;2-E
  27. 10.1523/JNEUROSCI.19-17-07486.1999
  28. M. DeTure L.-W. Ko C. Easson M. Hutton S.-H. Yen in Alzheimer's Disease; Advances in Etiology Pathogenesis and Therapeutics K. Iqbal S. S. Sisodia B. Winblad Eds. (Wiley New York 2001) pp. 651–660. (10.1002/0470846453.ch60)
  29. For electron microscopy brain and spinal cord were dissected and immersion-fixed in paraformaldehyde; thin sections of briefly fixed brain (4% paraformaldehyde) were trimmed to 1-mm 3 pieces fixed in 2.5% glutaraldehyde–0.1 M cacodylate buffer (pH 7.4) post-fixed in 1% osmium tetroxide and dehydrated in a graded series of alcohols and propylene oxide before infiltration and embedding in epoxy resin (Poly Bed 812; Polysciences Warrington PA). Ultrathin sections were stained with uranyl acetate and lead citrate for transmission electron microscopy using a Philips 208S.
  30. For supplemental data see Science Online (www.sciencemag.org/cgi/content/full/293/5534/1487/DC1).
  31. 10.1007/BF00692220
  32. Studies of the JNPL3 mice have indicated that there are sex differences in the development of NFT pathology. Female mice have higher tau expression in the central nervous system relative to males (in situ hybridization Northern and Western analysis) and develop tau pathology and motor disturbance at an earlier age. Similar higher levels of tau expression in female mice have also been observed in a second transgenic mouse line expressing the longest 4R tau isoform with the P301L mutation.
  33. 10.1016/S0002-9440(10)64064-3
  34. 10.1111/j.1600-0447.1987.tb02906.x
  35. 10.1002/ana.410300310
  36. C. H. Kawas R. Katzman in Alzheimer Disease R. D. Terry R. Katzman K. L. Bick S. Sisodia Eds. (Lippincott Williams & Wilkins Philadelphia ed. 2 1999) pp. 95–116.
  37. For Aβ measurements one hemisphere was dounce-homogenized in 70% formic acid and centrifuged at 100 000 g for 1 hour; the resulting supernatant was neutralized by a 1:20 dilution in 1 M tris base followed by dilution in buffer EC (0.02 M NaH 2 PO 4 0.002 M EDTA 0.4 M NaCl 0.2% BSA 0.05% CHAPS 0.04% BlockAce 0.05% NaN 3 pH 7.0). Aβ values were determined by sandwich enzyme-linked immunosorbent assay (ELISA) using the BAN50/BA27 and BAN50/BC05 ELISA systems for Aβ40 and Aβ42 respectively. The values presented were calculated by determining the amount of Aβ detected relative to synthetic human Aβ1-40 and Aβ1-42 and converting to pmol per gram of wet weight of brain.
  38. 10.1074/jbc.270.13.7013
  39. 10.1096/fj.00-0528fje
  40. For total soluble tau Western blots mouse brains were harvested and snap-frozen; immunoblotting was performed by homogenizing half of a mouse brain in buffer containing protease inhibitor (1 mM phenylmethylsulfonyl fluoride 20 μM aprotinin 10 μM leupeptin and 1 mM EGTA) and phosphatase inhibitor (5 mM sodium pyrophosphate 30 mM β-glycerophosphate and 30 mM sodium fluoride). Homogenates were dissolved in sample buffer and run on a 10% SDS–polyacrylamide gel electrophoresis (PAGE) gel transferred to nitrocellulose and stained with a human tau-specific antibody E1 and an antibody that recognizes mouse and human tau (WKS45). Equivalent sample loading was determined by protein assay and confirmed by probing the tau blots with an antibody to β-tubulin (Sigma). The antibody E1 was raised to amino acid residues 19 to 33 of human tau. WKS45 is a polyclonal antibody that recognizes mouse and human tau (amino acids 258 to 266). Quantitation of Western blots was performed by image analysis using MCID software (Research System Inc.). The ratio of tau to tubulin in different samples was compared.
  41. For Northern analysis total RNA was extracted from crushed whole mouse brains using Trizol (Life Technologies); RNA (15 μg) was electrophoresed on a denaturing gel transferred overnight onto a nylon membrane (Hybond-N + Amersham Life Science) and cross-linked. An oligomer designed to exon 11 of human/mouse tau (5′-AGATTTTACTTCCACCTGGCCACCTCCTG-3′) was used to assess transgenic mRNA levels. Probes were 3′ end-labeled with [α- 32 P]deoxyadenosine triphosphate. The membrane was hybridized in buffer [containing 4× SSC 1× Denhardt's solution 50% (w/v) deionized formamide 10% (w/v) dextran sulfate and herring sperm DNA (200 mg/μl)] with the labeled probe overnight at 42°C. The membrane was washed stringently (1× SSC/0.1% SDS at 55°C) and exposed to BioMax maximum-sensitivity film (Kodak) at –80°C and Amersham Phosphor Imaging Screens for 1 to 2 days. Band quantification was performed using a Storm Phosphor scanner and ImageQuant software (Molecular Dynamics). Membranes were stripped and reprobed with a 32 P-labeled histone cDNA probe to assess loading.
  42. For analysis of sarkosyl-insoluble tau brain tissue (sectioned into cortex-limbic and subcortical–basal ganglia–cerebellum fractions) was homogenized in tris-buffered saline (TBS); a small sample was removed for the analysis of total tau and the remainder was centrifuged at 100 000 g for 1 hour at 4°C. The pellet was homogenized in 0.8 M NaCl and 10% sucrose in TBS. After centrifugation at 150 000 g for 15 min the supernatant was brought to 1% sarkosyl and incubated at 37°C for 1 hour. The mixture was then centrifuged at 150 000 g for 30 min and the precipitate was collected as the sarkosyl-insoluble fraction. Equal amounts (v/w) of insoluble tau preparations were analyzed by SDS-PAGE and Western blotting using an antibody specific to human tau (E1 amino acids 19 to 33) and one that recognizes both human and mouse tau (WKS45 amino acids 258 to 266). Hyperphosphorylated tau was detected with PHF-1 (phospho-396/404).
  43. 10.1073/pnas.87.15.5827
  44. 10.1007/BF00230483
  45. 10.1016/S0197-4580(98)00071-2
  46. 10.1038/1565
  47. 10.1126/science.1062097
  48. We thank P. Davies (Albert Einstein College of Medicine) for antibodies V. Philips and L. Rousseau for histopathology and D. Forste E. Kirkham C. Ortega and F. Conkle for mouse maintenance. Supported by the National Institute on Aging (PO1 grant to D.W.D. S.-H.Y. J.H. M.H.) National Institute of Neurological Diseases and Stroke (M.H.) Mayo Foundation Smith Scholar Program (J.L. C.E.) John Douglas French Alzheimer's Foundation (J.L.) MetLife Foundation (D.W.D. M.H.) and Mayo Clinic Alzheimer's Disease Research Center (E.M.).
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:53 a.m.)
Deposited 1 year, 7 months ago (Jan. 9, 2024, 5:12 p.m.)
Indexed 46 minutes ago (Aug. 29, 2025, 3:08 p.m.)
Issued 24 years ago (Aug. 24, 2001)
Published 24 years ago (Aug. 24, 2001)
Published Print 24 years ago (Aug. 24, 2001)
Funders 0

None

@article{Lewis_2001, title={Enhanced Neurofibrillary Degeneration in Transgenic Mice Expressing Mutant Tau and APP}, volume={293}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.1058189}, DOI={10.1126/science.1058189}, number={5534}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Lewis, Jada and Dickson, Dennis W. and Lin, Wen-Lang and Chisholm, Louise and Corral, Anthony and Jones, Graham and Yen, Shu-Hui and Sahara, Naruhiko and Skipper, Lisa and Yager, Debra and Eckman, Chris and Hardy, John and Hutton, Mike and McGowan, Eileen}, year={2001}, month=aug, pages={1487–1491} }