Focally Elevated Creatine Detected in Amyloid Precursor Protein (APP) Transgenic Mice and Alzheimer Disease Brain Tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the β-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21–89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process. The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the β-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21–89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process. Alzheimer disease (AD) 6The abbreviations used are: ADAlzheimer diseaseAββ-amyloidAPP695amyloid precursor proteinNFTneurofibrillary tangleFTIRFourier transform infraredCrcreatinePCrphosphocreatineCKcreatine kinaseMRSmagnetic resonance spectroscopyTgtransgenic. is a progressive neurodegenerative disorder characterized by memory loss and dementia. The pathological hallmarks of AD include extracellular deposits of β-amyloid (Aβ) peptides derived from the amyloid precursor protein (APP695) and neurofibrillary tangles (NFTs) composed of intracellular bundles of hyperphosphorylated tau protein bound into paired helical filaments (1Selkoe D.J. Physiol. Rev. 2001; 81: 741-766Crossref PubMed Scopus (5168) Google Scholar). Reduced or altered brain metabolism, together with oxidative stress and elevated levels of Aβ, are believed to play a crucial role in AD pathology; however, their respective roles in the disease process are still not clear (2Beal M.F. Ann. Neurol. 1995; 38: 357-366Crossref PubMed Scopus (1264) Google Scholar, 3Blass J. Gibson G.E. Hoyer S. J. Alzheimer Dis. 2002; 4: 225-232Crossref PubMed Scopus (79) Google Scholar, 4Munch G. Schinzel R. Loske C. Wong A. Durany N. Li J.J. Vlassara H. Smith M.A. Perry G. Riederer P. J. Neural Transm. 1998; 105: 439-461Crossref PubMed Scopus (267) Google Scholar, 5Gibson G.E. Free Radic. Biol. Med. 2002; 32: 1061-1070Crossref PubMed Scopus (63) Google Scholar, 6Butterfield D.A. Castegna A. Appl. Genomics Proteomics. 2003; 2: 67-70Google Scholar). Alzheimer disease β-amyloid amyloid precursor protein neurofibrillary tangle Fourier transform infrared creatine phosphocreatine creatine kinase magnetic resonance spectroscopy transgenic. We are using Fourier transform infrared (FTIR) microspectroscopy to examine the brains of TgCRND8 and littermate control (non-Tg) mice (7Gough K.M. Rak M. Bookatz A. Del Bigio M. Mai S. Westaway D. Vib. Spectrosc. 2005; 38: 133-141Crossref Scopus (12) Google Scholar). FTIR microspectroscopy combines spatial resolution at the IR diffraction limit with molecular fingerprint information, providing a unique tool for the study of plaques and associated changes in situ. The TgCRND8 mouse (8Chishti M.A. Yang D. Janus C. Phinney A.L. Horne P. Pearson J. Strome R. Zuker N. Loukides J. French J. Turner S. Lozza G. Grilli M. Kunicki S. Morissette C. Paquette J. Gervais F. Bergeron C. Fraser P.E. Carlson G.A. St. George-Hyslop P. Westaway D. J. Biol. Chem. 2001; 276: 21562-21570Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) expresses a double mutant form of human APP695 (with two familial AD mutations, K670N/M671L and V717F) and has both diffuse and congophilic dense-cored plaques, with the cortex and hippocampus affected earliest. Dense-cored plaques are associated with dystrophic neurites and an inflammatory response accompanied by microglial activation. We report here the novel and unexpected discovery of large creatine (Cr) deposits in the hippocampi and cortex of transgenic AD mice and in post-mortem sections of human AD hippocampus. The creatine-phosphocreatine system plays a key role in energy metabolism in tissues such as neurons with high energy demand (9Wyss M. Kaddurah-Daouk R. Physiol. Rev. 2000; 80: 1107-1213Crossref PubMed Scopus (1943) Google Scholar). Phosphocreatine (PCr), synthesized from Cr and ATP in the mitochondria (site of ATP synthesis), is used to regenerate ATP in the cytosol (site of ATP use) by the mitochondrial and cytosolic isozymes of creatine kinase (CK), respectively, thus helping to maintain ATP homeostasis: Cr + ATP ↔ PCr + ADP + H+. CK is very sensitive to oxidative stress and appears to be one of the specific targets of post-translational oxidative modification in AD brain tissue (10Castegna A. Aksenov M. Aksenova M. Thongboonkerd V. Klein J.B. Pierce W.M. Booze R. Markesbery W.R. Butterfield D.A. Free Radic. Biol. Med. 2002; 33: 562-571Crossref PubMed Scopus (532) Google Scholar). CK activity is reduced in excised samples of human AD brain relative to age-matched controls (11David S. Shoemaker M. Haley B.E. Mol. Brain Res. 1998; 54: 276-287Crossref PubMed Scopus (121) Google Scholar, 12Aksenov M. Aksenova M. Butterfield A. Markesbery W.R. J. Neurochem. 2000; 74: 2520-2527Crossref PubMed Scopus (235) Google Scholar). Cultured neurons exposed to Aβ exhibit oxidative stress, including increase in protein carbonyl formation and a decline in CK activity (13Aksenov M.Y. Aksenova M.V. Markesbery W.R. Butterfield D.A. J. Mol. Neurosci. 1998; 10: 181-192Crossref PubMed Scopus (41) Google Scholar). Glucose transport is impaired, and ATP levels are reduced (14Mark R.J. Pang Z. Geddes J.W. Uchida K. Mattson M.P. J. Neurosci. 1997; 17: 1046-1054Crossref PubMed Google Scholar); mitochondrial function is disturbed (15Casley C.S. Canevari L. Land J.M. Clark J.B. Sharpe M.A. J. Neurochem. 2002; 80: 91-100Crossref PubMed Scopus (427) Google Scholar). Following our initial identification of Cr deposits in small IR maps of brain tissue from the four 47-week-old and one 21-week-old TgCRND8, we mapped the complete hippocampus in sections from sixteen animals (Tg and non-Tg littermates from two different mouse lines expressing the same double mutant form of APP695) and discovered extensive Cr load in all Tg mice. Large deposits of Cr were also identified in hippocampal sections from post-mortem AD human brain. These deposits are absent or significantly lower in age-matched control mice and in non-demented post-mortem human hippocampus; thus we speculate that their presence is an important and hitherto unsuspected marker of the disease process. Transgenic Mice—In the first study, Tg(K670N/M671L+V717F)8 (C3H/C57) mice and non-transgenic littermates were sacrificed at 21 weeks (three TgCRND8 and five non-Tg) and at 47 weeks (four TgCRND8 and four non-Tg). Following the detection of Cr in the transgenics, an additional 16 mice, aged 34–89 weeks, were examined. This set included six pairs of TgCRND8 and non-Tg littermates, as well as two pairs of Tg(K670N/M671L+V717F)19959 and their non-Tg littermates. This independent Tg19959 line derives from the same transgene construct as TgCRND8 (APP695, K670N/M671L+V717F) and has similar levels of APP holoprotein expression and associated pathology but is maintained on a different genetic background (129SvEv/Tac). All animals were bred at the Center for Research in Neurodegenerative Diseases. Tissue Preparation—Animals were killed by cervical dislocation; brains were removed and bisected at the midline. The left half of the brain was covered in OCT medium and frozen on dry ice; the right half was placed in cold 3% paraformaldehyde in 0.1 m phosphate-buffered saline for fixation, then dehydrated and embedded in paraffin. For general examination of tissue pathology prior to data collection, paraffin-embedded sections were stained with hematoxylin and eosin, modified Bielschowsky silver stain, and Congo red. Immunohistochemical staining with antibodies to tau and ubiquitin was also performed. The animals were treated in accordance with the guidelines of the Canadian Council on Animal Care, and the protocols were approved by the University of Toronto and University of Manitoba. For infrared and Raman microspectroscopy, the unfixed, flash-frozen tissues were cryosectioned at 8 μm thickness and mounted on either IR reflective slides (Low-e MirrIR™, Kevley Technologies, Chesterland, OH) or gold-coated silicon chips, fabricated in-house at the Synchrotron Radiation Center. Post-mortem Human Autopsy Tissue—In a pilot study, anonymized samples of hippocampus were acquired prospectively from hospital autopsies at the Health Sciences Center (Winnipeg, Canada) within the scope of the autopsy permission forms and the Autopsy Act. The study was approved by the tissue access committee. Initial assessments (two AD and two non-demented) were made from the available information: age, history of dementia or other neurological disease including seizures, and interval between death and autopsy. After dissection of the temporal lobe to expose the hippocampus, multiple coronal slices (3–4-mm-thick) were obtained by sharp dissection, frozen immediately by immersion of the sealed container in isopentane cooled in liquid nitrogen, and stored at –70 °C. The remaining brain was fixed by immersion in 10% buffered formalin for 10–14 days, then samples of the contralateral and remaining ipsilateral hippocampus, lateral frontal cortex, and middle temporal gyrus, as well as other brain regions, were obtained and embedded in paraffin. The sections were stained with the modified Bielschowsky method, and Congo red, to diagnose Alzheimer disease according to the NIA-Reagan criteria (16The National Institute on Aging and Reagan Institute Working Group on Diagnostic Criteria for the Neuropathological Assessment of Alzheimer's Disease Neurobiol. Aging. 1997; 18: S1-S2Crossref PubMed Scopus (289) Google Scholar). Both the plaque scores and the NFT percentages were considered in this classification. Following identification of acceptable normal and disease cases, frozen hippocampus sections were warmed to about –30 °C, sectioned for microspectroscopy as above, and air-dried. IR Microspectroscopy—All spectra were collected in reflectance mode from 4000–700 cm–1 at 4 cm–1 resolution with Happ-Genzel apodization and saved in log(1/R) format; thus these are raw data without any other postprocessing. Synchrotron data were recorded on a Nicolet Magna 500 FTIR with Nic-Plan IR microscope (Synchrotron Radiation Center, University of Wisconsin) or a Nicolet Magna 860 FTIR with Spectra Tech Continuum IR microscope (National Synchrotron Light Source, Brookhaven National Laboratories). Maps were analyzed with OMNIC/Atlμs software (ThermoNicolet). Additional survey maps were acquired on a Bruker Tensor 27 FTIR with Bruker Hyperion microscope (in-house) and analyzed with CytoSpec software (17Lasch P. Haensch W. Naumann D. Diem M. Biochim. Biophys. Acta. 2004; 1688: 176-186Crossref PubMed Scopus (363) Google Scholar). Pixel size in IR maps was 6 × 6 μm2 (National Synchrotron Light Source), 10 × 10 μm2 (Synchrotron Radiation Center), or 20 × 20 μm2 (in-house). IR Spectral Analysis—IR spectroscopy can be used to study protein aggregation, since the position of the amide I band is dependent on the secondary structure of the protein (7Gough K.M. Rak M. Bookatz A. Del Bigio M. Mai S. Westaway D. Vib. Spectrosc. 2005; 38: 133-141Crossref Scopus (12) Google Scholar, 18Gough K.M. Zelinski D. Wiens R. Rak M. Dixon I.M.C Anal. Biochem. 2003; 316: 232-242Crossref PubMed Scopus (57) Google Scholar, 19Choo L.P. Wetzel D.L. Halliday W.C. Jackson M. LeVine S.M. Mantsch H.H. Biophys. J. 1996; 71: 1672-1679Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 20Seshardi S. Khurana R. Fink A.L. Methods Enzymol. 1999; 309: 559-579Crossref PubMed Scopus (151) Google Scholar). Plaques were identified by the presence of a low frequency absorption (1635 cm–1) in the amide I band (7Gough K.M. Rak M. Bookatz A. Del Bigio M. Mai S. Westaway D. Vib. Spectrosc. 2005; 38: 133-141Crossref Scopus (12) Google Scholar). Reference spectra of pure Cr (Sigma) were recorded in reflectance mode on MirrIR slides. Raman Microspectroscopy—Spectra of the crystalline inclusions in TgCRND8 mouse brain and human AD brain sections, and of pure Cr (Sigma) mounted on gold-coated silicon chips, were recorded with a Renishaw Raman microscope (Saskatchewan Structural Sciences Centre, University of Saskatchewan); illuminated with 5 milliwatts of 785-nm light, at one-micron spatial resolution and 4 cm–1 spectral resolution. Paraffin-embedded sections from the brains of the TgCRND8 mice and non-transgenic littermate controls were first evaluated by histochemistry and immunohistochemistry (data not shown). No neurofibrillary tangles were apparent in either silver-stained or tau-immunostained sections. Subjectively, in the 21- and 47-week-old transgenic mice, plaques were most abundant in the cerebral cortex, corpus callosum, and hippocampus, slightly less abundant in the striatum and thalamus, and rare in the olfactory bulb, brainstem, and cerebellum. We found plaques in all seven TgCRND8 mice and none in the controls. We discovered spectral anomalies atypical of both normal and plaque tissue in several IR maps of TgCRND8 brain (Fig. 1A). The sharp spectral bands (Fig. 1A, red) indicated a high degree of order in the material, suggesting a crystalline nature. An IR spectral data base search targeted Cr as a candidate; we confirmed this identity by IR spectroscopy of pure Cr (Fig. 1A, green). The spectrum of the crystalline inclusion embedded in the tissue is readily seen to exhibit all the characteristic peaks of pure Cr, superimposed on the spectrum of normal tissue (Fig. 1A, blue). The identity was confirmed by Raman microspectroscopy of crystalline inclusions in human AD and TgCRND8 hippocampus, which are compared with the Raman spectrum of pure Cr in Fig. 1B. Given the specificity of the number, position, and relative intensity of the spectral bands in these samples, no other assignment is possible. Moreover, there is no evidence of the PCr form, as typical phosphate bands are absent. For Cr detection in the IR maps, we used the integrated intensity of the sharp band appearing at 1304 cm–1, since it lies in a region that is relatively free of interfering bands in normal tissue and can be readily identified. For each pixel that tested positive, the entire spectrum was reviewed to confirm that it was a complete match. Subsequent re-examination of all maps revealed some intriguing results. deposits were identified in the cortex of one of the 21-week-old TgCRND8 mice (Fig. Cr deposits were discovered in the cortex, and hippocampus of all four of the 47-week-old TgCRND8 mice. of these were associated with plaques (Fig. the as μm was found one of the rare dense-cored plaques in the of a 47-week-old No such deposits were identified in any of the maps from the control samples and 47 or in the other two 21-week-old TgCRND8 this Cr presence and with the transgenic mice, we a of These were to include of the entire hippocampal region in six pairs of TgCRND8 and non-Tg littermates and two pairs from a Tg Tg19959 (8Chishti M.A. Yang D. Janus C. Phinney A.L. Horne P. Pearson J. Strome R. Zuker N. Loukides J. French J. Turner S. Lozza G. Grilli M. Kunicki S. Morissette C. Paquette J. Gervais F. Bergeron C. Fraser P.E. Carlson G.A. St. George-Hyslop P. Westaway D. J. Biol. Chem. 2001; 276: 21562-21570Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar). The IR of Cr was detected in the hippocampus of Tg from both No Cr was detected in the non-transgenic TgCRND8 a small was identified in of four controls sacrificed Cr was detected the Tg19959 brain (Fig. and none be detected in the non-Tg littermate brain (Fig. and of were for Cr in the non-Tg brain. TgCRND8 mice, one Cr was found an of × μm in the with deposits the hippocampus (Fig. and small of Cr was detected the of the (Fig. and non-Tg we hippocampal sections from human brain (two AD and two The human hippocampus is the thus of the brain were of molecular and The IR of Cr was detected in sections from all but the and intensity of the were in the hippocampus of an advanced AD brain. maps of the Cr detected in of in a (Fig. and and in the non-demented (Fig. and the intensity of the IR is to the we can with that the of Cr in these AD hippocampus sections was elevated in a that was very similar to that found in the Tg mouse is a normal of brain tissue with an of in mouse brain C. J. H. A. S. 2001; PubMed Scopus Google Scholar). The system plays a crucial role in energy metabolism, maintaining ATP the additional or of phosphate (9Wyss M. Kaddurah-Daouk R. Physiol. Rev. 2000; 80: 1107-1213Crossref PubMed Scopus (1943) Google Scholar). The that the spectra to Cr and not PCr is since all PCr be at Cr as the there is no for Cr to be in the AD it is important to that reduced brain metabolism is associated with AD and other forms of dementia J. Gibson G.E. Hoyer S. J. Alzheimer Dis. 2002; 4: 225-232Crossref PubMed Scopus (79) Google Scholar). CK has identified as a of post-translational oxidative modification in AD brain tissue (10Castegna A. Aksenov M. Aksenova M. Thongboonkerd V. Klein J.B. Pierce W.M. Booze R. Markesbery W.R. Butterfield D.A. Free Radic. Biol. Med. 2002; 33: 562-571Crossref PubMed Scopus (532) Google Scholar). an with a crucial role in energy metabolism, Cr has as a for AD (9Wyss M. Kaddurah-Daouk R. Physiol. Rev. 2000; 80: 1107-1213Crossref PubMed Scopus (1943) Google Scholar, M.A. Ann. Neurol. 2001; PubMed Scopus Google Scholar, M. A. 2002; PubMed Scopus Google Scholar). the of has that Cr in and is in neurons J. M. J. Neurosci. PubMed Google Scholar, R. S. W. D. A. J. Neurochem. 1998; PubMed Scopus Google Scholar). The TgCRND8 mouse brain an inflammatory response accompanied by microglial (8Chishti M.A. Yang D. Janus C. Phinney A.L. Horne P. Pearson J. Strome R. Zuker N. Loukides J. French J. Turner S. Lozza G. Grilli M. Kunicki S. Morissette C. Paquette J. Gervais F. Bergeron C. Fraser P.E. Carlson G.A. St. George-Hyslop P. Westaway D. J. Biol. Chem. 2001; 276: 21562-21570Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar) that be associated with the Cr deposits found The FTIR detection here is to our These deposits be removed by the and in staining our the tissue is and the mounted sections are in the of the is in that has or We that there is no of tissues from control and AD brain were at the same The same was obtained sections from Tg and non-Tg mouse brain were mounted on different and gold-coated We used animals from two different lines such that the doubly mutant APP695 is maintained on different genetic The of Cr in a non-Tg mouse brain were relatively found in the aged animals and weeks, Fig. and in the littermate brain. This a increase in oxidative The in the human tissue is similar to that in the Tg it is associated with a plaque but is the tissue magnetic resonance spectroscopy the has used as an relative to which the of other and P. 2001; PubMed Scopus Google Scholar). a elevated levels of Cr in the and of and AD compared with controls A. D. 1999; PubMed Scopus Google Scholar). G. H. W. J. Alzheimer Dis. 2000; PubMed Scopus Google Scholar) and P. P. L. J. Biol. Med. 1998; Scholar) the of the and not between AD and aged controls on the Cr a of Cr was found in the five most AD in an in study W. G.E. L. 2001; PubMed Scopus Google Scholar) of 21 AD and age-matched controls. were × We found of Cr in the human autopsy samples, but the small size any on to the disease Cr is an of oxidative stress and thus be associated with different in The extensive of Cr found in the Tg mice and the small deposits found in the hippocampus of our most aged control mice the from the The spatial resolution of is to the of the deposits found relative are on the that the Cr is The and unexpected discovery here on this and other intriguing are by this including the form and of the Cr in and the that control this We M. for in and R. Strome for Tg19959 mice. We L. L. and N. (National Synchrotron Light and P. R. (Saskatchewan Structural Sciences Centre, University of Saskatchewan); S. A. F. A. S. S. M. C. and F. of for

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