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Impaired hypoxic tolerance and altered protein binding of NADH in presymptomatic APP23 transgenic mice
PII: S 0 3 0 6 - 4 5 2 2 ( 0 2 ) 0 0 2 8 0 - 4
Neuroscience Vol. 114, No. 2, pp. 285^289, 2002 M 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
Letter to Neuroscience IMPAIRED HYPOXIC TOLERANCE AND ALTERED PROTEIN BINDING OF NADH IN PRESYMPTOMATIC APP23 TRANSGENIC MICE º CHNER,a1 R. HUBER,a1 C. STURCHLER-PIERRAT,b M. STAUFENBIELb and M. W. RIEPEa M. BU a
Department of Neurology, University of Ulm, Steinho«velstraMe 1, 89075 Ulm, Germany b
Nervous System Research, Novartis Pharma Inc., 4002 Basel, Switzerland Key words : NADH, spectrum, Alzheimer, preconditioning, hypoxia.
cortices from patients with Alzheimer’s disease (Mutisya et al., 1994). The mitochondrial abnormalities seem to be associated early and intimately with the development of the disease (Hirai et al., 2001). With an appropriate time interval and dosage, primary hypoxic tolerance may be increased by chemical preconditioning (Riepe et al., 1997). Increased hypoxic tolerance is associated with improved energy metabolism during hypoxia, decreased posthypoxic free radical production, improved posthypoxic morphology, and preserved posthypoxic neuronal function (Kasischke et al., 1999; Riepe et al., 1997). Several mechanisms have been proposed to partake in preconditioning. Conservation of energy metabolism is one of the most consistent ¢ndings (Kasischke et al., 1999; Murry et al., 1990). NADH is an important parameter of oxidative phosphorylation. It is oxidized to NADþ by mitochondrial complex I. NADH is excited in UV and emits £uorescence in blue. Other than NADH its oxidation product, NADþ , is not £uorescent. An increase of UV-induced blue £uorescence therefore indicates an increase of the ratio of NADH to NADþ , which means a net shift in the pyridine nucleotide pool to the reduced state. NADPH may contribute to the total auto£uorescence signal since excitation and spectral £uorescence properties of NADPH/NADPþ are similar to NADH/NADþ . However, it has been demonstrated that the auto£uorescence signal upon UV excitation is predominantly generated from mitochondrial NADH (Duchen and Biscoe, 1992). The £uorescence from cytosolic sources contributes to a much lesser extent to the auto£uorescence signal due to much lower cytosolic concentrations and mitigation in favor of the mitochondrial signal. Time-resolved spectral analysis of NADH £uorescence o¡ers advantages over solely measurement of £uorescence intensity in than it is independent of probe concentration, sample absorption and scatter (Pradhan et al., 1995). The goal of the present study was to investigate hypoxic tolerance and oxidative energy metabolism in presymptomatic B6-Tg(ThylAPP)23Sdz (APP23) mice, a transgenic mouse model of Alzheimer’s disease.
It is being discussed whether impairment of energy metabolism is a ¢nal common pathway of neurodegeneration or initiates the neurodegenerative cascade. The goal was to investigate hypoxic tolerance and oxidative energy metabolism in 4-month-old, presymptomatic B6-Tg(ThylAPP)23Sdz (APP23) mice, a transgenic mouse model of Alzheimer’s disease. Posthypoxic recovery of the population spike amplitude in hippocampal region CA1 upon stimulation of Scha¡er collaterals in region CA3 (15 min hypoxia, 45 min recovery) was 43 . 46% (mean . S.D.) vs. 19 . 35% (P 6 0.05) in slices from wild-type and transgenic animals, respectively. Fluorescence lifetime sensitive spectroscopy of NADH in the CA1 pyramidal cell layer (gate set for detection of protein-bound NADH) showed a wavelength maximum at 455.3 . 1.6 nm (mean . S.D.) in controls and 453.5 . 2.4 nm (P 6 0.05) in mutants. We conclude that hypoxic tolerance is impaired in presymptomatic APP23 mice and occurs prior to extracellular deposition of amyloid plaques. Impaired energy metabolism may thus partake in initiating the neurodegenerative cascade in a transgenic model of Alzheimer’s disease. The blue shift of the spectrum of NADH in mutant mice indicates an altered protein microenvironment of energy metabolism under control conditions. 7 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Impairment of oxidative phosphorylation repeatedly and with increasing frequency has been assumed to partake in the pathophysiology of cell death in neurodegenerative diseases (Beal, 2000; Blass, 2000). Mitochondrial complex IV and, to a lesser general extent, mitochondrial complexes I and II^III have been found decreased in
1 These two authors contributed equally to the work. *Corresponding author. Tel.: +49-731-500-21430; fax: +49-731500-26745. E-mail address: [email protected] (M. W. Riepe). Abbreviations : 3NP, 3-nitropropionate ; NADH, nicotinamide adenine dinucleotide.
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M. Bu«chner et al. EXPERIMENTAL PROCEDURES
Animals The generation of APP23 transgenic mice has been described previously (Sturchler-Pierrat et al., 1997). These mice express the human APP751 cDNA with the Swedish double mutation under control of the neuron-speci¢c mouse Thy-1 promoter fragment. APP23 mice, established on a B6D2 background, have been continuously backcrossed to C57BL/6J. Four-month-old heterozygous mice and littermate controls were analyzed.
(pO2 in the recording chamber was below 10 kPa after 5 min of perfusion with hypoxic Ringer solution; for detailed time course of pO2 in the recording chamber see Kasischke et al. (1996)) superfused the slices for 15 min. After 15 min of hypoxia, slices were superfused with oxygenated Ringer solution until the end of the experiment. Scha¡er collaterals were synaptically activated at 0.1 Hz by bipolar electrodes placed in hippocampal region CA3. Population spikes were recorded in hippocampal region CA1 and analysis performed as described previously (Riepe et al., 1997). In brief, the amplitude of the population spike for each slice was determined after 45 min of recovery by averaging six subsequent responses.
Preparation of slices Fluorescence spectroscopy of NADH Hippocampal slices were prepared as described previously (Huber et al., 2000). Prior to further treatment, slices were incubated for 2 h at 35‡C in a static bath with Ringer solution containing: NaCl 126 mM, KCl 5 mM, KH2 PO4 1.3 mM, MgSO4 1.3 mM, CaCl2 2.4 mM, NaHCO3 26 mM, dextrose 10 mM, bubbled with 95% O2 and 5% CO2 . For hypoxia, Ringer solution bubbled with 95% N2 and 5% CO2 was used. Chemicals were obtained from Sigma Chemicals or RBI. Preconditioning Mild chemical inhibition of oxidative phosphorylation has previously been shown to increase cellular neuronal hypoxic tolerance. Similar to previous studies, chemical preconditioning was achieved by a single i.p. injection of 20 mg/kg 3-nitropropionate (3NP), a selective inhibitor of succinic dehydrogenase, 1 h prior to preparation of slices (Riepe et al., 1997). No overt clinical signs were observed in the animals treated with 3NP. 3NP was dissolved in 0.9% NaCl and the pH was corrected to 7.4. Electrophysiological recordings Recording of the population spike amplitude was performed as previously described (Riepe et al., 1997). In brief, slices were transferred to a recording chamber after preincubation. The recording chamber was perfused with Ringer solution at 6 ml/ min through a peristaltic pump. Slices with a population spike amplitude of less than 2 mV were not included in the analysis. Upon stabilization of the population spike amplitude, Ringer solution made hypoxic by bubbling with 95% N2 and 5% CO2
The NADH spectrum was measured as reported previously (Huber et al., 2000). In brief, a pulsed nitrogen laser (IOM, V-LF302, Berlin, Germany) with excitation wavelength Vexc = 337 nm and 0.35 ns pulse duration was used to excite the NADH. Before each slice was measured the £uorescence was normalized in Ringer solution. Corresponding with results reported in the literature (Lakowicz et al., 1992), £uorescence lifetime of free NADH was less than 1 ns. For the studies two gate positions were used similar to Huber et al. (2000). At gate 1 the detection gate was open from 0.5 to 2.5 ns following excitation. With this setting of the detection gate, total (that is, free and protein-bound) NADH is detected. At gate 2 the detection gate was open from 3.5 to 5.5 ns. With this setting only proteinbound NADH was detected due to the longer lifetime of protein-bound NADH. At each location a minimum of ¢ve measurements were taken. Analysis of the spectra was performed as described previously (Huber et al., 2000). In brief, a Gaussian function was ¢tted to the experimental data. All results were expressed in arbitrary units. Similar to a previous report (Huber et al., 2000), two parameters were analyzed: the wavelength at the spectral maximum and the bandwidth, i.e. the width of the spectrum at half maximum intensity. The bandwidth re£ects the number of degrees of freedom for vibration and rotation. Broadening of the spectra re£ects an increased number of interactions of the £uorophore, in this case NADH, with proteins. This may result from di¡erent conformation of NADH molecule, and/or di¡erent number of binding sites, and/or di¡erent or other binding interaction with the speci¢c proteins. An example of the NADH spectrum obtained in solution and in hippocampal slices is shown in Fig. 1.
Fig. 1. Spectrum of NADH (40 WM) in solution (dotted line) and in hippocampal slices from mutant mice (solid line) (a.u. : arbitrary units, cf. Experimental procedures). Bandwidth re£ects the width of the spectrum at half maximum intensity and re£ects degrees of vibration and rotation of the NADH (cf. Experimental procedures and a previous report (Huber et al., 2000).
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Statistics Each experiment was conducted with eight slices. The slices were prepared from four to ¢ve animals. The maximum number of slices prepared from one animal was two, in order to keep incubation time in vitro prior to the in vitro hypoxia comparable. Statistical testing was performed by Mann^Whitney U-test and for multiple comparisons the Newman^Keuls analog test. Statistical signi¢cance was accepted at P 6 0.05.
RESULTS
Posthypoxic recovery of population spike amplitude Posthypoxic recovery of population spike amplitude was 43 S 46% (mean S S.D.) and 19 S 35% (P 6 0.05) in hippocampal slices from wild-type littermates and transgenic animals, respectively (Fig. 2). No change of posthypoxic recovery of population spike amplitude was observed upon an attempt to increase hypoxic tolerance with chemical preconditioning with a single i.p. injection of 20 mg/kg body weight 3NP 1 h prior to slice preparation (Fig. 2). NADH spectroscopy The NADH spectra of transgenic and control animals were alike at gate 1, where both free and protein-bound NADH was detected. At gate 1 the wavelength maximum at onset of experiment was 461.0 S 3.2 nm (mean S S.D.) and 459.0 S 3.5 nm (n.s.) in wild-type and mutant mice. Bandwidth was 4329 S 179 and 4211 S 79 (n.s.). At gate 2, which is speci¢c for assessing protein-bound NADH (cf. Experimental procedures), the wavelength maximum was 455.3 S 1.6 nm and 453.5 S 2.4 nm (P 6 0.05 to control) in control and transgenic mice,
287
respectively. Bandwidth was 4209 S 166 nm in controls and 4176 S 121 nm (n.s.) in transgenic animals.
DISCUSSION
Recovery of population spike amplitude has been shown to be a good measure of neuronal integrity and function (Feig and Lipton, 1990). In the present study posthypoxic recovery of the population spike amplitude was signi¢cantly worse in slices from APP23 animals (Sturchler-Pierrat et al., 1997) at about 4 months of age. These results indicate that the hippocampus of young APP23 animals is more vulnerable to hypoxic episodes than the hippocampus of wild-type animals. A similar observation has been made in another transgenic model with overexpression of C-terminal fragments of L-amyloid precursor protein (Ghribi et al., 1999). In that model (Nalbantoglu et al., 1997), however, production of C-terminal fragments is not generated in a physiologic way and may reach levels that have a direct toxic e¡ect (Brera et al., 2000; Kim and Suh, 1996; Sopher et al., 1994). Neuronal loss and impairment of hypoxic tolerance in that model could thus have been a transgenic artifact speci¢c to that model. In the present animal model amyloid fragments are generated upon APP overexpression. Taken together the studies suggest that it is not APP overexpression but rather generation of C-terminal fragments (C99) or L-amyloid proteins that causes impairment of hypoxic tolerance at concentrations that may occur in human disease. It also shows that impairment of hypoxic tolerance in the current transgenic model (Sturchler-Pierrat et al., 1997) is not a result of the speci¢c mode of expression (Thy-1) or insertion of the cDNA. From previous studies in animals with the Swedish mutation it is known that amyloid plaques with neuritic
Fig. 2. Recovery of population spike amplitude upon hypoxia and recovery thereof in slices from untreated control animals (empty circles), untreated APP animals (empty squares), and slices prepared after pretreatment in vivo with a single injection of 20 mg/kg body weight 3NP 1 h prior to slice preparation from wild-type (¢lled circles) and APP23 animals (¢lled squares). (Population spikes were evoked with submaximal stimulation of Scha¡er collaterals in hippocampal region CA3 at 0.1 Hz and recorded in hippocampal region CA1 similar to Riepe et al. (1996).)
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changes and dystrophic cholinergic ¢bers and elevated tau phosphorylation are detected at about 6 months of age (Sturchler-Pierrat et al., 1997). The present results therefore indicate that the hippocampus of APP animals is more vulnerable to hypoxic episodes than the hippocampus of controls. Hypoxic tolerance is impaired prior to observation of typical Alzheimer-like pathology in general and of extracellular amyloid deposits in particular. In a previous study on hypoxic tolerance in a transgenic Alzheimer model no mechanism was identi¢ed mediating the impairment of hypoxic tolerance (Ghribi et al., 1999). We investigated oxidative metabolism in transgenic and wild-type animals. Since protein binding is required during the cascade of events in the respiratory chain, protein-bound NADH is more informative about the energy metabolism than free NADH. Free and protein-bound NADH di¡er regarding lifetime, wavelength at maximum and bandwidth of £uorescence maximum (Galeotti et al., 1970; Lakowicz et al., 1992). Also, the extent of the spectral shift and the broadening of the spectra depend on the speci¢c proteins to which NADH is bound (Galeotti et al., 1970; Huber et al., 2000). In the present study a signi¢cant blue shift of the NADH spectrum was observed in APP23 animals, indicating that the pattern or microenvironment of proteins to which NADH binds is altered. Previous and especially post-mortem human studies could not determine whether functional changes of the energy metabolism are primary causes or secondary consequences of neuronal degeneration. Taking the pathological features of the APP transgenic animal model into account (Sturchler-Pierrat et al., 1997; Sturchler-Pierrat and Staufenbiel, 2000), this study demonstrates that alterations of energy metabolism and hypoxic tolerance are impaired prior to neuronal degeneration and may thus
be a causative factor. This supports recent reports that mitochondrial abnormalities are associated early and intimately with the development of Alzheimer’s disease (Hirai et al., 2001). In previous reports the single i.p. application of 20 mg/ kg body weight 3NP improved neuronal hypoxic tolerance (Riepe et al., 1997, 1996). In the present study no such e¡ect was observed in either transgenic animals or the background strain. This again con¢rms previous ¢ndings that hypoxic tolerance is species- and strain-dependent (Fujii et al., 1997; Oli¡ et al., 1995; Prass et al., 2000; Yang et al., 1997). For future studies these observations raise the question as to what extent speci¢c pathologic characteristics of transgenic animals are in£uenced by the background strain or the interplay between background strain and speci¢c mutation. However, irrespective of this strain dependence of hypoxic tolerance it can be concluded that hypoxic tolerance is reduced in animals overexpressing APP with the Swedish mutation.
CONCLUSION
We conclude that hypoxic tolerance is impaired at presymptomatic stages prior to extracellular deposition of amyloid plaques in APP23 mice. The spectrum of NADH is slightly blue-shifted in mutant mice, indicating a di¡erential protein microenvironment in mutant mice. Reversion of impaired hypoxic tolerance may thus be an early target for future therapeutic strategies.
Acknowledgements$The work was supported by Grant Ri 583/ 2-2 from the Deutsche Forschungsgemeinschaft to M.W.R. The expert technical assistance of Mrs. M. Timmler is greatly appreciated.
REFERENCES
Beal, M.F., 2000. Energetics in the pathogenesis of neurodegenerative diseases. Trends Neurosci. 23, 298^304. Blass, J.P., 2000. The mitochondrial spiral: an adequate cause of dementia in the Alzheimer’s syndrome. Ann. NY Acad. Sci. 924, 170^183. Brera, B., Serrano, A., de Ceballos, M.L., 2000. beta-amyloid peptides are cytotoxic to astrocytes in culture : a role for oxidative stress. Neurobiol. Dis. 7, 395^405. Duchen, M.R., Biscoe, T.J., 1992. Mitochondrial function in type I cells isolated from rabbit arterial chemoreceptors. J. Physiol. 450, 13^31. Feig, S., Lipton, P., 1990. N-Methyl-D-aspartate receptor activation and Ca2þ account for poor pyramidal cell structure in hippocampal slices. J. Neurochem. 55, 473^483. Fujii, M., Hara, H., Meng, W., Vonsattel, J.P., Huang, Z., Moskowitz, M.A., 1997. Strain-related di¡erences in susceptibility to transient forebrain ischemia in SV-129 and C57black/6 mice. Stroke 28, 1805^1810. Galeotti, T., Rossum, G., Mayer, D.H., Chance, B., 1970. On the £uorescence of NAD(P)H in whole-cell preparations of tumours and normal tissues. Eur. J. Biochem. 17, 485^496. Ghribi, O., Lapierre, L., Girard, M., Ohayon, M., Nalbantoglu, J., Massicotte, G., 1999. Hypoxia-induced loss of synaptic transmission is exacerbated in hippocampal slices of transgenic mice expressing C-terminal fragments of Alzheimer amyloid precursor protein. Hippocampus 9, 201^205. Hirai, K., Aliev, G., Nunomura, A., Fujioka, H., Russell, R.L., Atwood, C.S., Johnson, A.B., Kress, Y., Vinters, H.V., Tabaton, M., Shimohama, S., Cash, A.D., Siedlak, S.L., Harris, P.L., Jones, P.K., Petersen, R.B., Perry, G., Smith, M.A., 2001. Mitochondrial abnormalities in Alzheimer’s disease. J. Neurosci. 21, 3017^3023. Huber, R., Buchner, M., Li, H., Schlieter, M., Speerfeld, A.D., Riepe, M.W., 2000. Protein binding of NADH on chemical preconditioning. J. Neurochem. 75, 329^335. Kasischke, K., Huber, R., Li, H., Timmler, M., Riepe, M.W., 1999. Primary hypoxic tolerance and chemical preconditioning during estrus cycle in mice. Stroke 30, 1256^1262. Kasischke, K., Ludolph, A.C., Riepe, M.W., 1996. NMDA-antagonists reverse increased hypoxic tolerance by preceding chemical hypoxia. Neurosci. Lett. 214, 175^178. Kim, S.H., Suh, Y.H., 1996. Neurotoxicity of a carboxyl-terminal fragment of the Alzheimer’s amyloid precursor protein. J. Neurochem. 67, 1172^ 1182.
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Lakowicz, J.R., Szmacinski, H., Nowaczyk, K., Johnson, M.L., 1992. Fluorescence lifetime imaging of free and protein-bound NADH. Proc. Natl. Acad. Sci. USA 89, 1271^1275. Murry, C.E., Richard, V.J., Reimer, K.A., Jennings, R.B., 1990. Ischemic preconditioning slows energy metabolism and delays ultrastructural damage during a sustained ischemic episode. Circ. Res. 66, 913^931. Mutisya, E.M., Bowling, A.C., Beal, M.F., 1994. Cortical cytochrome oxidase activity is reduced in Alzheimer’s disease. J. Neurochem. 63, 2179^ 2184. Nalbantoglu, J., Tirado-Santiago, G., Lahsaini, A., Poirier, J., Goncalves, O., Verge, G., Momoli, F., Welner, S.A., Massicotte, G., Julien, J.P., Shapiro, M.L., 1997. Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein. Nature 387, 500^505. Oli¡, H.S., Weber, E., Eilon, G., Marek, P., 1995. The role of strain/vendor di¡erences on the outcome of focal ischemia induced by intraluminal middle cerebral artery occlusion in the rat. Brain Res. 675, 20^26. Pradhan, A., Pal, P., Durocher, G., Villeneuve, L., Balassy, A., Babai, F., Gaboury, L., Blanchard, L., 1995. Steady state and time-resolved £uorescence properties of metastatic and non-metastatic malignant cells from di¡erent species. J. Photochem. Photobiol. B Biol. 31, 101^112. Prass, K., Wiegand, F., Schumann, P., Ahrens, M., Kapinya, K., Harms, C., Liao, W., Trendelenburg, G., Gertz, K., Moskowitz, M.A., Knapp, F., Victorov, I.V., Megow, D., Dirnagl, U., 2000. Hyperbaric oxygenation induced tolerance against focal cerebral ischemia in mice is strain dependent. Brain Res. 871, 146^150. Riepe, M.W., Esclaire, F., Kasischke, K., Schreiber, S., Nakase, H., Kempski, O., Ludolph, A.C., Dirnagl, U., Hugon, J., 1997. Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: ‘chemical preconditioning’. J. Cereb. Blood Flow Metab. 17, 257^264. Riepe, M.W., Niemi, W.N., Megow, D., Ludolph, A.C., Carpenter, D.O., 1996. Mitochondrial oxidation in rat hippocampus can be preconditioned by selective chemical inhibition of succinic dehydrogenase. Exp. Neurol. 138, 15^21. Sopher, B.L., Fukuchi, K., Smith, A.C., Leppig, K.A., Furlong, C.E., Martin, G.M., 1994. Cytotoxicity mediated by conditional expression of a carboxyl-terminal derivative of the beta-amyloid precursor protein. Mol. Brain Res. 26, 207^217. Sturchler-Pierrat, C., Abramowski, D., Duke, M., Wiederhold, K.H., Mistl, C., Rothacher, S., Ledermann, B., Burki, K., Frey, P., Paganetti, P.A., Waridel, C., Calhoun, M.E., Jucker, M., Probst, A., Staufenbiel, M., Sommer, B., 1997. Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology. Proc. Natl. Acad. Sci. USA 94, 13287^13292. Sturchler-Pierrat, C., Staufenbiel, M., 2000. Pathogenic mechanisms of Alzheimer’s disease analyzed in the APP23 transgenic mouse model. Ann. NY Acad. Sci. 920, 134^139. Yang, G., Kitagawa, K., Matsushita, K., Mabuchi, T., Yagita, Y., Yanagihara, T., Matsumoto, M., 1997. C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains : selective neuronal death in the murine transient forebrain ischemia. Brain Res. 752, 209^218. (Accepted 17 May 2002)
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Neuroscience Vol. 114, No. 2, pp. 285^289, 2002 M 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00
www.neuroscience-ibro.com
Letter to Neuroscience IMPAIRED HYPOXIC TOLERANCE AND ALTERED PROTEIN BINDING OF NADH IN PRESYMPTOMATIC APP23 TRANSGENIC MICE º CHNER,a1 R. HUBER,a1 C. STURCHLER-PIERRAT,b M. STAUFENBIELb and M. W. RIEPEa M. BU a
Department of Neurology, University of Ulm, Steinho«velstraMe 1, 89075 Ulm, Germany b
Nervous System Research, Novartis Pharma Inc., 4002 Basel, Switzerland Key words : NADH, spectrum, Alzheimer, preconditioning, hypoxia.
cortices from patients with Alzheimer’s disease (Mutisya et al., 1994). The mitochondrial abnormalities seem to be associated early and intimately with the development of the disease (Hirai et al., 2001). With an appropriate time interval and dosage, primary hypoxic tolerance may be increased by chemical preconditioning (Riepe et al., 1997). Increased hypoxic tolerance is associated with improved energy metabolism during hypoxia, decreased posthypoxic free radical production, improved posthypoxic morphology, and preserved posthypoxic neuronal function (Kasischke et al., 1999; Riepe et al., 1997). Several mechanisms have been proposed to partake in preconditioning. Conservation of energy metabolism is one of the most consistent ¢ndings (Kasischke et al., 1999; Murry et al., 1990). NADH is an important parameter of oxidative phosphorylation. It is oxidized to NADþ by mitochondrial complex I. NADH is excited in UV and emits £uorescence in blue. Other than NADH its oxidation product, NADþ , is not £uorescent. An increase of UV-induced blue £uorescence therefore indicates an increase of the ratio of NADH to NADþ , which means a net shift in the pyridine nucleotide pool to the reduced state. NADPH may contribute to the total auto£uorescence signal since excitation and spectral £uorescence properties of NADPH/NADPþ are similar to NADH/NADþ . However, it has been demonstrated that the auto£uorescence signal upon UV excitation is predominantly generated from mitochondrial NADH (Duchen and Biscoe, 1992). The £uorescence from cytosolic sources contributes to a much lesser extent to the auto£uorescence signal due to much lower cytosolic concentrations and mitigation in favor of the mitochondrial signal. Time-resolved spectral analysis of NADH £uorescence o¡ers advantages over solely measurement of £uorescence intensity in than it is independent of probe concentration, sample absorption and scatter (Pradhan et al., 1995). The goal of the present study was to investigate hypoxic tolerance and oxidative energy metabolism in presymptomatic B6-Tg(ThylAPP)23Sdz (APP23) mice, a transgenic mouse model of Alzheimer’s disease.
It is being discussed whether impairment of energy metabolism is a ¢nal common pathway of neurodegeneration or initiates the neurodegenerative cascade. The goal was to investigate hypoxic tolerance and oxidative energy metabolism in 4-month-old, presymptomatic B6-Tg(ThylAPP)23Sdz (APP23) mice, a transgenic mouse model of Alzheimer’s disease. Posthypoxic recovery of the population spike amplitude in hippocampal region CA1 upon stimulation of Scha¡er collaterals in region CA3 (15 min hypoxia, 45 min recovery) was 43 . 46% (mean . S.D.) vs. 19 . 35% (P 6 0.05) in slices from wild-type and transgenic animals, respectively. Fluorescence lifetime sensitive spectroscopy of NADH in the CA1 pyramidal cell layer (gate set for detection of protein-bound NADH) showed a wavelength maximum at 455.3 . 1.6 nm (mean . S.D.) in controls and 453.5 . 2.4 nm (P 6 0.05) in mutants. We conclude that hypoxic tolerance is impaired in presymptomatic APP23 mice and occurs prior to extracellular deposition of amyloid plaques. Impaired energy metabolism may thus partake in initiating the neurodegenerative cascade in a transgenic model of Alzheimer’s disease. The blue shift of the spectrum of NADH in mutant mice indicates an altered protein microenvironment of energy metabolism under control conditions. 7 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Impairment of oxidative phosphorylation repeatedly and with increasing frequency has been assumed to partake in the pathophysiology of cell death in neurodegenerative diseases (Beal, 2000; Blass, 2000). Mitochondrial complex IV and, to a lesser general extent, mitochondrial complexes I and II^III have been found decreased in
1 These two authors contributed equally to the work. *Corresponding author. Tel.: +49-731-500-21430; fax: +49-731500-26745. E-mail address: [email protected] (M. W. Riepe). Abbreviations : 3NP, 3-nitropropionate ; NADH, nicotinamide adenine dinucleotide.
285
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M. Bu«chner et al. EXPERIMENTAL PROCEDURES
Animals The generation of APP23 transgenic mice has been described previously (Sturchler-Pierrat et al., 1997). These mice express the human APP751 cDNA with the Swedish double mutation under control of the neuron-speci¢c mouse Thy-1 promoter fragment. APP23 mice, established on a B6D2 background, have been continuously backcrossed to C57BL/6J. Four-month-old heterozygous mice and littermate controls were analyzed.
(pO2 in the recording chamber was below 10 kPa after 5 min of perfusion with hypoxic Ringer solution; for detailed time course of pO2 in the recording chamber see Kasischke et al. (1996)) superfused the slices for 15 min. After 15 min of hypoxia, slices were superfused with oxygenated Ringer solution until the end of the experiment. Scha¡er collaterals were synaptically activated at 0.1 Hz by bipolar electrodes placed in hippocampal region CA3. Population spikes were recorded in hippocampal region CA1 and analysis performed as described previously (Riepe et al., 1997). In brief, the amplitude of the population spike for each slice was determined after 45 min of recovery by averaging six subsequent responses.
Preparation of slices Fluorescence spectroscopy of NADH Hippocampal slices were prepared as described previously (Huber et al., 2000). Prior to further treatment, slices were incubated for 2 h at 35‡C in a static bath with Ringer solution containing: NaCl 126 mM, KCl 5 mM, KH2 PO4 1.3 mM, MgSO4 1.3 mM, CaCl2 2.4 mM, NaHCO3 26 mM, dextrose 10 mM, bubbled with 95% O2 and 5% CO2 . For hypoxia, Ringer solution bubbled with 95% N2 and 5% CO2 was used. Chemicals were obtained from Sigma Chemicals or RBI. Preconditioning Mild chemical inhibition of oxidative phosphorylation has previously been shown to increase cellular neuronal hypoxic tolerance. Similar to previous studies, chemical preconditioning was achieved by a single i.p. injection of 20 mg/kg 3-nitropropionate (3NP), a selective inhibitor of succinic dehydrogenase, 1 h prior to preparation of slices (Riepe et al., 1997). No overt clinical signs were observed in the animals treated with 3NP. 3NP was dissolved in 0.9% NaCl and the pH was corrected to 7.4. Electrophysiological recordings Recording of the population spike amplitude was performed as previously described (Riepe et al., 1997). In brief, slices were transferred to a recording chamber after preincubation. The recording chamber was perfused with Ringer solution at 6 ml/ min through a peristaltic pump. Slices with a population spike amplitude of less than 2 mV were not included in the analysis. Upon stabilization of the population spike amplitude, Ringer solution made hypoxic by bubbling with 95% N2 and 5% CO2
The NADH spectrum was measured as reported previously (Huber et al., 2000). In brief, a pulsed nitrogen laser (IOM, V-LF302, Berlin, Germany) with excitation wavelength Vexc = 337 nm and 0.35 ns pulse duration was used to excite the NADH. Before each slice was measured the £uorescence was normalized in Ringer solution. Corresponding with results reported in the literature (Lakowicz et al., 1992), £uorescence lifetime of free NADH was less than 1 ns. For the studies two gate positions were used similar to Huber et al. (2000). At gate 1 the detection gate was open from 0.5 to 2.5 ns following excitation. With this setting of the detection gate, total (that is, free and protein-bound) NADH is detected. At gate 2 the detection gate was open from 3.5 to 5.5 ns. With this setting only proteinbound NADH was detected due to the longer lifetime of protein-bound NADH. At each location a minimum of ¢ve measurements were taken. Analysis of the spectra was performed as described previously (Huber et al., 2000). In brief, a Gaussian function was ¢tted to the experimental data. All results were expressed in arbitrary units. Similar to a previous report (Huber et al., 2000), two parameters were analyzed: the wavelength at the spectral maximum and the bandwidth, i.e. the width of the spectrum at half maximum intensity. The bandwidth re£ects the number of degrees of freedom for vibration and rotation. Broadening of the spectra re£ects an increased number of interactions of the £uorophore, in this case NADH, with proteins. This may result from di¡erent conformation of NADH molecule, and/or di¡erent number of binding sites, and/or di¡erent or other binding interaction with the speci¢c proteins. An example of the NADH spectrum obtained in solution and in hippocampal slices is shown in Fig. 1.
Fig. 1. Spectrum of NADH (40 WM) in solution (dotted line) and in hippocampal slices from mutant mice (solid line) (a.u. : arbitrary units, cf. Experimental procedures). Bandwidth re£ects the width of the spectrum at half maximum intensity and re£ects degrees of vibration and rotation of the NADH (cf. Experimental procedures and a previous report (Huber et al., 2000).
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Hypoxic tolerance in APP23 transgenic mice
Statistics Each experiment was conducted with eight slices. The slices were prepared from four to ¢ve animals. The maximum number of slices prepared from one animal was two, in order to keep incubation time in vitro prior to the in vitro hypoxia comparable. Statistical testing was performed by Mann^Whitney U-test and for multiple comparisons the Newman^Keuls analog test. Statistical signi¢cance was accepted at P 6 0.05.
RESULTS
Posthypoxic recovery of population spike amplitude Posthypoxic recovery of population spike amplitude was 43 S 46% (mean S S.D.) and 19 S 35% (P 6 0.05) in hippocampal slices from wild-type littermates and transgenic animals, respectively (Fig. 2). No change of posthypoxic recovery of population spike amplitude was observed upon an attempt to increase hypoxic tolerance with chemical preconditioning with a single i.p. injection of 20 mg/kg body weight 3NP 1 h prior to slice preparation (Fig. 2). NADH spectroscopy The NADH spectra of transgenic and control animals were alike at gate 1, where both free and protein-bound NADH was detected. At gate 1 the wavelength maximum at onset of experiment was 461.0 S 3.2 nm (mean S S.D.) and 459.0 S 3.5 nm (n.s.) in wild-type and mutant mice. Bandwidth was 4329 S 179 and 4211 S 79 (n.s.). At gate 2, which is speci¢c for assessing protein-bound NADH (cf. Experimental procedures), the wavelength maximum was 455.3 S 1.6 nm and 453.5 S 2.4 nm (P 6 0.05 to control) in control and transgenic mice,
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respectively. Bandwidth was 4209 S 166 nm in controls and 4176 S 121 nm (n.s.) in transgenic animals.
DISCUSSION
Recovery of population spike amplitude has been shown to be a good measure of neuronal integrity and function (Feig and Lipton, 1990). In the present study posthypoxic recovery of the population spike amplitude was signi¢cantly worse in slices from APP23 animals (Sturchler-Pierrat et al., 1997) at about 4 months of age. These results indicate that the hippocampus of young APP23 animals is more vulnerable to hypoxic episodes than the hippocampus of wild-type animals. A similar observation has been made in another transgenic model with overexpression of C-terminal fragments of L-amyloid precursor protein (Ghribi et al., 1999). In that model (Nalbantoglu et al., 1997), however, production of C-terminal fragments is not generated in a physiologic way and may reach levels that have a direct toxic e¡ect (Brera et al., 2000; Kim and Suh, 1996; Sopher et al., 1994). Neuronal loss and impairment of hypoxic tolerance in that model could thus have been a transgenic artifact speci¢c to that model. In the present animal model amyloid fragments are generated upon APP overexpression. Taken together the studies suggest that it is not APP overexpression but rather generation of C-terminal fragments (C99) or L-amyloid proteins that causes impairment of hypoxic tolerance at concentrations that may occur in human disease. It also shows that impairment of hypoxic tolerance in the current transgenic model (Sturchler-Pierrat et al., 1997) is not a result of the speci¢c mode of expression (Thy-1) or insertion of the cDNA. From previous studies in animals with the Swedish mutation it is known that amyloid plaques with neuritic
Fig. 2. Recovery of population spike amplitude upon hypoxia and recovery thereof in slices from untreated control animals (empty circles), untreated APP animals (empty squares), and slices prepared after pretreatment in vivo with a single injection of 20 mg/kg body weight 3NP 1 h prior to slice preparation from wild-type (¢lled circles) and APP23 animals (¢lled squares). (Population spikes were evoked with submaximal stimulation of Scha¡er collaterals in hippocampal region CA3 at 0.1 Hz and recorded in hippocampal region CA1 similar to Riepe et al. (1996).)
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changes and dystrophic cholinergic ¢bers and elevated tau phosphorylation are detected at about 6 months of age (Sturchler-Pierrat et al., 1997). The present results therefore indicate that the hippocampus of APP animals is more vulnerable to hypoxic episodes than the hippocampus of controls. Hypoxic tolerance is impaired prior to observation of typical Alzheimer-like pathology in general and of extracellular amyloid deposits in particular. In a previous study on hypoxic tolerance in a transgenic Alzheimer model no mechanism was identi¢ed mediating the impairment of hypoxic tolerance (Ghribi et al., 1999). We investigated oxidative metabolism in transgenic and wild-type animals. Since protein binding is required during the cascade of events in the respiratory chain, protein-bound NADH is more informative about the energy metabolism than free NADH. Free and protein-bound NADH di¡er regarding lifetime, wavelength at maximum and bandwidth of £uorescence maximum (Galeotti et al., 1970; Lakowicz et al., 1992). Also, the extent of the spectral shift and the broadening of the spectra depend on the speci¢c proteins to which NADH is bound (Galeotti et al., 1970; Huber et al., 2000). In the present study a signi¢cant blue shift of the NADH spectrum was observed in APP23 animals, indicating that the pattern or microenvironment of proteins to which NADH binds is altered. Previous and especially post-mortem human studies could not determine whether functional changes of the energy metabolism are primary causes or secondary consequences of neuronal degeneration. Taking the pathological features of the APP transgenic animal model into account (Sturchler-Pierrat et al., 1997; Sturchler-Pierrat and Staufenbiel, 2000), this study demonstrates that alterations of energy metabolism and hypoxic tolerance are impaired prior to neuronal degeneration and may thus
be a causative factor. This supports recent reports that mitochondrial abnormalities are associated early and intimately with the development of Alzheimer’s disease (Hirai et al., 2001). In previous reports the single i.p. application of 20 mg/ kg body weight 3NP improved neuronal hypoxic tolerance (Riepe et al., 1997, 1996). In the present study no such e¡ect was observed in either transgenic animals or the background strain. This again con¢rms previous ¢ndings that hypoxic tolerance is species- and strain-dependent (Fujii et al., 1997; Oli¡ et al., 1995; Prass et al., 2000; Yang et al., 1997). For future studies these observations raise the question as to what extent speci¢c pathologic characteristics of transgenic animals are in£uenced by the background strain or the interplay between background strain and speci¢c mutation. However, irrespective of this strain dependence of hypoxic tolerance it can be concluded that hypoxic tolerance is reduced in animals overexpressing APP with the Swedish mutation.
CONCLUSION
We conclude that hypoxic tolerance is impaired at presymptomatic stages prior to extracellular deposition of amyloid plaques in APP23 mice. The spectrum of NADH is slightly blue-shifted in mutant mice, indicating a di¡erential protein microenvironment in mutant mice. Reversion of impaired hypoxic tolerance may thus be an early target for future therapeutic strategies.
Acknowledgements$The work was supported by Grant Ri 583/ 2-2 from the Deutsche Forschungsgemeinschaft to M.W.R. The expert technical assistance of Mrs. M. Timmler is greatly appreciated.
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