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In the present study we have investigated the effect of prenatal hypoxia on expression of amyloid precursor protein (APP) and some metallopeptidases, which ...
Letters in Peptide Science, 10: 455–462, 2003. KLUWER /ESCOM  2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Effects of prenatal hypoxia on expression of amyloid precursor protein and metallopeptidases in the rat brain Natalia N. Nalivaeva1,2*, Lilia Fisk1, Rosa M. Canet Aviles1, Svetlana A. Plesneva2, Igor A. Zhuravin2 & Anthony J. Turner1 1

School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK; 2 Institute of Evolutionary Physiology and Biochemistry, RAS, 44 M. Thorez avenue, 194223 St. Petersburg, Russia (* Author for correspondence, e-mail: [email protected], Fax: +44-113-343-3157, Tel: +44-113343-2987)

Received 19 January 2004; Accepted 25 February 2004

Key words: alpha-secretase, APP, development, endothelin-converting enzyme, hypoxia, metallopeptidases, neprilysin, preconditioning

Summary In the present study we have investigated the effect of prenatal hypoxia on expression of amyloid precursor protein (APP) and some metallopeptidases, which regulate b-amyloid peptide (Ab) levels (neprilysin (NEP) and endothelin-converting enzyme (ECE-1)) in the cortex of rats during different periods of postnatal development. We have found that the level of APP in the sensorimotor cortex (SMC) of rats, analysed by Western blotting, increases from days 1 to 5 of postnatal development and then steadily decreases with age, with the most dramatic decline in the period from day 180 to 600. In the cortex of rats subjected to prenatal hypoxia on day 13.5 of embryogenesis, the postnatal levels of APP were higher than in the control. Secretion of the soluble form of APP (sAPP) by a-secretase was found to be the most active on day 30 of postnatal development and there was a significant decrease in the production of sAPP after prenatal hypoxia. NEP was found to be expressed in the cortex of rats only at the early stages of postnatal development and it was barely detectable in adult rats. The decline of NEP levels during ageing might contribute to accumulation of Ab in later life in humans. Prenatal hypoxia resulted in a significant decrease of NEP expression on day 10, but its level was recovered when animals were preconditioned to mild hypoxia. A similar phenomenon was observed when the expression of ECE-1 was analysed. Overall, prenatal hypoxia leads to significant changes in the levels of APP and expression of metallopeptidases involved in amyloid metabolism during all postnatal life and preconditioning to hypoxia appeared to be neuroprotective. Abbreviations: APP, amyloid precursor protein; Ab, b-amyloid peptide; NEP, neprilysin; ECE-1, endothelin-converting enzyme; E13.5, embryonic day 13.5; SMC, sensorimotor cortex.

Introduction It is well known that brain damage due to prenatal hypoxia, induced by vascular and metabolic disorders in the maternal organism, remains a major problem in children. The consequences of prenatal and early neonatal hypoxia include motor deficits and cognitive disorders in the post-

natal period [1, 2]. Animal models of prenatal hypoxia have been useful for the testing of potential neuroprotective agents, especially when conducted with concurrent physiological monitoring [3, 4]. In our previous studies we have shown that prenatal hypoxia on embryonic day 13.5 (E13.5) resulted in significant changes in the activity of enzymes providing biogenesis of cyclic

456 nucleotides, transmembrane ion transport and ganglioside content in brain hemispheres of embryos and adult rats [5, 6]. In behavioural studies we have found that rats subjected to prenatal hypoxia demonstrated slower development and locomotion than controls as well as having learning and memory deficits [7]. On the other hand, hypoxia and oxygen deprivation of the brain in the case of ischaemia in adults were shown to lead to pathological changes, characteristic of Alzheimer’s disease [8, 9]. In particular, transient hypoxic injury to cortical neurones resulted in mitochondrial dysfunction, generation of reactive oxygen species, damage of DNA and accumulation of phospho-tau proteins as well as of b-amyloid peptide (Ab)-immunoreactive products [10]. Although the role of the amyloid precursor protein (APP), a widely expressed transmembrane protein, in the functioning of the brain is still far from elucidated, it was proposed to be involved in stabilization of neuronal calcium fluxes, inhibition of the clotting cascade and cell– cell or cell–matrix adhesion [11] as well as in processes of learning and memory [12]. It is normally present in neurones at low levels but its expression in the brain is induced as part of the adaptive response of adult brain to hypoxia, ischaemia or head injury [13]. An increased level of APP was also shown to be a part of the acute adaptive response of the infant brain to neonatal hypoxia as well as of the brain of 14-day-old rat pups to unilateral ischaemic brain injury [14]. However, there are no data in the literature indicating how prenatal hypoxia affects APP expression in later life and affects its metabolism. Currently, one of the most intensively studied aspects of APP metabolism is its processing by specific proteases, resulting either in production of its soluble derivative sAPP (in case of the action of a-secretase) or the toxic 40–42 amino acid Ab peptides (produced by b- and c-secretases) [15]. While sAPP has neuroprotective properties [12], accumulation of Ab and its deposits leads to neuronal death and Alzheimer’s pathology. Although there are some data reporting that in human neuroblastoma cells hypoxia affects the activity of the metalloproteases involved in APP metabolism [16], there are no reports on the activity of APP metabolising enzymes in the brain of animals subjected to hypoxia.

Recently it became evident that accumulation of Ab in the brain is a dynamic process and depends not only on the activity of Ab producing enzymes but also on the availability of the peptidases able to cleave it. Several metallopeptidases were suggested to be able to hydrolyse Ab, including both neprilysin (NEP, neutral endopeptidase-24.11) and its homologue, endothelin-converting enzyme (ECE-1), which abolish Ab accumulation and pathogenic effects [17–19]. NEP is also one of the major endopeptidases responsible for the inactivation of substance P in the brain [20]. In the carotid body it plays an important role in maintaining levels of this neurotransmitter critical to transduction of hypoxic stimuli [21, 22]. Deletion of NEP was shown to cause marked alterations in both the magnitude and composition of the hypoxic ventilatory response and NEP was postulated to play an important role in modifying the expression of the ventilatory response to acute hypoxia [23]. Studying the factors responsible for increased pulmonary vascular permeability under hypoxic conditions in young rats, it was also shown that hypoxia leads to decreased activity of NEP and reduced expression of this peptidase, which contribute to increased pulmonary vascular leak via substance P and bradykinin receptors [24]. In the brain, intrastriatal administration of phosphoramidon, a dual inhibitor of NEP and ECE-1, resulted in a significantly increased infarct volume to hypoxia with a significant attenuation of cerebral blood flow [25]. However, intrastriatal administration of a selective NEP inhibitor thiorphan had no effect either on cerebral blood flow or infarct volume induced by the hypoxic challenge, suggesting that phosphoramidon-sensitive ECE-1 is functionally active in the modulation of cerebral blood flow in rats undergoing hypoxia [25]. In the aorta of animals subjected to hypoxia, expression of ECE-1 was shown to be increased, especially in the Weibel–Palade bodies, postulated storage sites of endothelin-1 (ET-1), indicating the enhancement of preproET-1 synthesis in the aortic endothelial cells as well as the acceleration of ET-1 processing [26]. Taking all these factors into account, the present study was undertaken to analyse the effects of prenatal hypoxia on the levels of APP, as well as production of its non-amyloidogenic derivative, sAPP, in the cortex of animals (rats) at

457 various stages of postnatal development. Also, we analysed the levels of the related amyloiddegrading peptidases, NEP and ECE-1, at certain periods of postnatal life of rats subjected to prenatal hypoxia, compared to the control group and the effect of preconditioning to hypoxia on these characteristics.

Methods Model of prenatal hypoxia Wistar rats from the vivarium of the Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences were used. Pregnant female rats were subjected to normobaric hypoxia on 13–14 days of gestation (the period of active proliferation of neuroblasts and the beginning of migration of nervous cells) as described previously [6, 27]. Hypoxic conditions were mimicked in a special chamber (100 L in volume) equipped with systems of thermoregulation, ventilation, CO2 absorption and gas analysis. To create hypoxic conditions the content of oxygen in the chamber was decreased linearly from 21 to 7% during the first 10 min by substitution with helium and then was maintained at this level (7%) for 3 h. Concentration of CO2 in the chamber was not higher than 0.1% and the temperature was kept at 22 C. Female rats from the control group were kept during the same period of time in the same room in a cage under normal oxygen content. In some experiments pregnant rats were preconditioned to hypoxia (15% O2, 2 h) on days 10–12 of gestation. The new-born animals were kept after birth under standard vivarium conditions and were sacrificed by decapitation on postnatal days 1 (P1), 5 (P5), 10 (P10), 20 (P20), 30 (P30), 180 (P180) and 600 (P600). The number of animals from the control and experimental groups were, respectively, for P1 – 14/14, P5 – 14/14, P10 – 10/10, P20 – 8/8, P30 – 8/8, P180 – 10/10 and P600 – 5/5. The sensorimotor cortex (SMC) was dissected from the brain according to a stereotaxic atlas [28] and to electrophysiological data on this strain of rats [29]. All experiments on animals were performed under the legal protocols on animal protection and welfare.

Preparation of the samples Analysis of the proteins of interest was performed in the low-salt soluble and membrane (detergent-soluble) fractions of the SMC, which were obtained following the protocol described in [30]. To obtain these fractions the brain tissue was cleared of the blood vessels and homogenised in 1 mL of 0.02 M Tris-HCl (pH 7.5), 0.01 M MgCl2 and 0.05 M NaCl and centrifuged at 500,000 g for 10 min in a Beckman model TL100 tabletop ultracentrifuge. The supernatant was collected and considered the low-salt soluble fraction. The pellet was re-suspended in 1 mL of 0.01 M phosphate buffer (pH 7.4) with 1% Triton X-100, incubated on ice for 1 h and centrifuged as above for 10 min to generate the detergent-soluble fraction. The amount of protein in the fractions obtained was analysed by the routine bicinchoninic acid (BCA) protein assay using the BCA kit (Sigma). For further immunoblotting analysis the fractions were lyophilised and dissolved to a final concentration of 2 mg/ mL in the sample buffer containing 125 mM Tris, 2% w/v SDS, 20% v/v glycerol, 2 mM EDTA, 2 mM EGTA, 5 lg/mL leupeptin, 5 lg/mL pepstatin A, 10 lg/mL aprotinin, pH 6.8 [31] with 1% bromophenol blue and 10% b-mercaptoethanol when required. Immunoblotting Samples containing equal amounts of protein (40–90 lg) were resolved on SDS-polyacrylamide gels [31] and blotted onto poly(vinylidene) difluoride membranes (Immobilon P, Millipore). Membranes were probed with adequate primary antibody as shown below followed by a secondary horseradish peroxidase-conjugated antibody (1:2000 dilution). Membranes were washed for 30 min with Tris-buffered saline pH 7.6 containing 0.1% (v/v) Tween 20 and proteins of interest were detected with the enhanced chemiluminescent detection system (Amersham Pharmacia Biotech Ltd., Bucks., UK). Analysis of APP and sAPP was performed using the selective antibody 22C11; ECE-1, Mab 32-(AEC32-236) (Dr. K. Tanzawa, Tokyo, Japan); NEP - polyclonal Rabbit anti-Rat antibody (US Biological); actin, anti–actin (20–33) IgG fraction of antiserum developed in rabbit (Sigma); horseradish

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peroxidase-linked secondary antibody was from Amersham Biosciences (UK). Quantification of the bands was performed by laser scanning densitometry using Bio-Rad Multi-AnalistTM/PC software, Version 1.1.1 (Bio-Rad, Hemel Hempstead, UK). All biochemical experiments were performed at least in triplicate and the statistical analysis was performed using t-test.

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Formation of the neocortex and its sensorimotor area (SMC) in the rat brain starts as early as days 12–13 of embryogenesis when intensive proliferation and then migration of neuroblasts into the vascular zone take place. Migration of neuroblasts into the SMC continues up to day 5 of postnatal life followed by active neuritogenesis and then synaptogenesis (on days 10–20) and completion of formation of main components of this brain structure by the end of the first month of life [32]. In our previous studies [6, 33] we have shown that prenatal hypoxia on day 13.5 of gestation results in morphological changes in the SMC, delayed physiological development and disturbed formation of motor responses during the first month of postnatal ontogenesis. During maturation of animals these abnormalities were in part compensated, however, in later life these rats still demonstrated lower ability to learn new complex instrumental reflexes compared to control animals [33]. In the present study, immunoblotting analysis of the levels of APP in the SMC of rat brain at various stages of postnatal life revealed detectable amounts of this transmembrane protein already on the first day after birth. In the process of development the amount of APP was then significantly increased (by 60%) by day 5 remaining at this level during the first month of postnatal ontogenesis (Figure 1). However, in the adult rats (6 and 20 months) the amount of APP detected in the SMC was found to be lower than in the brain of young animals. The fact that increased APP protein expression in the SMC correlates with the period of active migration of neuroblasts into the SMC and formation of neuronal network of this structure indicates that this protein might be involved in theses morphological processes. This

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Age (days) Figure 1. Developmental profile of APP levels in SMC of control rats and rats subjected to prenatal hypoxia on E13.5. Data are from image densitometric analysis of Western blot signals of the detergent-soluble fraction (APP doublet signal quantified). Solid line: control, dotted line: prenatal hypoxia). Ordinate: arbitrary units after densitometry of Western blots. Abscissa: Age of rats in days. Data are means ± S.D., *)P £ 0.05.

conclusion is also supported by the observations that APP is associated with specific astroglial cells of the neonatal and adult rat CNS that support axonal outgrowth [34]. After prenatal hypoxia we have found an increased APP expression in the SMC, which was the most dramatic on the first day after birth. Higher levels of APP were then detected practically at all periods analysed (Figure 1). This observation is in agreement with the data of Meng and co-authors who demonstrated that increased APP expression might be an early sign of axonal and neuronal lesions and that neuronal APP might function to repair cell damage [35]. An induction of APP expression is also believed to be a part of the acute adaptive response of the neonatal brain to hypoxia/ischaemia [14]. Production of a soluble form of APP in the SMC detected with monoclonal antibody 22C11 was found to be low during the first two weeks after birth but dramatically increased by the end of the first month. In the adult brain, secretion of sAPP was found to be reduced compared to the values observed in one-month old animals (Figure 2). The developmental profile of production of sAPP in the SMC was shown by us for the first time and is important for understanding the establishment of APP metabolism in the brain. Prenatal hypoxia was found to lead to a decrease in sAPP production, which was the most pronounced after day 10 of development

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Age (days) Figure 2. Developmental profile of secretion of sAPP in SMC of control rats and rats subjected to prenatal hypoxia on E13.5. Data are from image densitometric analysis of Western blot signals of the low-salt soluble fraction. Solid line: control, dotted line: prenatal hypoxia). Ordinate: arbitrary units after densitometry of Western blots. Abscissa: Age of rats in days. Data are means ±S.D., *)P £ 0.05.

and remained lower even on day 600 of animal life. These data indicate that despite the increase in APP expression induced by hypoxia it leads to

a deficit in its processing by the non-amyloidogenic pathway, which might lead to a shift in amyloid metabolism towards formation of Ab. On the other hand, taking into account the role of sAPP in memory formation [12], the deficit in production of sAPP might contribute to memory deficit and impaired reflex formation in the adult animals subjected to prenatal hypoxia. NEP and its homologue ECE-1, are two of the principal candidates for amyloid degrading peptidases in the brain. The analysis of expression of NEP demonstrated that it was mostly expressed at the very early stages of postnatal life, being barely detectable on day 20 after birth (Figure 3 a and b). Prenatal hypoxia resulted in a significant (40%) decrease in NEP expression when its analysis was performed on day 10 after birth, when we observed the highest level of NEP in the SMC (Figure 3c and d, lanes 1, 2). Importantly, after preconditioning of rats to mild hypoxia during three days prior to acute hypoxic conditions, we have not revealed any significant changes in NEP expression compared to control

Figure 3. Developmental profile of NEP levels detected by Western blotting in SMC of rats (a) and effect of prenatal hypoxia on E13.5 on subsequent NEP levels in SMC on postnatal day 11 (b). a: 1-postnatal day 5 (P5), 2-P10, 3-P20, 4-P30, 5–10 months old rats, 6-rat kidney NEP. b: 1: control; 2: prenatal hypoxia on E13.5; 3: prenatal hypoxia on E13.5 with preconditioning to hypoxia on E10, E11 and E12. c, d: Quantitative analysis of the changes of the amount of NEP in the cortex during development (c), 1–5 see the legend for part A; and after prenatal hypoxia and preconditioning (d), 1–3, see the legend for part B. Bars represent means ± S.D., *)P £ 0.05.

460 (Figure 3c and d, lane 3). Also in the cortex of rats subjected to prenatal hypoxia we observed a considerable decrease of the expression of ECE1, which was also rescued when animals were preconditioned to hypoxia (Figure 4a and b). The data of these experiments indicate that hypoxia leads to a deficit of the peptidases degrading Ab, which in turn might contribute to the changes in amyloid metabolism and accumulation of Ab, which was shown to be one of the hallmarks of the response of cortical neurones to ischaemia/hypoxia [9]. Our data on a decreased expression of NEP in the ageing SMC correspond to the observation of aged-related downregulation of NEP in transgenic mice Tg2576, which contain as transgene the Swedish double mutation in the human APP695 gene [36]. They also support the hypothesis of Iwata and coauthors, suggesting that reduction of NEP levels in mouse hippocampal neurones is likely to be associated with AD pathology [37]. The reduction of another NEP-related peptidase involved in Ab metabolism, namely ECE-1, in the SMC of 10-day old rats after prenatal hypoxia suggests that this might be a generic response to hypoxia of this family of metallopeptidases. NEP is located on chromosome 3, which is believed to be linked to the pathology of AD [38–40] and some recent studies suggest a link between the NEP gene and susceptibility to

Alzheimer’s disease [41, 42], although other studies have failed to find such a link [43, 44]. Supporting a direct role for NEP in amyloid accumulation is the report that NEP gene transfer reduces human amyloid pathology in transgenic mice [45]. Deficits in ECE-1 expression have been shown to be involved in accumulation of Ab in ECE-1 deficient transgenic mice [46]. The effect of preconditioning on the recovery of the expression both of NEP and ECE-1 observed in our work is further evidence that this type of preconditioning treatment might be neuroprotective. Although the effect of preconditioning to hypoxia has been studied by several groups leading to a number of hypotheses of the mechanism by which preconditioning might reprogramme the cell response to hypoxia [47–49], this is the first observation of the effect of preconditioning on brain metallopeptidases. Further analysis of the effect of hypoxia and preconditioning on expression of metalloproteases and amyloid metabolism might be beneficial for suggesting a strategy for prevention of AD.

Conclusions This study provides evidence that prenatal hypoxia results in a significant change in the levels of amyloid precursor protein and production

Figure 4. Effect of prenatal hypoxia on levels of ECE-1 in SMC of rats on 10th day of postnatal development. a: 1: control; 2: prenatal hypoxia on E13.5; 3-prenatal hypoxia on E13.5 with preconditioning to hypoxia on E10, E11 and E12. b: Quantitative analysis of the changes of the amount of ECE-1 in the cortex after prenatal hypoxia and preconditioning. 1-3, see the legend for part A. Bars represent means ± S.D., *)P £ 0.05.

461 of its soluble fragment (sAPP) detectable throughout all animal life. Although an increased level of APP in the SMC might play a compensatory role, a decreased activity of the enzymes participating in production of neuroprotective sAPP (e.g., a-secretase) might result in a higher risk of neuronal death under various pathological conditions. In addition, a deficit of the enzymes capable of hydrolysing Ab might result in the disruption of the steady state of amyloid production and catabolism, resulting in accumulation of Ab with consequent neuronal loss and higher probability of formation of amyloid aggregates and plaques. Preconditioning to hypoxia seems to have a protective effect against the reduction in the levels of NEP and ECE-1, which might have some therapeutic implications. Finally, the natural decrease in NEP concentration in the brain during ageing might contribute to the accumulation of Ab in later life. Acknowledgements This work was supported by INTAS-01-245, The Biochemical Society (Mrs. L. Fisk) and RBRF02-04-49385. References 1. Nyakas, C., Buwalda, B. and Luiten, P.G., Prog. Neurobiol., 49 (1996) 1. 2. Salchner, P., Engidawork, E., Hoeger, H., Lubec, B. and Singewald, N., J. Investig. Med., 51 (2003) 288. 3. Mahmoudian, M., Siadatpour, Z., Ziai, S.A., Mehrpour, M., Benaissa, F. and Nobakht, M., Acta. Physiol. Hung., 90 (2003) 313. 4. Nunez, J.L., Alt, J.J. and McCarthy, M.M., Exp. Neurol., 181 (2003) 270. 5. Nalivaeva, N., Klementyev, B., Plesneva, S., Chekulaeva, U. and Zhuravin, I., J. Evol. Biochem. Physiol., 34 (1998) 339. 6. Lavrenova, S.M., Nalivaeva, N.N. and Zhuravin, I.A., J. Evol. Biochem. Physiol., 39 (2003) 203. 7. Zhuravin, I.A., Tumanova, N.L., Nalivaeva, N.N., Plesneva, S.A., Dubrovskaya, N.M., Potapov, D.O. and Turner, A.J., Adv. Gerontol., 6 (2001) 73. 8. Blass, J.P., J. Neurosci. Res., 66 (2001) 851. 9. Jendroska, K., Poewe, W., Daniel, S.E., Pluess, J., Iwerssen-Schmidt, H., Paulsen, J., Barthel, S., Schelosky, L., Cervos-Navarro, J. and DeArmond, S.J., Acta Neuropathol. (Berl), 90 (1995) 461. 10. Chen, G.J., Xu, J., Lahousse, S.A., Caggiano, N.L. and de la Monte, S.M., J. Alzheimers Dis., 5 (2003) 209. 11. Dodart, J.C., Mathis, C. and Ungerer, A., Rev. Neurosci., 11 (2000) 75.

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