CHEMICAL INTERVENTION IN METABOLISM OF SENESCENCE ACCELERATED MICE FOR MODELING NEURODEGENERATIVE DISEASES: AN OVERVIEW

Alexander BOLDYREV 1,2), Tatiana FEDOROVA 2), Sergey STVOLINSKY 2),

Consuelo BORRAS 3), Juan SASTRE 3) and Jose VINA 3)

M.V. Lomonosov Moscow State University, Lenin’s Hills, 119992 Moscow, Russia.

Institute of Neurology, Russian Academy of Medical Sciences, 123367 Moscow, Russia

University of Valencia, Ave Blasco Ibanez 17, 46010 Valencia, Spain

Abstract

SAMP1 is a line of inbred mice with a pronounced misbalance between generation and neutralization of reactive oxygen species (ROS) in brain and other tissues. This results in accumulation of molecular defects in lipids, proteins and DNA moieties. The metabolic disorders appear at a very early step of ontogenic development and induce morphological and behavioral defects manifesting from the 4th month after birth. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatment of these mice induced specific changes that closely resembled Parkinsonian syndrome. Neuropeptide carnosine prevented toxic effects of MPTP and protected the animals against experimental parkinsonism.

Keywords: SAMP1, oxidative stress, NMDA-receptors, MPTP, carnosine

Abbreviations

Introduction

Senescence Accelerated Mice (SAM) is a group of inbred (derived from AKR/J mice) strains consisting of series of SAMP (prone) and SAMR (resistant) lines [1]. All SAMP lines (from SAMP1 to SAMP11) are characterized by accelerated accumulation of senile features, earlier onset and faster progress of age-associated pathological phenotypes, including disorders of immune functions, weakening of learning and memory. Control lines (SAMR1-5) have longer lifespan and more stable metabolism. However in some of them (SAMR1, SAMR4) non-thymic lymphomas and histiocytic neoplasm were shown. Many neurochemical studies showed that accumulation of senile features in SAMP were accompanied with the rise in protein carbonyls, lipid peroxides, 8-OH dihydroguanosine, and chromosomal aberrations in different tissues [2]. Thus, the conclusion was made that senescence process is developed in parallel with increased generation of ROS and metabolic disorders induced by oxidative stress.

Among different SAMP lines, SAMP1 has one of the shortest life span, multiple defects in immune system, senile metabolic disorders, and apparent failure in learning ability [3]. Data presented in this review focused on SAMP1 and SAMR1, with the latter being used as a control strain.

 

2. Neurochemical evidence for oxidative stress in SAMP1

There are a number of metabolic defects in SAMP1 brain characteristic of oxidative stress [4,5]. The stationary level of lipid hydroperoxides in brain is 1.5 times higher and the resistance of membrane lipids to oxidation is 3 times lower in SAMP1 compared to SAMR1 of the same age while the stationary rate of lipid oxidation is similar in brains of both lines. The content of protein carbonyls in whole brain homogenates and in mitochondrial fractions was the same even in old (10-12 month) SAMP1 и SAMR1. At the same time, total activity of superoxide dismutase (SOD) was lower by 40%, being inhibited preferably due to mitochondrial Mn-SOD and, to a lower extent, – to cytosolic (not peroxisomal) Cu/Zn-SOD.

During SAMP1 ontogeny the Na/K-ATPase activity and NMDA-receptors population in brain gray matter underwent complex changes. In the first months of ontogeny, SAMP1 brain is characterized by lower Na/K-ATPase activity and enormously high level of NMDA binding compared to the level in SAMR1. Later, the levels of АТРase rose steadily and NMDA binding decreased, so in brains of 4 month old SAMP1 mice these parameters were equal to that in SAMR1. After 4th month, Na/K-ATPase was 2-2.5 times higher than control, while the NMDA binding decreased and corresponded to about 50% of that in SAMR1 mice. This period was accompanied by a progressive increase in MAO B activity in brain mitochondrial fractions of SAMP1.

In addition, we found a pronounced increase in brain excitotoxic amino acids, glutamate and aspartate (180% and 151% to SAMR1 brain respectively), and a sharp decrease in the content of some biogenic amines in SAMP1 striatum, typically dopaminergic structure: nor-epinephrine was decreased by 28% and dopamine – by 10 times (!) compared to SAMR1 of the same age. It is of interest that levels of serotonine were similar in both lines.

All these changes were associated with an increase in reactive oxygen species (ROS) in SAMP1 tissues that was demonstrated in previous studies [6]. We also found differences in the ROS levels in bone cells of old animals and in neurons of pups of SAMP1 and SAMR1 (Fig. 1). This increased ROS level correlated with several cellular defects – chromosomal aberrations in bone cells, and accumulation of membrane damage in the neurons. The increased ROS level was also detected in SAMP thymocytes, and correlated with an increase apoptosis, when compared to SAMR1.

 

Fig. 1. The generation of ROS and molecular defects in SAMP1/SAMR1 cells. A – stationary (gray) and NMDA-induced (stripped) ROS levels in cerebellum granule cells of 10 day old mice; B – stationary (gray) and PMA-induced (stripped) ROS level in bone cells of 8 months old mice; C – chromosomal aberrations in bone cells of the mice of different age

An increased generation of ROS was also noted in liver [REF]. It was shown for 12 month old SAMP1. Older animals (18-20 months) were characterized by an increase in production of H2O2 (Fig. 2 А). The old animals also had lower level of expression of some brain enzymes that participate in antioxidant defense mechanisms - Mn-SOD, cytochrome C oxidase (Cyt C Oxidase), and glutathione peroxidase (GPx) (Fig. 2 B). Conditions and methods of these experiments were previously described [7,8]. These data demonstrated the change in balance between the generation and neutralization of ROS in SAMP1 that began at the time when morphological changes in SAMP only began to appear (before 4 month).

Fig. 2. Hydroperoxide production (A) and expression of several enzymes (B) in tissues of old SAMP1/SAMR1. A - ** p<0.01 (18-20 months vs 12 months), ## p<0.01 (SAMP vs SAMR); B - * >2 fold change (SAMR vs SAMP)

 

3. Behavioral features

The behavioral parameters characterizing adult (10-12 months old) SAMP1 and SAMR1 were determined using "Hole board", "Open Field" and "Elevated plus maze’ tests [9]. The SAMP1 were found to have slightly lower anxiety (statistically not significant) and decreased locomotion, compared to the SAMR1. The exploratory activity in both groups was found to be similar. At the same time, significant weight loss and enhancement of rigidity were observed in SAMP1 mice compared to SAMR1. The results of various tests suggested that SAMP1 were characterized by disorder in dopaminergic system in the brain [10]. This was in agreement with the neurochemical data discussed above.

4. Chemical intervention in SAMP1 metabolism

The metabolic pathway of SAMP1 is believed to be under constant pressure of oxidative stress, which might be one of the causes for accelerated ageing. In brain tissue, this results in a specific alteration of dopaminergic system, typical for Parkinsonian syndrome. The combination of specific neurotoxins with oxidative stress inducers is frequently used for simulation of neurodegenerative diseases. For example, a combination of experimental brain ischemia with 3-nitropropionate treatments results in total energy deficit [11].

Such chemical intervention in rat metabolism was found to induce neurological symptoms similar to Huntington disease [12]. In the case of SAMP1, we suggested to use MPTP, a known neurotoxin to induce symptoms similar to Parkinsonian syndrome [13]. The detailed protocol of treatments of SAMP1 and SAMR1 with MPTP was described previously [14].

When MPTP was used to treat these mice, we noticed behavioral differences between the two groups (Fig. 3). One can see from Fig. 3 that locomotion function was suppressed in SAMP1, compared to that in SAMR1, and underwent further suppression after MPTP treatment. Grooming, which is one of the features of anxiety, was elevated in SAMP1 and suppressed in SAMR1, which made the difference in this parameter more pronounced. At the same time, exploratory activity (measured as rearing and head dipping) was not changed by MPTP.

Shortly after MPTP injections, SAMP1 but not SAMR1 animals demonstrated short-term passing tremor. Loss of the body weight and rigidity levels during experimental period was neglected in SAMR1 both with and without MPTP but in the case of SAMP1 the loss of the body weight (from 31.2± 0.1 till 27.7± 0.2 g) and pronounced rigidity (19.4% decrease in the distance between the basis of tail and a neck vertebra) were found.

Fig. 3. Individual changes of locomotion (A) and grooming (B) in SAMR1 и SAMP1 animals measured in Hole board test before and after MPTP administration (Tbefore and Tafter, respectively). * - P<0,05 - differences between SAMP and SAMR (non-parametric Mann-Whitney-Wilcoxon U-test). White bars correspond to control, gray bars – to MPTP treated animals

Thus, chemical treatment by MPTP resulted in further alteration of SAMP1 brain metabolism: mitochondrial MAO B was further increased (compared to that found before MPTP administration), whereas SOD decreased [14].

Table 1. Several biochemical parameters in brain mitochondrial fraction of 10-month old SAMR1 and SAMP1

SAMR1

SAMP1

Control

Control

After MPTP treatment

Protein carbonyls, nmoles/mg protein

1.50± 0.02

1.50± 0.01

3.10± 0.01

MAO B, nmoles/mg protein per hr

85± 3

131± 6

154± 7

Cu/Zn-SOD, u/mg protein

58± 8

31± 6

22± 4

Lipid hydroperoxides, arb.u.

99.5± 19.1

115.0± 10.6

127.0± 15.3

Rate of lipid oxidation, tg a

1.24± 0.13

1.10± 0.14

2.03± 0.30

Norepinephrine (in striatum), nmoles/g

2.55± 0.4

1.80± 0.50

0.33± 0.10

Serotonine (in striatum), nmoles/g

2.76± 0.1

2.71± 0.20

2.80± 0.80

Protein carbonyls and lipid hydroperoxides were elevated, while the rate of their oxidation was doubled (see Table 1). The level of nor-epinephrine and dopamine underwent further decrease, but serotonine level was the same as in animals not treated with MPTP. These results suggest that dopaminergic system was involved in metabolic and behavioral disorders as a result of MPTP treatment.

5. May antioxidants be useful?

Because one of the important aspects of MPTP effect is acceleration of oxidative stress in dopaminergic structures of SAMP1 brain [13], one can suggest that an antioxidant therapy may be useful in repair of metabolic defects in tissues of animals with MPTP induced Parkinsonian syndrome. Recently, an attempt to improve antioxidant status of SAMP8 by vitamin E or ROS spin trap PBN (N-tert-butyl-a -phenylnitrone) was described. It resulted in partial protection of brain lipids and proteins against oxidative damage [15]. In our experiments we have used hydrophylic neuropeptide carnosine and demonstrated strong protecting ability against experimental brain ischemia [11,16]. Carnosine was also found to increase the lifespan of SAMP1 when used as a food additive at a daily dose of 100 mg/kg [17]. In our experiments, carnosine was administered in the same dose (100 mg/kg) throughout all experimental period (14 days). At the end of experiments, several physiological parameters were tested and after decapitation a number of biochemical parameters in brain were measured.

 

In carnosine-treated animals MPTP did not induce the loss of body weight and did decrease the rigidity to the much lower level. Moreover, carnosine prevented suppression of locomotion induced by MPTP. Sharp increase in protein carbonyls (from 13.0 to 17.5 nmoles/mg protein) was also arrested by carnosine. Lipid hydroperoxides in that were elevated from 95± 7 units till 127± 15 units as a result of MPTP treatment, were lowered by carnosine to the levels that were lower than that in controls (75± 5 units), and the resistance to oxidation, which was increased by MPTP, treatment returned to the control levels.

Fig. 4. Changes in activity of MAO B (A) and SOD (B) in brain of SAMP1 after treatment of animals with MPTP alone (2) or with carnosine (3) compared to that before MPTP administration

MPTP-treated SAMP1, MAO B increased and SOD – decreased, whereas carnosine prevented these changes. All these data demonstrated that oxidative stress was a permanent factor for SAMP1 and a specific acceleration of oxidative damage of dopaminergic structures of brain by MPTP may result in neurological deficit similar to Parkinsonian syndrome. The natural neuropeptide carnosine may be successfully used to protect SAMP1 mice against oxidative damage and to improve the neurological state of these animals.

Acknowledgements. The work was supported by Russian Foundation of Basic Research (03-04-48767 and 03-04-48947), "Zoetic Neuroscience" Ltd., UK (Steven Gallant, The Founder) and "Medtechnika Ltd.", Russia (Ildar Bagaoutdinov, The President). Authors thank Elena Bulygina, Maria Yuneva, Galina Kramarenko, Natalia Bastrikova, Elena Sorokina, and Vasily Kazey for experimental help and useful discussion.

References

[1] T. Takeda, Ed., The SAM model of senescence, Excerpta Medica, Amsterdam, 1994.

[2] A. Mori, K. Utsumi, J. Liu, M. Hosokawa, Oxidative damage in the Senescence Accelerated Mice, Ann. NY Acad. Sci., 854 (1998) 239-250.

[3] M. Hosokawa, A higher oxidative status accelerates senescence and aggravates age-dependent disorders in SAMP strains of mice, Mech. Ageing Dev., 123 (2002) 1553-1561.

[4] Y. Okuma, T. Murayama, T. Uehara, M. Miyamoto, Y. Nomura, Senecence-accelerated mice (SAMP8) as an animal model of senile dementia: neurochemical features of the brain and drug action,

[5] A. Boldyrev, M. Yuneva, E. Sorokina, G. Kramarenko, T. Fedorova, G. Konovalova, V. Lankin, Antioxidant systems in tissues of Senescence Accelerated Mice (SAM) characterized by accelerated ageing, Biochemistry Moscow, 66 (2001) 1157-1163.

[6] E. Bulygina, S. Gallant, G. Kramarenko, S. Stvolinsky, M. Yuneva, A. Boldyrev, Characterization of the age changes in brain and liver enzymes of senescence accelerated mice SAM), J. Anti-Aging Med., 2 (1999) 43-49.

[7] D. Rickwood, M.T. Wilson, V.M. Darley-Usmar, Isolation and characteristics of intact mitochondria. In: Mitochodria: a practical approach (V.M. Darley-Usmar, Wilson, M.T., Rickwood, D., Eds.) IRL Publisher Press Ltd., Oxford (1987), pp. 1-16.

[8] G. Barja, 1999. Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity and relation to aging and longevity. J. Bioenerg. Biomembr., 31 (1999) 347- 366.

[9] J. Bures, O. Buresova, J.P. Huston, Techniques and Basic Experiments for the study of brain and behavior. Elsevier, Amsterdam – New York, 1983.

[10] N. Bastrikova, E. Sorokina, V. Kazey, T. Fedorova, A. Boldyrev, MPTP-induced changes in Senescence Accelerated Mice, J. Soc. Phys. Chem., (2003) in press.

[11] T. Fedorova, S. Stvolinsky, D. Dobrota, A. Boldyrev, Therapeutic action of carnosine under experimental brain ischemia, Problems of Biol. Med. Pharm. Chem. (in Russian), 1 (2002) 41-44.

[12] M.F. Beal, Neurochemistry and toxin models in Huntington’s disease, Curr. Opinion Neurol., 7 (1994) 542-547.

[13] M. Wu, S.M. Brudzynski, G.J. Mogenson, Functional interaction of dopamine and glutamate in the nucleus accumbens in the regulation of locomotion. Can. J. Physiol. Pharmacol., 71 (1993) 407-413.

[14] N.A. Bastrikova, E.V. Sorokina, V.I. Kazey, T.N. Fedorova, S.L. Stvolinsky, A.A. Boldyrev, Model of parkinsonism caused by neurotoxin MPTP administration in Senescence Accelerated Mice Prone 1 (SAMP1) Neurochemistry Moscow (in Russian), 129 (2002) 247-253.

[15] D.A. Butterfield, T. Koppal, B. Howard, R. Subramaniam, N. Hall, K. Hensley, S. Yatin, K. Allen, M. Aksenov, M. Aksenova, J. Carney, Structural and functional changes in proteins induced by free radical-mediated oxidative stress and protective action of the antioxidants N-tert-butyl-phenylnitrone and vitamine E, Ann. NY Acad. Sci., 854 (1998) 448-462.

[16] S. Gallant, M. Kukley, S. Stvlinsky, Bulygina, A. Boldyrev, Effect of carnosine on rats under experimental brain ischemia, Tohoku J. Exp. Med., 191 (2000) 85-99.

[17] S. Gallant, M. Yuneva, M. Semenova, Carnosine as a potential anti-senescence "drug", Biochemistry Moscow, 65 (2000) 866-868.

Start Rejuvenating your body for less than £1 a day...

 Can you really afford not to?

 L-carnosine

 *Buy Now*

 L-Carnosine

[ Home ] [ The Facts ] [ The Story ] [ Story Contd. ] [ Testimonials ]

[ Discount Endymion ] [ FREE Endymion ] [ Bright Eyes ]

[ Bright Eyes NAC Drops ] [ Bright Eyes Capsules ]

[ Lose Weight ] [ Fight Cellulite ] [ Parkinson's]

[ Ethos Weight Loss Plan ] [ Info. ]