A Computational Mathematical Model of Neuronal Death Caused by Oxidative Stress in Alzheimers Disease

Reis, A. E. S. 1, Vianna, G. K. 2, Barreto, F. 3, Carvalho, L. A. V. 4 1,3,4 Systems Engineering and Computer Science Program, COPPE / UFRJ. Centro de Tecnologia, Bloco H, sl. 319. Cidade Universitria, Ilha do Fundo, RJ, Brasil. CP 68511. CEP 21945-970. Tel.: +[55](21)2562-8658. 2Mathematics Department, UFRRJ, P1, Seropdica, RJ, Brasil. 1 artur_reis@yahoo.com, 2gkupac@gmail.com, 3fbarreto@cos.ufrj.br, 4alfredo@cos.ufrj.br.

Abstract
This paper presents part of a mathematical model constructed to study oxidative stress actions in Alzheimers disease. There will be shown the production, oxidation, reduction and loss of proteins caused by oxidants and antioxidants substances. These processes, among others, were formalized into mathematical equations that were converted into a computer program, which, from some known initial condition, simulates the system evolution along the time. Finally, the results of some simulations are qualitatively analyzed and discussed.

1. Introduction
The increasing advances in technology and medicine have made possible a significant augment in the population lifespan, specially in the industrialized and indevelopment countries. Unfortunately, this lifespan augment is bringing an increase in the occurrence of many typically elderly diseases. Among those, Alzheimers Disease (AD) is the most common, being the most frequent form of dementia and the forth cause of mortality in industrialized countries. In the United States, AD affects from 7 to 11% of people older than 65 years and almost 50% of people older than 85 years [3, 4, 5]. In Brazil, approximately 1.5 million people suffer from this disease [6]. AD is a progressive neurodegenerative disorder, still not completely understood, that leads to patients death within 8-10 years after clinical onset [2]. AD is associated with progressive neuronal death in the central nervous system. At advanced stages, it is possible to perceive changes in patients cognition and memory [7, 8]. The majority of AD cases is sporadic and has no apparent genetic causes, but a small part of the cases (less than 30%) shows a familiar relation, associated with different genetic components. The sporadic form of this disease is not known yet and it is considered that it may have

heterogeneous origin, with several factors leading to the same final stage [9]. An important pathological process in DA is the Oxidative Stress, characterized by an oxidation / reduction imbalance in the cerebral cells leading to the oxidation of important neuronal components. This will cause many alterations in normal physiology, culminating in neuronal malfunction and death. During normal aging, there are many neurobiological alterations on several brain areas, causing synapse loss and biochemical changes, which progressively deteriorate the brain over the years, in a natural aging process [10]. In 1960, Harman proposed the free radical theory of aging, where the vulnerability of certain neurons could be caused by free radicals (FR). This theory suggests that brain aging is the final result of several years of accumulation of neuronal malfunctions induced by free radicals, which are highly reactive substances[11]. FR are highly unstable chemical species and, inside the brain, they can react with a vast amount of substances. These reactions may produce other FR, starting chain reactions that generate several intermediates, possibly toxic products, modifying physiological substances [10]. The majority of FR species are derived from oxygen metabolism and, as they are very reactive, these substances are called Reactive Oxygen Species (ROS). The impairments caused by ROS start when their production is higher than normal, exceeding the natural capacity of organisms to neutralize them. In normal conditions, ROS production is maintained under control by an efficient antioxidant system. However, in certain pathological conditions, this balance is not achieved and the antioxidant defense system cannot efficiently eliminate these toxic substances. An excess of ROS, or a reduction in the natural capacity of the antioxidant system, causes oxidative stress, leading to neuronal malfunction and death [12, 13]. Oxidative stress is associated with more than 50 diseases. The most important are atherosclerosis, asthma, pulmonary emphysema, AIDS and cancer. Because the central nervous system has intense metabolism and a low degree of cellular renovation, it is particularly sensible to

oxidant substances, and oxidative stress contributes to the beginning and progress of neurodegenerative diseases. Several neural disorders are related to ROS generation and oxidative stress, like Parkinsons disease, amyotrophic lateral sclerosis and Alzheimers disease, with is the object of study of this work [14, 12]. To study the oxidative stress action in AD, a mathematical model was developed. This model uses mathematical equations to describe the variation, with respect to time, of the concentration of substances that participate in the following processes related to AD: production of an aggregated form of -amyloid peptide; production and recycling of antioxidant substances; protection of cellular proteins and lipids; neuronal energy metabolism; control of extracellular levels of the excitatory neurotransmitter glutamate; and neuronal death caused by excitotoxicity. Those processes are complex for direct simulation and were recursively subdivided into smaller and more detailed ones, until all processes could be represented as sets of algebraic and ordinary differential equations. In this text, the following processes related to oxidative stress in AD will be briefly discussed: production of an aggregated form of -amyloid peptide, starting the pathological process; the inflammatory response, initiated with the activation of brain defense cells against the pathological peptide; and oxidative modifications of proteins, caused by oxidant substances produced in the inflammatory process. To illustrate the modeling, a mathematical representation of the oxidative modifications of proteins will be showed. The developed model is for an idealized, homogeneous medium, subdivided into the following compartments: neuron, microglia, astrocyte and extracellular space. Cells were not simulated individually, but as a group of cells of the same type1.

generation of more potent oxidants, leading to neuronal loss [15]. The -amyloid peptide, cleaved from Amyloid Precursor Protein (APP), is found in AD brains and can initiate an inflammatory response by forming insoluble aggregates. When aggregated, this peptide may activate the brain immunological cells. Once activated, these cells generate oxidant substances that are normally used against a possible invader organism, leading to the death of nearby neurons [16, 17].

3. Inflammation
In the last 15 years, several substances associated with inflammation were discovered in the brain of AD patients. However, those inflammation signals are very distinct from those that occur in other pathologies, like redness, swelling or pain, which are classical signals of the inflammatory activity and are not present in AD. In AD, the inflammation is characterized by the activation of glial cells, which are responsible for brain defense against strange organisms. The inflammation is not an AD cause, but an internal response to cerebral tissue injury and chronic presence of insoluble material deposits, like plaques formed by -amyloid aggregation (known as senile plaques). Nevertheless, the inflammation is very important in AD, as the inflammatory activity observed may cause many injuries to healthy cells [18]. Astrocytes and microglia, or microglial cells, are responsible for the inflammatory process against strange organisms in the brain. Microglial cells function as a highly sensible detector for strange substances, changing to an activated state when it gets in contact with certain substances, while astrocytes are activated by products liberated by the activated microglia [19, 20]. Once activated, astrocytes and microglia proliferate and release various substances, known as cytokines, which function as chemical messengers, allowing communication between microglial cells, astrocytes and neurons [21]. It is also through the cytokines that microglial cells cause the activation of surrounding astrocytes at the beginning of the immunological response [20].

2. Production of -Amyloid Peptide

The production of oxidant species may have several causes, as practically all brain cells use oxygen and are capable to produce oxidants, like superoxide and hydrogen peroxide. However, these two substances are not very toxic by themselves, and the formation of effectively toxic substances depends on many factors. Thus, for oxidative stress to occur, it is necessary that some process cause the generation of oxidant substances more dangerous than superoxide and hydrogen peroxide [15]. Several studies indicate that the generation of toxic oxidant substances is not simply caused by metabolism impairment, but by an active response of the brain immunologic system. This immunologic activation configures an inflammatory process, with subsequent production of superoxide, nitric oxide and other substances that may react with each other, causing
1 The complete model can be found in [23] (other two closely related mathematical models on Alzheimers Disease are detailed in [24] and [25]).

4. Changes Caused by Oxidants and Antioxidants in Proteins

The regulation of protein structure and function gives cells the capacity to adapt themselves to a dynamic environment. There are many ways to do this regulation: interactions between proteins, changes promoted by other substances, proteolytic processing and various chemical modifications [22]. In this section, we will work with protein modifications caused by oxidants and antioxidants that may affect temporarily or permanently the proteins, and their mathematical representation. Figure 1 gives a general view of these processes (thick border boxes represent processes, while thin border boxes represent substances produced or consumed in these processes). The production velocity of a protein (rProdProtP) may be defined as:

rProdProt P = cProdProt P cSaturProt P [TotalProt P ]

In this equation, cProtProdP represents the greatest increase in concentration that a protein can have in an unity of time; cSaturProtP, varying between 0 and 1, is used to limit the production of the protein, simulating a saturation effect, or negative feedback; and TotalProtP represents the total concentration of protein P, i.e., the concentrations of the reduced protein plus the concentrations of the temporarily oxidized protein. In this way, de novo production of protein will not cover a possible loss caused by temporary oxidations. It is important to mention that protein concentrations are distinct among astrocytes and neurons, as well as the process they participate in, but they are not simulated for microglial cells.

previous process, is reduced back to its functional state. Thus, antioxidants as well as temporarily oxidized proteins are consumed in this process, producing oxidized antioxidants and reduced proteins. To calculate the velocity of this process, the following equation was used:
rReducTempOxProt P , A = cReducTempOxProt P , A [ Antiox A] [TempOxProt P ]

The term cReducTempOxProtP,A represents the affinity between the temporarily oxidized protein P and the antioxidant A; AntioxA represents the concentration of the antioxidant A; and TempOxProtP represents the concentration of the temporarily oxidized protein P. A process of enzymatic reduction of temporarily oxidized proteins is also considered. This process resembles the previous, non-enzymatic, reduction, except by the participation of an enzyme E that is temporarily transformed during the process and cannot participate on other process at the same time. Thus, we need to represent the concentrations of the free enzyme (EnzymeE) as well as the concentrations of the enzyme that occupied with
E substrates P and A ( ). This reaction was divided in two parts, [A] and [B]: the combination of reagents and enzyme, forming an intermediate product (the enzyme is occupied by its substrates); and the transformation of this enzymatic complex into the final products, releasing the occupied enzyme. Thus, the first equation [A] is:

Enzyme

P, A

rEnzReducTempOxProt [ A] P, A, E = cEnzReducTempOxProt [ A]P , A, E [ Antiox A] [TempOxProt P] [ Enzyme E ]

Fig. 1 Production, oxidation, reduction and loss of proteins

We also have a process for temporary oxidation of some protein P by some oxidant O (rTempOxidProtP,O), that consumes protein P as well as the oxidant O, producing a temporarily oxidized form of the protein P and a non-radical (this later is not controlled in the model). However, protein P itself is consumed, for it is temporarily oxidized. The velocity of this process is given by:

The term rEnzReducTempOxProt[A]P,A,E represents the equation [A] for the velocity of the enzymatic reduction of a temporarily oxidized protein P by the antioxidant A and the enzyme E; cEnzReducTempOxProt[A]P,A,E represents the affinity among the initial reagents; AntioxA represents the concentrations of the antioxidant A; and EnzymeE represents the concentration of the free, nonoccupied, enzyme E. For the second part of this reaction, [B], we have the following equation:
rEnzReducT empOxProt [ B ]P , A, E = cEnzReducT empOxProt [ B ] P , A, E
P, A [ Enzyme E ]

rTempOxidP rot P ,O = cTempOxidP rot P ,O [Oxid O ] [ Prot P ] where cTempOxidProtP,O represents the affinity between protein P and oxidant O, which is proportional to the concentrations of oxidant O (OxidO) and protein P (ProtP). Another process represents the reduction of a temporarily oxidized protein P by an antioxidant A (rReducTempOxProtP,A). In this process, the temporarily oxidized protein P, which production is represented in the

where the term rEnzReducTempOxProt[B]P,A,E represents the velocity of part [B] of this enzymatic reduction, i.e., the transformation velocity of the enzymatic complex into the final products of the reaction; cEnzReducTempOxProt[B]P,A,E is used to adjust the velocity of this reaction with respect to other reactions; and
, A EnzymeP E

represents the enzymatic complex

concentrations, i.e., the concentrations of the occupied enzyme E. Finally, there is a permanent oxidation and loss of a protein P, caused by an oxidant O. This happens when an oxidant reacts with an already oxidized protein, i.e., a temporarily oxidized protein, making it permanently oxidized. The permanently oxidized protein cannot be recycled or reutilized and must be eliminated from the cell. As this permanent oxidized protein does not participate on the modeled processes, its concentrations are not covered in the model. The following equation calculates the velocity of permanent oxidation for a protein P:
rPermOxidProt P ,O = cPermOxidProt P,O [Oxidant O ] [TempOxProt P ]

The temporarily oxidized protein P (TempOxProtP) has its concentrations increased accordingly to the temporary oxidation reactions where the reduced protein P participates, but these concentrations decrease with all the reductions, enzymatic or not, and with all permanent oxidations on which this protein participates:
d [TempOxProt P ] dt

rTempOxidProt rReducTempOxProt rEnzReducTempOxProt rPermOxidProt

P ,O O P, A A A E P ,O O

[ A] P , A, E

where rPermOxidProtP,O represents the velocity of permanent oxidation of protein P by oxidant O; the term cPermOxidProtP,O represents the affinity between the oxidant O and the temporarily oxidized protein P; OxidantO represents the concentrations of oxidant O; and TempOxProtP represents the concentrations of temporarily oxidized protein P. In this manner, the velocity of the permanent oxidation of a protein is proportional to its concentrations as well as the oxidants concentration. Once the velocity of each process is defined in terms of algebraic equations, we can use ordinary differential equations to calculate the concentration difference between the actual time instant t and the next time instant t + 1 for each involved substance. The variation on the concentration of a protein P at its reduced state is equal to the production of this protein plus the sum of its reductions by all antioxidants A that reduce this protein when it is temporarily oxidized,. However, it is necessary to decrease the protein concentration accordingly to the sum of all temporary oxidations that this protein participates on. Thus, the variation of protein P concentration with respect to time d [ Prot ] P may be defined by: dt
d [ Prot P ] dt

The concentrations of the antioxidant A decrease with protein reductions, while the concentrations of the oxidant O decrease with protein oxidations, temporary or permanent. In addition, in the reduction reactions, the oxidized antioxidant A (OxAntioxidantA) has its concentrations raised:
d [ Antioxidant A] dt

rReducTempOxProt rEnzReducTempOxProt
P, A P P E
P ,O

[ A] P , A, E

d [Oxidant O] dt

rTempOxidProt
P

rPermOxidProt
P

P ,O

d [OxAntioxidant A] dt

rReducTempOxProt + rEnzReducTempOxProt
P, A P P E

[ A ] P , A, E

The concentrations of the enzyme E vary accordingly to the enzymatic reductions where it participates: in the first part of the reaction [A], the free enzyme is occupied by the protein P and the antioxidant A; in the second part or the reaction [B], the occupied enzyme is changed to its free state again:

= rProdProt P + +
A E

rReducTempOxProt
A P, O

P, A

rEnzReducTempOxProt rTempOxidProt
O

d [ Enzyme E ] dt

[ B ] P , A, E

rEnzReducTempOxProt rEnzReducTempOxProt
P A P A

[ B ] P , A, E [ A] P , A, E

This equation uses several already defined terms, plus

.. .
some other constructions: the sum over A ( A ) indicates that the corresponding expression must be evaluated for all antioxidants A; the other sums, over O and E, are similar to the oxidants O and enzymes E.

P, A d [ EnzymeE ] = dt

rEnzReducTempOxProt rEnzReducTempOxProt
P A P A

[ A] P , A, E [ B ] P , A, E

5. Results and Discussion

Following the model, we made a computer program to simulate those pathological processes, obtaining the concentration variation of the substances involved through time, allowing the testing of oxidative stress hypothesis in AD. For this purpose, the consequences of pathological -amyloid addition to the medium were simulated, corresponding to in vitro experiments, as well as the permanent production of the peptide, like an in vivo situation. The simulations showed that the proposed model is coherent with the studied references, reinforcing the event sequence that was proposed for this disease: production of the pathological -amyloid peptide [19]; production of free radicals and other oxidant species; malfunction of the neuronal energy metabolism [26, 27, 28, 33], loss of glutamate homeostasis; excitotoxicity; and neuronal death [29]. In the first set of simulations, the aggregated amyloid peptide was added to the medium. The peptide caused a rapid activation of microglial cells. As these cells were activated, they became phagocytic and degraded the peptide, decreasing its concentrations (Figure 2).

of -amyloid peptide, so there was not a permanent state of inflammation.

Fig. 3 Free radical formation after -amyloid addition.

To approach an in vivo situation, however, it is more interesting to have a continuous production of -amyloid peptide. So, in the next simulations, the peptide production was linearly increased from 0 to 0.1nM/s, between 50 and 60 seconds. After that, the peptide production remained constant (it is important to note that it is not the peptide concentration that remained constant, but its production). This scenario allowed a better comparison between simulation data and real situations. With the -amyloid production, occurred microglial cells activation to do the peptide phagocytosis, producing cytokines and causing astrocytes activation, similar to the previous simulation. However, as there was a continuous peptide production, the peptide degradation by glial cells leaded the system to a new equilibrium with production and consumption of this substance, but the system does not returned to its normal state (Figure 4).

Fig. 2 Microglial response to added -amyloid.

After activation, microglial cells released cytokines, leading to the activation of nearby astrocytes. This is an important consequence, because microglial cells, as well as astrocytes, produce oxidant substances that negatively affect neuronal function. These oxidant substances are primarily superoxide and nitric oxide, but they participate on reactions that produce more toxic substances, like the radical hydroxyl and other potent free radicals. Figure 3 shows the observed augment in concentration of these substances. In this new environment, favorable to oxidation reactions, antioxidants were used for cellular defense against free radicals and their concentrations decreased slightly, but as -amyloid peptide was degraded by microglial cells, the oxidants and antioxidants concentrations slowly returned to their normal levels. This happened because there was not a continuous production

Fig. 4 Immunologic activation caused by -amyloid production

Activated astrocytes and microglial cells produced superoxide and nitric oxide, leading to the formation of other oxidant substances. However, as -amyloid peptide was continuously produced, concentrations of these oxidants did not return to normal levels and we can compare the system before and after the peptide production.

After starting the production of -amyloid, as the oxidant levels of the system were above normal, the antioxidant levels remained below normal. This decrease in antioxidant levels continued until about 200 minutes of simulation, when these concentrations stabilized and the system reached the equilibrium again, but in a situation more prone to oxidation than before peptide production. Figure 5 shows a comparison between the levels of an important cellular antioxidant, glutathione (GSH), before and after -amyloid production, both in neurons and in astrocytes (the last having higher physiologic concentrations of this substances than the former). The observed changes in -tocopherol (Vitamin E) and ascorbate (Vitamin C) concentrations, which are two other major antioxidants, were not significant, because the concentrations of these substances were maintained by other antioxidants, like glutathione and thioredoxin, and by their dietary intake, represented in the model as a production.
14000

100% 90%

C o n c e n tra tio n (% )

80% 70% 60% 50% 40% 30% 20% 10% 0% Before After ATP (Astrocytes) 100,00% 83,57% ATP (Neurons) 100,00% 73,27%

Fig. 6 Energy decrease in astrocytes and neurons.

12000

10000

8000

6000

4000

2000

0 Before After

GSH (Astrocytes) 11489,8217 10905,7210

GSH (Neurons) 7005,6678 6394,4635

Fig. 5 Comparison between GSH levels before and after amyloid production.

It is interesting to note, however, that the recycling of these more basic antioxidants requires energy (ATP). Therefore, in an oxidation-prone environment, there are higher demands for energy. Not only there are higher energy demands to recycle the more basic antioxidants, but the oxidant substances oxidize several proteins involved in cellular energy metabolism, including glucose transporters and ATP production enzymes. Thus, in an oxidative stress situation, the cellular ATP levels decreased (Figure 6). As energy concentrations decrease, the ion pumps, which are responsible for the maintenance of electrochemical gradient across the cell membrane, and that need ATP to work, did not function properly. This caused an augment in the concentrations of positive ions inside the cell, leading to a decrease in the viability of the neurons, which may die by necrosis in this situation.

The transporters of the excitatory amino acid glutamate, responsible for retaking this substance from the extracellular space, also suffered from oxidation and were deactivated by oxidants, thus aggravating the damage. This leaded to an increase in the extracellular glutamate levels, causing an abnormal activation of glutamate receptors on neurons. As the activation of these receptors allows the entry of more positive ions into the cell, mainly Calcium and Sodium ions, the cell may die by necrosis, characterized by its membrane rupture. This process is knows as excitotoxicity, because there is the participation of glutamate, which is an excitatory amino acid. Figure 7 shows a simulated decline on neuronal viability caused by this process. Although there was a fast reduction on neuronal viability, after about 15 minutes neuronal viability stabilizes around 87%. However, if we define the peptide maximum production rate as 0.2nM/s, two times the previous value, the neuronal viability decrease to zero after approximately 14 minutes of simulation, representing cell death (Figure 8). Analyzing the simulations qualitatively, the results obtained with the proposed model were coherent with the hypothesis and observations about the oxidative stress in Alzheimers Disease encountered in the researched literature2. This happen even considering the extension and heterogeneity of the modeled processes: stimuli at certain parts of the system cause responses at other parts of the system, without contradicting the biochemical experiments on which the model was based. As an example, consider the increase on -amyloid production and its effect over positive ions inside the neurons, going through process like inflammation, production and elimination of oxidant substances, mal-function of several transport systems, protein modifications, etc.

C o n c e n t ra tio n ( M )

For a comparison, see references 30 to 40.

Fig. 7 Neuronal viability decrease.

Fig. 8 Neuronal death.

6. Acknowledgements
This work was supported by CAPES. So, we would like to acknowledge their help.

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