Energy Metabolism in Conformational Diseases

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7Energy Metabolism in Conformational DiseasesJudit Ovádi and Ferenc Orosz

Abstract

Increasing evidence indicates that accumulation of unfolded/misfolded proteinsresults in protofibril and protein aggregate formation, inducing mitochondrialdysfunction and failure of synaptic, transport and other crucial physiologicalprocesses. Our knowledge on molecular defects of the energy metabolism in thepathogenesis of neurodegenerative diseases is scarce. One problem that investi-gators face is distinguishing primary from secondary events: is the impairmentof energy production a consequence of the development of neurodegeneration, orit is an active contributor? Genomic and proteomic data provide useful informa-tion for understanding the structural and functional perturbations during the de-velopment of neurodegeneration, but the metabolomic alterations are much lessdisclosed. This paper reviews the direct and indirect role of the damage of energymetabolism, focusing on the metabolism of glucose as major energy source of thebrain. Special attention is paid to the functional consequences of the associationsof glycolytic enzymes with unfolded proteins that specifically determine the natureof the so-called “conformational diseases”. This review shows that the available dataare largely inconclusive for the evaluation of the pathomechanism of energy pro-duction defects in human brains of patients of conformational diseases at systemlevel, and the genuine need for development of rational models (biosimulation) ofneuropathological conversion of glucose to ATP via glycolysis and oxidative phos-phorylation.

7.1What is the Major Energy Source of the Brain?

The brain is an organ with high energy demands. Glucose has been considered thealmost exclusive energy source utilized by the brain. The glucose taken up by braincells is metabolized by glycolytic enzymes, the mitochondrial respiratory chain andthe oxidative phosphorylation system. In addition to glucose, neurons might rely

bertau v.2007/03/15 Prn:29/05/2007; 10:44 F:BERTAU07.tex; VTEX/Danute p. 1

Biosimulation in Drug Development. Martin Bertau, Erik Mosekilde, and Hans V. WesterhoffCopyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-31699-1

2 7 Energy Metabolism in Conformational Diseases

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on lactate to sustain their activity. Activation of brain areas is induced by the arrivalof different impulses, which release glutamate into the synaptic cleft. Neurotrans-mitter molecules diffuse across the cleft and stimulate the postsynaptic cell, caus-ing Na+ channels to open (Fig. 7.1), resulting in membrane depolarization. Thisleads to the initiation of action potentials as a consequence of the movement ofions along their electrochemical gradients. Na+ entry and K+ release during electri-cal activity (action potential) initiate increased energy metabolism within neurons.The movement of ions activates the Na+/K+ ATPase pump which restore the po-larization of the membrane supplying ATP. These processes imply rapid supply ofenergy produced by glycolysis (conversion of glucose to pyruvate) and by the tricar-boxylic acid (TCA) cycle and mitochondrial oxidative phosphorylation. Activation ofglycolysis lowers the glucose level, causing enhanced flux of glucose into neurons.According to the conventional hypothesis, neurons metabolize glucose nearly fullyin an aerobic manner: The generated ATP is largely used to restore Na+/K+ bal-ance via Na+/K+ ATPase. The rapid increase of glycolysis promotes NADH/NAD+,as well as pyruvate production, leading to the accumulation of lactate that dimin-ishes the utilization of lactate produced by astrocytes [1].

Astrocytes, sub-type of the glial cells in the brain, outnumber neurons by ten toone and play a number of active roles in the brain. Astrocytes have a critical role asthe main energy stores and transporting materials and small molecules betweenblood and brain tissue as well as within the brain tissue. Even the supportive activ-ity of astrocytes is driven by neuronal activity and interplay. Interactions betweenneurons and astrocytes are critical for signaling, energy metabolism, extracellularion homeostasis, volume regulation, and neuroprotection in the central nervoussystem. In astrocytes, oxidative glucose metabolism is activated by uptake of gluta-mate and the basic activation process is similar to that in the neuron (cf. Fig. 7.1)[1]. Therefore, the conventional hypothesis asserts that glucose is the primary sub-strate for both neurons and astrocytes and that lactate is produced during neuralactivity and can be utilized by neurons as an energy source. However, the ques-tion is whether neurons do utilize glial-produced lactate as their principal energysubstrate during activity, as proposed by the astrocyte–neuron lactate shuttle hy-pothesis (ANLSH).

The ANLSH challenged the classic view [2, 3]. It postulates compartmental-ization of brain lactate metabolism between neurons and astrocytes: the activity-induced uptake of glucose takes place predominantly in astrocytes, which metabo-lize glucose anaerobically. Lactate produced from anaerobic glycolysis in astrocytesis then released from astrocytes and provides the primary metabolic fuel for neu-rons. The increased lactate in the neurons is converted to pyruvate via lactate de-hydrogenase (LDH), which enters the TCA cycle, and increases ATP production inthe neurons via oxidative phosphorylation (Fig. 7.1). This view is highly discussed,pro [4, 5]) and contra [1, 6].

A modeling approach was used to determine which mechanisms are appropri-ate to explain typical brain lactate kinetics observed upon activation [7]. The modeltakes into account the mechanisms that are known to determine extracellular lac-tate concentration and includes a systematic study of the effects of changing para-


7.1 What is the Major Energy Source of the Brain? 3

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Fig. 7.1 Glucose metabolism in coupledneuron and astrocyte system. ATP is producedvia oxidative energy metabolism (glycolysis,TCA cycle and oxidative phosphorylation) inneurons and in astrocytes. Na+ entry duringelectrical activity initiates increased oxidativeenergy metabolism within neurons. Theactivation of neuronal Na+-K+ ATPase in theplasma membrane leads to reduced levels ofATP, which rapidly activates glycolysis. Thisprocess requires an elevated glucose level,which is transported via the neuronal glucosetransporter (GT). The generated ATP canrestore the Na+/K+ balance via Na+-K+

ATPase. The rapid increase of glycolysis resultsin increased NADH/NAD+ and increasedcytoplasmic pyruvate. In astrocytes, the uptake

of glutamate via high-affinity glutamatetransporters activates oxidative glucosemetabolism, which provides ATP. The basicactivation process is considered similar to thatin the neuron. According to theastrocyte–neuron lactate shuttle hypothesis,the lactate is transported across the glialmembrane and enters into the neuron via theextracellular space, where it is converted topyruvate by LDH to produce ATP by the TCAcycle and oxidative phosphorylation (O.P.) [1].It is hypothesized that, in the case of damagedglucose uptake or ATP production of astrocytes(e.g., due to inclusion formation), lactate couldbe transported from the extracellular spaceinto the damaged astrocytes.

meters, such as the effect of cellular production or consumption of lactate, regionalcerebral blood flow, exchanges through the blood–brain barrier, and extracellularpH variation. The modeling of the glucose flux was based on a similar model. Theestimated contribution of lactate (versus glucose) to neuronal extra pyruvate sup-ply was found to be between 30 and 60%. The model enabled a simulation of theearly dip in extracellular lactate concentration and its subsequent increase (“over-shoot”) found experimentally by Hu and Wilson [8], thus the validity of ANLSHwas suggested.



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The ANLSH is based largely on in vitro experiments, and it postulates that lac-tate is derived from the uptake of synaptically released glutamate by astrocytes.However, the time course of changes in lactate, derived from in vivo experiments,appeared to be incompatible with the ANLSH. Neuronal activation leads to a de-layed rise in lactate, followed by a slow decay which greatly outlasts the period ofneuronal activation. This led to the proposition proposed that the uptake of stim-ulated glutamate released from astrocytes, rather than that of synaptically releasedglutamate, is the source of lactate released following neuronal activation [6]. Theincrease of lactate occurs too late to provide energy for neuronal activity. Further-more, it was considered that there is no evidence that lactate undergoes local ox-idative phosphorylation.

Positron emission tomography (PET) and certain other techniques require longsampling times to obtain a sufficient signal and are therefore more likely to detectlong-lasting signals of the glucose uptake. Thus, these images generated primarilyreflect astrocytic activity rather than neuronal activity, even though the activation ofboth cell types is correlated in the majority of cases [9]. Similarly, results obtainedby magnetic resonance spectroscopy (MRS), which monitors changes in glucoseand lactate levels with low temporal resolution, are more likely to reveal the latecomponent of the metabolic response associated with astrocytes [10, 11]. Thus, theincrease observed in lactate concentration would correspond to enhanced glycol-ysis in astrocytes with associated sustained production of lactate. In contrast, useof MRS with higher temporal resolution [12] allows much faster sampling timesenabling detection of the early lactate “dip”. The detected “dip” is consistent withthe rapid activation of oxidative phosphorylation followed by lactate oxidation viathe TCA cycle in neurons, before lactate production by astrocytes fill up the extra-cellular lactate pool [9].

Recently, functional images using two-photon fluorescence signal of NADHsuggested the existence of the astrocyte–neuron lactate shuttle [13]. (It is wellknown that the lactate/pyruvate ratio critically depends on the NADH/NAD ratio[14].) These data showed that initial consumption of NADH in dendrites causedby oxidative metabolism in mitochondria is followed by glycolytic (nonoxidative)metabolism in astrocytes. The latter process generates lactate, which is shuttledout of the astrocyte and into the neuron, where it can then be “burned” within themitochondria (see Fig. 7.1). Therefore, experimental evidence suggested the cou-pling between oxidative metabolism in the dendritic shaft and glycolytic activity inthe astrocyte to provide sustained neuronal energy in the form of ATP.

Astrocytes face the synapses and form an extensive network interconnected bygap junctions. They participate in synaptic signaling by providing metabolic sup-port to the active neurons. Disturbances of these neuron–astrocyte interactionsare likely to play an important role in neurologic disorders. Understanding thebrain energetics might be critically important because several neurodegenerativediseases exhibit diverse alterations in energy metabolism.

There are virtually no data whether neurons use ambient glucose, and/or glial-derived lactate under neoropathological conditions. It has been proposed thatglycolytic enzymes such as phosphofructokinase (PFK), and glyceraldehyde-3-


7.1 What is the Major Energy Source of the Brain? 5

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Table 7.1 Major characteristics of selected conformational diseases (selected data from [54]).

Disease Diseased proteins Major affected region Pathology

AD Aβ andhyperphosphorylated tau

Cortex, hippocampus,basal forebrain

Neuritic plaques andneurofibrillary tangles

PD α-Synuclein Substantia nigra, cortexand Lewy neurites

Lewy bodies

HD Huntingtin withpolyglutamin expansion

Striatum, basal ganglia,cortex

Intranuclear inclusionsand cytoplasmicaggregates

phosphate dehydrogenase (GAPDH) are inhibited by interacting proteins, such astau, β-amyloid, mutant huntingtin, α-synuclein, that play crucial role in the patho-genesis of conformational diseases (see Table 7.1). As consequences of these het-eroassociations, glycolytic enzymes are impeded, resulting in less ATP and pyru-vate. The reduced pyruvate production decreases the ATP production via the oxida-tive energy metabolism. The appearance of these aberrant protein–protein associ-ations in the cytoplasm and/or the formation of insoluble aggregates due to theaccumulation of these heteroassociates within inclusions of neurons or glia cellscould impede glycolysis. ATP production could slow down, then lactate could betransferred between neurons and astrocytes depending on the request, and utilizedas oxidative substrate during neuronal activity as suggested by the ANLS concept(Fig. 7.1). If inclusions are formed in glial cells, like in the case of multiplex systematrophy, and the ATP supply cannot be fulfilled by the damaged glia cells, the en-ergy (ATP) can be fuelled by the extracellular lactate released from the healthy gliaor neurons. Indeed, there may be no restriction for a glial cell to take up lactate andproduce pyruvate by its LDH. One can hypothesize that the transfer of lactate fromneurons to astrocytes might occur under pathological conditions. All these possi-bilities could be the target of biosimulation studies considering also the reducedactivities of key glycolytic enzymes, hexokinase (HK), PFK, GAPDH or pyruvatekinase (PK) according to the experimental data measured in diseased tissues (seeSection 7.2). However, the data referring to the brain samples including those fromthe well-studied Alzheimer’s disease (AD) brain are so far inconsistent and contro-versial. The activities varied depending on the sampling techniques [15], on thedifference of the brain sections [16], and on the origin of the disease, e.g. decreaseof HK activity was found exclusively in the case of familial AD [17]. Nevertheless,a part of the differences of the findings was explained by technical reasons, thedifferent conditions and the instability of the key glycolytic enzymes [18].

Another important difficulty in the evaluation of the correct activity values for en-zymes involved in energy metabolism in neurons resides in the fact that oppositeprocesses may occur simultaneously, like the reduction of neuronal metabolismand compensatory enhancement in glial activity. The altered activities of some gly-colytic enzymes in brain homogenates represent a net-effect, and it is unclear howthe energy state (ATP level) of the neurons is perturbed if the metabolic activation is



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restricted to astrocytes. This explains why the extensive search for well-establishedinformation on the involvement of glycolytic enzymes and energy metabolism inneurodegenerative processes gave limited amount of in vitro and in vivo data. Eventhe results obtained by determination of glycolytic activities in normal control andpathological brains of patients did not provide straightforward results [16].

7.2Unfolded/Misfolded Proteins Impair Energy Metabolism

The major part of the intracellular proteins has well defined secondary and tertiarystructures; however, a number of in silico and experimental data have been accumu-lated indicating that a part of the proteins does not bear well defined 3D structure[19]. These proteins denoted as intrinsically unstructured or natively unfolded pro-teins, are rather common in living cells and fulfil essential physiological functions[20]. These functions are linked with their structural states and interactions withother cellular targets. They are sometimes form fibrils or protein aggregates withdistinct ultrastructures. Many of them, termed as “neurodegenerative proteins”,are known to form intracellular inclusions, tightly linked to the development of dif-ferent neurodegenerative diseases [21]. It has become apparent that unfolded andmisfolded proteins are extensively involved in different unrelated neurodegenera-tive diseases such as AD, Parkinson’s disease (PD), and Huntington’s disease (HD).These diseases are frequently termed conformational diseases because the initia-tion of the diseases is likely caused by unstructured proteins, like α-synuclein, tauor mutant huntingtin protein (Table 7.1) and other unfolded/misfolded proteins,such as a recently isolated new brain-specific protein, termed TPPP/p25, which in-duces aberrant tubulin aggregates and is enriched in the inclusions characteristicsfor PD and other synucleinopathies (for a review, see [22]).

The development of neurodegeneration is a multistep process: the unfolded/misfolded proteins enter into aberrant protein-protein interactions that finally leadto the formation of inclusions in different area of the brain producing character-istic pathology. However, the exact molecular connections between these steps areunclear yet. “Precisely when in the disease process such interactions occur is un-clear, as is any structural understanding of how altered protein conformations oraggregates trigger neuronal dysfunction” [23]. In addition, there is no clear dis-tinction concerning the primary and secondary nature of the events [24], such asaccumulation of unfolded or misfolded proteins, protofibril formation, differentstress effects, failure of axonal and dendritic transport, mitochondrial dysfunctioncoupled with the damage of energy metabolism. A combination of these and otherevents that impair normal neuronal function can cause chronic neurodegenerativedisorders such as the most common ones: AD, PD, and HD. The major character-istics of these conformational diseases are summarized in Table 7.1.

The impaired glucose turnover can cause not only ATP deficit in the cells in-volved but may induce other deleterious effects [25]. For example, a low glucoseturnover can highly accelerate progress leading to serious dementia. Relevant data


7.3 Interactions of Glycolytic Enzymes with “Neurodegenerative Proteins” 7

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originate from the use of different imaging techniques for monitoring glucose con-sumption in vivo [26]. These data showed that the low glucose turnover resultedin cholinergic deficit as a consequence of decreased acetyl-coenzyme A (AcCoA)synthesis and reduced acetylation of choline to acetylcholine [27]. Pyruvate dehy-drogenase multienzyme complex, which is responsible for the synthesis of Ac-CoA, was inhibited due to its phosphorylation by mitochondrial tau protein kinaseI/glycogen synthase kinase-3beta activated by the amyloid β peptide (Aβ) [28, 29].Hypometabolism of glucose can also lead to alterations in homeostasis of ions, tounfolding of proteins, altered protein synthesis, disturbance of the transport anddegradation of proteins, loss of cell potentials, all of which could be relevant in thedevelopment of conformational diseases [30–33].

7.3Interactions of Glycolytic Enzymes with “Neurodegenerative Proteins”

AD associated with progressive impairment of memory and cognition is character-ized by three pathological lesions: senile plaques, neurofibrillary tangles, and lossof synapses. Plaques are mostly extracellular and consist of deposits of a fibrillousprotein referred to as Aβ surrounded by dying neurites. Aβ is heterogeneous andproduced from a precursor protein (amyloid precursor protein; APP; 130 kDa) bytwo sequential proteolytic cleavages that involve β- and γ -secretases. In fact, sec-retase represents a potentially attractive drug target since it dictates the solubilityof the generated Aβ fragment by creating peptides of various lengths, namely Aβ

(40) and Aβ (42). Tangles are intracellular and are abnormally altered deposits oftau, a microtubule-associated protein, whose normal function involves intracellularaxonal transport. Senile plaques develop extracellularly with the main componentbeing aggregated Aβ, whereas neurofibrillary tangles (NFTs) develop intracellularlywith the main component being aggregated forms of phosphorylated tau. However,recently evidence has been presented that Aβ bind strongly to tau in solution thatmay be a precursor event to later self-aggregation of both molecules [34]. Sincethe insoluble Aβ and tau appear simultaneously in the same neurons, even if thedominant fraction of Aβ plaques are extracellular particles, it indicates that bothmolecules can be aggregated intracellularly at an early stage of the disease. Thismight represent an intermediate state when Aβ could be associated with glycolyticenzymes causing their inhibition.

APP functions as a neuronal receptor and stimulates dendritic and synaptic out-growth. This transmembrane protein is split under non-physiological conditionwhen the glucose turnover-generated and oxidative phosphorylation-generated ATPsupply is insufficient [35]. A critical level of glucose and ATP turnover is crucial forthe proper insertion of APP into the cellular or synaptic membranes. The accumu-lation of Aβ initiates an undesired cycle by binding to the glycolytic enzyme, PFK[36], that decreases glucose turnover by inhibition of the catalytic activity of PFK,resulting in reduced ATP production (Table 7.2). The decreased PFK activity in se-



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Table 7.2 Involvement of glycolytic enzymes in impairing theenergy metabolism of conformational diseases.

Enzyme AD PD HD TPI deficiency

HK Activation [46, 116] Activation [81]PFK Binding to Aβ:

inactivation [36]Aldolase Activation [81]TPI Nitration [32, 63,

66]Inactivation? [75] Binding to MT:

inactivation [79, 86]GAPDH Binding to APP

[37]; inactivation[39]; nitration [67]

Binding toα-synuclein [49, 52]

Binding to CAGrepeat: inactivation[39, 45, 46]

Activation [81]

Enolase Nitration:inactivation [32, 66]

PGM Inactivation [70];nitration:decreased proteinexpression andactivity [66]

LDH Activation [16] Activation [81]PK Activation [16] Activation [16] Activation [81]

nile demented brain cortex resulting from PFK-Aβ interaction can decline glucoseturnover that accelerates Aβ generation.

Association of APP to an other glycolytic enzyme, GAPDH has been also re-ported [37]. This enzyme, GAPDH displays multiple functions in eukaryotic cells.It binds to nucleic acids, translocates into the nucleus affecting transcriptionprocesses, exhibits pro-apoptotic function, binds to cytoskeleton filaments, tubu-lin and actin, and associates with other glycolytic enzymes ([38] and referencestherein). Functional role other than the glycolytic activity was identified for GAPDHin conformational diseases which is related to its specific binding to proteinssuch as APP and huntingtin implicated in neurodegeneration. The association ofGAPDH with these proteins, similarly to the PFK-Aβ interaction, reduced the en-ergy production in diseased cells and tissues. Mazzola and Shirover [38, 39] foundthat there was no measurable difference in GAPDH glycolytic activity in crudewhole-cell sonicates of AD and HD fibroblasts as compared to normal controls.However, they could not exclude the disruption of macromolecular associations ofGAPDH resulting in diminution of GAPDH glycolytic activity. In fact, studies onthe glycolytic activity and subcellular distribution of GAPDH protein in a num-ber of cells revealed the impairment of GAPDH glycolytic function in AD and HDsubcellular fractions despite unchanged gene expression. In the nuclear fraction,deficits of 27% and 33% in GAPDH function were observed in AD and HD, re-spectively. This finding supports a functional role for GAPDH in neurodegenera-tive diseases; the reduced GAPDH activity in the cytoplasm of AD cells can causea significant deficit in energy production. This would greatly contribute or possibly


7.3 Interactions of Glycolytic Enzymes with “Neurodegenerative Proteins” 9

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even initiate the disease process [40–42]. This idea was supported by in vivo data aswell which demonstrated that glucose utilization is decreased by 20–40% in bothAD and HD brain tissue (see Table 7.2) [43].

HD is an inherited neurodegenerative disorder caused by an insertion of multi-ple CAG repeats in huntingtin gene resulting in N-terminal polyglutamine (polyQ)expansion of the large huntingtin protein similarly to other polyQ-related confor-mational diseases. Huntingtin expression is ubiquitous, the greatest level is in neu-rons, occurring in membrane, cytosol, nucleus, mitochondrium. In mouse modelsthe N-terminal fragments of mutant huntingtin caused neurodegeneration, sug-gesting the toxicity of the caspase-produced polyQ fragment [44]. The mutant hunt-ingtin protein-induced neurodegenerative process is associated with increased in-tracellular calcium level that triggers the cleavage of huntingtin and the transloca-tion of the polyQ into the nucleus.

GAPDH was found to bind extensively to the polyQ tract of huntingtin protein[45]. The direct effect of the polyQ tract on the catalytic function of the glycolyticenzymes are unclear yet, however, reduced catalytic activity of GAPDH was foundin HD fibroblasts [46] and brain samples (see above). Since GAPDH plays an im-portant role in the glucose metabolism of the brain, the inhibition of this enzymecausing the defective energy metabolism at system level, could contribute to thedevelopment of the neurodegenerative diseases [47]. The activity of HK in fibrob-lasts expressing mutant huntingtin was higher as compared to the control at 1–3weeks after withholding fresh medium [46]. The finding that the activity of the keyglycolytic enzyme, HK, is altered in fibroblasts expressing mutant huntingtin maysupport connection between the impairment of glycolysis and the disease as well.

PD is the most common neurodegenerative movement disorder caused by selec-tive and progressive degeneration of dopaminerg neurons. One important featureof PD is the presence of cytoplasmic inclusions of fibrillar misfolded proteins, Lewybodies, in the affected brain areas (see Table 7.1). The exact composition of Lewybodies is unknown, but they contain ubiquinated α-synuclein, parkin, synphilin,neurofilaments, synaptic vesicle proteins (for a review, see [24]), and a recently iden-tified protein, TPPP/p25 [48]. Immunohistochemical studies provided evidence forthe co-localization of α-synuclein with GAPDH in Lewy bodies [49, 50]. A punctu-ate pattern of GAPDH distribution for other inclusions of PD nigral section hasalso been described [51]. There are in vitro binding data for the direct associationof GAPDH with both α-synuclein [52] and TPPP/p25 protein [50], however, neitherthe counteraction of these associations nor their effect on the ATP production hasbeen established. Interaction between α-synuclein and TPPP/p25, and TPPP/p25promoted assembly of α-synuclein into filaments within Lewy bodies was revealedas well [53]. The data referring to the impairment of energy metabolism in PD arevery limited; virtually only mitochondrial injury and altered cellular transport havebeen proposed [54].

Indeed, not very much data have been accumulated that effectively contribute toour understanding how the appearance/accumulation of the unfolded/misfoldedproteins affects the energy metabolism of cells, whether the expression of theseproteins directly or indirectly interfere with the glucose metabolism. One of these



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studies was performed on a stable SK-N-MC neuroblastoma cell line expressingTPPP/p25 (denoted as K4 cells), and the energy metabolism was studied at cellularand molecular levels in the transfected cells as compared with the controls [55].

Mitochondrial membrane potency, which regulates the production of high-energy phosphate, is an important parameter determining the fate of the cells [56].For this reason, the hyperpolarization of mitochondrial membrane was determinedby immunfluorescence microscopy monitoring the accumulation of a fluorescencemarker dye. The comparison of the fluorescence images of the control (SK-N-MC)and transfected (K4) cells showed that the expression of TPPP/p25 did not dam-age, rather stimulated, the production of the intracellular high-energy phosphateas long as the cells preserved their morphology similar to the control. Quantitativedata was obtained by determination of the cellular ATP level. The ATP concentra-tion was found 1.5-fold higher in the extract of the K4 clone as compared withthat of the control cells (26.6±3.0 µM/g protein and 17.2±1.8 µM/g protein, re-spectively). Analysis of the activities of the key glycolytic enzymes revealed that,except aldolase, the activities of other enzymes from the upper part of glycolysiswere significantly higher in the case of K4 cells than in the control. The flux of glu-cose consumption, computed using a mathematical model with kinetic parametersdetermined experimentally and by well established rate equations of individual en-zymatic reactions, showed that it was stimulated by the presence of TPPP/p25. Thecellular, biochemical, and computational results suggested that the expression ofthe unfolded TPPP/p25 in K4 cells could be tolerated by responding with extensiveenergy production. This situation may mimic a very early stage of the accumulationof the undesired toxic proteins.

7.4Post-translational Modifications of Glycolytic Enzymes

The impairment of glucose utilization could result from the modification of theglycolytic enzymes under oxidative stress effects. Oxidative stress is an importantfactor leading to the pathophysiologcal alterations in conformational diseases. Ox-idative stress is manifested in protein oxidation, lipid peroxidation, DNA oxidation,and advanced glycation end-products, as well as reactive oxygen species (ROS), andreactive nitrogen species (RNS) formation. Either the oxidants or the products ofoxidative stress could modify the proteins or activate other pathways that may leadto additional impairment of cellular functions and to neuronal loss [57, 58].

Nitration of some glycolytic enzymes has been reported in AD [32], PD [59], andother neurodegenerative diseases (see Table 7.2) [60, 61]. Nitric oxide (NO) is, in-deed, implicated in the pathophysiology of a number of neurodegenerative dis-eases. NO reacts with the superoxide anion producing highly reactive peroxynitrite[62], which causes protein nitrotyrosination, a marker of cell damage reported inneurons and glial cells from AD brains [32]. Vascular amyloid deposits correlatewith nitrotyrosination in brain vessels from AD patients [63]. Aβ fibrils act as asource of superoxide anion [64], which can react with the basal levels of NO, owing


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to the high affinity of NO for the superoxide anion [65], thereby triggering nitroty-rosination.

The redox proteomics approach utilized extensively for the identification of ox-idized proteins has provided data consistent with the biochemical and pathologi-cal alterations in AD ([66] and references therein). Among other proteins, the gly-colytic enzymes GAPDH, triosephosphate isomerase (TPI), phosphoglycerate mu-tase (PGM), and enolase were identified as the targets of nitration in AD hippocam-pus. Perturbation in energy metabolism and mitochondrial functions by specificprotein nitration could be one of the mechanisms for the onset and progression ofAD [67].

In AD, neuronal and synaptic loss occurs in a region-specific manner. Regionsof the brain (for example the hippocampus) where oxidized forms of the glycolyticenzymes occurred showed an extensive deposition of Aβ, compared with the age-matched control brains. Oxidation of α-enolase and TPI was found not only inthe hippocampus but also in the inferior parietal lobule, which implies a commonmechanism operating in the two different regions of brain [66]. Why some brainregions are more sensitive in AD is unclear yet. The identification of commontargets of oxidative damage would help us to understand the pathogenesis of thedisease and could assist the development of more specific therapeutic strategies.

Enolase produces phosphoenolpyruvate, the second of the two high-energy in-termediates that generate ATP in glycolysis. A proteomics analysis of AD brainshowed that the protein level of the α-subunit of enolase is increased as comparedto control brain [66, 68], and α-enolase as a nitrated protein was identified [66, 69].Due to oxidation of the two isoforms of enolase existing in brain tissue (α and γ ),the activity of this enzyme in AD brain was decreased [66, 70]. Enolase is also atarget for carbonylation in AD hippocampus, which implies changes in cellular pHand bioenergetics that could impair enzyme activities leading to neurodegenera-tion.

PGM catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate.A significant increase in oxidation of PGM and a decrease in protein expressionwere observed in AD hippocampus [66] that is consistent with the reported de-creased expression and activity of PGM in AD brain, as compared with the age-matched controls [70, 71].

GAPDH is an excessively nitrated protein in AD hippocampus [?] Aβ-inducedoxidative disulfide bond formation of GAPDH, promoting the nuclear accumula-tion of the detergent-insoluble form of the enzyme in brain samples of AD patientsand transgenic AD mice, was also observed [72]. Oxidation and subsequent loss offunction of these glycolytic enzymes result in decreased ATP production, a findingconsistent with the altered glucose tolerance and metabolism of patients sufferingfrom neurodegenerative diseases [73, 74].

TPI, another glycolytic enzyme that catalyzes the interconversion of dihydroxy-acetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate in glycolysis, wasalso oxidatively modified [32, 63, 66]. However, no change of TPI activity wasobserved [66], probably because the carbonyl groups added were localized awayfrom the catalytic site of this enzyme. TPI was strongly inhibited by endogenous



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β-carbolines due to their binding to TPI under in vivo conditions. β-Carbolines areknown as potent inhibitors of mitochondrial respiration and cause neurodegener-ation after injection into the substantia nigra [75]. Increased level of β-carbolinederivatives was observed in the cerebrospinal fluid of patients with de novo PD [76].Since it is known that TPI deficiency (originated by mutations of the isomerase)manifests itself in severe progressive extrapyramidal disorder and other neurolog-ical symptoms [77], it was thus suggested that inhibition of TPI by β-carbolinescould contribute to neurodegeneration [75]. It is worth noting from this aspectthat the structure of β-carbolines closely resembles that of the neurotoxin MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) that is routinely used to mimic PDin animals.

7.5Triosephosphate Isomerase Deficiency, a Unique Glycolytic Enzymopathy

The deficiency of the glycolytic enzyme, TPI, results not only in hematologicalsymptoms, as in the case of the majority of the glycolytic enzymes, but in extrapyra-midal neurodegeneration [78]. It is a rare disease with childhood death. It has beensuggested that the disease is developed due to the mutation of TPI causing proteinmisfolding which leads to the partial inactivation of the isomerase [79]. Thus TPIdeficiency could be considered as a “conformational disease”. The extent of the lossof activity depends on the position of the mutation as well as on the types of bloodcells ([78] and references therein). It is has to be noted that no brain samples havebeen available for research, even from post mortem brain tissues. However, the in-vestigation of glycolytic pathway in the erythrocytes of patients rendered it possibleto evaluate the effect of mutation on the energy metabolism.

The classic function of TPI is to adjust the rapid equilibrium between the twotriosephosphates, glycerinealdehyde-3-phosphate and DHAP. Patients with TPI de-ficiency have unimpressive alterations in glucose utilization, ATP and lactate pro-duction. Modeling studies and experimental data suggested that the physiologicalATP level was maintained due to the activation of enzymes involved in the pen-tosephosphate and glycolytic pathways (Fig. 7.2) [80, 81]. The interconnection ofthe two pathways with increased activities can compensate for the reduced TPIactivity of deficient cells in TPI-deficient erythrocytes [80, 81].

Dramatic change in erythrocytes is characterized by the marked increase inDHAP and in a much lesser increase in fructose-1,6-bisphosphate levels [78,81]. An inverse relationship between TPI activity and DHAP concentration wasdetected in all TPI-deficient patients. Computer models were evaluated to sim-ulate this inverse relationship in normal and deficient red blood cells [82–84].However, the low TPI activity-derived DHAP accumulation was not supportedby the previous models unless a very low TPI activity was introduced which wasnot supported by experimental data [82, 84]. Recently, a realistic model of hu-man erythrocyte glycolysis was elaborated on the basis of experimentally deter-mined kinetic parameters [81]. It was shown that the mutation-derived activity


7.5 Triosephosphate Isomerase Deficiency, a Unique Glycolytic Enzymopathy 13

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Fig. 7.2 Glycolysis and related pathways.Glycolysis is a central metabolic machinery inwhich one mole of glucose is catabolized totwo moles of pyruvate, NADH, and ATP. Underaerobic conditions, pyruvate is further oxidizedby mitochondrial system. In erythrocytes DHAPis a dead-end product; however, in brain it canbe converted into direction of lipid synthesis.Glycolysis and the pentose phosphate pathway(pentosePP) are interconnected viafructose-6-P and glyceraldehyde-3-P. A highlevel of NADPH favors lipid synthesis viapentose phosphate shunt (pentosePP). At TPIinhibition (TPI deficiency), glyceraldehyde-3-Pcan be produced via G6PDH as well, tocontribute to the glycolytic flux. α-GDHcatalyzes the oxidation of NADH to NAD+ and

the reduction of DHAP to glycerol-3-P, which isa substrate of biosynthesis of glycerolipids(i.e., phospholipids, triacylglyceride). At lowlevel of NADPH, DHAP can be transformed toplasmalogens. The reverse situation can occurat reduced TPI activity and neurologicaldisorders [77]. Enzymes: HK = hexokinase, GPI= glucosephosphate isomerase, PFK =phosphofructokinase, TPI = triosephosphateisomerase, GAPDH =glyceraldehyde-3-phosphate dehydrogenase,PGK = phosphoglycerate kinase, PGM =phosphoglycerate mutase, ENO = enolase, PK= pyruvate kinase, LDH = lactatedehydrogenase, α-GDH = α-glycerolphosphatedehydrogenase, G6PDH = glucosephosphatedehydrogenase.

decrease of TPI was associated with an increase in the activity of the other gly-colytic enzymes, including those (HK and PFK) which are known to control pre-dominantly the glycolytic flux in erythrocytes. The mathematical modeling of theglycolytic flux required a consideration of changes in the kinetic parameters of



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the glycolytic enzymes, in addition to that of TPI, to obtain relevant informationon the energy state of TPI-deficient erythrocytes. For the modeling of the wholeglycolytic pathway (see Fig. 7.2), the Vmax values of the glycolytic enzymes aswell as the Kequ (= [DHAP]/[GAP]) and Km values of the TPI reaction were de-termined experimentally in the hemolysates from both the control and the dis-eased cells (see http://www.BiochemJ.org/bj/392/bj3920675add.htm). Simulationdata showed that the rate of glucose conversion into lactate was higher for the mu-tant cell, and the steady-state flux was increased by a 2.5-fold factor due to the en-hanced activities of HK, PFK, and GAPDH. The mathematical model correspondedto the pathological criterion, namely, this realistic model was appropriate to mimicthe higher DHAP level in the hemolysate of the patient. In fact, a new ratio oftriosephosphate concentrations was adjusted for the steady-state flux of glycolysisin the diseased cell, due to the altered activity of the other enzymes of the glycolyticpathway. It was proposed that the TPI mutation-derived alterations of key glycolyticenzymes might represent a “repairing mechanism” [80] that could ensure a virtu-ally normal energy state in erythrocytes [85].

A number of experimental and theoretical data has been accumulated relatedto the mutant TPI-connected metabolism in diseased cells, however, it is still amystery which extent the TPI mutation is responsible for the development of neu-rodegenerative disorder. Recently, Bonnet and co-workers [75] suggested a quali-tative model, in which they incorporated results published mostly by Hollán andher co-workers [77, 86]. The compensation of reduced activity of TPI by activationof the pentose phosphate shunt [80] leads to the accumulation of NADPH, whichis mainly utilized for the synthesis of fatty acids in brain cells (Fig. 7.2). Thus, in-hibition of TPI likely stimulates indirectly the biosynthesis of fatty acids. DHAPis a substrate of glycerol-3-phosphate dehydrogenase, an oxidoreductase which ac-cepts both NADH and NADPH as co-substrates, depending on the supply. Theproduct of the enzymatic reaction is glycerol-3-phosphate, a common substrateof various glycerolipids such as phospholipids and triacylglyceride. Furthermore,DHAP also serves as a precursor for the formation of etherlipids among themthat of plasmalogens (see Fig. 7.2). High levels of NADPH shift the utilization ofDHAP to glycerol-3-phosphate and cause a reduction in plasmalogens, specificallyin phosphatidyl-ethanolamines [77]. These changes lead to an imbalance betweenbilayer- and nonbilayer-forming lipids in membranes, severely affecting the activ-ity of several enzymes and the association of protein kinase C and G proteins tothe membrane. Thus, the decrease in plasmalogens impairs the protection againstoxidative stress with consecutive worsening of the neurodegenerative process.

The classic interpretation of the development of neurodegeneration in the caseof TPI deficiency is based upon the extensive inhibition of TPI activity by mutation,which prevents the adjustment of the physiological ratio of triosephosphates. In-deed, in erythrocytes, it results in the accumulation of DHAP. However, this maynot be valid for neurons or astrocytes. Even in the case of lymphocytes, which dis-play some similarity to brain cells, only slight elevation of DHAP concentration wasdetected [79]. Therefore, further investigations are necessary to support or excludethe direct role of the mutation of TPI in the development of neurodegeneration.


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Recently, significant changes in mRNA levels of nitric oxide synthase and prolyloligopeptidase were reported in the lymphocytes of a patient suffering TPI defi-ciency [81]. These two enzymes have been suggested to be indirectly involved inneurodegenerative diseases [87, 88]. The nitration of TPI has been documented inAD (see Table 7.2), and a 15-fold increase in the urinary 3-nitrotyrosine was foundin a TPI-deficient patient as compared with that of the control [89].

7.6Microcompartmentation in Energy Metabolism

Most of the proteins involved in neuronal homeostasis are synthesized within thebody of the neuronal cell. Many of these proteins are required in the axon or at thenerve terminal far from the point of their synthesis. Most synapse-bound cargoesare transported directly to their destinations through the action of microtubulesand actin-based motors [90]. The bulk of neuronally synthesized proteins destinedfor the axon is transported about 100 times slower than the vesicular traffic of fastaxonal transport (100 mm/day). Two slow axonal transport components, SCa andSCb, have been identified [91]. The SCb is primarily made of actin, but it includesglycolytic enzymes with other known and not yet identified components. The bind-ing of the slowly transported proteins to the motor or some intermediate couldbe in a “piggy-back” manner. Such piggy-back binding of sequential glycolytic en-zymes along sarcomeres of Drosophila flight muscles has been demonstrated [92].The co-localization of a set of six glycolytic enzymes, aldolase, glycerol-3-phosphatedehydrogenase, GAPDH, TPI, 3-phosphoglycerate kinase (PGK), and PGM, thatcatalyse consecutive reactions along the glycolytic pathway was revealed. It becameclear that the presence of active enzymes in the cell is not sufficient for musclefunction; a highly organized cellular system of the specific isoforms of enzymes isrequired ensuring mechanisms by which ATP is supplied to the filament ATPase[93].

In brain tissues, specific isoforms of glycolytic enzymes are also expressed; thereare specific brain isoforms for PFK (PFK-C), fructose-1,6-bisphosphate aldolase (al-dolase C), enolase (enolase γ ), but not for GAPDH. The isoforms bear the same cat-alytic functions; however, they could be specialized to form different ultrastructuralentities. For example, muscle PFK (a dissociable tetrameric form) binds to micro-tubules and bundle them [94, 95], however, the brain isoenzyme (stable tetramer)does not [96].

Glucose is not only the major energy source, but it is essential for maintainingnormal neuronal function. Recent evidence suggests a direct correlation betweenglucose utilization and cognitive function ([97] and reference therein). Reductionof glucose levels results in pathophysiological state, however, this occurs long be-fore significant alteration in tissue ATP levels is detected. Substitution of pyruvatefor glucose does not support normal evoked neuronal activity, although tissue ATPlevel returns to normal. All these results suggest that glycolysis or glycolytic inter-



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mediate(s) are necessary for normal synaptic transmission independently of globalcellular ATP levels.

Microcompartmentation of a metabolite is defined as a substrate having morethan one distinct pool. It has been documented, for example, for glutamatemetabolism at the multi- and single-cellular level in brain. Glycogen metabolismof astrocytes was investigated by exposing it to (U-13C) glucose in the absenceand presence of a glycogen phosphatase inhibitor [98]. The results indicated dis-tinct compartments for lactate derived from glycogenolysis and that derived fromglycolysis, and suggested a linkage between glycogen turnover and glycolytic andTCA cycle activity [99]. It has been proposed that the TCA cycle intermediates andTCA cycle-derived amino acids are preferentially generated from the glucose me-tabolized by glycolysis, whereas the glutamine mainly originated from glycogenol-ysis. Since glutamine is a precursor of the neurotransmitter glutamate, glycogenturnover could play a role in glutamatergic activity [99]. In addition, mitochondrialheterogeneity within single astrocytes was revealed.

Importance of glucose metabolism in maintaining neural activity was investi-gated by measuring the effect of lowering the concentration of glucose in perfusionmedium on synaptic activity (population spikes, PS) and on the level of high energyphosphates in various regions of the hippocampus of pigs. Lowering the glucoseconcentration, ATP and creatine phosphate levels were well preserved [100]. How-ever, significant differences were detected in the activity of PFK, in contrast to HKthat showed no significant differences in the different regions of the hippocampus.The sensitivity in maintaining synaptic activity at lowered glucose concentrationsvaried parallel with the activity of PFK in the different regions of the hippocam-pus, indicating that the glycolytic metabolism regulated by PFK is crucial for themaintainance of synaptic activity.

The synaptic vesicles are capable of accumulating the excitatory neurotransmit-ter glutamate by harnessing ATP. It has been also demonstrated that GAPDH andits sequential glycolytic enzyme, PGK, are enriched in synaptic vesicles, forming afunctional complex [101]. The vesicle-bound GAPDH/PGK can produce ATP at theexpense of their substrates. The inhibition of mitochondrial ATP synthesis, how-ever, has minimal effect on any of these parameters. This suggests that the coupledGAPDH/PGK system, which converts ADP to ATP, ensures maximal glutamate ac-cumulation into presynaptic vesicles.

The maximum rate of some enzyme activities related to glycolysis, Krebs cycle,acetylcholine catabolism, and amino acid metabolism were determined in differ-ent types of synaptosomes obtained from rat hippocampus. Characterization of theenzyme was performed on two synaptosomal populations defined as “large” synap-tosomes mainly containing the glutamatergic nerve endings and “small” synapto-somes, which typically contains the cholinergic nerve endings mainly arising fromseptohippocampal fiber synapses involved in cognitive processes [102]. In controlanimals, the activities of enzymes participating in energy metabolism were differ-ent in the two synaptosomal populations. It was proposed that this energetic micro-heterogeneity of the synaptosomes may underlay their different behavior during


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both physiopathological events and pharmacological treatment due to the differentsensitivity of neurons confirming the existence of different metabolic machinery.

The postsynaptic density (PSD) organelle is a dense concentration of proteinsattached to the cellular surface of the postsynaptic membrane in dendritic spineheads. It contains a large number of proteins; for example in the cerebral cortexmore than 30 kinds of proteins were identified by characteristic 2D electrophoreticmobility, immunoblotting with specific antibodies, and N-terminal and peptide se-quencing [103]. Recently, much more (>1000) protein constituents of the PSD wereidentified by proteomic analysis [104–110], of which 466 were validated by their de-tection in two or more studies [110]. These proteins include the receptors and ionchannels for glutamate neurotransmission, proteins for maintenance and modu-lation of synaptic architecture, sorting and trafficking of membrane proteins, gen-eration of anaerobic energy, scaffolding and signalling, local protein synthesis, andcorrect protein folding and breakdown of synaptic proteins.

PSD is enriched for spectrin, actin, tubulin and microtubule-associated proteinII, myosin, glycolytic enzymes, creatine kinase, elongation factor 1 alpha, receptor,and neurofilament proteins. In fact, all glycolytic enzymes were found to be presentin the PSD fraction. Among them GAPDH and PGK, directly involved in the gen-eration of ATP, lactate dehydrogenase (LDH) used for the regeneration of NAD+,were identified [111]. The association of GAPDH with synaptosomal particles butnot with the synaptosomal plasma membranes was revealed as well [112]. A physi-cal association of GAPDH with endogenous actin in synaptosomes and PSDs wasdemonstrated very early [111]. These data suggest a role of glycolysis in synapto-somes, as well as the involvement of GAPDH in nonglycolytic functions, such asactin binding and actin filament network organization.

The presence of GAPDH and fructose-bisphosphate aldolase was validated bytheir detection in all of the above-mentioned studies. It was postulated that theseproteins provide immediate availability of glycolytic source of ATP to the synapse.This is especially important during acute increase in synaptic activity when themitochondrial supply of energy does not meet the transient and highly localizedincreased demand in energy. Indeed, the glycolytic generation of ATP at the iso-lated PSD was demonstrated. In addition to the proteins involved in glycolysis,other proteins also involved in energy generation (i.e. creatine kinase-B) or in thephosphate transfer system (i.e. adenylate kinases) were detected. These proteinsplay important roles in providing the site-specific burst of high-energy phosphate.The ATP utilized by kinases (Ca2+/calmodulin-dependent protein kinase, proteinkinase A and C) was also found in the PSD. Therefore, it is likely that localized ATPis supplied not by the mitochondria, but by the glycolytic complex at the PSD [113].

Compartmentation of energy-producing systems in the central nervous systempoints to their crucial role in cell communication. ATP produced by mitochondriain the dendritic shaft may travel into the dendritic spine, the site of the synapse,because there are no mitochondria in the spine itself. However, there is anotherenergy-producing system forming ATP in the spine itself, produced by glycolyticenzymes localized in the PSD at the postsynaptic membrane of the synapse [113].The significance of these findings is that the ATP generated at this important



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synapse site can be used at ion channels, by various protein kinases involved insignal transduction, and also for protein synthesis in the spine. Thus, there couldexist two energy-producing systems, one existing in the astrocyte and in the den-dritic shaft in the production of energy for general “housekeeping” metabolic func-tions, and the other at the postsynaptic PSD site for the processing of nerve signaltransduction. The communication requirement of the central nervous system al-most invites a specialized, localized, structural site for its most important mission.These results show that the activity-dependent glycolytic and oxidative metabolicresponses in the central nervous system are highly compartmentalized, on onehand, between astrocytes and neurons, on the other hand, in the neuron itself.

7.7Concluding Remarks

Numerous studies have provided intriguing evidence for the role of energy pro-duction in the resting and activated function of the neuronal system. Much less is,however, known about the molecular mechanism for coupling between energy pro-duction and the development of conformational diseases that include neurodegen-erative disorders, such as AD, PD, and HD, and TPI deficiency (a new candidate en-tering this family of diseases). This paper reviews the interrelations among glucoseand lactate consumption, ATP production and utilization, physiological neuronalactivation and aberrant protein–protein associations. It is clear that the interrela-tionships among glycolysis, ATP supply, and synaptic transmission is an importantissue to understand the potential role of the damage of energy metabolism in theneurodegenerative processes. One of the major difficulties is, however, that twoopposite processes may occur simultaneously: a reduction of neuronal metabolismand compensatory enhancement in glial activity [16]. The altered activities of someglycolytic enzymes in brain homogenates represent a net effect and it is unclear, ifthe metabolic activation is restricted to astrocytes, how the energy state (ATP level)of the neighboring neurons is being perturbed. Energy metabolism that uses lac-tate as an oxidizable energy substrate, produced by either neurons or astrocytes,is an important pathway for neuronal energy production under prolonged stimu-lation and may be under pathological conditions [114]. The appearance of clinicalsymptoms such as protein aggregates, fibres, inclusions, is frequently initiated byconformationally unfolded/misfolded proteins that enter aberrant protein–proteininteractions. In these interactions well characterized “neurodegenerative proteins”(Aβ, α-synuclein, hyperphosphorylated tau, TPPP/p25) and metabolic enzymes in-volved in energy production are participating, resulting in nonphysiological ATPlevels by slowing down the glucose consumption and lactate oxidation.

Microcompartmentation of metabolic enzymes has been shown to be a power-ful way to control energy production in living cells. PSD is an excellent examplefor the ATP utilized by ion channels or protein kinases involved in signal trans-duction. Microcompartmentation can alter the flux of the whole pathway by pro-ducing high local metabolite concentration, resulting in different kinetic parame-


Acknowledgments 19

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ters as compared with that determined for individual enzymes. The formation ofsuch organized enzyme structures (metabolon) [114] creates non-ideal behavior ofthe systems, which can be described in a phenomenological manner. However, forunderstanding the mechanism of energy metabolism in normal and pathologicalconditions, the evaluation of rational models is needed, including relevant data thatdetermine metabolic flux and well established mathematical equations of the con-secutive enzymatic reactions and transport processes. Coupling of more extendedmodeling data obtained for physiological and pathological situations is expected toimprove the chances of early diagnosis as well as proposing novel drugs for thetreatment of different stages of the diseases.

Acknowledgments

This work was supported by FP6-2003-LIFESCIHEALTH-I: Bio-Sim and Hungar-ian National Scientific Research Fund Grants OTKA T-046071 (to J.O.) and T-049247 (to F.O.), and NKFP-MediChem2 1/A/005/2004 (to J.O.).

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Energy Metabolism in Conformational Diseases