Effect of coenzyme Q10 and vitamin E on brain energy metabolism

in the animal model of Huntington’s disease

Svatava Kasparova a,*, Zuzana Sumbalova b, Peter Bystricky a, Jarmila Kucharska b,Tibor Liptaj a, Vladimır Mlynarik c, Anna Gvozdjakova b

a Central Laboratory of NMR Spectroscopy, Faculty of Chemical and Food Technology, Slovak University of Technology,

Bratislava, Radlinskeho 9, SK-812 37 Bratislava, Slovakiab Pharmacobiochemical Laboratory of the 3rd Department of Internal Medicine, School of Medicine,

Comenius University, Bratislava, Slovakiac Laboratory for Functional and Metabolic Imaging, EPFL, Lausanne, Switzerland

Received 12 July 2005; accepted 15 September 2005

Available online 14 November 2005

Abstract

The neuropathological and clinical symptoms of Huntington’s disease (HD) can be simulated in animal model with systemic administration of

3-nitropropionic acid (3-NP). Energy defects in HD could be ameliorated by administration of coenzyme Q10 (CoQ10), creatine, or nicotinamid.

We studied the activity of creatine kinase (CK) and the function of mitochondrial respiratory chain in the brain of aged rats administered with 3-

NP with and without previous application of antioxidants CoQ10 + vitamin E. We used dynamic and steady-state methods of in vivo phosphorus

magnetic resonance spectroscopy (31P MRS) for determination of the pseudo-first order rate constant (kfor) of the forward CK reaction, the

phosphocreatine (PCr) to adenosinetriphosphate (ATP) ratio, intracellular pHi and Mgi2+ content in the brain. The respiratory chain function of

isolated mitochondria was assessed polarographically; the concentration of CoQ10 and a-tocopherol by HPLC.

We found significant elevation of kfor in brains of 3-NP rats, reflecting increased rate of CK reaction in cytosol. The function of respiratory chain

in the presence of succinate was severely diminished. The activity of cytochromeoxidase and mitochondrial concentration of CoQ10 was unaltered;

tissue content of CoQ10 was decreased in 3-NP rats. Antioxidants CoQ10 + vitamin E prevented increase of kfor and the decrease of CoQ10 content

in brain tissue, but were ineffective to prevent the decline of respiratory chain function.

We suppose that increased activity of CK system could be compensatory to decreased mitochondrial ATP production, and CoQ10 + vitamin E

could prevent the increase of kfor after 3-NP treatment likely by activity of CoQ10 outside the mitochondria. Results of our experiments contributed

to elucidation of mechanism of beneficial effect of CoQ10 administration in HD and showed that the rate constant of CK is a sensitive indicator of

brain energy disorder reflecting therapeutic effect of drugs that could be used as a new in vivo biomarker of neurodegenerative diseases.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Creatine kinase; In vivo saturation transfer 31P NMR; Rats; 3-Nitropropionic acid; Oxidative phosphorylation; Antioxidants

www.elsevier.com/locate/neuint

Neurochemistry International 48 (2006) 93–99

1. Introduction

Huntington’s disease (HD) is a hereditary neurodegenerative

disorder, which is characterized by psychiatric symptoms,

movement disorder and progressive dementia (Browne et al.,

1999). The mutant gene, located on the short arm of

chromosome 4, results in an expanded CAG trinucleotide

repeat (Beal, 1995). The function of the huntingtin protein is

unknown. Several different etiological processes may play

* Corresponding author. Tel.: +421 2 5292 6018; fax: +421 2 5292 6018.

E-mail address: kasparov@cvt.stuba.sk (S. Kasparova).

0197-0186/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2005.09.002

roles, and strong evidence from studies in both humans and

animal models suggest the involvement of energy metabolism

dysfunction, excitotoxic processes, and oxidative stress. The

gene defect may cause a subtle impairment of energy

metabolism that ultimately leads to neuronal degeneration,

initially in the striatum and later in other brain regions (Jenkins

et al., 1998). Impaired energy production may lead to increases

in intracellular calcium, and generation of free radicals but

exact mechanism of energy impairment in HD is unclear.

Accidental human ingestion of 3-nitropropionic acid (3-NP),

a potent blocker of succinate dehydrogenase (SDH), complex II

of mitochondrial electron transport chain, results in chorea and

dystonia and in basal ganglia degeneration (Ludolph et al., 1992).

S. Kasparova et al. / Neurochemistry International 48 (2006) 93–9994

Systemic administration of 3-NP to both rats and primates

produces age-dependent striatal lesions, which are strikingly

similar to those seen in HD (Brouillet et al., 1995). Chronic 3-NP

treatment reproduces selective loss of spiny projection neurons

containing the inhibitory neurotransmitter g-aminobutyric acid

(GABA) and sparing of cholinergic neurons that are observed

typically in the HD brain (Beal et al., 1993). It is well known that

GABA is an important source of energy in the brain

(GABA = 1.6 mmol/g, glucose = 1–1.5 mmol/g), thus we sup-

pose that an impairment of GABAergic neurons by 3-NP could

cause changes in the brain energy metabolism leading to increase

of BB-CK activity. SDH inhibitor causes a rapid depletion of

intracellular ATP in neurons, leading to impairment of Ca2+-

ATPase and Na+/K+-ATPase and to progressive membrane

depolarization as a result of intracellular sodium overload

(Brouillet et al., 1999). BB-CK has been found to be functionally

coupled with the plasma membrane-bound Na+/K+-ATPase (Lim

et al., 1983; Wallimann et al., 1985) thus the creatine/

phosphocreatine (Cr/PCr) shuttle is supposed to be a major

component of the neuronal Ca2+ reconstitution mechanism

(Hemmer and Wallimann, 1993; Wallimann and Hemmer, 1994).

Although the main steps leading to 3-NP toxicity have been

recognized, the reasons for the vulnerability of striatal

GABAergic neurons in contrast to the resistance of cholinergic

interneurons to chronic 3-NP application remain not fully

understood.

3-NP lesions formation in rats that have been reported to be

associated with elevated lactate levels (Brouillet et al., 1995)

could be blocked by removal of glutamatergic excitatory

striatal inputs by decortication, by glutamate release inhibitors,

or by glutamate receptor antagonists (Jenkins et al., 1996).

Quantitative localized proton magnetic resonance spectro-

scopy (1H MRS) and proton decoupled 31P MRS are sensitive to

�10% alterations in key cerebral metabolites, and may be of

value in noninvasive monitoring of appropriate therapies in

neurological diseases (Hoang et al., 1998). 1H MRS revealed an

increase of lactate concentration in the occipital cortex of HD

patients that correlated with duration of illness (Jenkins et al.,

1993). A decrease in the phosphocreatine to inorganic

phosphate ratio (PCr/Pi) in HD muscle suggests that the defect

in energy metabolism extends also to non-neural tissue

(Koroshetz et al., 1997). However, measurements of steady-

state high-energy phosphate levels do not reflect energy

metabolism of brain tissue in chronic pathological states

satisfactorily (Kasparova et al., 2000, 2005; Sumbalova et al.,

2002). On the other hand, the rates of synthesis and/or

consumption of high-energy phosphate compounds, i.e.

adenosinetriphosphate (ATP) and phosphocreatine (PCr) could

be more reliable indicators of brain function under these

conditions. Creatine kinase (CK) is an important enzyme that

catalyses the reversible exchange of phosphate group between

PCr and ATP according to the equation:

PCr2� þMgADP� þHþ , MgATP2� þCr (1)

CK in brain exists in multiple forms, and together with Cr and

PCr constitute a system that seems to be critical in regulation of

energy homeostasis in the brain and other organs with high and

fluctuating energy demands (Hemmer and Wallimann, 1993).

Over the last years, the important role of creatine and CK

reaction in various pathological states have been highlighted

(Kasparova et al., 1998, 2000, 2005; Sumbalova et al., 2002;

Wyss and Schultze, 2002). Changes of CK activity may reflect

the energy disorder of the brain as well as therapeutic effect of

drugs (Wyss and Schultze, 2002). Novel therapeutic strategy to

ameliorate mitochondrial-induced dysfunction is to increase

intracellular energy stores. The administration of Cr, the sub-

strate of CK reaction, stimulates mitochondrial respiration and

PCr synthesis, which may help to sustain ATP levels under

oxidative stress (Matthews et al., 1998). There is evidence that

impaired energy metabolism contributes to neuronal death in

HD and the most rapid event after addition of 3-NP is a decrease

in PCr/Cr ratio (Erecinska and Nelson, 1994).

Measurements of lactate production in brains patients with

HD and animals with modeled HD by 1H MRS revealed that

creatine, cyclocreatine, coenzyme Q10 (CoQ10) and nicotina-

mide—compounds increasing energy metabolism could exert

neuroprotective effect in this disease (Koroshetz et al., 1997;

Matthews et al., 1998; Beal, 1999). Antioxidants vitamin E and

CoQ10 form an essential redox-couple (Kagan et al., 2000).

Pretreatment with a-tocopherol had no neuroprotective effect in

animal model of HD (Beal et al., 1988); and treatment with high

doses of a-tocopherol was effective only in patients in early

course of the disease (Peyser et al., 1995). On the other hand,

pretreatment with CoQ10 exerted neuroprotective effect in a

variety of animal models of HD and oral administration of CoQ10

significantly decreased elevated lactate levels in patients with

Huntington’s disease (Beal, 1999). CoQ10 serum levels of HD

patients were significantly lower than that of healthy controls and

HD patients treated with CoQ10 (Andrich et al., 2004). It was also

proposed that supplementation of HD patients with CoQ10, an

essential cofactor of respiratory chain, could reduce their

impaired mitochondrial function (Andrich et al., 2004).

The purpose of the present study was to study the CK

reaction in 3-NP model of HD by kinetic parameter of in vivo31P MR spectroscopy and to elucidate the mechanism of

beneficial effect of the pretreatment with CoQ10 + vitamin E on

brain energy metabolism in 3-NP model of HD.

2. Materials

Male Wistar rats 20–24 months old were injected with 3-NP (10 mg/kg/

every 12 h, i.p.) for 11 days to develop chronic model of HD (Matthews et al.,

1998). Group QE + 3-NP received coenzyme Q10 and vitamin E by the use of

gastric tube (hydrosoluble Q-GEL1, Tishcon Corp., USA) (250 mg CoQ10

+ 530 mg vit. E/kg/day) during 10 days before application of 3-NP. The control

group (C) received corresponding vehicle. At the end of the experiment all the

animals were measured by 31P MRS. The last dose of 3-NP was given to animals

in the evening before their sacrifying.

3. Experimental procedures

3.1. In vivo 31P MRS technique

In vivo 31P MRS experiments were performed at 4.7 T on a SISCO 200/300

imaging spectrometer equipped with horizontal bore magnet for measurements

on animals. 31P MR spectra were collected using 16 mm surface coil. The static

S. Kasparova et al. / Neurochemistry International 48 (2006) 93–99 95

Graph 1. Unidirectional rate constant of CK reaction, kfor, calculated from 31P

MRS saturation transfer experiment. C, control aged rats; 3-NP, aged rats after

administration of 3-nitropropionic acid; QE + 3-NP, rats pretreated with

CoQ10 + vitamin E. Number of animals in groups was 4–6. *p < 0.05 vs. C.

magnetic field was shimmed using the proton signal of water that showed a

typical line width of 20–35 Hz. Relative concentrations of phosphate metabo-

lites were determined from integrals of their signals in 31P MR spectra using

program MESTRE-C 1.5.1. Values of intracellular pHi and Mgi2+ concentration

were calculated using equations given by Jelicks and Gupta (1992).

Time-dependent 31P MRS saturation transfer was applied to determine the

pseudo-first order rate constant of forward CK reaction (kfor) as described

previously (Clark et al., 1991; Mlynarik et al., 1998). Time-dependent satura-

tion transfer allows one to measure simultaneously two parameters, T1 and kfor.

Saturation of the g-ATP resonance for increasing time periods induces an

exponential decay of the PCr resonance to a new steady state. The evolution of

longitudinal magnetization as a function of time is described by the equation

(Forsen and Hoffman, 1963):

dMPCr

dt¼ �½kfor þ ðT1PCrÞ�1�ðMPCr �M0

PCrÞ þ krevðMATP �M0ATPÞ (2)

The dependence of the longitudinal magnetization of the PCr signal, MPCr, on

the time t of the g-ATP signal saturation is given by the equation:

MPCr ¼ M0PCr

�1 � kforT1sPCr

�1 � exp

��t

T1sPCr

���(3)

where M0PCr is the magnetization of PCr in the absence of g-ATP saturation, kfor

the forward CK reaction rate constant, T�11sPCr ¼ kfor þ T�1

1PCr the apparent

longitudinal relaxation rate in the presence of g-ATP saturation, and t is the

irradiation time. The T1sPCr value was calculated as a slope of the semiloga-

rithmic plot of MPCr �M1PCr against t where M1

PCr ¼ M0PCrT

�11PCr=ðkfor þ T�1

1PCrÞ is

the steady-state magnetization of PCr after a long-term irradiation of the g-ATP

signal. The pseudo first-order rate constant kfor was calculated according to the

equation:

kfor ¼1 �M1

PCr=M0PCr

T1sPCr

(4)

In place of the magnetization values, corresponding PCr signal intensities were

used in these calculations. M1PCr was read from the spectrum with 10-s

irradiation of the g-ATP resonance, and M0PCr was obtained from the reference

spectrum measured with the irradiation offset in the mirror position relative to

the PCr resonance and with the irradiation time of 1 s. The saturation was

accomplished by an on-resonance DANTE sequence consisting of a series of

10 ms radio frequency pulses with interpulse delays of 400 ms. The time of

irradiation of the g-ATP resonance was varied from 0.3 to 10 s (Clark et al.,

1991). As a check of validity of the results, the T1PCr values were calculated

using the following equation:

T1PCr ¼ T1sPCr

M0PCr

M1PCr

(5)

3.2. Biochemical analyses

The animals were sacrificed by decapitation. Brain mitochondria were

isolated by differential centrifugation (Sarma et al., 1976). The functions of

respiratory chain in the presence of glutamate as a NAD-substrate or succinate/

rotenone as a FAD-substrate were assessed in isolated mitochondria by means

of Oxygraph Gilson 5/6H (USA) using an oxygen Clark-type electrode. The

activity of cytochromeoxidase (COX) was determined from oxygen consump-

tion after addition of cytochrome c to mitochondria pre-incubated with 0.1%

Triton X-100 (Muscatello and Carafoli, 1969). Mitochondrial proteins were

determined by the method of Lowry et al. (1951) using bovine serum albumin as

a standard. The concentrations of a-tocopherol, CoQ9 and CoQ10 in brain tissue

and mitochondria were determined by the use of high-performance liquid

chromatography (Lang et al., 1986; Kucharska et al., 1998).

3.3. In vitro CK determination

The brain tissue was homogenized with Ultra Turrax in 14 volumes of the

medium containing: 10 mM HEPES, 137 mM NaCl, 4.6 mM KCl, 1.1 mM

KH2PO4, 0.6 mM MgSO4, and protease inhibitors pepstatin (0.7 mg/ml) and

phenyl-methylsulphonyl fluoride (PMSF, 40 mg/ml) (pH 7.4) (Horakova et al.,

2000). The samples were frozen and stored at �20 8C. The activity of CK was

determined using the Creatine Phosphokinase kit (Procedure no. 661) from

Sigma Diagnostic.

3.4. Statistics

The differences between groups were evaluated using Student’s t-test,

p < 0.05 was considered statistically significant. Values in tables are mean

� S.E.M.

4. Results

Using in vivo 31P MRS saturation transfer experiment, we

observed a significant elevation of the rate constant, kfor in brain

of 3-NP rats versus control values (Graph 1). The increase of

kfor was prevented by 10-day pretreatment of rats with high

doses of the antioxidants CoQ10 and vitamin E. On the other

hand, no significant differences between groups were found by

steady-state 31P MR spectroscopy in the ratio of high-energy

metabolites concentrations, intracellular pHi or Mgi2+ content

(Table 1). Also the activities of CK measured by biochemical

method in brain homogenates were similar in all experimental

groups (Table 1).

The measurement of respiratory activities of isolated brain

mitochondria revealed that 3-NP administration induced

pronounced decrease of the rate of ATP production

(�75.3%) as well as of ADP-stimulated (�71.4%) and basal

respiration (�68.8%) in the presence of succinate, i.e. through

complex II of mitochondrial respiratory chain (Table 2). The

rate of ATP production as well as state 3 respiration in the

presence of glutamate were decreased in 3-NP brain

mitochondria (�28.2 and �25.1% versus C), however, these

changes were not statistically significant (Table 2). Antiox-

idants CoQ10 + vitamin E given to rats before induction of HD

were not effective in preventing of 3-NP induced changes in

function of respiratory chain in the presence of succinate and

resulted in the decrease of parameters of oxidative phosphor-

ylation in the presence of glutamate (Table 2). Concentrations

S. Kasparova et al. / Neurochemistry International 48 (2006) 93–9996

Table 1

The activity of creatine kinase reaction in homogenates determined in vitro

using Creatine Phosphokinase kit from Sigma Diagnostics (in sigma units per

mg protein)

C 3-NP QE + 3-NP

CK (SU/mg protein) 292.5 � 9.2 269 � 21.3 315 � 12.6

pHi 7.105 � 0.015 7.118 � 0.016 7.153 � 0.014

Mgi2+ 478.7 � 34.7 432.4 � 12.6 398.9 � 8.8

PCr/b-ATP 2.032 � 0.128 2.164 � 0.145 2.212 � 0.117

Number of assays in groups was 4–9. Intracellular pHi, magnesium concentra-

tion, and PCr/b-ATP ratio calculated from in vivo 31P MR spectra of the rat

brain. Assignment of experimental groups is as in Graph 1. Number of animals

in groups was 4–6.

Table 3

The concentration of lipophilic antioxidants in the brain tissue (in nmol/g w.w.)

and mitochondria (in nmol/mg protein)

C 3-NP QE + 3-NP

Tissue

a-Tocopherol 28.14 � 1.59 31.39 � 5.35 25.77 � 2.52

CoQ9-OX 23.70 � 1.56 14.95 � 1.41*** 19.01 � 1.11

CoQ10-OX 10.26 � 1.09 6.95 � 0.70* 14.36 � 0.92###

Mitochondria

a-Tocopherol 0.324 � 0.016 0.248 � 0.011** 0.397 � 0.019*,###

CoQ9-OX 0.701 � 0.048 0.717 � 0.03 0.747 � 0.042

CoQ10-OX 0.331 � 0.03 0.327 � 0.014 0.335 � 0.028

Assignment of experimental groups and p-values as in Table 2. Number of

assays in groups was 4–9.* p < 0.05 vs. C.

** p < 0.01 vs. C.*** p < 0.001 vs. C.### p < 0.001 vs. HD.

of CoQ9 and CoQ10 in brain mitochondria and complex IV

(cytochromeoxidase) activity were not affected by treatment

with 3-NP or with antioxidants (Tables 2 and 3). The level of

vitamin E in brain mitochondria was decreased in 3-NP rats and

increased in the pretreated rats (Table 3).

On the other hand, the tissue concentration of vitamin E was

similar in all the groups of animals, while that of CoQ9 and

CoQ10 were decreased in 3-NP group and unchanged or

increased, respectively, in QE + 3-NP group (Table 3).

5. Discussion

HD is an autosomal, dominantly inherited neurodegenera-

tive disorder that is characterized by abnormal involuntary

movements (chorea), intellectual impairment and selective

neuronal loss. Human and animal studies show that energy

metabolism is selectively disrupted in brain regions targeted

in HD. One hypothesis is that the gain of function of the

huntingtin protein leads either directly or indirectly to a defect

Table 2

Parameters of oxidative phosphorylation in the rat brain mitochondria with

NAD-substrate glutamate and FAD-substrate succinate, and cytochromeoxidase

(complex IV of mitochondrial respiratory chain) activity (in

nAtO mg prot�1 min�1)

Parameter C 3-NP QE + 3-NP

Complex I

QO2(S4) 25.45 � 3.33 26.13 � 1.53 20.02 � 2.00#

QO2(S3) 55.30 � 6.65 41.43 � 1.67 34.54 � 2.88*

OPR 103.73 � 7.03 87.36 � 5.29 63.67 � 5.55**,#

Complex II

QO2(S4) 108.98 � 11.72 33.99 � 4.16*** 28.40 � 6.30***

QO2(S3) 127.75 � 14.47 36.53 � 3.97*** 39.51 � 3.8**

OPR 119.70 � 16.21 29.62 � 14.41** 32.27 � 5.23**

Complex IV

Cytochromexidase 1148.0 � 54.7 1128.6 � 68.3 1280.0 � 54.8

QO2(S4), basal respiration (state 4) in nAtO mg prot�1 min�1; QO2(S3), ADP-

stimulated respiration (state 3) in nAtO mg prot�1 min�1; OPR, oxidative

phosphorylation rate in nmol ATP mg prot�1 min�1. Assignment of experi-

mental groups is as in Graph 1. Number of assays in groups was 4–6.* p < 0.05 vs. C.

** p < 0.01 vs. C.*** p < 0.001 vs. C.

# p < 0.05 vs. HD.

in mitochondrial energy metabolism. These mitochondrial

defects may result from oxidative damage to cellular elements,

or may themselves induce oxidative stress via increased free

radical production. From clinical and pre-clinical studies, it is

clear that oxidative stress and its consequence—oxidative

damage of lipids, proteins, nucleic acids—may be the cause, or

at least a contributory factor, in an expanding number of

neurodegenerative disorders including HD (Coyle and Putt-

farken, 1993). The pattern of neurodegeneration in the striatum

in HD is consistent with an excessive activation of glutamate-

gated ion channels (Beal, 1992). In quinolinic acid, animal

model of HD, the most significant indicators of the lesion were

changes consistent with oxidative stress: the decrease in

reduced glutathione (GSH), increases in creatine and lactate

levels (Tkac et al., 2000).

One of proposed mechanisms, by which the HD gene defect

could lead to impaired energy metabolism, is that of interaction

between huntingtin and glyceraldehyde-3-phosphate dehydro-

genase, a critical component of the glycolytic pathway (Burke

et al., 1996). Glucose metabolism is markedly reduced in basal

ganglia and cerebral cortex of symptomatic HD patients

(Browne and Beal, 1994) as well as in presymptomatic HD

patients (Beal, 1995). Other study reported decreased oxygen

utilization in the cortex and basal ganglia of HD patients

(Kuwert et al., 1990), and reduced pyruvate dehydrogenase

activity in the basal ganglia and hippocampus (Butterworth

et al., 1985). Increased lactate concentrations in the occipital

cortex (Jenkins et al., 1993), frontal cortex and cerebellum of

HD patients (Martin et al., 1996; Harms et al., 1997) suggest

enhanced glycolytic activity likely as a compensation of

impaired oxidative phosphorylation in HD (Jenkins et al., 1993;

Koroshetz et al., 1997).

Two recent studies in postmortem HD human brain tissues

demonstrated a 55–60% decrease in mitochondrial complex

II–III activity in basal ganglia, no changes in complex I and

citrate synthase activity, and less consistent reductions in

mitochondrial complex IV activity (Gu et al., 1996; Browne

et al., 1997). Hence, the most consistent alterations in electron

transport activity in the basal ganglia of HD patients appear to

S. Kasparova et al. / Neurochemistry International 48 (2006) 93–99 97

be the decrease in complex II–III activity. Genetic defects

affecting complex II–III activity are associated with basal

ganglia degeneration (Bourgeron et al., 1995), comparable

defects in experimental animals result in striatal degeneration

(Brouillet et al., 1995).

3-NP toxicity produces an energy defect in vivo and has

gained acceptance as an animal model of HD. In the present

study, we confirmed that rats injected with 3-NP exhibit

significant disturbance in the brain energy metabolism. As

expected, 3-NP administration resulted in severe inhibition of

respiration and ATP production in brain mitochondria in the

presence of succinate. We observed also a slight decrease in

state 3 respiration and rate of oxidative phosphorylation in the

presence of glutamate. The activity of complex IV was not

affected. Klivenyi et al. (2004) reported approximately 30%

decrease of state 3 respiration supported with pyruvate plus

malate, and 60% decrease in state 3 respiration supported with

a-ketoglutarate in brain mitochondria as well as decreased

a-ketoglutarate dehydrogenase activity in brain tissue of 3-NP-

treated mice. Henry et al. (2002) reported decreased

tricarboxylic acid (TCA) cycle rate and increased exchange

rate between a-ketoglutarate and glutamate in the rat brain 24 h

after single dose of 3-NP. Our results of decreased parameters

of oxidative phosphorylation in the presence of glutamate are

consistent with works of Henry et al. (2002) and Klivenyi et al.

(2004) and could be a result of decreased activity of complex I

or of decreased activity of TCA cycle and particularly of

decreased a-ketoglutarate dehydrogenase activity. GABA is

formed from glutamate in ‘‘GABA shunt’’ bypassing the step of

oxidative decarboxylation of a-ketoglutarate in TCA cycle.

Intrasynaptosomal levels of glutamate and GABA increase

after addition of 3-NP to synaptosomes (Erecinska and Nelson,

1994). The neurotransmitter efflux from synaptosomes is

supposed to be an early step of 3-NP toxicity occurring at

terminal level (Marti et al., 2003).

The lactate–pyruvate ratio is a measure of the redox state of

the cell and it parallels the NADH/NAD+ ratio. On the basis of

oxidative stress, as a hypothesized mechanism for neuronal

vulnerability in HD, we carried out a study on the simultaneous

effect of antioxidants CoQ10 + vitamin E on brain energy

metabolism in 3-NP model of HD. Concerted action of vitamin

E and CoQ10 provides for an effective antioxidant protection of

membranes and lipoproteins (Kagan et al., 2000). Antioxidative

effectiveness of vitamin E is enhanced by CoQ10, which links

recycling process of vitamin E to major electron transport

systems. Treatment with high doses of D-a-tocopherol was not

effective in whole group of HD patients, however, it had

significant selective therapeutic effect on neurological symp-

toms of patients in the early course of the HD disorder (Peyser

et al., 1995). In quinolinic acid (QA) model of HD adm-

inistration of a-tocopherol for 3 days prior to QA had no effect

on striatal lesions (Beal et al., 1988); melatonin suppressed

changes induced by QA (Southgate et al., 1998). a-Lipoic acid,

a potent antioxidant and coenzyme for pyruvate dehydrogenase

and a-ketoglutarate dehydrogenase increased survival of rats in

two transgenic models of HD (Andreassen et al., 2001). The

lactate concentrations in occipital cortex decreased when

patients with HD were taken CoQ10 (Koroshetz et al., 1997).

Pretreatment with CoQ10 exerted neuroprotective effect in a

variety of HD animal models (Beal, 1999). Oral administration

of CoQ10 during 2 months increased its cerebral cortex

concentration in adult rats (Beyer, 1992).

Our steady-state in vivo 31P MRS measurements showed that

concentrations of high-energy phosphate compounds do not

reflect brain function with sufficient sensitivity under this

chronic pathological condition. However, our kinetic results of

in vivo 31P MRS saturation transfer experiment reveal a

significant elevation of kfor in brains of 3-NP rats and provide

evidence for an impairment of energy metabolism in this model

of HD. In vivo 31P NMR transfer measurements showed a

correlation between CK flux and brain 2-deoxyglucose uptake

leading to changes of the brain activity (Sauter and Rudin,

1993; Corbett and Laptook, 1994). Our result of increased CK

activity corresponds well with reported increased 2-deoxyglu-

cose uptake in basal ganglia of HD patients (Beal, 1998).

Furthermore, it has been shown that excitation of the brain

activity (measured by EEG) by bicuculline—a specific

antagonist of GABA effect on GABA-A receptor—induced a

pronounced increase of the rate constant of CK in the rat brain

(Sauter and Rudin, 1993). Thus, we suppose that chronic

administration of 3-NP resulting in selective loss of GABAer-

gic neurons could induce similar increase in kfor.

The study of the brain ATP metabolism, including its

synthesis and consumption, is complicated by regional and

cellular heterogenity. However, it has been demonstrated that

cytosolic isoforms and only minority of mitochondrial isoforms

of CK are monitored in the brain tissue in our experimental

setup of in vivo 31P MRS saturation transfer (Jost et al., 2002).

Thus, the unidirectional rate constant of CK, kfor, is a parameter

predominantly reflecting activity of the cytosolic isoforms of

CK in the rat brain. Our in vitro biochemical study of CK in

brain homogenates showed no significant changes in 3-NP

model of HD. The discrepancy between our in vivo and in vitro

results will be subject of further studies.

Pretreatment of 3-NP-treated rats with high doses of the

antioxidants CoQ10 + vitamin E prevented the increase of kfor.

In our study, the time course of the pretreatment—10 days,

had stimulating effect on content of a-tocopherol predomi-

nantly in the brain mitochondria, without changes in its tissue

level. On the other hand, contents of CoQ9 and CoQ10 were not

changed in brain mitochondria, but were decreased in HD brain

tissue and restored to/over control values in brain tissue of rats

pretreated with antioxidants (Table 3). CoQ is present

ubiquitously in all types of membranes. As an essential

cofactor of mitochondrial electron transport chain CoQ is

inevitable for ATP production. Its antioxidant properties are

associated with major metabolic electron transport systems

(Kagan et al., 2000). Increased trans-plasma-membrane

electron transport has been reported to accompany increased

content of CoQ10 in plasma membrane (Villalba et al., 1995).

The trans-plasma-membrane electron transport chain (Morre,

2005) regenerating cytosolic NAD+ has been reported to play a

key role for viability cells lacking functional mitochondria

(Lawen et al., 1994). In our study, neither the content of CoQ10

S. Kasparova et al. / Neurochemistry International 48 (2006) 93–9998

in mitochondria, nor the mitochondrial function was improved

in rats pretreated with CoQ10 + vitamin E. Nevertheless, the

elevation of the activity of CK in cytosol was prevented.

Jenkins et al. (1996) reported decrease of lactate level in brains

of rats after intrastriatal injection with malonate, which were

pretreated with CoQ10 for 10 days in similar doses as we used in

our experiment. Interestingly, the lactate level was decreased

also when the animals were pretreated with nicotinamide—a

precursor of NAD (Jenkins et al., 1996). We suppose that the

positive effect of CoQ10 + vitamin E pretreatment on brain

energy metabolism of 3-NP rats in our experiment could be

attributed to non-mitochondrial activity of CoQ10 and could be

mediated by cytosolic NADH/NAD+ ratio, which could be

affected by activity of CoQ10 in trans-plasma-membrane

electron transport chain.

Literature data indicate that GABAergic neurons are

especially sensitive to energy deficit (Marti et al., 2003).

Mitochondrial defect produced by 3-NP could be ameliorated

by increasing intracellular energy stores. Administration of

creatine to cultured striatal tissue protected GABAergic

neurons against 3-NP toxicity (Andres et al., 2005). In our

experiment, the energy deficit was reduced by increasing

concentration of CoQ10 in the brain tissue. We suppose that

CoQ10 + vitamin E pretreatment improving brain energy

metabolism could protect GABAergic neurons against 3-NP

toxicity as well.

In summary, our investigation confirmed that systemic 3-NP

administration produces energy defects in brain mitochondria

and affects activity of the CK system as well. We suppose that

increased activity of CK system could be a compensatory

mechanism to decreased ATP production in brain mitochondria

of 3-NP-treated rats.

Results of our 31P MRS experiment confirm importance of

CK/PCr system in the regulation of brain energy metabolism

and contribute to elucidation of mechanisms of a beneficial

effect of CoQ10 administration in HD. Our findings may have

significant implications for clinical treatment of HD, because

we confirmed sensitivity of the method of in vivo 31P MRS

saturation transfer in measurement of CK activity. The

parameter kfor reflects changes in brain energy metabolism

in the 3-NP model of HD and its response to treatment with

antioxidants CoQ10 + vitamin E. We suppose that measurement

of the rate of CK reaction by 31P MR saturation transfer could

be useful for diagnostics of brain metabolic changes in

Huntington’s disease.

Acknowledgments

NMR part of this work was facilitated by the support of the

Slovak State Program of Research and Development no.

2003SP200280203. The financial support from Grant of

Ministry of Education, Slovak Republic and Slovak Grant

Agency for Science (Grants No 1/7547/20 and 1/0546/03) are

gratefully acknowledged. We thank very much for technical

assistance to: Ing. E. Benko, Ing. P. Zauskova, V. Jeskova, A.

Stetkova, and Ing. D. Ivanicka.

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