ALTHOUGH THE ABNORMAL gene for Friedreich’s ataxia(FA), an autosomal recessive disorder, was localizedin 1988 to chromosome 9 (Chamberlain et al., 1988),identification of the gene has not yet been achievedand the primary defect underlying this spinocerebel-lar degeneration remains unknown. As summarizedby Beal (1992), cell death occurring in a variety of neurodegenerative illnesses could be due in part to

defective energy metabolism which might render thecells more susceptible to damage. In the case of FA,much evidence, albeit controversial, suggests thatsome patients with this disorder have abnormalmetabolism of pyruvate (Kark et al., 1974; Blass et al.,1976; Barbeau et al., 1976; Livingstone et al., 1980;Dijkstra et al., 1984; Purkiss et al., 1981; Bertagnolio etal., 1980, but see Stumpf and Parks, 1979). Attempts toexplain this abnormality have focused primarily ontwo enzyme complexes, the α-ketoglutarate dehydro-genase complex (αKGDHC), which is composed ofmultiple copies of its three constituent enzyme subunits (α-ketoglutarate dehydrogenase [E1], dihy-drolipoamide succinyltransferase [E2] and dihy-drolipoamide dehydrogenase [E3]) and the pyruvatedehydrogenase complex (PDHC). Activities of one or

Immunoreactive Levels of α-ketoglutarateDehydrogenase Subunits in Friedreich’s Ataxia and

Spinocerebellar Ataxia Type 1Frank Mastrogiacomo,1 Jacques LaMarche,2 Slobodan Dozic3, GordonLindsay,4 Lucien Bettendorff,5 Yves Robitaille,6 Lawrence Schut7 and

Stephen J. Kish1

1Human Neurochemical Pathology Laboratory, Clarke Institute of Psychiatry, Toronto, Canada;2Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Quebec, Canada; 3Institute ofPathology, University of Belgrade, Yugoslavia; 4Division of Biochemistry and Molecular

Biology, University of Glasgow, Scotland; 5Laboratory of Neurochemistry, University of Liège,Liège, Belgium; 6Department of Neuropathology, University of Montreal, Quebec, Canada;

7Department of Neurology, University of Minnesota, Minneapolis, Minnesota, U.S.A.

Enzyme activities of α-ketoglutarate dehydrogenase complex (αKGDHC) and one of its con-stituent subunits, dihydrolipoamide dehydrogenase (E3), are reported to be reduced in non-CNStissues of some patients with Friedreich’s ataxia (FA); however, the results are highly conflicting.To determine whether an enzyme abnormality occurs in brain, we measured immunoreactivelevels of the three αKGDHC subunits, namely, α-ketoglutarate dehydrogenase (E1), dihydro-lipoamide succinyltransferase (E2) and E3 in postmortem frontal, occipital and cerebellar corticesof 18 control subjects, 9 patients with FA and, for comparison, 12 patients with spinocerebellarataxia type 1 (SCA1). Decreased (220 to 231%) levels of E3 were observed in all three examinedareas of the patients with FA with the changes statistically significant in cerebellar and frontal cor-tices. The E3 reduction could be explained by a loss of αKGDHC or other dehydrogenase com-plexes (e.g. pyruvate dehydrogenase complex) which utilize this subunit. In SCA1, enzymechanges were limited to E2 in cerebellar (226%) and frontal (219%) cortices. Although the E3 andE2 reductions are only slight, and may represent secondary events, the changes in this key Krebscycle enzyme could exacerbate degenerative processes in both of the spinocerebellar ataxia dis-orders.

© 1996 Academic Press Limited

Key words: α-ketoglutarate dehydrogenase complex, Friedreich’s ataxia, spinocerebellar ataxia type 1, olivopontocerebellar atrophy, cerebellum

27

NEURODEGENERATION, Vol. 5, pp 27–33 (1996)

Correspondence to: Dr Stephen J. Kish, Human NeurochemicalPathology Laboratory, Clarke Institute of Psychiatry, 250 College Street,Toronto, Ontario, Canada M5T 1R8

Received 8 September 1995; revised and accepted for publication 17October 1995

© 1996 Academic Press Limited1055-8330/96/0100027 1 7 $18.00/0

both of these enzyme complexes have been reportedby some groups to be reduced in non-CNS tissue of atleast a subgroup of patients with FA (Blass et al., 1976;Livingstone et al., 1980; Barbeau et al., 1978; Rodriguez-Budelli and Kark, 1978; Kark et al., 1978; Sorbi et al.,1989) although other laboratories have reported normal enzyme levels (Stumpf and Parks, 1978;Constantopoulos et al., 1980; Filla et al., 1980; 1978).Since a defect in E3, which is a component subunit ofboth αKGDHC and PDHC, could explain thedeficiency of both enzymes, much effort has beendevoted to investigations of E3 in FA. However, thestatus of E3 activity in various non-CNS tissues in FAis still uncertain (reduced: Livingstone et al., 1980;Rodriguez-Budelli and Kark, 1978; Kark et al., 1978;Filla et al., 1978 [serum], Kark et al., 1980; Kark et al.,1981); normal: Stumpf and Parks, 1979; Filla et al., 1978[platelets], Evans, 1983; Melancon et al., 1978).

The relevance of the above biochemical findings,obtained in muscle and blood, to the spinocerebellardegeneration of FA is uncertain. Since no informationis available on the status of either αKGDHC or E3 inCNS of patients with this disorder, we measured inautopsied brain of patients with FA, immunoreactivelevels of E1 and E2, the subunits specific to αKGDHC,and E3, the subunit shared by αKGDHC and PDHC.The concentration of antigenic cross-reactive materialprovides an estimate of the actual amount of proteinof the subunit present in tissue. For simplicity, we willrefer to these measurements as ‘levels’ of E1, E2 andE3. Unlike activity measurements of αKGDHC inautopsied human brain, which show high scatter and are markedly affected by agonal status (seeMastrogiacomo & Kish, 1993), immunoreactive levelsof the αKGDHC subunits appear to be much lessaffected by confounding pre- and postmortem factors,and therefore provide a more useful index of the status of the enzyme in autopsied human brain studies. The enzyme data in FA were compared withthose from a matched control group as well as from agroup of patients with spinocerebellar ataxia type 1(SCA1; Orr et al., 1993), a disorder in which decreasedcerebellar αKGDHC activity has previously beendescribed (Mastrogiacomo & Kish, 1994).

Patients and Methods

Patients

Frontal (Brodmann area 10), occipital (area 17) and cerebel-lar cortices were obtained from autopsied brain of clinicallyend-stage patients with FA (n 5 9) and SCA1 (n 5 12) and

neurologically and histopathologically normal controls (n 518) matched (one way analysis of variance [ANOVA], P .0.05) with respect to age (FA: 32 6 3 years; SCA1: 40 6 3years; controls: 39 6 3 years), postmortem interval (intervalbetween death and freezing of one half of the brain at 280°C;FA: 9 6 2 h; SCA1: 9 6 2 h; controls: 13 6 1 h), and, as anindex of premortem agonal status (see Mastrogiacomo et al.,1993), cerebral (frontal) cortical pH (FA: 6.2 6 0.1; SCA1: 6.2 6 0.1; controls: 6.3 6 0.1).

All patients of the FA group fulfilled the clinical diagnos-tic criteria for FA (Lamarche et al., 1984) and had onset beforethe age of 17 (range 2–16), progressive ataxia of limbs andgait, and deep tendon areflexia in lower limbs. Cardiopathy(n 5 9), pes cavus (n 5 7), and scoliosis (n 5 7) wereobserved at last examination in all or most of the patientswith FA. A positive family history consistent with autoso-mal recessive inheritance was observed in eight of the nineFA patients with one patient having an apparently sporadicoccurrence. Neuropathological analysis disclosed the typi-cal features of FA (Lamarche et al., 1984) with severe degen-eration of the posterior columns and neuronal loss inClarke’s nucleus, and degeneration of the lateral corti-cospinal tract and posterior spinocerebellar tract. The pos-terior roots and ganglia were affected in all cases examined.The cerebellar cortex showed, at most, mild, patchy, neu-ronal loss in the Purkinje cell layer whereas the dentatenucleus was mildly to severely affected by neuronal loss.The cerebral cortex appeared normal in all cases with theexception of one patient who had mild cortical atrophy. Thediagnosis of SCA1 was based upon the presence of autoso-mal dominant inheritance in families known to have theSCA1 gene defect (Orr et al., 1993) and the characteristic clin-ical (limb and gait ataxia, dysarthria, dysphagia) and neu-ropathological features with severe neuronal loss and gliosisin the Purkinje and molecular layers of the cerebellar cortex,basal pontine nuclei, and inferior olives. Detailed neu-ropathological findings in nine of the patients with SCA1have recently been described in detail (Robitaille et al., 1995).

Biochemical analyses

For the estimation of E1, E2 and E3 levels, frozen brain tis-sue was homogenized (20% solution) in ice-cold deionizedwater (dH2O) using a Bronwill Biosonik sonicator (1 3 20strokes on ice, probe intensity 70). Samples were then dena-tured (667 µg protein/ml for E1; 833 µg protein/ml for E2,333 µg protein/ml for E3) in sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) sample buffer(5 M urea, 173 mM SDS, DTT [65 mM for E1 and E2, 649 mMfor E3], 5% Tris Buffer pH 8.0, and bromophenol blue),heated for 10 min in boiling water, and subjected to SDS-PAGE in slab gels (1.5 mm thick, 7.5% mini-gels). 20 µg pro-tein per test were loaded for E1 detection, 25 µg protein forE2, and 10 µg protein for E3. After electrophoresis, proteinsin the resolving gel were transferred electrophoretically (3V/cm, 16 h, room temperature) to nitrocellulose sheets. Thenitrocellulose sheets were stained with Ponceau S, destainedbriefly in TBS-T (137 mM sodium chloride, 20 mM Tris BasepH 7.5, 0.1% Tween-20) and then incubated in blocking solu-tion (5% nonfat dry milk, 0.5% BSA, in dH2O) for 1 h. Theblocking solution was drained and the nitrocellulose sheets

28 F. Mastrogiacomo et al.

Brain αKGDHC in FA and SCA1 29

were incubated for up to 4 h with primary antibodies (anti-E1, 1:150 (v/v); anti-E2, 1:200 (v/v); anti-E3 1:150 (v/v) ) inTBS-T (containing 5% non-fat dry milk, 0.5% BSA), rinsed,then incubated for 1 h with peroxidase-conjugated proteinA (Sigma Chemical Co., St. Louis, MO) in TBS-T, and thenrinsed again. Immunoreactivity was visualized by exposureto autoradiographic film (ReflectionTM, NEN) after incu-bation for 1 min with chemiluminescence reagents(RenaissanceTM, NEN). Immunoreactivity associated withthe 96-kDa E1 band, 48-kDa E2 band, and 56-kDa E3 bandwere quantified densitometrically using computer-basedimaging (Imaging Research Inc., St. Catherines, Canada).The primary antisera used in this study (rabbit anti-bovineheart E1, E2; rabbit anti-porcine heart E3) are high-titre,monospecific, polyclonal antisera raised against the threeconstituent enzyme subunits of αKGDHC purified frombovine (E1, E2) and porcine (E3) heart and were previouslycharacterized in detail with respect to specificity and reac-tivity (Hunter & Lindsay, 1989; Lindsay, 1989). Five concen-trations of purified porcine αKGDHC standard (SigmaChemical Co.) were run on each gel (15 well) together witheight samples, and a standard curve (optical density 3 areaunits vs ng purified αKGDHC standard protein) was plot-ted for each gel. The amount of immunoreactivity of E1, E2and E3 in each lane was calculated as ng purified αKGDHCstandard/µg protein by interpolation from the standardcurve. Immunoreactivity was present as a prominent singleband for each protein subunit (E1, 96-kDa; E2, 48-kDa; E3,56-kDa) from which protein levels were quantified.

Activity of citrate synthase, an enzyme marker for mito-chondrial mass (see Mastrogiacomo et al., 1993), was mea-sured by a spectrophotometric assay (Mastrogiacomo et al.,1993).

Statistical analyses

Statistical analyses were performed using a one way analy-sis of variance (ANOVA [P , 0.05 criterion]) followed byFisher’s Least Significant Difference comparison test and thePearson correlation coefficient.

Results

Representative immunoblots of control and patientbrain levels of E1, E2 and E3 are shown in Figure 1. Asexpected (Hunter and Lindsay, 1986; Lindsay, 1989),immunoreactivity was present as a prominent singleband for each protein subunit (E1, 96-kDa; E2, 48-kDa;E3, 56-kDa) in all of the patients and controls.Statistically significant positive correlations betweenage and enzyme subunit levels were observed for E1and E3 in frontal and cerebellar cortices of the controls(r 5 0.53 to r 5 0.66, P , 0.05) and for E1 in the frontal(r 5 0.77, P, 0.05) and occipital (r 5 0.71, P , 0.05)cortices of the patients with FA. Significant correla-tions between postmortem time and subunit levels

were limited to E1 in cerebellar cortex in the FA group(r 5 20.81, P , 0.01).

As compared with the controls (see Table 1 andFigures 1 and 2), E3 levels in the FA group werereduced by 20–31% in all three cortical areas with thedifferences statistically significant for frontal and cerebellar cortices. The other statistically significantchanges in the FA group were limited to E1 in frontalcortex (234%) and E2 in occipital cortex (116%).Employing age as a covariate in an analysis of covari-ance the statistically significant changes in FA werelimited to E1 and E3 in frontal (E1: 228%, P , 0.01; E3:225%, P , 0.001) and cerebellar cortices (E1: 26%, P , 0.05; E3: 222%, P , 0.001). In the SCA1 group sta-tistically significant changes in αKGDHC subunit con-centrations were limited to a reduction of E2 incerebellar (226%) and frontal (219%) cortices.

Statistically significant (P , 0.05 or less) positivecorrelations (r 5 0.52 to 0.87) were observed betweenlevels of E1 and E2 (control frontal cortex, SCA1 frontal

Table 1. Immunoreactive levels of aKGDHC subunits andcitrate synthase activity in postmortem brain of control sub-jects and patients with Friedreich’s ataxia and spinocerebel-lar ataxia type 1

E1 E2 E3 CS

Frontal cortexControls 108 6 7 115 6 4 235 6 13 179 6 6FA 71 6 5 121 6 4 162 6 10 177 6 5

% change 234b 15 231b 21SCA1 121 6 5 93 6 5 240 6 13 176 6 2

% change 112 219b 12 22Occipital cortex

Controls 109 6 8 123 6 4 224 6 15 206 6 7FA 97 6 3 143 6 6 179 6 8 180 6 10

% change 211 116a 220 213a

SCA1 128 6 9 112 6 6 249 6 22 164 6 4% change 117 29 111 220b

Cerebellarcortex

Controls 84 6 4 112 6 4 237 6 19 197 6 5FA 76 6 4 116 6 5 166 6 10 151 6 9

% change 210 14 230a 223b

SCA1 87 6 5 83 6 4 242 6 19 166 6 3% change 14 226b 12 216b

Values represent mean 6 SE of 17–18 control subjects, 9 patientswith Friedreich’s ataxia (FA) and 12 patients with spinocerebellarataxia type 1 (SCA1). αKGDHC 5 α-ketoglutarate dehydrogenasecomplex. E1 5 immunoreactive levels (ng αKGDHC/µg protein) of α-ketoglutarate dehydrogenase; E2 5 immunoreactive levels ofdihydrolipoamide succinyltransferase; E3 5 immunoreactive levels of dihydrolipoamide dehydrogenase; CS 5 activity(nmol/min/mg protein) of citrate synthase; P , 0.05, bP , 0.001(Fisher’s LSD test).

30 F. Mastrogiacomo et al.

Figure 1. E1 (α-ketoglutarate dehydrogenase), E2 (dihydrolipoamide succinyltransferase, and E3 (dihydrolipoamide de-hydrogenase) immunoreactivity in a sample of purified porcine α-ketoglutarate dehydrogenase complex (αKGDHC) stan-dard (St) and (left side) in cerebellar cortex of four patients with Friedreich’s ataxia (F) and four control subjects (C) and(right side) in cerebellar cortex of four patients with spinocerebellar ataxia type 1 (S) and two control subjects (C). A singleband of immunoreactivity is present for each of the αKGDHC subunits in both the control and patient samples. The mole-cular weight markers were β-galactosidase (116 kDa), phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa),and carbonic anhydrase (29 kDa).

E1

E3

E2

and cerebellar cortices), E1 and E3 (control and SCA1frontal and cerebellar cortices, FA frontal and occipi-tal cortices) and E2 and E3 (FA cerebellar cortex, allbrain areas of patients with SCA1).

As shown in Table 1, activities of citrate synthasewere significantly decreased by 13–23% in occipitaland cerebellar cortices of the FA and SCA1 patients butwere normal in frontal cortex of both groups ofpatients.

Discussion

To our knowledge, this is the first investigation ofαKGDHC in brain of patients with FA. Our majorfinding is that brain immunoreactive levels of E3 aredecreased in FA. This investigation also extends ourprevious report of decreased cerebellar αKGDHCactivity in SCA1 (Mastrogiacomo & Kish, 1994) byshowing that this change is accompanied by reducedlevels of the E2 component of the enzyme.

Our finding in FA of reduced brain levels of E3 isinteresting in view of the extensive, albeit contradic-tory, literature documenting decreased activity of this enzyme in muscle and blood elements of somepatients with FA (see Introduction). The fact that thebrain E3 change was only slight with complete over-lap between control and patient values (see Fig. 2), isconsistent with the literature showing that in FAreduced activity of lipoamide dehydrogenase in non-CNS tissue is not a constant finding, being reducedonly modestly and only in some patients. In our inves-tigation, all of the individual patient values for brainE3 fell below the control mean value, but within the

control range. In this regard, the distribution of brain E3 levels in the control and FA groups is similar to that obtained for enzyme activity determined in muscle of patients with FA (see Fig. 3in Evans, 1983).

The decreased brain αKGDHC levels in FA andSCA1 could be explained by a number of factorsincluding loss of neurones enriched in differentαKGDHC subunits. In the case of E3, there could havebeen a loss of neurones enriched in other enzyme complexes (PDHC, branched chain α-ketoacid de-hydrogenase complex, glycine decarboxylase com-plex) which also utilize the E3 component. Althoughthe neuronal localization of αKGDHC and PDHC inhuman brain is not known, in the rodent both enzymecomplexes are moderately enriched in Purkinje cellsof the cerebellar cortex (Calingasan et al., 1994), an areaof the brain which is variably and severely affected inFA and SCA1, respectively. No information appears tobe available regarding the localization of αKGDHC orPDHC in terminals of the spino- or ponto-cerebellarneurones which degenerate in FA and SCA1, respec-tively. In the rodent both enzyme complexes are alsohighly enriched in the nucleus basalis of Meynert(Calingasan et al., 1994) which contains the cell bodieswhich provide the major cholinergic innervation tocerebral cortex. However, we found no statisticallysignificant correlation between levels of αKGDHC and cerebral cortical activities of choline acetyltrans-ferase (ChAT), the cholinergic marker enzyme, eitherin SCA1 in which cerebral cortical ChAT levels aremarkedly reduced (Kish et al., 1987), or in FA, in whichChAT activities are normal (Mastrogiacomo & Kish,unpublished observations). The decrease of brainαKGDHC might also be consequent to downregula-tion of enzyme synthesis due to loss of input to thecerebellar cortex from neurones originating in thepons and inferior olives (in SCA1) and spinal cord (inFA). Alternatively, the changes could be explained bydestabilization of the enzyme complex as a conse-quence of a chronic deficiency of a cofactor such as thiamine diphosphate. However, brain thiaminediphosphate levels are normal in FA and only slightly(and not statistically significantly) reduced in SCA1(Bettendorff et al., in press). We think it likely that thebrain enzyme changes in the two spinocerebellarataxia conditions are non-specific results of variousneurodegenerative events (e.g. excessive free radicalgeneration and/or proteolysis) associated with dif-ferent neurodegenerative events. This is strongly sug-gested by the previous demonstration of decreasedimmunoreactive and/or activity levels of αKGDHC in

Brain αKGDHC in FA and SCA1 31

Figure 2. Immunoreactive levels of αKGDHC subunits (ngαKGDHC/µg protein) in cerebellar cortex of 17 control sub-jects (C), 9 patients with Friedreich’s ataxia (FA) and 11patients with spinocerebellar ataxia type 1 (SCA1). E1 5 α-ketoglutarate dehydrogenase; E2 5 lipoyl succinyltrans-ferase; E3 5 lipoamide dehydrogenase. aP , 0.05, bP ,0.0001 (Fisher’s LSD test).

Parkinson’s disease (Mizuno et al., 1994), Wernicke-Korsakoff syndrome (Butterworth et al., 1993), and inAlzheimer’s disease (see Mastrogiacomo et al., 1993)and the observation that in the latter condition braincerebral cortical E3 immunoreactive levels are, like FA, also reduced (Mastrogiacomo et al., in press).

The decreased levels of the αKGDHC subunits inFA and SCA1 might not have any biologicalsignificance since the changes were only slight and, in cerebellar cortex, failed to achieve statisticalsignificance when values were adjusted for loss ofmitochondrial mass (as estimated by citrate synthaseactivity). In addition, given the fact that E1 is the rate-limiting component of the enzyme complex, the de-creases in levels of the E3 and E2 subunits might notresult in any actual decrement in activity of αKGDHC.Nevertheless, the possibility has to be considered thata 30% decrease in concentration of these αKGDHCsubunits in cerebellum might lead to a modest reduc-tion in activity of the enzyme, considered to be the rate-limiting enzyme of the Krebs cycle (see Sheu et al.,1994). The decreased levels could further impair brainenergy metabolism. In principle this might be relatedto the positron emission tomography findings ofdecreased glucose metabolism (Junck et al., 1994) andblood flow (Giroud et al., 1994) in the cerebellum ofclinically advanced patients with FA and reduced cere-bellar glucose metabolism in SCA1 (Matthew et al.,1993).

In conclusion, we found diminished immunoreac-tive levels of αKGDHC subunits in postmortem brainof patients with FA and SCA1. We suggest that thequantitatively modest enzyme reduction is probablya non-specific consequence of the neurodegeneration.Nevertheless, decreased levels of this key energymetabolizing enzyme in the cerebellum could con-tribute to the brain metabolic defects in both spino-cerebellar conditions.

Acknowledgements

This study was supported by U.S. NINDS #NS26034 to SKand by a Studentship award from the Ontario Mental HealthFoundation to FM.

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