neuropathological, biochemical and molecular findings in a glutaric
TRANSCRIPT
doi:10.1093/brain/awh401 Brain (2005), 128, 711–722
Neuropathological, biochemical and molecularfindings in a glutaric acidemia type 1 cohort
Christopher B. R. Funk,1,5 Asuri N. Prasad,6 Patrick Frosk,3 Sven Sauer,7 Stefan Kolker,7
Cheryl R. Greenberg3,4 and Marc R. Del Bigio1,2,5
Correspondence to: Marc R. Del Bigio MD PhD FRCPC,
Canada Research Chair in Developmental Neuropathology,
Department of Pathology, University of Manitoba,
D212-770 Bannatyne Avenue, Winnipeg MB,
R3E 0W3, Canada
E-mail: [email protected]
Departments of 1Pathology, 2Human Anatomy and Cell
Science, 3Biochemistry and Medical Genetics and4Pediatrics and Child Health, Faculty of Medicine,
University of Manitoba, 5Manitoba Institute of Child
Health, Winnipeg, 6Department of Pediatrics, University
of Western Ontario, London, Ontario, Canada and7Department of General Pediatrics, Division of Metabolic
and Endocrine Diseases, University Children’s Hospital,
Heidelberg, Germany.
SummaryGlutaric acidemia type 1 (GA-1) is an autosomal recessive
disorder characterized by a deficiency of glutaryl-CoA
dehydrogenase (GCDH) activity. GA-1 is often associated
with an acute encephalopathy between 6 and 18 months
of age that causes striatal damage resulting in a severe dys-
tonicmovement disorder. Tenautopsy cases havebeenpre-viously described. Our goal is to understand the disorder
better so that treatments can be designed. Therefore, we
present the neuropathological features of six additional
cases (8months – 40 years), all NorthAmerican aboriginals
with the identical homozygous mutation. This cohort dis-
plays similar pathological characteristics to those previ-
ously described. Four had macroencephaly. All had
striatal atrophy with severe loss of medium-sized neurons.Wepresent several novel findings. This natural time course
study allows us to conclude that neuron loss occurs shortly
after the encephalopathical crisis and does not progress.
In addition, we demonstratemild loss of large striatal neur-
ons, spongiform changes restricted to brainstem white
matter and amild lymphocytic infiltrate in the early stages.
Reverse transcriptase-PCR to detect the GCDH mRNArevealed normal and truncated transcripts similar to those
in fibroblasts. All brain regions demonstrated markedly
elevated concentrations of GA (3770–21 200 nmol/g pro-
tein) and3-OH-GA (280–740nmol/g protein),with no evid-
ence of striatal specificity or age dependency. The role of
organic acids as toxic agents and as osmolytes is discussed.
The pathogenesis of selective neuronal loss cannot be
explained on the basis of regional genetic and/or metabolicdifferences. A suitable animal model for GA-1 is needed.
Keywords: autopsy; glutaric acid; 3-hydroxyglutaric acid; striatum; molecular genetics
Abbreviations: ChAT = choline acetyltransferase; DAB = diaminobenzidine; H&E = haematoxylin and eosin; GA = glutaric
acid; GA-1 = glutaric acidemia type 1; GCDH = glutaryl-CoA dehydrogenase; GFAP = glial fibrillary acidic protein;
HLA-DR = human leucocyte antigen-DR; NMDA = N-methyl-D-aspartate; 3-OH-GA = 3 hydroxyglutaric acid;
RT-PCR = Reverse transcription polymerase chain reaction
Received July 14, 2004. Revised December 10, 2004. Accepted December 13, 2004. Advance Access publication
February 2, 2005
IntroductionGlutaric acidemia type 1 (GA-1) is an autosomal recessive
disorder of amino acid metabolism caused by the deficiency
of functional glutaryl-CoA dehydrogenase (GCDH) activity
(Christensen, 1993), an essential enzyme in the catabolic path-
ways of L-tryptophan, L-lysine and L-hydroxylysine (Goodman
et al., 1977; Baric et al., 1998; Goodman and Frerman, 2001).
Lack of functional GCDH activity typically leads to the
accumulation of glutaric acid (GA), 3-hydroxyglutaric acid
(3-OH-GA) and glutaconic acid in the blood, urine, CSF and
brain tissue (Stokke et al., 1975; Goodman et al., 1977; Baric
et al., 1998). GA and 3-OH-GA might induce an imbalance in
glutamatergic and GABAergic neurotransmission (Wajner
et al., 2004) and 3-OH-GA might act through excitotoxic
NMDA receptors to produce a neurotoxic effect (Kolker
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et al., 2004a,b), although this is not supported by all experi-
mental data (Freudenberg et al., 2004; Lund et al., 2004). In
one autopsy case, 3-OH-GA was relatively more abundant in
the striatum (Kolker et al., 2003), where the neuronal damage
is most severe.
GA-1 affected children are clinically characterized by
macrocephaly appearing at, or shortly after, birth and initial
normal development interrupted by abrupt onset of dystonia
and choreoathetosis, which then remain relatively static.
Neurological abnormalities usually appear between 6 and
18 months of age, often in conjunction with a febrile illness.
Intellect seems to be relatively preserved (Goodman and
Frerman, 2001). The profound neurological sequelae may
lead to death in early childhood; however, some individuals
survive for many years. A minority of biochemically affected
individuals may remain asymptomatic or experience an insi-
dious onset of mild neurological abnormalities. The brains of
children affected with GA-1 exhibit wide Sylvian fissures and
enlarged frontal ventricles due to caudate atrophy. There are
only 10 published autopsy reports of GA-1 (Goodman et al.,
1977;Leibeletal., 1980;Bennettetal., 1986;Chowetal.,1988;
Bergman et al., 1989; Soffer et al., 1992; Kimura et al., 1994;
Kolker et al., 2003). Atrophy and severe neuronal loss affecting
the caudate and putamen are always present. Spongiform
change in the white matter has been frequently described.
This disease is over-represented among North American
aboriginals (Ojibway-Cree) in a genetic isolate in north-
eastern Manitoba and north-western Ontario in central
Canada (Haworth et al., 1991). In this population, the carrier
frequency is �1 in 10. Twenty-eight affected children, many
the products of consanguineous unions, have been identified
since 1970; 21 individuals have suffered an encephalopath-
ical crisis with severe striatal damage (unpublished data).
Although the phenotype is severe, the amount of GA and
3-OH-GA in blood and urine tends to be very low (Haworth
et al., 1991). All affected individuals are homozygous for a
splicing mutation, a G to T transversion at the +5 position
of intron 1 in the gene encoding GCDH (IVS-1 +5 G>T)
(Greenberg et al., 1995; Goodman et al., 1998). This splicing
mutation allows for some normal splicing with a transcript of
the expected size, as well as a truncated transcript resulting
from activation of a cryptic splice site 26 base pairs (bp)
upstream in exon 1; the cryptic splicing leads to a frame
shift and premature termination.
There are many gaps in the present understanding of the
neuropathogenesis of striatal injury in this disorder. Here we
describe the neuropathological findings in the brains of five
children and one adult with GA-1, all with the same mutation.
This is of particular interest because the range of survival times
and the availability of frozen tissue for genetic analysis
might offer additional insight into pathogenesis of the disorder.
Material and methodsThis was a retrospective neuropathological study of six individuals
of North American aboriginal background diagnosed with GA-1.
All patients in this study were examined and diagnosed by clinical
geneticists at the Children’s Hospital/Health Sciences Centre in
Winnipeg, Canada. There have been 13 known deaths in this cohort
between 1978 and 2004; complete autopsies have been performed on
six cases. The age of death ranged from 8 months to 40 years. In all
cases, the medical records were reviewed in detail. Original brain
imaging studies were available for review from only three cases. For
each case, histological samples from one or two anonymized control
cases with no neurological disease and matched for age and gender
were obtained from the autopsy archives. Frozen control tissues were
more limited and not as closely comparable. This study was con-
ducted with approval of the University of Monitoba Biomedical
Research Ethics Board as well as the Pathology Access Committee
for Tissue.
Archived paraffin blocks, glass slides and hospital records were
retrieved for all cases. The brains had been reasonably well sampled
with 8–20 tissue blocks per brain available for microscopic exam-
ination. All slides were examined by one neuropathologist (M.R.D.).
Age- and sex-matched controls were identified and similar levels of
the striatum were sampled for each case. Sections from striatal
blocks were stained with haematoxylin and eosin (H&E). Immuno-
histochemical staining was performed to detect glial fibrillary acidic
protein (GFAP) (polyclonal anti-GFAP; 1/1200 dilution; DakoCyto-
mation (Carpinteria CA, USA), activated microglia (anti-HLA-DR;
1/250 dilution; Dako), lymphocytes (anti-CD3; 1/100 dilution;
Dako) and synaptic vesicle protein synaptophysin (1/25 dilution;
Dako). Neurons were identified with the use of anti-NeuN (neuronal
nuclei) (1/800; Chemicon International). To identify inhibitory
interneurons, antibodies to calbindin (1/100 dilution; Chemicon)
and g-aminobutyrate (anti-GABA; 1/125; Chemicon) were used.
To identify noradrenergic axons, anti-tyrosine hydroxylase (dilution
1/75; Chemicon) was used. Choline acetyltransferase (ChAT)
(anti-ChAT; 1/250 dilution; Chemicon) Chemicon International
(Temecula CA, USA) was used to identify large cholinergic neurons.
GFAP, human leucocyte antigen-DR (HLA-DR), CD3 and synapto-
physin antibodies were detected using the Envision detection sys-
tem. GABA, ChAT, NeuN and tyrosine hydroxylase antibodies were
detected with biotinylated secondary antibodies, streptavidin horse-
radish peroxidase and 3,30- diaminobenzidine (DAB). A fluorescent
secondary antibody (Cy-3) was used with the calbindin primary
antibody. Appropriate negative controls were used in all cases.
Neuron counts were performed on H&E stained sections. This was
done because the neurons have a fairly characteristic morphology
and because we found the immunostaining to be inconsistent in the
autopsy material. Counts were made in the dorsal and ventral regions
of both the caudate and putamen at an ocular magnification of 4003.
The size of the ocular reticule counting square was 250mm 3 250mm.
The counts consisted of neurons contained within six adjacent focal
areas in each of the four regions stated above. Only neurons that
could be unambiguously identified based on cytological details were
counted. National Institutes of Heath (NIH) image analysis software
was used to measure the density of DAB precipitation—as an indic-
ator of the magnitude of immunoreactivity—with antibodies against
GFAP, HLA-DR and synaptophysin. Images used for NIH analysis
were taken in the dorsal and ventral regions of the caudate and
putamen at 103 objective magnification. Two images were obtained
from each region; their densities were then averaged to give a better
representation of immunoreactivity in each area. Neuron counts and
immunohistochemical labelling data were tested for normal distribu-
tion. Paired t-tests were then used to compare differences between
cases and age-matched controls using StatView 5 Software (SAS;
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Cary, NC, USA). Regression analysis was used to test for age-
dependent effects.
Reverse transcription PCR (RT-PCR) was performed on total
RNA isolated from frozen brain tissue stored at �70�C following
autopsy of four cases. The method has been previously described for
analysis of fibroblasts and lymphoblasts from this cohort (Greenberg
et al., 1995). Briefly, following reverse transcription of RNA from
homogenized tissue, two overlapping fragments of the complete
GCDH cDNA were generated by separate standard PCR reactions.
PCR conditions were 2 ml cDNA in 50 ml of 50 pmol of each primer,
200 mM of each dNTP, 5 ml of 103 PCR reaction buffer (Perkin
Elmer, Boston, MA, USA) and 1ml AmpliTaq (8 units) (Applied
Biosystems, Foster City CA, USA) for 35 cycles at 95�C/3 min,
95�C for 1 min, 58�C for 1 min, and 72�C for 1 min with a final 10
min cycle at 72�C. Products were analysed on 6% acrylamide mini-
gels with ethidium bromide (10 mg/ml) staining. RT-PCR for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used
as a positive confirmation of mRNA integrity and isolation. Similar
analyses were performed on brain tissues obtained from non-age
matched patients (because the supply of control material was lim-
ited) without known neurological disease and with similar post-
mortem delay. All analyses were done blinded.
Analysis of organic acid (GA and 3-OH-GA) concentrations was
performed on frozen brain tissue from four GA-1 and three control
cases. The samples were shipped by courier on dry ice to Germany.
The analyses were performed in a blinded manner. The methods for
this analysis are described in detail in previous studies (Schor et al.,
2002; Kolker et al., 2003). Again due to limited supply, the controls
were not age matched.
ResultsThe ages of the individuals with GA-1 ranged from 8 months
to 40 years. Body weights and heights, GA levels in urine
(Seargeant et al., 1992) and GCDH activity are shown in
Table 1. In the youngest four cases, the brains were much
larger than expected for age. A description of clinical and
neuropathological findings follows in order of ascending age.
Case 1This male had six out of seven older siblings also affected by
GA-1. He is a member of pedigree B described in the clinical
report of this cohort (Haworth et al., 1991). At full term birth
in 1993, he was said to be jittery. A brief seizure occurred at
3 months. Developmental delay, mild hypotonia and head
bobbing were noted at 4 months. At 6 months, a CT scan
showed slightly enlarged ventricles, temporal fossa fluid
collections and mild widening of frontal sulci. The head
circumference was on the 99.9 percentile at birth, 2 months
and 8 months. At 8 months, he presented with vomiting,
diarrhoea, fever and severe dehydration. He became rapidly
Table 1 Clinical and autopsy data of patients with glutaric acidemia
Case # 1 2 3 4 5 6
Gender Male Male Male Male Female MaleAge at neurologicalcrisis
6.5 months 10.5 months 7 months 5.5 months 6 months 4 months
Age at death 8 months 12 months 16 months 18.5 months 7 years 7 months 40 yearsHeight/length(percentile)
73 cm (50) 75 cm (25) 79 cm (25) 74 cm (<5) 114 cm (5) Not known
Body weight(percentile)
9.4 kg (75) 9.1 kg (15) 7.7 kg (<5) 8.1 kg (<5) 21 kg (25) 43 kg (<5)
Brain weight(deviation fromexpected weight)
1320 g >+9 SD 980 g >+2 SD 1176 g >+2 SD 1104 g +1 SD 1300 g >median 1635 g >median
GCDH mutationpresent
+ / + Not done + / + + / + + / + + / +
GCDH lymphocyteenzyme activity(% of normal)
Not done Not done 4 % Not done 9 % 7 %
Urine GA(mmol/mmolcreatinine)(normal < 10)y
33–450 Not done 58 99–118 6–97 13
Urine 3-OH-GA(mmol/mmolcreatinine)
Small amount Not done Small amount Slight increase Trace
Delay in autopsy 20 h 11 h 48 h 18 h 40 h 5.5 hFrozen braintissue sampled
Cerebellum None Caudate,cerebellum,frontalcerebrum
Frontalcerebrum
Striatum,frontalcerebrum
None
yNone of these individuals had GA or 3-OH-GA assayed in blood or CSF. Other members of the cohort who were tested had normal orundetectable levels.
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comatose. A CT scan showed haemorrhage in the right tem-
poroparietal region and he died 3 days later. Autopsy revealed
widespread ischaemic neuronal damage in the cerebrum and
cerebellum. Haemorrhagic infarction in the right parietal and
temporal cerebrum was due to venous sinus thrombosis,
apparently a complication of sepsis and dehydration. Mild
symmetric dilation of the lateral ventricles accompanied
atrophy of the striatum. There was severe loss of medium-
sized neurons in the caudate and putamen, with dorsal areas
more severely affected than ventral. Immunohistochemical
stains demonstrated astrocyte hypertrophy and microglial
activation in the striatum. There were rare scattered and a
few perivascular clusters of CD3 immunoreactive lymphocytes
in the putamen. No white matter vacuoles were identified.
Case 2Following full term birth in 1977, this male sibling of Case 1
had mild respiratory distress requiring incubation. He is
the presumed first GA-1 case of pedigree B described in
the clinical report of this cohort (Haworth et al., 1991). At
10 months of age, acute bacterial (E.coli) pneumonia was
accompanied by fever and lethargy. Cerebrospinal protein
was elevated. Despite antibiotic treatment, the fever contin-
ued and 3 weeks later he developed episodes of opisthotonus,
limb stiffening and lethargy. A CT scan was reported to show
enlarged lateral ventricles and Sylvian fissures. On Pheno-
barbital, there was a slight improvement of his neurological
status. He suffered respiratory arrest following aspiration of
vomit �6 weeks after presentation. Autopsy revealed pneu-
monia. The head circumference was on the 71.9 percentile.
The external surface of the brain appeared normal, but the
ventricles were mildly enlarged. The caudate and putamen
exhibited widespread loss of medium-size neurons with
marked astrocytic proliferation, microglial activation and
focal dystrophic calcification. Mild diffuse lymphocyte infilt-
ration and rare perivascular cuffing (up to 5 cells thick) was
present in the putamen. The tail of the caudate adjacent to the
hippocampus appeared normal. Rare vacuoles were seen in
the white matter of a single gyrus and vacuoles were fairly
abundant in the central tegmental tract of the brainstem, but
not elsewhere.
Case 3This male’s parents were known heterozygotes for the GA-1
mutation. He is Case 12 in pedigree C described in the clinical
report of this cohort (Haworth et al., 1991). Following full-
term birth in 1989, he presented at 7 months of age with
developmental delay and relatively sudden onset of dystonia
and seizures. A CT scan showed hypointensity of the caud-
ate and putamen, and widening of the Sylvian fissures. He
became severely impaired, was treated with Phenobarbital
and required placement of a feeding tube. He was placed in
a chronic care institution. At 16 months, he developed fever
and died suddenly. Autopsy showed acute glottitis and pneu-
monitis. His head circumference was on the 22 percentile.
The external appearance of the brain was unremarkable. The
caudate and putamen were atrophic and the lateral ventricles
were mildly enlarged. Microscopically, the striatum exhibited
loss of medium-size neurons, plump reactive astrocytes,
reactive microglia, scattered calcospherites and rare CD3
immunoreactive lymphocytes in the caudate. The tail of the
caudate adjacent to the hippocampus appeared normal. There
were scattered pyknotic neurons in the cerebral cortex.
Case 4This male was born at 39 weeks by Caesarean section and was
found, on genetic screening in 1999, to have GA-1 (he is
Case 3 in Greenberg et al., 2002). His development was
delayed slightly. At 5.5 months of age, he developed fever
with onset of dystonia and athetoid limb movements as well
as seizure activity. A CT scan showed enlarged frontal horns
of the lateral ventricles and widened Sylvian fissures, but no
generalized atrophy (Fig. 1). Caudate atrophy was worse
at 10 months. He was treated with Phenobarbital and
Fig. 1 Photographs showing a CT scan of the head in thehorizontal plane obtained 8 months before death (upper) and acoronal slice of the brain (lower) from Case 4. Both exhibitflattening of the head of the caudate nucleus along the wall of thelateral ventricle (arrows) and mild enlargement of the lateralventricles. Note that the cortical thickness is essentially normal.
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topiramate, but failed to thrive and had multiple respiratory
infections. At 15 months, he was unable to sit but had some
head control and visual interaction. During a febrile illness at
18 months, he stopped breathing. Autopsy revealed laryngitis
and dehydration. His head circumference had been on the
52 percentile at birth, 89 percentile at 3 months, 63 percentile
at 8 months and was on the 50 percentile at the time of death.
The brain exhibited mild widening of temporal and frontal
sulci, and pronounced widening of the Sylvian fissures. No
histological abnormalities were apparent in the cerebral cor-
tex of the temporal lobe tips or frontal lobes. The caudate
nuclei were small, yellowish and firm. There was symmetric
lateral ventricle enlargement (Fig. 1). The head of the caudate
and the putamen exhibited severe neuronal loss with pro-
nounced reactive astrocytes. Only rare reactive microglia
were identified. The tail of the caudate adjacent to the hip-
pocampus appeared normal. The cerebral white matter exhib-
ited no vacuoles, no damaged axons were identified using
amyloid precursor protein immunohistochemistry, and no
myelin debris could be demonstrated with the Marchi
method. Rare vacuoles were present in the central tegmental
tract at the level of the midbrain.
Case 5This female born by forceps delivery at 37 weeks gestation,
in 1983, had generalized hypotonia in infancy. She is Case 1
in pedigree A described in the clinical report of this cohort
(Haworth et al., 1991). At 6 months of age, she developed
seizures and choreoathetoid movements. Thereafter she
developed severe spastic quadriparesis. She was unable to
walk and developed severe scoliosis. A CT scan at 5 years
showed enlarged frontal horns and slightly wide Sylvian
fissures. She died of acute pneumonia at age 7 years. Her
head circumference had been on the 25 percentile at birth,
52 percentile at 6 months, 3 percentile at 19 months and
25 percentile at the time of death. Her brain exhibited mild
gyral flattening, slightly enlarged ventricles and severe striatal
atrophy. The caudate and putamen had a near complete loss of
medium size neurons (Fig. 2) and some GFAP immunoreactive
astrocytes; the nucleus accumbens was spared. Scattered
hypertrophic astrocytes and very rare HLA-DR immuno-
reactive microglia were present in the internal capsule—
although there were no vacuoles. There were small foci of
spongiform change in the frontal and insular cortex.
Case 6This male born at 40 weeks gestation, in 1953, was apparently
slow to breathe. He had three siblings that died in infancy
from pneumonia. He is Case 14 in pedigree E described in
the clinical report of this cohort (Haworth et al., 1991). At
12 months of age, he developed a high fever and was admitted
to hospital with dystonia. At 6 years, he was documented
to have severe flexor spasticity of the arms and legs,
choreoathetoid movements and oral dyskinesia. Seizures
were reported only early in life. He resided in a nursing
care facility for his entire life. At no time was brain imaging
conducted and head circumference information was not
available. He required many hospitalizations for aspiration
pneumonia. On one such admission at age 40 years, the
treatment was complicated by pseudomembranous colitis
from which he died. His brain had a normal external appear-
ance. The frontal horns of the lateral ventricles were moder-
ately enlarged. There was marked atrophy of the caudate and
putamen with neuronal loss and abundant corpora amylacea.
There was no significant immunoreactivity for GFAP or
HLA-DR. The anterior nucleus accumbens was spared.
The white matter of the cerebrum and brainstem was well
myelinated and no vacuoles were identified. Subtle chronic
astrogliosis, demonstrable with modified phosphotungstic
acid haematoxylin (PTAH) stain, was evident in the inferior
olivary nuclei but there was no obvious neuron loss. Very rare
empty basket cells were present in the cerebellum, suggestive
of mild Purkinje cell loss. Frontal cortex tissue was examined
in all cases, none provided evidence of obvious cell loss or
reactive changes.
Comparative analysisQuantitative comparison of the striatum in GA 1 cases and
controls showed statistically significant (P < 0.05) loss of
Fig. 2 Photomicrographs of the striatum showing (A) normalneuron density (neurons indicated by arrows) in an age-matchedcontrol and (B) severe loss of neurons in the same area of GA-1Case 5. (H&E stained sections, 403 objective magnification,bar = 20 mm).
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medium sized neurons in the dorsal caudate, ventral caudate,
dorsalputamenandventralputamen(Fig.3).Thedorsal regions
of the caudate and putamen were more severely affected,
although this was not statistically significant. There was no
age-dependent trend (Fig. 4) in the quantity of neurons in
the GA-1 cases, suggesting that maximal neuron loss had
occurred within 2 months of onset of symptoms, which was
the time of death after encephalopathical crisis in Case 1. Large
neurons, which are normally much less abundant than the
medium-sized neurons, were significantly fewer in the ventral
putamen of the GA1 patients with similar trends in all areas of
the striatum (Fig. 5). Immunostaining for GFAP (Figs 6 and 7)
demonstrated the presence of reactive astrocytes in all areas of
the striatum, with a tendency to greater staining in the dorsal
striatum. Analysis of reactive microglial activation (Fig. 8)
demonstrated significant HLA-DR immunoreactivity in
only the three youngest cases and mild infiltrate in the fourth
case, suggesting that it only persists a few months after the
acute episode. Overall, the differences approached statistical
significance (P = 0.0725 and 0.0699, respectively; paired t-test
versus age-matched controls) only in the dorsal and ventral
putamen.
Loss of GABA and calbindin immunoreactivity confirmed
that the population of medium sized neurons is decreased in
GA-1 (data not shown). The relative absence of NeuN label-
ling confirmed that neurons were lost and not simply atrophic.
Qualitative inspection of ChAT and tyrosine hydroxylase
immunoreactivity in large cholinergic and dopaminergic
neurons indicated little, if any, difference between GA-1
Fig. 3 Counts of medium sized neurons (mean 6 SE) in fourregions of the striatum. A significant loss of medium sized neuronswas seen in dorsal and ventral areas of the caudate and putamen;paired t-test versus age-matched controls, *P < 0.008.
Fig. 6 Photomicrographs showing scant immunoreactivityfor GFAP in dorsal caudate of a control case (A) and abundantGFAP-positive reactive astrocytes in GA-1 case 1(B). (DAB detection of anti-GFAP with haematoxylincounterstain; bar = 50 mm).
Fig. 4 Neuron density as a function of age. Note that themagnitude of neuron loss is similar regardless of the age of theGA-1 patient.
Fig. 5 Counts of large neurons (mean 6 SE) in the striatum.There was a marginally significant loss in the ventral caudate;paired t-test versus age-matched controls, P = 0.0422.
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cases and controls (data not shown). Synaptophysin immun-
oreactivity in striatum was not significantly different between
cases and controls (data not shown), indicating the preserva-
tion of input axons to the striatum.
The presence of GAPDH amplification product in both
GA-1 cases and controls indicated the presence of undegraded
mRNA despite long post-mortem delays to autopsy and the
lengthy interval between autopsies and molecular study.
However, there was limited frozen tissue stored and striatal
tissue was not recoverable. Thus, proper quantification of the
relative proportion of normal and mutant GCDH transcripts
was not possible. Nonetheless, we observed abundant mutant
and normal sized GCDH transcripts in the frontal cortex of
Cases 3 and 5 (the most abundant frozen tissue available)
(Fig. 9). A similar pattern has been seen in fibroblasts and
lymphoblasts of affected patients (Greenberg et al., 1995).
Organic acid analysis demonstrated marked elevations of
GA and 3-OH-GA compared with controls. There was no
evidence of striatal specificity or age dependency. There
was a slight elevation of GA in one control brain, which
likely can be considered a non-specific change related to
agonal events (Table 2).
Fig. 7 Proportionate area with DAB precipitate (measured by NIHimage analysis; mean 6 SE) when labelled with primary antibodyagainst GFAP; paired t-test versus age-matched controls*P < 0.016, **P < 0.005.
Fig. 8 Photomicrographs showing HLA-DR immunolabellingof reactive microglia in the putamen. The age matched controlcase (A) exhibits no cells while the GA-1 sample (Case 1, B)exhibits abundant activated cells. (DAB detection of anti-HLA-DR with haematoxylin counterstain, bar = 50 mm).
Fig. 9 PCR amplification products from frontal lobe samplesincubated without (–RT) and with reverse transcriptase ( + RT),respectively, are shown from Case 3 (lanes 1 and 2), Case 5 (lanes3 and 4), Control 1 (lanes 5 and 6) and Control 2 (lanes 7 and 8).The upper band in lanes 2, 4, 6 and 8 represents a normal sizedfragment of 393 bp and the lower band in lanes 2 and 4 representsthe truncated message 26 bp shorter than the normalsized-fragment. Lane 9 is the l00 bp ladder.
Table 2 Analysis of organic acids in brain tissue samplesfrom four GA-1 and three control cases
Brain sample areas Age atdeath
GA(nmol/gprotein)
3-OH-GA(nmol/gprotein)
Case 1 – cerebellum 8 months 21 240 740Case 3 – cerebellum 16 months 8020 550
– thalamus 10 960 360– caudate 5960 360– frontal 7760 280
Case 4 – frontal 18 months 3770 360Case 5 – frontal 7 years
7 months5990 290
– caudate/internal capsule
8310 420
Control – frontal 3 weeks n.d. n.d.Control – frontal 6 months n.d. n.d.Control – frontal 5 months 170 n.d.
n.d.= not detected (i.e. <10).
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DiscussionThe neuropathological changes associated with GA-1
described here in six individuals are essentially similar to
those previously described in 10 affected children of other
ethnic backgrounds ranging in age from 10 months to
15 years (Goodman et al., 1977; Leibel et al., 1980; Bennett
et al., 1986; Chow et al., 1988; Bergman et al., 1989; Soffer
et al., 1992; Kimura et al., 1994; Kolker et al., 2003). Atrophy
involving the striatum is consistently described, with sparing
of the caudate in one case (Kimura et al., 1994). We observed
sparing of the nucleus accumbens and tail of the caudate
nucleus. In seven out of ten previously published cases,
spongiform change of the white matter was observed. An
additional autopsy by Kolker and colleagues (unpublished)
on a female aged 3 years and 3 months, with disease onset
at 15 months, demonstrated severe neuron loss and gliosis in
the basal nuclei, widespread moderate neuron loss in the
cerebral cortex, brainstem (including substantia nigra) and
cerebellum and diffuse spongiform changes in white matter.
There was minimal spongiform white matter change in the
cases described here and it was largely confined to the brain-
stem. Whether this reflects a cohort difference or simply a
timing difference is not clear. We did not find clear evidence
of axon damage or demyelination in any case, the exception
being subtle microglial activation in the internal capsule of
Case 5. This suggests that the white matter changes could
represent a fluid shift into myelin rather than a destructive
change. The anatomical restriction in these cases cannot
readily be explained. Imaging studies suggest that white matter
alterations are rare in the Amish cohort (Strauss et al., 2003).
However, inaboywith GCDHdeficiency presenting at15years
age without major neurological abnormality, magnetic reson-
ance spectroscopy suggested increased myelin turnover in
conjunction with a reduction of neuronal integrity (Bodamer
et al., 2004). In contrast, a 19-month old child studied by the
same technique exhibited changes suggestive of neuroaxonal
damage and demyelination (Kurul et al., 2004). Obviously, the
only way to truly understand such studies is to perform them on
individuals who shortly thereafter undergo autopsy.
We have previously reported both normally spliced and
deleted cDNA in fibroblasts and lymphoblasts of our affected
patients (Greenberg et al., 1995) and we now observe similar
findings in brain tissue. Thus, there does not appear to be a
direct correlation between the presence of wild type GCDH
transcript in the regions of the brains available for study and
clinical outcome. Unfortunately, cDNA could not be prepared
from the regions of the basal nuclei to assess expression
in these vulnerable areas of the brain. Nonetheless, it seems
reasonable to assume that GCDH intron 1 splicing is probably
comparable in these areas of the brain. Our results suggest
that splicing alone is probably not the sole determinant of
clinical outcome. It is possible that, in the target regions,
aberrantly spliced mRNA is unstable or is prematurely
degraded; other genetic factors influencing higher order
RNA structure or RNA protein interactions are likely to affect
the final clinical outcome.
Biochemically, the most intriguing result of our study
is the discrepancy between high concentrations of GA and
3-OH-GA in brain and low concentrations in urine and plasma
(Haworth et al., 1991). The concentrations are much higher
than in one previously described patient who was treated with
a lysine-restricted diet (Kolker et al., 2003). However, GA
concentrations are comparable with those found in a post-
mortem examination of another published case (Goodman
et al., 1977) and in a frontal cortex biopsy of an untreated
adult who excreted high levels of GA (S. Kolker, unpublished
data). We failed to confirm the prior observation that con-
centrations of GA and 3-OH-GA are highest in the striatum
(Kolker et al., 2003). The high gradient of organic acids
towards the brain and the low permeability of the blood–
brain barrier for dicarboxylic acids in general (Hoffmann
et al., 1993) support the notion that organic acids are probably
generated in the brain. However, this has not yet been expli-
citly proven for GA or 3-OH-GA, and requires detailed
further investigation, including permeability studies of the
blood–brain barrier and blood–CSF barrier. Unfortunately,
we do not have data concerning CSF concentrations of GA
and 3-OH-GA in this cohort of patients (Haworth et al., 1991;
Greenberg et al., 2002).
Despite genetic homogeneity and a wide range in duration
of survival following encephalopathical crisis, all cases had
near complete loss of medium neurons from the striatum,
with the exception of the nucleus accumbens and tail of the
caudate. This likely occurs within, at most, a few weeks of the
first encephalopathical crisis. It is important to emphasize
the apparent lack of progression over the lifespan because
this observation supports the idea that a single severe insult
during infancy creates the bulk of striatal injury. The three
individuals that died soonest after the encephalopathical crisis
(Cases 1, 2 and 3) had small collections of lymphocytes in the
cerebrum. This is not a common incidental finding in child-
hood death; it might simply reflect a septic state rather than a
specific component of GA-1 brain damage or it could be
interpreted as evidence that mild encephalitis can precipitate
the striatal destruction. Reactive microglia, which can
accompany neuron loss or encephalitis, also dissipated within
6 months of the encephalopathical crisis. Because the neuro-
toxic effect of 3-ON-GA per se is weak (Freudenberg et al.,
2004; Lund et al., 2004), it has been suggested that additional
amplifying mechanisms are necessary to initiate neuronal
damage. Among these, induction of nitric oxide, synthase
and indoleamine 2,3-oxygenase by inflammatory cytokines
have been considered as relevant, increasing the formation of
nitric oxide and quinolinic acid, respectively. The occurrence
of infections in early life may provide the trigger for a cas-
cade of injurious events. Reactive astrocytes, which are activ-
ated acutely perhaps as a protective response (Porciuncula
et al., 2004), persist many years post injury.
Quantification of large cholinergic neurons has never been
performed in cases of GA-1; these cells have been reported as
unaffected. If this population of neurons was undamaged, one
would demonstrate an increased density in atrophied striatum
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whose medium neurons had been lost. Our results indicate
that there is a significant loss of large neurons from some
regions of the striatum. They receive glutamatergic input
from the thalamus that is mediated by NMDA, AMPA and
mGluR receptors (Haber and Gdowski, 2004) and therefore
should be vulnerable. Quinolinic acid has been shown to
damage cholinergic neurons (Rossato et al., 2002; Guidetti
and Schwarcz, 2003; Kumar, 2004). The observed dorso-
ventral gradient of neuron loss might be explained in different
ways. First, the neuron characteristics as well as the afferent
and efferent connections to the striatum exhibit dorso-ventral
and rostro-caudal differences (Haber and Gdowski, 2004),
which are likely to influence vulnerability. Secondly, blood
flow through lenticulostriate arteries could, in the face of the
excitotoxic stress/hyperactivity, be diverted away from the
dorsal regions leading to a additive hypoxic/ischaemic injury.
There are other conditions with overlapping neuropatho-
logical features including familial infantile striatal necrosis
(Straussberg et al., 2002), Huntington disease (Portera-
Cailliau et al., 1995), neuroacanthosis and Wilson disease
(Nelson, 1995), most of which have a gradual progressive
course. The presentation of the acute encephalopathical crisis
in GA1 resembles that of a ‘metabolic stroke’ (i.e. acute
neuronal damage induced by toxic agents), in contrast to
vascular occlusion or hypoxia/ischaemia as in peripartum
neonatal encephalopathy (Johnston, 2001). The result is a
rapid selective loss of vulnerable neurons rather than an
infarct. Our results support the prior assertion that GA-1
preferentially targets striatal medium-spiny neurons, whereas
cholinergic neurons are affected to a lesser extent. The mech-
anisms underlying this particular vulnerability have been the
subject of much debate and have been well investigated in
models for adult neurodegenerative disorders (Bates, 2003)
and for a few paediatric neurological disorders (hypoxia
ischemia in the term infant, bilirubin encephalopathy); they
may well share final overlapping pathways. Several variables
should be considered in a hypothesis to explain the selective
striatal damage. A neuro-anatomical basis might play a role.
The striatum receives strong glutamatergic corticostriatal and
thalamostriatal input as well as dopaminergic input (Haber
and Gdowski, 2004). Within the striatum, GABAergic
medium-spiny neurons are more vulnerable than cholinergic
aspiny interneurons to energy compromise (Nishino et al.,
2000). Specific membrane ion channels, glutamate receptor
subtypes and subunits, and intracellular enzymatic activities
are involved in the events responsible for differential vulner-
abilities to oxygen or glucose deprivation and to glutamate
receptor-mediated toxicity (Calabresi et al., 2000). Suscept-
ibility to nitric oxide mediated cell damage also strongly
differs among striatal neurons, with medium-spiny neurons
being the most vulnerable (Dawson, 1995). Quinolinic acid
production via shunting through the kynurenine pathway was
postulated to play a role in GA-1 some years ago (Heyes,
1987) and has been reiterated as a mechanism through which
intercurrent infection could precipitate damage (Varadkar
and Surtees, 2004). Recent pathophysiological models for
GA-1 refer to many of the above mentioned aspects, hypo-
thesizing a role for accumulating organic acids acting via
direct or indirect overactivation of glutamatergic receptors
(in particular NMDAr) resulting in increased influx of
calcium and increased generation of reactive oxygen species
(Kolker et al., 2004a,b). Notably, concentrations of 3-OH-
GA in the present post-mortem CNS investigations are at the
same level as the lowest concentrations of 3-OH-GA that
cause neuronal damage in vitro (Kolker et al., 2004a).
Alternate mechanisms have been also considered. Strauss
and Morton (2003) speculated on alternative mechanisms
of organic acids, such as damage to the microvasculature
with modulation of blood–brain barrier permeability, altera-
tion of astrocyte function (Frizzo et al., 2004; Muhlhausen
et al., 2004). Integrity of the blood–brain barrier and
functional status of astrocytes cannot be assessed by our
methods; there is no morphological evidence for changes
in the vasculature.
The timing of injury needs to be considered; most children
who suffer an encephalopathical crisis do so by �1 year of
age and rarely if ever after 5 years (Goodman and Frerman,
2001; Strauss et al., 2003). Some of the vulnerability might be
explained on the basis of postnatal developmental changes.
Several studies have examined the concentration of glutama-
tergic receptors in human forebrain using 3H-glutamate and3H-MK801 binding assays. In the cerebral cortex, the con-
centration appears to be relatively low in newborns and peaks
sometime between 5 months and 1–2 years, thereafter declin-
ing. Changes in the modulatory sites of the NMDA receptor
can also be demonstrated by pharmacological studies
(Kornhuber et al., 1988, 1989; D’Souza et al., 1992; Piggott
et al., 1992; Slater et al., 1993; Chahal et al., 1998). The
quantity of aspartate binding sites also peaks at around
5 months postnatal in the cortex; this peak in glutamatergic
synapses could be related to plasticity (Slater et al., 1992).
Fewer studies have directly examined the human striatum; the
quantity of glutamate binding sites is roughly similar in puta-
men and frontal cortex (Kornhuber et al., 1988) and the
concentrations of glutamate and aspartate increase rapidly
during the first postnatal year (Kornhuber et al., 1993). Stud-
ies in the postnatal rat during the first month of life, an age
that roughly corresponds to human infancy, have defined at
the molecular level the shifts that occur in NMDA receptor
subtypes (Monyer et al., 1994; Portera-Cailliau et al., 1996;
Gurd et al., 2002). Particular NMDA receptor subtypes may
make neurons especially vulnerable in infancy (McQuillen
and Ferriero, 2004). Additionally and in relation to the
infectious aspect discussed above, the onset of a vulnerable
period could reflect the loss of passive immunity after which
damage is initiated by exposure to, presumably ubiquitous,
infectious agents.
The gross brain findings also need to be addressed. The
temporal fossa fluid collections have been interpreted by
some as an indicator of atrophy. However, at least two
patients in this cohort (not described in this report) identified
by newborn screening had the abnormality at 1 and 6 weeks
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age, without significant neurological impairment. This has also
been described by others (Neumaier-Probst et al., 2004). The
one case where we were able to examine the temporal lobe tip
exhibited no obvious abnormalities and there were no insular
cortex abnormalities in any case. Therefore, this represents a
developmental abnormality, i.e. temporal hypotrophy or arach-
noid cyst-like anomaly, rather than atrophy. The absence of
generalized subarachnoid space enlargement suggests that this
is not a disorder of CSF absorption, as has been suggested
previously (Martinez-Lage, 1996). Concerning the macroen-
cephaly and megalencephaly, only two out of six individuals
had enlarged heads during infancy, while brain weight deviated
substantially from the median only in the three youngest cases
(up to 16 months). In a clinical follow-up study of Nordic
patients, the head circumference relative to normal values
peaks at 6 months of age and tends to normalize thereafter
(Kyllerman et al., 2004). Together, the observations suggest
that the brain is most enlarged around the time that encepha-
lopathical crises occur. If the organic acids act as osmotic
agents, brain enlargement could partly be accounted for largely
by water accumulation; however, our observation that concen-
tration of the organic acids is not correlated with age to some
extent negates the idea. Alternative mechanisms, such as a
role for GA or 3-OH-GA as osmoregulators (in analogy to
N-acetylaspartate in aspartoacylase deficiency) have not yet
been investigated (Baslow, 2003). Because our data do not
indicate that the concentrations decrease with age, ‘normal-
ization’ of head size and brain weight might be accounted
for by mild atrophy beyond the striatum, which can be almost
impossible to detect histologically.
With the advent of screening among vulnerable popula-
tions, GA-1 can now be identified presymptomatically at
birth. Current treatments for newborns identified presympto-
matically with GA-1 involve dietary modifications and very
aggressive treatment of intercurrent infections (Strauss et al.,
2003; Naughten et al., 2004). Outcome in some children
treated presymptomatically appears to be improved, but
acute brain injury and its devastating sequelae still occur
in others (Greenberg et al., 2002). Our evidence of a single
insult and data indicating that excitatory synapses peak in the
first year of life opens up the possibility that presymptomatic
detection and the use of neuroprotective agents could be tried
to limit brain injury. Anti-inflammatory agents might also be
of value. Whether preservation of neurons (e.g. with a phar-
maceutical agent) in infancy could allow them to mature into
a less vulnerable phenotype is not known. An animal model
of GA-1 that mimics the human neuropathology is required to
test possible treatment interventions. However, a useful
animal model is not yet available (Funk et al., 2004; Koeller
et al., 2004). Further comparative clinical studies of individu-
als with GA1 in this cohort who do not suffer an encephalo-
pathical crisis could provide some insight. For example, if
GA levels in the CSF simply correlated with enzyme activity
and with clinical severity, a dose-response relationship could
be invoked. However, when considering all mutations, there
is no simple correlation between genotype and phenotype;
there is some evidence that low excretors tend to be more
impaired (Christensen et al., 2004). The significance of this
finding is not yet known. One might speculate that low
urinary excretion of organic acids in some patients reflects
high tissue retention. Alternately, accumulating organic acids
might influence the expression of relevant proteins, such as
neurotransmitter receptors, thereby altering the susceptibility
to neuronal damage—analogous to chemical preconditioning
(Riepe et al., 1997; Ravati et al., 2001; Kolker et al., 2002).
More work and a representative animal model are required
to fully understand the pathogenesis of this disorder
(Goodman, 2004).
AcknowledgementsWe wish to thank members of the Winnipeg GA-1 study
group and especially Louise Dilling for helping to identify
patients. We wish to thank Melissa Caswill for help with
review of the records and Sharon Allen, Susan Janeczko,
Patrick Feyh and Christy Pylypjuk for technical assistance.
This work was funded by grants to M.D., A.P. and C.R.G.
from the Garrod Association of Canada and the Manitoba
Medical Service Foundation and a grant to S.K. from
the German Research Community (DFG KO 2010/2-1).
Dr Del Bigio holds the Canada Research Chair in Develop-
mental Neuropathology.
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