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Cellular Distribution of Torsin A and Torsin B in Normal Human Brain
Marina Konakova, PhD;
Duong P. Huynh, PhD;
William Yong, MD;
Stefan M. Pulst, MD
Arch Neurol. 2001;58:921-927.
ABSTRACT
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Background Early-onset torsion dystonia is a hyperkinetic movement disorder caused
by a deletion of 1 glutamic acid residue in torsin A protein, a novel member
of the AAA family of adenosine triphosphatases. No mutation has been found
so far in the closely related torsin B protein. Little is known about the
molecular basis of the disease, and the cellular functions of torsin proteins
remain to be investigated.
Objective To study the regional, cellular, and subcellular distribution of the
torsin A and torsin B proteins.
Methods Expression of torsin proteins in the central nervous system was analyzed
by Western blot analysis and immunohistochemistry in human postmortem brain
tissues.
Results We generated polyclonal antipeptide antibodies directed against human
torsin A and torsin B proteins. In Western blot analysis of normal human brain
homogenates, the antibodies specifically recognized 38-kd endogenous torsin
A and 62-kd endogenous torsin B. Absorption controls showed that labeling
was blocked by cognate peptide used for immunization. Immunolocalization studies
revealed that torsin A and torsin B were widely expressed throughout the human
central nervous system. Both proteins displayed cytoplasmic distribution,
although torsin B localization in some neurons was perinuclear. Strong labeling
of neuronal processes was detected for both proteins.
Conclusions Torsin A and torsin B have similar distribution in the central nervous
system, although their subcellular localization is not identical. Strong expression
in neuronal processes points to a potential role for torsin proteins in synaptic
functioning.
INTRODUCTION
PRIMARY EARLY-ONSET torsion dystonia is an autosomal dominant inherited
disorder that has been linked to the genetic locus DYT1 on human chromosome 9q34.1, 2
Two genes from this locus (TOR1A and TOR1B) have been recently identified through positional cloning.3, 4 The TOR1A
gene encodes a 37-kd protein called torsin A. The adjacent TOR1B gene encodes a closely related protein called torsin B of unknown
molecular weight. A GAG deletion at codon 302 in torsin A results in loss
of 1 glutamic acid and causes disease, although penetrance of this mutant
allele is only 30%.
Sequence analysis of the complementary DNA encoding torsin A and torsin
B indicated that these proteins belong to the AAA family of adenosine triphosphatases
(ATPases), associated with diverse cellular activities. This family of ATPases
is defined by a conserved ATPase domain that contains Walker homology sequences.
Since AAA-type ATPases are implicated in a wide variety of functions, it is
difficult to predict what cellular processes involve the torsin proteins.
Despite significant progress in understanding the genetic basis of early-onset
torsion dystonia, the molecular mechanism by which this mutation results in
the disease phenotype is not well understood. The process is thought to involve
a deficiency in dopamine release in the substantia nigra.5, 6
To address the molecular mechanisms of early-onset dystonia, several studies
have examined the anatomical distribution of torsin proteins in the central
nervous system (CNS). One of these studies focused on the expression of torsin
A and torsin B messenger RNA (mRNA) expression in the normal human brain7—torsin A mRNA was found in many brain regions,
including the dopamine neurons of the substantia nigra; however, no expression
of torsin B mRNA was detected in the same regions. One study examined the
expression of torsin A protein in the frontal cortex, cerebellum, and substantia
nigra of rat and human brains.8 Localization
was nuclear and cytoplasmic in neurons; the distribution of torsin B protein
was not analyzed. Nuclear localization of torsin A in this study did not correspond
to cytoplasmic localization of normal and mutant torsin A in cultured cells.9
To address the discrepancies in previous studies and to compare torsin
A and torsin B protein distribution, we generated and characterized torsin
A– and torsin B–specific antibodies. Using these antibodies, we
performed a comprehensive comparative analysis of the cellular expression
of torsin A and torsin B in the human CNS. We also made the first estimate
of the molecular weight of torsin B.
METHODS
PREPARATION OF ANTIPEPTIDE ANTIBODIES
Antisera against human torsin A and B were generated by immunizing rabbits
with synthetic peptides conjugated to keyhole limpet hemocyanin. These peptides
were synthesized with a COOH-terminal cysteine. Synthetic peptides used for
immunization were as follows: peptide 1 (GQKRSLSREALQK [residues 51-65]) and
peptide 2 (SGKQREDIKLKDIE [residues 224-237]) of human torsin A and peptide
3 (HEQKIKLYQDQLQK [residues 77-90]) of human torsin B.
AFFINITY PURIFICATION OF ANTIBODIES
Individual peptides were coupled to cyanogen bromide–activated
Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ) in 0.1-mol/L carbonate-bicarbonate
buffer (pH 11) overnight. Antiserum samples were passed through the columns
in a slow, regulated flow (0.5 mL/min). The columns were then washed with
phosphate buffer and peptide-specific antibodies were eluted with 0.5-mol/L
glycine hydrochloride buffer (pH 2.5). The eluants were immediately neutralized
with 10% (vol/vol) Tris base buffer (pH 8.0), desalted, and concentrated with
Centriprep 30 columns (Amicon Pharmaceuticals Inc, Beverly, Mass).
PROTEIN EXTRACTION
Nondenaturing Triton X-100 lysates of human trabecular bone (HTB) cells
were made by resuspending cell pellets in ice-cold lysis buffer (100-mmol/L
Tris hydrochloride, pH 7.5; 150-mmol/L sodium chloride; 5-mmol/L EDTA; 1%
Triton X-100; and a cocktail of protease inhibitors). Lysates were clarified
by centrifuging in a microcentrifuge at full speed for 15 minutes. Brain homogenates
were prepared as described previously.10
GEL ELECTROPHORESIS AND WESTERN BLOTTING
Cell lysates were mixed with an equal volume of 2X loading buffer (135-mmol/L
Tris hydrochloride, pH 6.8; 20% glycerol; 4% sodium dodecyl sulfate; 5% 2-mercaptoethanol;
and 0.0025% bromphenol blue) and subjected to one-dimensional sodium dodecly
sulfate–polyacrylamide gel electrophoresis (4%-15% acrylamide gels)
according to the method of Laemmli.11
After electrophoresis, proteins were transferred to a nitrocellulose
membrane (Amersham Pharmacia Biotech) in transfer buffer (25-mmol/L Tris,
192-mmol/L glycine, and 20% methanol) using a tank blot apparatus. Blots were
stained with Ponceau S to verify equal protein loading per lane. After 1 hour
of blocking using 5% nonfat milk powder in Tris-buffered saline with Tween
(20-mmol/L Tris-hydrochloride, pH7.6; 137-mmol/L sodium chloride; and 0.05%
Tween 20), blots were probed with antibodies (1 or 2 µg/mL diluted in
Tris-buffered saline with Tween containing 5% milk powder) overnight. The
primary antibodies were detected with horseradish peroxidase–conjugated
antirabbit antibodies (1:5000 dilution in Tris-buffered saline with Tween
containing 5% milk powder) and an ECL detection system (Amersham Pharmacia
Biotech).
IMMUNOHISTOCHEMISTRY
All immunohistological staining was performed on 7-µm sections.
Tissue sections were dewaxed in xylene and rehydrated through washes in decreasing
concentrations of ethanol (100%, 95%, and 70%). Sections were subsequently
treated with a protease cocktail (Biomeda Corp, Hayward, Calif) and avidin-biotin
and blocked with 3% normal goat serum. Sections were then incubated with torsin
A and B antibodies, 20 µg/mL, overnight at 4°C. Primary antibodies
were detected using a Vector avidin-biotin complex elite peroxidase kit (Vector
Laboratories, Burlingame, Calif), enhanced by diaminobenzidine enhancer (Biomeda
Corp), and visualized with diaminobenzidine. Sections were counterstained
with aqueous hematoxylin (Xymed Co, San Francisco, Calif). All dilutions and
washes were performed in 0.1-mol/L phosphate-buffered saline containing 0.01%
Triton X-100. The specificity of immunostaining was confirmed by preabsorption
of the primary antibodies with the specific peptides used for immunization,
100 µmol/L.
RESULTS
PRODUCTION AND EVALUATION OF POLYCLONAL ANTIBODIES
We generated affinity-purified rabbit polyclonal antibodies using synthetic
peptides derived from the predicted amino acid sequences of torsin proteins.
Peptides were chosen based on the analysis of hydrophilicity (Kyte-Doolittle
scale), antigenic index (Jameson-Wolf method), and surface probability (Emini
formula). The chosen peptide epitopes did not show significant homology to
known proteins. Two torsin A–specific antibodies (A1 and A2) and 1 torsin
B–specific (B1) antibody were prepared. The A1 and A2 antibodies were
generated using an N-terminal (residues 51-65) and a C-terminal (residues
224-237) torsin A peptide, respectively. The B1 antibody was raised against
an N-terminal torsin B peptide (putative residues 77-90).
Expression of torsin A and B proteins in the brain was examined by Western
blot analysis of whole human brain homogenates (Figure 1A). Analysis using A1and A2 antibodies revealed that torsin
A migrates as a single band with an apparent molecular mass of approximately
38 kd (Figure 1A, lanes 1 and 4),
which is in agreement with the molecular weight of 37.8 kd predicted from
the torsin A complementary DNA sequence. Both anti–torsin A antibodies
recognized the same protein band, indicating the specificity of the generated
antibodies. The specificity was further verified by preabsorption of antibodies
with peptides used for immunization. Thus, immunorecognition of the 38-kd
band was completely blocked by preabsorption of A1 antibody with the amino-terminal
peptide (peptide 1), 100 µmol/L, and A2 antibody with the carboxy-terminal
peptide (peptide 2), 100 µmol/L (Figure
1A, lanes 2 and 5). Preabsorption of antibodies with unrelated peptides
did not alter the specific immunorecognition of the 38-kd torsin A protein
(Figure 1A, lanes 3 and 6).
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Figure 1. Characterization and specificity
of anti–torsin A and anti–torsin B antibodies. A, Western blot
detection of torsin A and B in human brain homogenates with A1, A2, and B1
antibodies. Preabsorption of antibodies with the cognate peptide (lanes 2,
5, and 8) precluded staining, but not preabsorption with an unrelated peptide
(lanes 3, 6, and 9). B, Western blot detection of torsin A and B in human
neuroblastoma (HTB10) cell lysates with A1, A2, and B1 antibodies.
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Immunoblotting analysis using B1 antibody revealed that torsin B underwent
electrophoresis as a 62-kd protein (Figure
1A, lane 7). This is the first estimate of the molecular weight
of endogenous torsin B. As with torsin A antibodies, the immunoreactivity
of torsin B–specific B1 antibody could be completely blocked by preabsorption
with homologous peptide (peptide 3), 100 µmol/L (Figure 1A, lane 8). Preabsorption of the B1 antibody with the same
amount of an unrelated peptide (peptide 1) did not affect its ability to recognize
torsin B (Figure 1A, lane 9).
To gain additional information about specificity, we performed a Western
blot analysis using Triton X-100 (Sigma Chemical Co, St Louis, Mo) extracts
of human neuroblastoma HTB10 cells. This cell line expressed a single immunoreactive
band when probed with anti–torsin A antibodies (Figure 1B, lanes 1 and 2). Both antibodies (A1 and A2) showed similar
reaction patterns. In HTB10 cells, torsin A migrated as a protein with an
apparent molecular weight of 35 kd. In contrast to torsin A, torsin B was
not detectable in HTB10 cells (Figure 1B,
lane 3).
IMMUNOHISTOCHEMICAL ANALYSIS
Torsin A Expression in the CNS
To determine the regional and cellular distribution of torsin A and
B, we performed immunohistochemical analyses of normal human brain using paraffin-embedded
sections. These studies demonstrated a widespread distribution of torsin proteins
in the central nervous system. Both torsin A–specific A1 and A2 antibodies
and torsin B–specific B1 antibody demonstrated strong immunoreactivity
in all brain regions studied. In all regions examined, labeling for torsin
A and B staining was neuronal and cytoplasmic. Expression was not restricted
to neuronal cell bodies, and strong to moderate staining was observed in the
neuropil in all areas of the human brain. Preabsorption of antibodies with
the homologous peptide, 100 µmol/L (Figure 2A and
Figure 3A),
significantly reduced specific staining, confirming the specificity of antibodies
on Western blots.
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Figure 2. Immunolocalization of torsin A
in the normal human brain. The distribution of torsin A immunoreactivity was
determined with A1 antibody, 20 µg/mL. A-C, Patches and matrix compartments
of the caudate nucleus. D, Dopaminergic neurons of the substantia nigra. E,
Molecular and Purkinje cell layers of the cerebellum. F and K, Purkinje neurons
of the cerebellum. G and H, Large pyramidal neurons of layer III of the cerebral
cortex. I and J, Pyramidal neurons of layer CA1 of the hippocampus. L, Glomeruli
in the granular layer of the cerebellum. M and N, Neurons of the oculomotor
nucleus of the midbrain. O and P, Neurons of the anterior nuclear group of
the thalamus. Q and R, Motor neurons of the spinal cord. In part A, antibody
preabsorbed with peptide 1, 100 µmol/L, was used for staining (all sections
were counterstained with hematoxylin; original magnification x100 [parts
A, B, G, O, and R], x250 [parts C, F, H, J, K, L, N, and P], and x50
[parts D, E, I, M, and Q]).
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Figure 3. Immunolocalization of torsin B
in the normal human brain. The distribution of torsin B immunoreactivity was
analyzed with B1 antibody, 20 µg/mL. A-C, Patches and matrix compartments
of the caudate nucleus. D, Dopaminergic neurons of the substantia nigra. E
and F, Motor neurons of the spinal cord, G-I, Large pyramidal neurons of layer
III of the cerebral cortex. J-L, Neurons of the anterior nuclear group of
the thalamus. M and N, Oculomotor neurons of the midbrain. O and P, Pyramidal
neurons of layer CA1 of the hippocampus. Q, Molecular and Purkinje cell layers
of the cerebellum. R, Purkinje neurons of the cerebellum. In part A, antibody
preabsorbed with peptide 1, 100 µmol/L was used for staining (all sections
were counterstained with hematoxylin; original magnification x100 [parts
A, B, G, K, and M], x250 [parts C, H, L, N, P, and R], x50 [parts
D, E, J, O, and Q], and x500 [parts F and I]).
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In the basal ganglia, weak torsin A–immunoreactive labeling was
observed in the caudate and putamen (Figure
2B and
Figure 2C). In the matrix regions, torsin A– specific staining
was detected in numerous cell populations although it was almost absent in
the fibers. In contrast, no patch neurons were labeled, and torsin A immunolabeling
was restricted to fibers. Moderate torsin A immunoreactivity was also observed
in the globus pallidus (data not shown). In the substantia nigra, the cell
bodies of dopaminergic neurons were weakly labeled with staining extending
to the cell processes
(Figure 2D).
In the cerebellum, the most intense labeling was observed in Purkinje
cells and in cells in the dentate nucleus. In the Purkinje cells, torsin A–specific
immunoreactivity was localized in cell bodies and dendrites (Figure 2E and
Figure 2F).
No staining was observed in the glial cells of the molecular layer. Torsin
A was not expressed in the cell bodies of granule neurons.
In the frontal cortex, torsin A labeling was detected in all layers
and was especially prevalent in the pyramidal neurons of layers III and V
(Figure 2G and
Figure 2H). Staining was not restricted to the cell bodies and extended
into axons and dendrites. Light staining was also observed in the neuropil
of all cortical areas and in the white matter.
Torsin A–positive cells were detected in all hippocampal subfields,
including the dentate gyrus and subiculum as well as the CA1 and CA3 regions
(Figure 2I and
Figure 2J). In the Purkinje cells, torsin A–specific immunoreactivity
was localized in dendrites (Figure 2K).
Some Golgi cells of the granular layer were faintly labeled. Interestingly,
strong torsin A immunoreactivity was found in cerebellar glomeruli (Figure 2L), which consist of mossy fiber
terminals, Golgi cell axons, and granule cell dendrites. The neurons of the
dentate nuclei were moderately immunopositive whereas the surrounding fibers
were strongly torsin A–positive.
The highest intensity of staining was found in the pyramidal neurons
of CA3 and in the granule cells of the dentate gyrus. Light staining was also
observed in the neuropil of the hippocampal formation.
In the midbrain, the most abundant torsin A–immunoreactive neurons
were detected in the oculomotor nucleus (Figure 2M and
Figure 2N).
Regions that exhibited moderate expression of torsin A were the nucleus of
the geniculate body, the reticular formation, and the superior colliculus.
No obvious labeling of pigmented neurons of the substantia nigra was observed.
It is notable that strong to moderate expression of torsin A was observed
in the fibers throughout all the midbrain areas, with the most intense signals
detected in the brachium of the superior colliculus and the mesencephalic
trigeminal nucleus.
In the thalamus, clusters of torsin A–positive cells were found
in the anterior nuclear group and in the dorsomedial nucleus (Figure 2O
and Figure 2P).
Immunostaining was also detected in neuronal fibers of the ventromedial nucleus.
In the spinal cord, high immunoreactivity was seen in almost all regions (Figure 2Q and
Figure 2R). Prominent labeling was observed in the motor neurons
of the anterior horn, which extended throughout the axons. Dense punctate
torsin A immunolabeling was seen in the neuropil.
Torsin B Expression in the CNS
Immunohistochemical analysis of torsin B distribution revealed a pattern
of expression similar to that of torsin A. Torsin B–immunopositive cells
were detected in all brain regions studied. Torsin B exhibited a slightly
different subcellular localization compared with torsin A; it was found exclusively
in the cytoplasm, and unlike torsin A, its expression was polarized toward
the cell edge. Torsin B labeling was completely abolished by addition of the
immunizing peptide, confirming the specificity already shown on Western blots
(Figure 3A).
In the basal ganglia, neurons in the caudate and putamen were strongly
labeled (Figure 3B and Figure 3C). As with torsin A, differential
staining of patch and matrix was observed. The matrix region contained numerous
torsin B–immunopositive cells but was nearly devoid of immunoreactive
fiber bundles, whereas the patch region exhibited intense immunolabeling of
fibers with no cellular staining. In the globus pallidus, strong expression
of torsin B was detected in some neuronal cells (data not shown). Pigmented
dopamine-containing cells of the substantia nigra were moderately stained
in their cell bodies and proximal processes. Stronger staining was observed
in the cellular fibers in the neuropil (Figure
3D).
In the spinal cord, torsin B immunoreactivity was present in the cell
bodies of motor neurons as well as in the surrounding neuropil (Figure 3E and Figure 3F).
Torsin B was strongly expressed in all cortical layers, including the pyramidal
neurons of layers III and V (Figure 3G, Figure 3H, and Figure 3I).
Moderate torsin B immunoreactivity was present throughout the thalamus.
Significant staining was observed in the neurons of the anterior nuclear group
(Figure 3J, Figure 3K, and Figure 3L), while neurons of the dorsomedial nucleus were only weakly stained. In the
midbrain, strong labeling was detected in the oculomotor neurons and in the
medial geniculate body (Figure 3M and Figure 3N). Similar to torsin
A, intense fiber staining was seen throughout all areas of the midbrain. Torsin
B expression was also observed throughout the hippocampal formation (Figure 3O and Figure 3P). In this structure, labeling was prevalent in the pyramidal
neurons of CA3 and the granule cells of the dentate gyrus. There was also
significant staining within the neuropil.
Little staining for torsin B was seen in the cerebellum (Figure 3Q and Figure 3R).
In contrast to torsin A labeling, Purkinje cells as well as neurons of the
dentate nucleus did not show any significant immunoreactivity for torsin B.
However, there was some staining in the glomeruli of the granular layer (not
shown).
COMMENT
Torsins A and B are physically close on chromosome 9 and share significant
homology (72% identity at the nucleotide level and 69% identity in the predicted
amino acid sequence). We generated 3 different antibodies against torsin proteins,
and several lines of evidence suggest that the antibodies are specific to
the individual proteins.
SPECIFICITY
Two anti–torsin A antibodies, designated A1 and A2, specifically
detected a single protein band of approximately 38 kd. This finding was in
concordance with the predicted molecular weight of torsin A calculated from
its deduced amino acid sequence. The 38-kd protein was not recognized by the
torsin B antibody. This antibody recognized a single band of 62 kd, which
in turn was not recognized by the torsin A antibodies. Since a full-length
torsin B complementary DNA sequence has not yet been isolated, we cannot compare
the observed with the predicted molecular weight. The finding of a larger
torsin B protein, however, is consistent with the observation that the torsin
B mRNA seen by Northern blotting is approximately 1 kb larger than the torsin
A mRNA.3 The torsin B antibody did not recognize
any proteins in HTB10 cells, which apparently only express torsin A. In addition,
staining of Western blots and immunocytochemical labeling were completely
eliminated when the antibodies were preincubated with their respective peptides
(Figure 1, Figure 2, and Figure 3).
This absorption reaction was specific, because absorption with unrelated peptides
did not affect labeling (Figure 1).
In HTB10 cells, torsin A migrated slightly faster, demonstrating an
apparent molecular weight of 35 kd. This may be a result of different posttranslational
modifications of torsin A in cultured cells. Another torsin A antibody raised
against a different C-terminal epitope has recently been described.8 This antibody detected a protein in human tissue with
an apparent molecular weight of 48 kd. The recombinant torsin A protein, however,
showed the predicted electrophoretic migration. Interestingly, this discrepancy
in the migration pattern of endogenous and recombinant torsin A was eliminated
if tissue homogenates were incubated with ATP; yet in another study, 3 rabbit
polyclonal antibodies and mouse monoclonal antibody against torsin A were
generated and shown to recognize a protein with a predicted size ranging from
37 to 39 kd in different cell types.12
EXPRESSION MORE WIDESPREAD THAN DISEASE
Torsins A and B were widely expressed in all human brain regions studied,
including areas affected by disease (the extrapyramidal system) and areas
not implicated in the disease process (the cortex, cerebellum, and spinal
cord). Torsin A and B expression was neuronal, with no detectable expression
in glial cells. This expression pattern is in accordance with expression analysis
of torsin A mRNA.7 The main expression site
of torsin A and B in neurons was in the cytoplasm, which contrasts with a
recent study that also detected nuclear labeling.8
No neuronal cytoplasmic labeling was observed in the patch regions of
the caudate and putamen. The striking contrast between patch and matrix compartments
probably relates to their distinct input-output organization. Thus, the matrix
area, containing cholinergic neurons, receives input from sensory and motor
cortical layers and projects to the substantia nigra pars reticulata. The
patch area, which is rich in enkephalin and substance P, receives input from
the prelimbic cortex and hippocampus while it projects to the substantia nigra
pars compacta. The dopaminergic neurons of the substantia nigra were faintly
labeled with both torsin A– and torsin B–specific antibodies.
However, the neuropil of the substantia nigra consistently showed stronger
staining as compared with the neuronal cell bodies.
The distribution of torsin A and B labeling was almost identical except
for the cerebellum. While strong torsin A immunoreactivity was observed in
the Purkinje cells and dentate nucleus, immunolabeling for torsin B was significantly
weaker in these structures. We also observed a slightly different pattern
in the intracellular distribution of these 2 proteins. Torsin A staining was
diffusely cytoplasmic, whereas torsin B staining was considerably polarized
in the cytoplasm of some neurons. No nuclear torsin A or torsin B immunoreactivity
was observed. This finding was in contrast to a recent study in which torsin
A immunoreactivity was localized to both the nucleus and cytoplasm.8 However, the cytoplasmic localization of torsin A
and its extension to neurites described herein appear to be similar to the
previous findings obtained through immunofluorescence analysis of transfected
cells.9, 12 In these studies, torsin
A was found throughout the cytoplasm with a high degree of colocalization
with the endoplasmic reticulum.
Findings by other research groups in rodents and by us in the human
brain clearly demonstrate the presence of torsin A and B proteins in brain
structures unaffected by disease.9, 8
Why these regions (which are apparently spared in disease) have high levels
of torsin A and B expression will remain unclear as long as the cellular functions
of these proteins continue to be elusive. The widespread and abundant distribution
of the torsin proteins detected in the present study suggests their involvement
in a number of important functions within the human brain. Our data are consistent
with the positron emission tomography scan analyses of dystonia patients that
described increased metabolic activity in the midbrain, cerebellum, and thalamus.13 Of particular interest will be the analysis of human
brains of dystonia patients, especially those with DYT1 gene mutations and those with sporadic dystonia, to determine whether
there are any differences in the expression of these proteins between normal
and diseased brains.
Both torsin A and torsin B contain 1 ATP-binding site and have sequence
homology with heat-shock proteins of the Hsp100/Clp family.14, 15
Moreover, their sequences include a conserved module of approximately 220
amino acids, which allows their classification as new members of the AAA protein
family (ATPase associated with diverse activities).16, 17
Members of this family include spastin and the PEX proteins.18, 19, 20, 21
Mutations in these proteins cause spastic paraplegia or peroxisome biogenesis
disorders, respectively.
Staining for torsins A and B was granular, suggesting labeling of synaptic
vesicles. Torsin A may control dopamine release by regulating the transport
of dopamine-containing vesicles or vesicle fusion with the plasma membrane,
or both.16 In eukaryotic cells, several AAA
ATPases have been shown to be involved in different aspects of intracellular
vesicle trafficking.22, 23, 24
The distribution of torsins A and B in the human brain has a striking resemblance
to that of parkin.25 Although parkin is a neuronal
cytoplasmic protein, most of the labeling was detected in fibers. Similar
to torsin A and B, parkin labels glomeruli in the granule cell layer of the
cerebellum. These are regions of intense synaptic connection, suggesting that
all 3 proteins may play a part in synaptic transmitter release. The conserved
AAA ATPase domains further suggest that this class of proteins may be involved
in regulating transmitter release in response to cellular ATP.
AUTHOR INFORMATION
Accepted for publication December 15, 2000.
This work was supported by the Carmen and Louis Warschaw Endowment Fund
for Neurology, by FRIENDs of Neurology, and by grant R01-NS33123 (Dr Pulst)
from the National Institutes of Health, Bethesda, Md.
From the Rose Moss Laboratory for Parkinson's and Neurodegenerative
Diseases, Burns and Allen Research Institute (Drs Konakova, Huynh, and Pulst),
the Department of Surgical Pathology (Dr Yong), and the Division of Neurology
(Dr Pulst), Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles,
Calif.
Corresponding author and reprints: Stefan M. Pulst, MD, Division
of Neurology, Cedars-Sinai Medical Center, UCLA School of Medicine, 8700 Beverly
Blvd, Los Angeles, CA 90048 (e-mail: stefan.pulst{at}cshs.org).
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