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Evidence for Decreased DARPP-32 in the Prefrontal Cortex of Patients With Schizophrenia
Katherine A. Albert, MD, PhD;
Hugh C. Hemmings, Jr, MD, PhD;
Anna I. B. Adamo, RT;
Steven G. Potkin, MD;
Schahram Akbarian, MD, PhD;
Curt A. Sandman, PhD;
Carl W. Cotman, PhD;
William E. Bunney, Jr, MD;
Paul Greengard, PhD
Arch Gen Psychiatry. 2002;59:705-712.
ABSTRACT
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Background The neurotransmitters dopamine and glutamate have been implicated in
the prefrontal dysfunction associated with schizophrenic illness. Studies
suggest that the D1 subclass of dopamine receptor and the N-methyl-D-aspartate subclass of glutamate receptor are involved in
this prefrontal dysfunction. These 2 receptors regulate, in opposing directions,
the amount of phosphorylated activated DARPP-32, a potent inhibitor of protein
phosphatase 1 that modulates the activity of several classes of receptors
and ion channels. Thus, DARPP-32 occupies a key regulatory position, and may
play an important role in the pathophysiological changes in dopamine and glutamate
function reported in patients with schizophrenia.
Methods The amounts of DARPP-32, synapsin I, and the  subunit of calcium/calmodulin-dependent
protein kinase II were measured by immunoblotting in postmortem samples from
14 schizophrenic subjects and their age-, gender-, and autolysis time–matched
control subjects. Possible confounding influences of neuroleptic treatment
were analyzed by comparing subjects with Alzheimer disease who were and were
not treated with neuroleptic agents.
Results DARPP-32 was significantly reduced in the dorsolateral prefrontal cortex
in more schizophrenic subjects relative to matched controls. The ratios of
2 other synaptic phosphoproteins, synapsin I and the subunit of calcium/calmodulin-dependent
protein kinase II, did not differ between schizophrenic and control subjects,
nor between subjects with Alzheimer disease who were and were not treated
with neuroleptic agents.
Conclusions Our findings are consistent with a selective reduction in DARPP-32 levels
in schizophrenic subjects. This may be involved in the prefrontal dysfunction
associated with schizophrenia.
INTRODUCTION
DARPP-32 IS specifically localized to neurons containing dopamine receptors.1-5
It is a potent inhibitor of protein phosphatase 1, which plays a central role
in dopaminergic and glutamatergic signaling and in integrating the activity
of these 2 pathways (Figure 1). (Greengard
et al provide a review.6) Dopamine, through
a pathway involving D1 receptor activation of cyclic adenosine monophosphate–dependent
protein kinase, stimulates the phosphorylation of DARPP-32.5, 7
Dopamine, through activation of the D2 receptor, and glutamate, through activation
of the N-methyl-D-aspartate (NMDA) glutamate receptor,
increase the activity of calcineurin, which results in the dephosphorylation
of DARPP-32.8-10 In
its phosphorylated, but not dephosphorylated, state, DARPP-32 potently inhibits
the major serine/threonine protein phosphatase 1.11
Through its inhibition of protein phosphatase 1, DARPP-32 controls the state
of phosphorylation and the physiological activity of several key proteins,
including ion channels, ion pumps, neurotransmitter receptors, and transcription
factors; thus, DARPP-32 controls the physiological characteristics of neurons
containing dopamine receptors.6, 12
The importance of DARPP-32 in the regulation of dopaminoceptive neuron function
is clearly demonstrated by the alterations in dopaminergic signaling evident
in mice with a targeted deletion of DARPP-32.13
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Figure 1. Central role of DARPP-32 as a
molecular integrator of dopaminergic and glutamatergic signaling. Multiple
first messengers acting through the second messengers cyclic adenosine monophosphate
(cAMP), cyclic guanosine monophosphate (cGMP), and calcium (Ca2+)
regulate the phosphorylation of DARPP-32 at threonine 34, which in its phosphorylated
form (pDARPP-32) inhibits protein phosphatase 1 (PP-1). Phosphorylation of
threonine 34 is regulated through protein kinase A (PKA) (and protein kinase
G [PKG]) by various transmitters, principally by dopamine acting at D1 receptors.
DARPP-32 phosphorylated at threonine 34 is dephosphorylated by protein phosphatase
2B (PP-2B) (also known as calcineurin), a Ca2+/calmodulin-dependent
phosphatase, which is activated by several transmitters, principally following
Ca2+ influx produced by glutamate acting at N-methyl-D-aspartate (NMDA) and L--amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid (AMPA) receptors. The psychostimulants cocaine and amphetamine increase
DARPP-32 phosphorylation by increasing dopaminergic transmission. Neuroleptic
drugs achieve certain of their clinical effects by antagonism of dopamine
D2 receptors, an action that leads to an increase in DARPP-32 phosphorylation
indirectly by reducing the intracellular Ca2+ concentration. Through
the regulation of PP-1 activity, which has a broad substrate specificity,
various first messengers are able to modulate the function of receptors (NMDA,
AMPA,  -aminobutyric acid A [GABAA], and neurokinin A [NKA]),
ion channels (L-, N-, and P-type Ca2+ channels; and sodium [Na+] channels), and transcription factors (cAMP response element binding
protein [pCREB] and fos-related antigens [FRAs]). CCK indicates cholecystokinin;
A2A, adenosine 2A; 5HT4, serotonin 4; VIP, vasoactive
intestinal polypeptide; NO, nitric oxide; and p, phosphorylated.
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Much evidence implicates abnormalities of dopaminergic and glutamatergic
neurotransmission in the pathophysiological features of schizophrenia.14-16 Compounds that increase
synaptic dopamine levels (eg, amphetamines and related compounds) induce or
exacerbate psychotic behavior in a significant subgroup of schizophrenic,
but not healthy, subjects,17 while dopamine
receptor antagonists ameliorate symptoms.18-19
Similarly, noncompetitive NMDA receptor antagonists (eg, phencyclidine, ketamine,
and dizocilpine) can induce prolonged psychotic episodes in schizophrenic
subjects in contrast to short episodes in nonschizophrenic subjects.20 These findings provide pharmacological evidence compatible
with altered dopaminergic and glutamatergic function in patients with schizophrenia.
One of the critical cognitive-affective parallel circuits in the brain
is the dorsolateral prefrontal cortex (DLPFC) circuit.21
Cognitive, imaging, neuropathological, and neurochemical evidence supports
compromised function of the DLPFC in patients with schizophrenia. (Bunney
and Bunney provide a review.22) The present
study was conducted to determine whether changes in the total protein levels
of DARPP-32 in the DLPFC occur in patients with schizophrenia.
MATERIALS AND METHODS
ACQUISITION, STORAGE, AND PREPARATION OF BRAIN TISSUE
Brain specimens of schizophrenic patients and control subjects, individually
matched for age, gender, and autolysis time, as described in detail in Table 1, were used. Although patients were
not matched for cause of death, no patients received mechanical ventilation
before death. The matching process was finished before all experimental procedures
were undertaken. The diagnosis of schizophrenia was made before and after
the deaths of the schizophrenic patients and control subjects by 2 board-certified
psychiatrists (S.G.P. and W.E.B.). Informed consent was obtained from the
next of kin and, in one third of the cases, from the patients themselves before
death. Diagnoses were made according to DSM-IV criteria
by using the best-estimate diagnostic procedure from all available interview
and medical record sources. These sources included interviews with the subjects
and/or postmortem interviews with a family member, a significant other, and/or
a treating professional. Exclusion criteria for the schizophrenic patients
and control subjects included a history of neurological illnesses with the
exception of neuroleptic-induced seizures; positive test results for the human
immunodeficiency virus; neuropathological evidence of stroke, Alzheimer disease
(AD), brain tumor, or vascular anomalies; and a history, or evidence at autopsy,
of severe drug abuse (eg, needle tracks or a positive toxicological profile
for substances of abuse). Moderate marijuana or alcohol use or occasional
recreational drug use were not exclusionary criteria, unless they confounded
the Axis I diagnosis (eg, cocaine or amphetamine abuse resulting in psychosis).
All schizophrenic patients had a long-term nonremitting pattern of illness.
Neuroleptic drugs taken by the patients included butyrophenones, phenothiazines,
and/or thioxanthenes. Atypical neuroleptic agents, such as benzamides (eg,
sulpiride), dibenzazepines (eg, clozapine), or risperidone, had not been taken
by any of the schizophrenic patients. For religious reasons, schizophrenic
patient 6 had taken no neuroleptic drugs for 10 years before death.
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Table 1. Demographic Information for Schizophrenic Patients and Matched
Control Subjects*
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The second set of brain specimens was obtained from 9 patients with
AD who were treated with haloperidol and 9 patients with AD who were not treated
with any neuroleptic agents. They were matched for age and postmortem delay,
as described in detail in Table 2.
Postmortem delay, the time from death to autopsy, was listed in Table 2 rather than autolysis time, the time from death to freezing
of the brain, because autolysis time was unavailable for the patients with
AD. The patients with AD were included to determine whether differences observed
in the schizophrenic cohort could be attributable to neuroleptic treatment.
Patients with AD often receive lower doses of antipsychotic medication for
shorter periods.
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Table 2. Demographic Information for Subjects With Alzheimer Disease*
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The brain specimens of the control subjects were obtained from the coroner's
office or the eye bank. The coroner's office or eye bank investigators determined
that there had been no psychiatric illness in the subjects based on the findings
from their investigative review. The absence of a psychiatric illness was
further confirmed by the results of a telephone interview with a family member,
a review of available medical records, and/or consultation with the control
subject's family physician. Statistical analyses from previous studies23 identified no confounding influences of the matching
variables.
Patients and controls were individually matched by using the following
order and criteria: (1) gender; (2) age difference (mean difference, 5.0 ±
4.9 years); (3) autolysis time of 3 hours (mean time, 2.7 ±
2.1 hours); and, if possible, (4) cause of death. For the schizophrenic group
(n = 14), the age was 58.3 ± 21.5 years, the autolysis time was 15.8
± 10.5 hours, and the disease duration was 30.6 ± 18.7 years.
For the control group (n = 14), the age was 61.5 ± 22.3 years and the
autolysis time was 15.5 ± 10.3 hours. In each group of schizophrenic
and control subjects, there were 4 women and 10 men. For the haloperidol-treated
patients with AD (n = 9), the age was 76.4 ± 4.8 years, the postmortem
delay was 5.0 ± 2.4 hours, and the disease duration was 7.9 ±
2.2 years. There were 5 women and 4 men. For the untreated patients with AD
(n = 9), the age was 73.9 ± 12.0 years, the postmortem delay was 4.6
± 1.5 hours, and the disease duration was 10.0 ± 5.4 years.
There were 4 women and 5 men. (Data are given as mean ± SD unless otherwise
indicated.)
A computer program was used to identify potential control subjects as
possible matches for patients in the schizophrenic cohort. The brain specimens
of the control subjects were taken from a control brain repository that consisted
of more than 100 cases. Of those cases identified by the computer, the composite
records of the possible matches were carefully reviewed to satisfy criteria
for schizophrenia and normality, following which the best matches were selected.
Examination of the brain tissue by a board-certified neuropathologist excluded
tumors and vascular and other abnormalities in the analyzed brain specimens.
Procedures for removal and storage of the brain specimens have been
described previously.24 Briefly, each brain
specimen was cut into coronal slices of about 0.9-cm thickness, and slices
were flash frozen between 2 supercooled aluminum plates. In all sampling,
tissue from each subject's brain was processed together with tissue from the
matched control brain. From the left side of each brain, several blocks of
frozen gray matter (size, 2 cm2) were obtained from the DLPFC at
the tip of the superior frontal gyrus, corresponding to the rostral part of
Brodmann area 9. Blocks were first cut from coronal slices of about 0.9-cm
thickness; extreme care was taken not to include any subcortical white matter
that, if present, was trimmed off. The samples were coded, paired, and sent
on dry ice to the processing laboratory. The investigators in the processing
laboratory were blinded to the sample identities.
IMMUNOBLOTTING
Frozen samples (0.25-0.50 g of tissue) were homogenized in 20 volumes
of boiling 1% wt/vol sodium dodecyl sulfate containing 1 µg/mL of leupeptin
(Chemicon International, Inc, Temecula, Calif), sonicated, and boiled for
5 minutes. The samples were centrifuged at 1000g
for 5 minutes; the protein concentration in the supernatants was determined
by the bicinchoninic acid method (Pierce Biotechnology, Inc, Rockford, Ill).
Protein (100 µg per lane) was subjected to 10% sodium dodecyl sulfate–polyacrylamide
gel electrophoresis, as described.5 Samples
from each pair of subjects and matched control samples were run in quadruplicate
on the same gel to minimize variations in transfer and immunolabeling. The
proteins were transferred to 0.2-µm nitrocellulose (Schleicher &
Schuell Bioscience, Inc, Keene, NH) at 200 mA for 18 hours.25
After transfer of the proteins onto nitrocellulose, the gels were stained
with Coomassie blue to visualize protein loading and transfer efficiency.
If this assessment demonstrated protein degradation in any sample, the sample
and its match were not included in the analysis. All blotting steps were performed
at room temperature in blotting buffer (a combination of 86mM sodium phosphate,
pH 7.3; 150mM sodium chloride; 0.05% vol/vol polyethylenesorbitan monolaurate
[Tween 20] [Sigma-Aldrich Corp, St Louis, Mo]; and 0.02% wt/vol sodium azide).
Nonspecific binding was blocked by incubation in blotting buffer containing
2.5% wt/vol nonfat dry milk (Carnation) for 8 hours. After 2 rinses (15 minutes
each), blots were cut into sections containing the proteins of interest. The
blot sections were incubated for 2 hours with primary antibodies to DARPP-32
(mouse monoclonal antibody C24-6a, 1:1000 dilution), synapsin I (rabbit polyclonal
antibody G-472 [1:200 dilution], G454/455 [1:2000 dilution], or G486 [1:2000
dilution]), or the subunit of calcium/calmodulin-dependent protein
kinase II (CaMK II) (rabbit polyclonal antibody RU16, 1:1000 dilution).
None of the antibodies used was selective for the phosphorylation state of
the proteins, ie, phosphorylated and dephosphorylated forms should be detected
equally. After 2 rinses (15 minutes each), the DARPP-32 blot sections were
incubated for 1 hour with rabbit anti–mouse IgG (1:500 dilution) (Pierce
Biotechnology, Inc). After 2 rinses (15 minutes each), all blotsections were
incubated for 90 minutes in iodine 125 labeled protein A (1:1000 dilution)
(Amersham Holdings, Inc, Arlington Heights, Ill). After two 15-minute and
two 5-minute rinses, the blot sections were dried, wrapped in plastic, and
subjected to imaging analysis (PhosphorImager; Molecular Dynamics, Sunnyvale,
Calif).
Multiple experiments were conducted. Within each experiment, 4 replicate
lanes from each member of a matched pair were run together on a single gel
to minimize variability between blots. The data were expressed in arbitrary
units obtained from the imaging instrument (PhosphorImager), and a mean value
of the 4 lanes for each experiment was calculated. For each pair, the ratios
of the mean values from each experiment (schizophrenic/control subjects and
haloperidol-treated/untreated subjects with AD) were computed; the mean of
the ratios calculated for the matched pairs across experiments was the final
value used for statistical comparisons.
STATISTICAL ANALYSES
Results were analyzed statistically by the binomial exact test, which
was used to test the hypothesis that the ratio of matched data pairs came
from a binomial population with a specified probability of an event. The binomial
analysis was selected because of the assumption that each paired comparison
(schizophrenic vs matched control) must be treated as a separate experiment
as opposed to comparing the differences between the groups. This assumption
is required because the absolute values for the immunoblotting procedures,
although highly reliable within a pairwise comparison, are not comparable
across experiments because of variability in transfer efficiency, immunolabeling
efficiency, 125I-specific radioactivity, and imaging instrument
(PhosphorImager) values, factors that cannot be completely controlled. Thus,
absolute values from one pairwise comparison are independent from the same
pairwise comparison at another time. The calculation of the pairwise ratio
and the computation of means across experiments control for the inherent variability.
Thus, the ratio across experiments (which summarizes the differences between
the members of each pair) is a reliable estimate. The ratios were converted
to dichotomous variables, with one category of values equal to or greater
than 1 (ie, schizophrenic subjects have higher values than controls) and the
second category of values less than 1 (ie, schizophrenic subjects have lower
values than controls). With a cut point of 0.5, the null hypothesis that there
was an equal chance that the ratio will sort into either of the 2 categories
was tested. The binomial exact test in this case measures and tests the significance
of the number of pairwise comparisons in which schizophrenic subjects have
either higher or lower values than matched controls.
RESULTS
DARPP-32 IN THE DLPFC OF SCHIZOPHRENIC VS CONTROL SUBJECTS
DARPP-32 protein levels were analyzed by immunoblotting in the DLPFC
from 14 pairs of schizophrenic and individually matched control subjects.
The present study addressed the total amount of DARPP-32 protein and not its
level of phosphorylation. Figure 2 shows representative immunoblots of DARPP-32 from a single experiment for
each pair used in the analysis. The average ratios of DARPP-32 in schizophrenic
compared with control brain specimens for each pair are shown in Figure 3A. The relative amount of DARPP-32
in the DLPFC was significantly lower among schizophrenic patients compared
with their matched control subjects (P<.04). There
were 11 pairs in whom the ratio of the value in the schizophrenic patient
to that in the control was less than 1, 2 pairs in whom the ratio was not
significantly different from 1, and 1 pair in whom the ratio was greater than
1. To ensure confidence in the values, each sample was analyzed in quadruplicate.
There were no significant correlations between DARPP-32 ratios and the mean
age of pairs ( = -0.07), the mean pair autolysis time ( = -0.20),
gender ( = -0.08), or duration of illness ( = -0.01).
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Figure 2. Immunoblots prepared from schizophrenic
and control dorsolateral prefrontal cortex brain tissue. Autoradiograms of
immunoblots from a single experiment (n = 4) are shown for the region around
32 kd containing DARPP-32. Matched pairs are organized from the lowest (upper
left) to the highest (lower right) ratio of schizophrenic to control DARPP-32,
as determined by the average ratios determined by imaging analysis (PhosphorImager;
Molecular Dynamics, Sunnyvale, Calif) for the 4 independent experiments.
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Figure 3. Ratios of phosphoprotein levels
in the dorsolateral prefrontal cortex from matched pairs of schizophrenic
and control subjects. The average ratios for each protein were determined
by immunoblotting, as described in the "Materials and Methods" section (n
= 4). Each ratio represents the protein level from a schizophrenic patient
divided by the protein level from a matched control patient. A, DARPP-32.
B, The subunit of calcium/calmodulin-dependent protein kinase II.
C, Synapsin I.
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CaMK II AND SYNAPSIN I IN SCHIZOPHRENIC VS CONTROL SUBJECTS
For comparison with DARPP-32, the levels of 2 synaptic phosphoproteins,
CaMK II and synapsin I, were analyzed by immunoblotting in the DLPFC
from 14 pairs of schizophrenic and matched control subjects (Figure 3B and C, respectively). The ratios of CaMK II and
synapsin I were not different between schizophrenic and control brain specimens.
For synapsin I, there were 4 pairs in whom the ratio of the value in the schizophrenic
subjects to that in the controls was less than 1, 6 pairs in whom this ratio
was not different from 1, and 4 pairs in whom this ratio was greater than
1. For CaMK II, there were 3 pairs in whom the ratio of the value in
the schizophrenic subjects to that in the control was less than 1, 10 pairs
in whom this ratio was not significantly different from 1, and 1 pair in whom
this ratio was greater than 1.
DARPP-32, CaMK II, AND SYNAPSIN I IN PATIENTS WITH AD
It is possible that the differences observed in the schizophrenic cohort
could be attributable to neuroleptic treatment. To address the issue, we examined
the effect of such treatment by comparing the relative amounts of DARPP-32,
CaMK II, and synapsin I in 9 patients with AD who were treated with
haloperidol with those in matched patients with AD who were not treated with
neuroleptic agents (Figure 4). The
ratios of each of these proteins in the DLPFC was not different (P>.05) for haloperidol-treated patients with AD vs untreated patients
with AD. For DARPP-32, there were 4 pairs in whom the ratio of the value in
the treated patient divided by the value in the control was less than 1, 1
pair in whom this ratio was not significantly different from 1, and 4 pairs
in whom this ratio was greater than 1. For CaMK II, there were 3 pairs
in whom this ratio was less than 1, 4 pairs in whom this ratio was not significantly
different from 1, and 2 pairs in whom this ratio was greater than 1. For synapsin
I, there were 3 pairs in whom this ratio was less than 1, 4 pairs in whom
this ratio was not significantly different from 1, and 2 pairs in whom this
ratio was greater than 1.
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Figure 4. Ratios of phosphoprotein levels
in the dorsolateral prefrontal cortex from pairs of patients with Alzheimer
disease. The average ratios for each protein were determined by immunoblotting,
as described in the "Materials and Methods" section (n = 4). Each ratio represents
the protein level from a patient treated with the antipsychotic haloperidol
divided by the protein level from a matched patient not treated with any antipsychotic
medication. A, DARPP-32. B, The subunit of calcium/calmodulin-dependent
protein kinase II. C, Synapsin I.
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In all cases, the relative molecular mass values of DARPP-32, synapsin
I, and CaMK II were the same in all pairs (schizophrenic/control subjects
and subjects with AD with/without haloperidol treatment) (data not shown).
The relative molecular mass values of DARPP-32 (32 kd) and synapsin I (a doublet
of 86 and 80 kd) were previously reported to be the same in the postmortem
human brain as in the rat brain.26 The relative
molecular mass value of CaMK II in our human samples was 50 kd, the
same as in the rat brain.27
COMMENT
This study demonstrates that the protein level of DARPP-32 is decreased
in the DLPFC of significantly more schizophrenic subjects relative to matched
controls. The reduction in DARPP-32 does not seem to be generalized to all
brain regions, because a significant alteration in DARPP-32 was not observed
in a separate study of caudate nucleus tissue from 12 schizophrenic subjects
and their matched controls (K.A.A., H.C.H., and W.E.B., unpublished data,
1999). Similar reductions were not observed for synapsin I or CaMK II,
indicating that the decrease in DARPP-32 was not attributable to a nonspecific
reduction in neuronal phosphoproteins, although the possibility of a type
II error must be considered. Synapsin I has been analyzed in other brain regions,
and found to be altered in schizophrenic subjects. Synapsin Ia and Ib messenger
RNA (mRNA) levels were elevated in the superior and left middle gyri of schizophrenic
subjects younger than 75 years,28 while in the
hippocampus, synapsin I protein levels were significantly lower.29
The levels of the  subunit of calcium/calmodulin-dependent protein kinase
II mRNA were elevated in the frontal cortex of schizophrenic subjects30; the interesting possibility that this represents
a selective increase in the subunit of calcium/calmodulin-dependent
protein kinase II expression and not in CaMK II (this study) requires
further study.
The reduction in DARPP-32 seen in schizophrenic subjects is unlikely
to be the result of neuroleptic treatment. DARPP-32 in the DLFPC was not reduced
in patients with AD who were treated with haloperidol compared with patients
with AD who had not been treated with neuroleptic agents. Moreover, DARPP-32
was not decreased in the rat frontal cortex by long-term treatment with the
neuroleptic agent raclopride.31 Studies using
positron emission tomography have reported that dopamine D1 receptor binding
is reduced in the prefrontal cortex,14, 32
but not in the striatum,14 of drug-naive schizophrenic
patients. In addition, a decrease in dopamine D1 receptors was observed in
the monkey prefrontal cortex in response to neuroleptic agents.33-34
Although DARPP-32 and dopamine D1 receptor expression are closely linked in
the central nervous system,35 dopamine D1 receptor–mutant
mice exhibit normal DARPP-32 expression on gross examination.36
The observed reduction in DARPP-32 could be due to a selective loss
of DARPP-32–containing neurons, or their processes. An increased density
of neurons with a reduction in neuropil has been reported in Brodmann area
9 of schizophrenic patients.37 However, no change
in DLPFC cell number was found in a separate study38
using the same brain specimens examined in this study. We cannot exclude a
selective loss of a small subpopulation of DLPFC neurons that contain DARPP-32.
There are several limitations to this and other studies that use postmortem
tissue analysis. A principal limitation is the small sample size for rigorous
statistical analysis, although this represents one of the largest samples
for this area of neuropsychiatry. Ideally, prospective enrollment of subjects
should be used, an approach that is under way. Extreme efforts were made to
control for the many biological parameters between subjects by individual
matching of patients and control subjects. The limitations inherent in quantitative
immunoblotting are discussed in the "Immunoblotting" subsection of the "Materials
and Methods" section.
Further studies will be needed to determine whether the reduction in
DARPP-32 is integral to the cause of schizophrenia or is a compensatory adaptation
to the pathological features. Changes in DARPP-32 levels in experimental animals
have been reported only following neurotoxic lesions of the striatum or transections
of striatonigral fibers in rats5 or targeted
deletion of the gene for DARPP-32 in mice.13
It will be of interest to compare schizophrenic patients with matched controls
for protein levels of various downstream physiological effectors, known to
be regulated by the DARPP-32/protein phosphatase 1 cascade, including voltage-dependent
sodium channels39; voltage-dependent L-, P-,
and N-type calcium channels40; the electrogenic
ion pump of Na+,K+–adenosine triphosphatase41; the NR1 class of NMDA receptors42;
L--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors43; and -aminobutyric acid A receptors.44 Another component of the DARPP-32 signal transduction
pathway, the NMDA receptor, may also be altered in patients with schizophrenia,
because the NR2D subunit mRNA was increased in the DLPFC, but not in the parietotemporal
cortex or the cerebellum, of postmortem schizophrenic brain specimens.45 However, studies of mRNA levels may not indicate actual
levels of protein present and, therefore, must be interpreted cautiously.
Other recent studies have reported alterations in NMDA, L--amino-3-hydroxy-5-methyl-4-isoxazolepropionic
acid, and kainate receptor mRNA levels in various cortical regions in schizophrenic
subjects. (Meador-Woodruff and Healy20 provide
a review.) However, these findings have been inconsistent.
In summary, our major finding suggests a selective decrease of DARPP-32
protein levels in the DLPFC of schizophrenic patients. DARPP-32 is a key regulatory
phosphoprotein involved in the control of receptors, ion channels, and transcription
factors; it is reciprocally activated and deactivated by reversible phosphorylation
regulated by the 2 neurotransmitter systems most consistently implicated in
the pathophysiological features of schizophrenia (Figure 1). The DLPFC has alterations in white matter interstitial
neuron number and abnormalities in the glutamate and -aminobutyric
acid systems in schizophrenia.22 The observed
abnormality in DARPP-32 in the DLPFC could contribute to the compromised function
of the cognitive-affective parallel DLPFC circuit in patients with schizophrenia.
Thus, decreased availability in the compromised DLPFC circuit of the DARPP-32
protein required for the regulation of critical downstream receptor and ion
channel functions could contribute to the profound frontal cognitive deficits
observed in patients with schizophrenia.
AUTHOR INFORMATION
Submitted for publication April 27, 1998; final revision received August
16, 2001; accepted September 11, 2001.
This study was supported by grants NS 01715 (Dr Albert), MH 40899 (Drs
Hemmings and Greengard), and MH 44188 and MH 60398 (Dr Bunney) from the National
Institutes of Health, US Public Health Service, Bethesda, Md; a fund established
in The New York Community Trust by DeWitt-Wallace, New York (Dr Albert); Young
Investigator Awards from National Alliance for Research on Schizophrenia and
Depression (NARSAD), Great Neck, NY (Drs Albert and Akbarian); and a Cornell
Scholar Award in Biomedical Sciences from Cornell University Medical College,
New York, NY (Dr Hemmings).
Presented at the NARSAD Scientific Symposium, New York, NY, October
17, 1997.
We thank Andrew Czernik, PhD, for the antibodies to synapsin I and CaMK
II; Edward G. Jones, MD, PhD (who received grant NS 21377 from the
Public Health Service and a Senior Investigator Award from NARSAD), for dissecting
samples and for his support of the brain bank; the patients and families who
participated in these studies; The California Alliance for the Mentally Ill;
James Beisner, Jacques Berndt, and the staff of the Orange County Sheriff/Coroner's
Office; Merle Wingate and Kathleen Burke of the Orange County Eye and Tissue
Bank; Warren Lovell, MD, Ventura County medical examiner/coroner; the Los
Angeles Coroner/Medical Examiner's Office; and Harbor View House.
The brain repository study coordinators were Michael Nilsson, Florence
Huang, and Preston Cartagena, PsyD. Resources for processing brain specimens
and the initial storage were provided by Wallace W. Tourtelotte, MD, PhD,
and Iris Rosario of the National Neurological Research Specimen Bank, West
Los Angeles Veteran's Affairs Hospital, Los Angeles, Calif, which is sponsored
by the National Institute of Neurological Disorders and Stroke/National Institute
of Mental Health, the National Multiple Sclerosis Society, the Hereditary
Disease Foundation, the Comprehensive Epilepsy Program, the Tourette Syndrome
Association, the Dystonia Medical Research Foundation, and the Veterans Health
Services and Research Administration, Department of Veterans Affairs.
Corresponding author and reprints: Hugh C. Hemmings, Jr, MD, PhD,
Department of Anesthesiology, Weill Medical College of Cornell University,
525 E 68th St, Campus Box 50, Room LC-203, New York, NY 10021 (e-mail: hchemmi{at}med.cornell.edu).
From the Laboratory of Molecular and Cellular Neuroscience, the Rockefeller
University (Drs Albert, Hemmings, and Greengard), the Departments of Psychiatry
(Dr Albert), Neurology and Neuroscience (Dr Albert), and Anesthesiology and
Pharmacology (Dr Hemmings and Ms Adamo), Weill Medical College of Cornell
University, New York, NY; and the Departments of Psychiatry and Human Behavior
(Drs Potkin, Akbarian, Sandman, and Bunney), Neurology (Dr Cotman), and Psychobiology
(Dr Cotman), University of California, Irvine. Dr Akbarian is now with the
Department of Psychiatry, Massachusetts General Hospital/Harvard Medical School,
Boston.
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