13 February 2007. The signaling molecule dopamine- and cAMP-regulated protein of 32 kilodaltons, or DARPP-32, has been the baby of Paul Greengard and colleagues at the Rockefeller University, New York, for over 2 decades; his work on this molecule led to the Nobel Prize in Physiology or Medicine in 2000. Greengard’s in vitro, animal, and postmortem brain tissue work has been pivotal in demonstrating that DARPP-32 acts as a central molecule switch integrating multiple information streams from the dopamine, glutamate, and serotonin pathways.
Now, Daniel R. Weinberger and colleagues from the NIH’s Genes, Cognition and Psychosis Program have published the first human functional data on DARPP-32, linking genetic variation at the locus to brain structure, cognitive performance, and schizophrenia. They present three lines of evidence, including cognitive testing, mRNA testing in postmortem brain tissue, and magnetic resonance imaging, to show that a common haplotype is associated with better performance on cognitive tests that call upon circuits linking frontal cortex and the striatum, perhaps as a benefit of more efficient information processing within the striatum. They also show that this haplotype is associated with schizophrenia.
DARPP-32: A Central Molecular Switch
Over the years, Greengard’s group has shown that DARPP-32 acts as a central molecular switch integrating the dopamine, glutamate, and serotonin pathways. This is accomplished through the use of different phosphorylation sites. When phosphorylated at Thr34, DARPP-32 acts as an amplifier of PKA- and PKG-mediated signaling, which leads to a potent inhibition of protein phosphatase-1 (PP-1) (Hemmings et al., 1984) PP-1 is one of the few phosphatases in the mammalian cell, and it has been implicated in the pathogenesis of neurodegenerative diseases, particularly Parkinson's disease (see Alzheimer Research Forum related news story). On the other hand, when phosphorylated at Thr75, DARPP-32 becomes an inhibitor of PKA. Thus, DARPP-32 regulates a variety of phosphorylation cascades within the cell that control many key proteins, including ion channels, neurotransmitter receptors, and transcription factors.
Greengard and colleagues have linked DARPP-32 function to schizophrenia with both animal and human studies. Following up on evidence that schizophrenia patients, as a group, display impaired sensorimotor gating (the processing and filtering of stimuli and information) they found that DARPP-32 knockout mice do not lose sensorimotor gating in response to the psychotomimetic drugs D-amphetamine, D-lysergic acid diethylamide (LSD), and phencyclidine (PCP), as wild-type mice do. This supports the idea that DARPP-32 activity mediates the actions of these agents on sensorimotor gating (see SRF related news story). In addition, Greengard’s group found that DARPP-32 protein levels were significantly reduced in the dorsolateral prefrontal cortex in postmortem brain tissue from patients with schizophrenia (Albert et al., 2002).
PPP1R1B Haplotype Influences Cognitive Performance and Schizophrenia Risk
In the current study, first author Andreas Meyer-Lindenberg and colleagues first documented genetic variability at the DARPP-32 locus (PPP1R1B). DNA from 105 white and 44 African-American patients with schizophrenia was sequenced, including all seven exons, all introns, and 2 kb upstream of the transcription start site. Within this region, the investigators found 17 single nucleotide polymorphisms (SNPs) that were present in greater than 3 percent of the white patients. None of these SNPs were coding or splice site mutations.
Next, these SNPs were genotyped in a large sample of white subjects from the Clinical Brain Disorders Branch Sibling Study (CBDB/NIMH), which is a study of neurobiological abnormalities related to genetic risk for schizophrenia (Egan et al., 2001). The sample included 257 white patients with schizophrenia, 327 siblings, 397 parents, and 243 controls. From the 17 SNPs, seven were chosen as a 7-SNP haplotype. About 75 percent of the sample carried a very frequent haplotype (CGCACTC), and 15 percent carried the haplotype GATGTCA. There were three other, rarer haplotypes present in the sample as well, at frequencies of 3.5 percent, 2.1 percent, and 1.6 percent.
Weinberger’s group then searched the CBDB/NIMH dataset for associations between PPP1R1B haplotypes and cognitive function. Cognitive function in the CBDB/NIMH sample has previously been tested using a variety of standard, validated cognitive tests. The investigators found that the most common PPP1R1B haplotype, CGCACTC, was associated with higher IQ and better scores on tests of working memory, sequencing, response alternation, and attention. In other studies, scores on these tests have been related to the function of circuits linking frontal cortex and neostriatum (caudate and putamen) via the thalamus (Pantelis et al., 1997). No association between any PPP1R1B haplotype and scores on tests of episodic memory, verbal learning, or logical memory was found. These tests are traditionally related to function of temporal-diencephalic circuitry.
Of interest to the schizophrenia community, the PPP1R1B haplotype CGCACTC was not only associated with higher scores on tests related to cortical striatal loop function, but it was also associated with schizophrenia. “Our data lead to the provocative observation that a
frequent haplotype in PPP1R1B predicts increased frontostriatal interactions that appeared beneficial (as evidenced by relatively better performance on a wide range of cognitive tasks) yet contributed to risk for schizophrenia,” write Meyer-Lindenberg and colleagues. “This raises the question of whether a genetic advantage in normal subjects may translate into a disadvantage in the context of other functional impairments also associated with schizophrenia, such as abnormal function of the prefrontal cortex.”
PPP1R1B Haplotype Associated with DARPP-32 Brain Biochemistry and Structure
Next, DARPP-32 mRNA levels were tested in postmortem brain tissue from the prefrontal cortices of 16 white patients with schizophrenia and 22 controls. The data from these tests suggested that variations in levels of full-length mRNA are associated with the PPP1R1B haplotype. In particular, full-length DARPP-32 mRNA was expressed at the highest levels in subjects who were homozygous for the most common haplotype of PPP1R1B, the CGCACTC haplotype. This is the same haplotype that was associated with cognitive gains as well as schizophrenia in the earlier experiments. Expression of the full-length mRNA was lowest for carriers of at least one copy of the less frequent GATGTCA haplotype, and it was intermediate in two subjects who were heterozygous for CGCACTC in combination with a rarer haplotype. The results from this analysis were controlled for sex, smoking status, diagnostic status, postmortem
interval, age, and RNA quality. As a control, the authors performed the same analysis with a well-characterized genetic variant of the catechol-O-methyltransferase (COMT) gene, and, as expected, they found no significant effect upon the expression levels of DARPP-32 mRNA.
Building their case brick by brick, Meyer-Lindenberg and colleagues next demonstrated an association between PPP1R1B haplotype and neostriatal volume using voxel-based
morphometry (VBM). A cohort of 96 white, healthy volunteer subjects was selected for imaging after screening for lifetime history of psychiatric or neurological illness, psychiatric treatment, or drug or alcohol abuse. This cohort was independent from those selected for the other tests. The VBM data indicated that there was a bilateral relative decrease in neostriatal volume in subjects carrying the frequent PPP1R1B haplotype CGCACTC when compared to the volume in subjects carrying all other haplotypes.
PPP1R1B Haplotype Associated with Functional Connectivity in the Brain
Lastly, the investigators found an association between the PPP1R1B haplotype and the functional connectivity between the striatum and the prefrontal cortex using archival functional MRI (fMRI) datasets. Subjects had been given two tasks: the N-back task, a working memory task that robustly activates the dorsolateral prefrontal cortex (Callicott et al., 2000), and the emotional face-matching task using threatening visual stimuli, which engages the ventrolateral prefrontal and lateral orbitofrontal cortices (Pezawas et al., 2005). The authors noted that while neither of these tasks was specifically designed to probe striatal function, both of them show differential activation in neostriatum in the context of prefrontal cortex involvement.
Although percentage correct response and reaction time on these tasks was not correlated with PPP1R1B haplotype, the frequent haplotype was associated with other parameters. During the N-back test, carriers of the frequent CGCACTC haplotype showed significantly less reactivity in the bilateral putamen, and significantly increased functional connectivity of the bilateral striatum. During the emotional face-matching task, carriers of the frequent haplotype again showed decreased reactivity of the putamen. They also appeared to have increased functional connectivity between the left dorsolateral prefrontal cortex and striatum.
As a control for the imaging studies, the regional volume and functional activation experiments were also conducted in a sample of healthy white volunteers for which the COMT genotype was known, and no genotype associations were found for either striatal volume or activation.
“Taken together, the neuroimaging data therefore show that the frequent haplotype is associated in healthy individuals with more efficient intrastriatal processing combined with an increase of prefrontal cortical input onto a smaller striatum,” write Meyer-Lindenberg and colleagues. “We present convergent evidence in three independent
datasets implicating DARPP-32 in a frontostriatal neural system for executive cognition and response selection in humans.”—Jillian Lokere.
Meyer-Lindenberg A, Straub RE, Lipska BK, et al. Genetic evidence implicating
DARPP-32 in human frontostriatal structure, function, and cognition. J Clin Invest. Published online Feb. 8, 2007. Abstract