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DARPP-32 Isoform Elevated in Psychiatric Disorders

23 January 2013. A truncated isoform of the phosphoprotein DARPP-32 is elevated in schizophrenia, bipolar disorder, and major depressive disorder, according to a new postmortem study published online January 8 in Molecular Psychiatry. Led by Barbara Lipska of the National Institutes of Mental Health in Bethesda, Maryland, the study also finds that higher transcript expression of the truncated isoform was correlated with genetic variants in DARPP-32 linked to poor cognitive functioning.

DARPP-32, or dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa, is mainly expressed in dopaminergic neurons, and is a critical mediator of dopamine function through its inhibition of protein phosphatase 1. It has also been implicated in glutamatergic signaling, and is proposed to act as an integrator of the two neurotransmitter signals (Svenningsson et al., 2004; see SRF related news story). DARPP-32 has multiple splice variants, including the full-length isoform as well as a truncated version (t-DARPP-32) that lacks the sequences critical for dopamine signaling.

Several postmortem studies have examined levels of full-length DARPP-32 in schizophrenia with mixed results—increased, decreased, and unchanged levels have all been reported (Feldcamp et al., 2008; Zhan et al., 2011)—but levels of the truncated isoform have not yet been studied. In the current report, first author Yasuto Kunii and colleagues used real-time quantitative PCR to assess the gene expression of both DARPP-32 isoforms in the dorsolateral prefrontal cortex (DLPFC), hippocampus, and caudate of a large cohort of over 700 controls and subjects with schizophrenia, bipolar disorder, and major depressive disorder.

DARPP-32 in disease and development
In the DLPFC, levels of full-length DARPP-32 transcript were increased in major depression, while the expression of t-DARPP-32 was elevated in all three psychiatric illnesses. In contrast, in the hippocampus, both isoforms were significantly elevated only in bipolar disorder. There was no change in DARPP-32 levels in the caudate in either schizophrenia or bipolar disorder, although subjects with major depression were not studied.

Although smoking increased expression levels of both isoforms in the caudate, it had no effect in the other two brain regions. There were also no effects of antipsychotics, antidepressants, or lithium in any of the brain regions, suggesting that the findings reflect the disease process and are not a consequence of treatment.

An examination of DARPP-32 expression in control brain samples from different stages of human lifespan revealed that, in the DLPFC, the full-length transcript decreased during the prenatal period, but increased throughout postnatal life. In contrast, DLPFC t-DARPP-32 increased throughout both fetal and postnatal life, similar to the trajectory of both transcripts in the hippocampus.

SNPs vary with expression
Kunii and colleagues next investigated whether single nucleotide polymorphisms (SNPs) in the DARPP-32 gene (PPP1R1B) could affect its mRNA expression. Of the 58 examined, six SNPs varied with the expression of t-DARPP-32 in the DLPFC, although no associations were found in the hippocampus or caudate. Three of the identified SNPs have previously been associated with cognitive performance and frontostriatal functioning (Meyer-Lindenberg et al., 2007; see SRF related news story). In fact, the minor alleles that were associated with higher t-DARPP-32 expression had been linked to worse cognitive functioning. Since the truncated isoform lacks domains critical for dopamine signaling, it is possible that elevated levels of t-DARPP-32 may result in a reduction of dopamine signaling that impairs cognitive function.

The authors also report that a seven-SNP haplotype that they previously associated with risk for schizophrenia in a single, family-based association analysis is linked to t-DARPP-32 but not full-length DARPP-32 transcript expression. However, this finding contradicts their earlier report that the full-length isoform expression levels were associated with the haplotype (Meyer-Lindenberg et al., 2007).

In summary, the current study provides evidence that the truncated isoform of DARPP-32 is increased in subjects with schizophrenia, bipolar disorder, and major depression in a region-specific manner and is associated with SNPs previously implicated in cognitive functioning. According to the authors, because the association of SNPs with t-DARPP-32 is found in the DLPFC but not the other brain regions, “genetic structure, although perhaps necessary, is not a sufficient factor for regulating t-DARPP-32 expression in the brain.” They suggest that epigenetic mechanisms may be involved.—Allison A. Curley.

Reference:
Kunii Y, Hyde TM, Ye T, Li C, Kolachana B, Dickinson D, Weinberger DR, Kleinman JE, Lipska BK. Revisiting DARPP-32 in postmortem human brain: changes in schizophrenia and bipolar disorder and genetic associations with t-DARPP-32 expression. Mol Psychiatry . 2013 Jan 8. Abstract

Comments on News and Primary Papers
Comment by:  Jamal NasirNirmal Vadgama
Submitted 8 March 2013
Posted 8 March 2013

Kunii et al. used two different TaqMan Assays (Applied Biosystems) to compare expression levels of full-length (FL) and truncated DARPP-32 (t-DARPP-32) in various regions of the brain, and detected increased expression of t-DARPP-32 in DLPFC in both schizophrenia and bipolar disorder samples compared to controls. Overexpression of genes can cause developmental abnormalities in the brain. For example, increased LIS1 expression can lead to significant brain abnormalities in humans and mice (Bi et al., 2009).

We previously showed increased expression of DARPP-32 in human DLPFC tissue from both schizophrenia (n = 33) and bipolar disorder (n = 32) samples using the same TaqMan assay (Hs00259967_ml) as above (this detects both FL-DARPP-32 and t-DARPP-32), after excluding brain weight, age of onset, postmortem interval, time in hospital, duration of illness and antipsychotics, gender, race, smoking, alcohol, drugs, suicide status, family history, insight and psychotic features as potential confounding factors (Zhan et al., 2011). After applying Bonferroni corrections to account for multiple comparisons, our findings remained significant, and after correcting for brain pH our p-values became much more significant (p <0.001 for both schizophrenia and bipolar disorder samples vs. controls [n = 34]).

Hierarchical clustering analysis of our data revealed a distinct pattern for DARPP-32 expression in comparison to other dopamine signaling genes and dopamine receptors D1-D5, although the expression of these genes appeared to be co-regulated with the exception of dopamine receptors and D2, in particular (Zhan et al., 2011). DARPP-32 expression in relation to D2 expression is strikingly different in controls but remarkably similar in schizophrenia and bipolar samples, suggesting aberrant D2-regulated expression of DARPP-32 may be an important trigger in pathogenesis.

We found increased DARPP-32 expression in DLPFC of schizophrenia and bipolar samples by using an assay that detects both FL-DARPP-32 and t-DARPP-32. We are, therefore, unable to say whether this is attributable to increased expression of FL-DARPP-32, t-DARPP-32, or both. Kunii et al. failed to find any differences in expression levels using this assay, but since they found increased expression of t-DARPP-32, this would indicate that FL-DARPP-32 expression levels have gone down in schizophrenia and bipolar samples. However, there is inevitably considerable variability between postmortem brain samples as shown in the data presented by Kunii et al. and in other studies, so this could also account for the results. Finally, it would be useful to compare the relative expression of both isoforms of the gene in the brain during various stages of development in the same patients. This might shed useful light on their respective functions.

References:

Zhan L, Kerr JR, Lafuente M-J, Maclean A, Chibalina MV, Liu B, Burke B, Bevan S, Nasir J. (2011) Altered expression and coregulation of dopamine signalling genes in schizophrenia and bipolar disorder. Neuropathology and Applied Neurobiology 37, 206-219. Abstract

Bi W, Sapir T, Shchelochkov OA, Zhang F, Withers MA, Hunter JV, Levy T, Shinder V, Peiffer DA, Gunderson KL, Nezarati MM, Shotts VA, Amato SS, Savage SK, Harris DJ, Day-Salvatore DL, Horner M, Lu XY, Sahoo T, Yanagawa Y, Beaudet AL, Cheung SW, Martinez S, Lupski JR, Reiner O. (2009) Increased LIS1 expression affects human and mouse brain development. Nat Genet. 41:168-177. Abstract

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Related News: SfN Atlanta: Paul Greengard on DARPP-32 and p11

Comment by:  Karl-Ludvig Reichelt (Disclosure)
Submitted 7 November 2006
Posted 7 November 2006

Serotonin Transmission in Mental Disorders
As always, Greengard makes outstanding contributions. Very, very interesting.

We, as well as several other groups, have demonstrated peptide increases in schizophrenia (Hole et al., 1979; Drysdale et al., 1982; Idei et al., 1982; Cade et al., 2000) and also in several other disorders (e.g., Cade et al., 2000; Reichelt and Knivsberg, 2003). This confirms older data from Sweden (Lindstrom et al., 1986), where opioids were found, but measured as receptor binding total level. Unfortunately they named these endorphins, too, while we find that these are probably exorphins.

Opioids affect uptake and release of monoamines, and long ago we could demonstrate uptake inhibition of dopamine and serotonin (Hole et al., 1979), and later a serotonin uptake stimulating tripeptide, which in oocytes from frog stimulate the transport protein in a bell-shaped dose response (hormetic) manner (Pedersen et al., 1999; Keller, 1997). There has been some dispute about the structure of the tripeptide, and we are re-running mass spectrometry as soon as possible to see if we made any mistake. The structure we arrived at was pyroglu-trp- glyNH2 and in depression (in press) pyroglu-trp-gly.

Peptides are a bit tricky because of their considerable tendency to aggregate (Reichelt, in press), which might explain some of the problems. We use tri-fluoroacetic acid (TFA) on HPLC, therefore, and offline mass spectrometry in methanol formic acid (10mM). Formic acid 10mM is not electrometrically as strong and dissociating as TFA. (The mass spectrometry does not tolerate TFA well). In our hands, formic acid 10 mM does not deaggregate all peptide complexes.

Be that as it may, peptides regulating uptake and release of transmitters have been neglected too long. Also, the immune data on peptides in brain should by and large have been confirmed by independent methods such as HPLC and also, preferably, mass spectrometry. Immuno-like does not really ensure identity. For an overview of schizophrenia in this regard, see Reichelt et al, 1996.

We do not know what percentage of the schizophrenics show peptide increases, but a fairly large untreated cohort does. (We have great problems in getting untreated patient urine, 10 ml of the first morning urine (frozen) of carefully diagnosed cases). Our data seem able to explain the onset and suggest reasonable treatment, as shown for autism (Knivsberg et al., 1995; Knivsberg et al., 2002). It does not apply to all, of course, but a large percentage. The curse of medicine is that diagnosis is usually based on symptoms and not aetiology, almost like Morbus febris once was a diagnosis, but with a thousand different causes. We have suffered considerable opposition as would be expected, but now seem to get support from many experiments carried out properly.

References:

Cade RJ, Privette M, Fregly M, Rowland N, Sun Z, Zele V, Wagemaker H and Edelstein C (2000) Autism and schizophrenia: intestinal disorders. 3: 57-72.

Keller J. (1997) Impact of autism-related peptides and 5-HT system manipulations on cortical development and plasticity -Ist Ann report for EU proj. BMH4-CT96-0730 pp 1-10.

Knivsberg A-M, Reichelt KL, Nödland M and Höien T. (1995) Autistic syndromes and diet :a follow up study. Scand J Educat. Res 39: 225-236.

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Related News: DARPP-32 Haplotype Affects Frontostriatal Cognition and Schizophrenia Risk

Comment by:  Jonathan Burns
Submitted 14 February 2007
Posted 14 February 2007

This study provides hard empirical evidence for the hypothesis that psychosis (and schizophrenia in particular) represents a costly "byproduct" of complex human (social) brain evolution. Interestingly, the activation paradigms in the fMRI study (N-back and emotional face-matching tasks) are both testing social cognition. And the demonstrated changes in frontostriatal connectivity support the hypothesis that schizophrenia is a disorder of evolved intrahemispheric circuits comprising the Social Brain in our species.

I would suggest that further candidates (conferring vulnerability to psychosis) should be sought from amongst those genes known to have played a significant role in human brain evolution.

References:

Burns J. (2007) The Descent of Madness: Evolutionary Origins of Psychosis and the Social Brain. Routledge Press: Hove, Sussex.

Burns J. The social brain hypothesis of schizophrenia. World Psychiatry. 2006 Jun;5(2):77-81. Abstract

Burns JK. Psychosis: a costly by-product of social brain evolution in Homo sapiens. Prog Neuropsychopharmacol Biol Psychiatry. 2006 Jul;30(5):797-814. Epub 2006 Mar 3. Review. Abstract

Burns JK. An evolutionary theory of schizophrenia: cortical connectivity, metarepresentation, and the social brain. Behav Brain Sci. 2004 Dec;27(6):831-55; discussion 855-85. Review. Abstract

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Related News: DARPP-32 Haplotype Affects Frontostriatal Cognition and Schizophrenia Risk

Comment by:  Daniel Durstewitz
Submitted 8 June 2007
Posted 8 June 2007
  I recommend the Primary Papers

The phosphoprotein DARPP-32 occupies a central position in the dopamine-regulated intracellular cascades of cortical and striatal neurons (Greengard et al., 1999). It is a point of convergence for multiple signaling pathways, is differentially affected by D1- vs. D2-class receptor activation, and mainly through inhibition of protein-phosphotase-1 mediates or contributes to a number of the dopaminergic effects on voltage- and ligand-gated ion channels. These, in turn, by regulating intracellular Ca2+ levels, themselves influence phosphorylation of DARPP-32 and thereby interact with dopamine-induced processes.

Given its central, vital role in dopamine-regulated signaling pathways, it is quite surprising that (to my knowledge) only a few studies exist on the implications of DARPP-32 variations for cognitive functions and brain activity. Therefore, this comprehensive series of studies by Meyer-Lindenberg et al. combining human genetics, structural and functional MRI, and behavioral testing represents an important milestone. Meyer-Lindenberg et al. identified different functionally relevant DARPP haplotypes, associated with differential DARPP mRNA activity in postmortem studies, and found that these were linked to significant differences on a number of cognitive tests probing “executive functions,” as well as to differences in putamen volume and activity, and structural and functional covariation between striatal and prefrontal cortical areas. Thereby, they paved the way for detailed investigations of the role of DARPP-32 in human cognition.

Since DARPP-32 is so intricately interwoven into so many intracellular and physiological feedback loops, as with dopamine itself (Durstewitz and Seamans, 2002), mechanistic accounts for the functional involvement of DARPP-32 variations in neural network dynamics may be hard to obtain. “Linear” causal thinking usually breaks down in such complex functional networks constituted of so many interacting positive and negative feedback loops on different time scales. Thus it may still be a while until we gain a deeper, biophysically based understanding of the neural processes that mediate the influence of DARPP variations on cognition, and integrative computational approaches may be required to help resolving these issues. Given the complexity of DARPP-regulated networks, I also would expect that fine-grained behavioral testing and analysis of error types of human subjects on different cognitive tasks may ultimately reveal quite subtle and differential effects of DARPP polymorphisms. Moreover, the effects on neural network dynamics may be such (e.g., changing the temporal organization of spiking patterns) that they may not always be detectable by current neuroimaging methods, meaning that while the most dramatic effects were found on activation and volume of striatum, where DARPP-32 is most abundantly expressed, a significant contribution of other brain areas in DARPP-associated cognitive differences may not be ruled out. Regardless of these difficulties in unraveling the underlying neural mechanisms, the work by Meyer-Lindenberg et al. allows us to tackle the question of how the balance in dopamine-regulated intracellular networks relates to cognition in humans, and points toward the neural structures and interactions most interesting to look at.

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