Forward genetic screen of mouse reveals dominant missense mutation in the P/Q-type voltage-dependent calcium channel, CACNA1A.

Dominant mutations of the P/Q-type Ca(2+) channel (CACNA1A) underlie several human neurological disorders, including episodic ataxia type 2, familial hemiplegic migraine 1 (FHM1) and spinocerebellar ataxia 6, but have not been found previously in the mouse. Here we report the first dominant ataxic mouse model of Cacna1a mutation. This Wobbly mutant allele of Cacna1a was identified in an ethylnitrosourea (ENU) mutagenesis dominant behavioral screen. Heterozygotes exhibit ataxia from 3 weeks of age and have a normal life span. Homozygotes have a righting reflex defect from postnatal day 8 and later develop severe ataxia and die prematurely. Both heterozygotes and homozygotes exhibit cerebellar atrophy with focal reduction of the molecular layer. No obvious loss of Purkinje cells or decrease in size of the granule cell layer was observed. Real-time polymerase chain reaction revealed altered expression levels of Cacna1g, Calb2 and Th in Wobbly cerebella, but Cacna1a messenger RNA and protein levels were unchanged. Positional cloning revealed that Wobbly mice have a missense mutation leading to an arginine to leucine (R1255L) substitution, resulting in neutralization of a positively charged amino acid in repeat III of voltage sensor segment S4. The dominance of the Wobbly mutation more closely resembles patterns of CACNA1A mutation in humans than previously described mouse recessive mutants (tottering, leaner, rolling Nagoya and rocker). Positive-charge neutralization in S4 has also been shown to underlie several cases of human dominant FHM1 with ataxia. The Wobbly mutant thus highlights the importance of the voltage sensor and provides a starting point to unravel the neuropathological mechanisms of this disease.

Increased expression of calcium/calmodulin-dependent protein kinase IIbeta in frontal cortex in schizophrenia and depression.

In searching for genes dysregulated in schizophrenia, we measured the expression of the two splice variants of calcium/calmodulin-dependent protein kinase II (CaMKIIalpha and CaMKIIbeta) in postmortem frontal cerebral cortex tissues from patients who had died with schizophrenia, bipolar disorder, or severe depression. The mRNA levels of expression of these two splice variants were measured by real-time Quantitative PCR, using an Mx4000 instrument. The values for the expression of CaMKIIalpha and CaMKIIbeta were normalized by the expression of beta-glucuronidase in the tissues. The expression of CaMKIIalpha was significantly elevated in the depression tissues by 29%. The expression of CaMKIIbeta was significantly elevated in the schizophrenia tissues by 27%, and in the depression tissues by 36%. Because CaMKIIbeta influences the expression of many neuroreceptors and influences neural outgrowth and pruning, its altered expression in the cerebral cortex in schizophrenia or depression may contribute to these diseases.

Dopamine receptor contribution to the action of PCP, LSD and ketamine psychotomimetics.

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2High, their dissociation constants (Ki) were obtained on [3H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2High with a Ki of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a Ki of 313 nM, as labeled by [3H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2High with a Ki of 55 nM, an affinity higher than its 3100 nM Ki for the NMDA sites. Dizocilpine had a Ki of 0.3 nM at D2High, but a Kd of 1.8 nM at the NMDA receptor. LSD had a Ki of 2 nM at D2High. Because the psychotomimetics had higher potency at D2High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis.

Schizophrenia and Nogo: elevated mRNA in cortex, and high prevalence of a homozygous CAA insert.

Schizophrenia is a major psychiatric disorder which is hypothesized to result from abnormal neurodevelopment or neural changes in adulthood and possibly associated with altered gene expression. To search for genes overexpressed in schizophrenia, cDNA library subtractive hybridization experiments between post-mortem human frontal cerebral cortices from schizophrenia individuals and neurological controls were carried out. One of the genes over-expressed in schizophrenia was identified as Nogo (also known as reticulon 4, RTN4, NI 250, or RTN-X), a myelin-associated protein which inhibits the outgrowth of neurites and nerve terminals. The elevated expression of Nogo mRNA in schizophrenia was confirmed by quantitative reverse transcription-polymerase chain reaction studies: 16.5 pg Nogo cDNA/microg total RNA in schizophrenia, and 10.2 pg Nogo cDNA/microg total RNA in controls (n=7; P=0.01, t-test for n<30). To identify possible polymorphisms in this gene, the Nogo nucleotide sequence was determined in a series of schizophrenia and control samples. The Nogo mRNA was found to contain a CAA insert polymorphism in the 3'-untranslated region. The prevalence of individuals homozygous for this CAA insert was significantly higher in schizophrenia compared to controls in genomic DNA samples extracted from post-mortem brain and blood samples: 17/81 or 21% in schizophrenia and 2/61 or 3% in controls (P=0.0022, chi(2)- and Fisher's exact-tests). Because the 3'-untranslated regions of eukaryotic genes are known to regulate gene expression, the increased frequency of the Nogo CAA insert in schizophrenia may contribute to abnormal regulation of Nogo gene expression, and may indicate a role for Nogo in disturbed neurodevelopment in schizophrenia.

Schizophrenia: elevated mRNA for dopamine D2(Longer) receptors in frontal cortex.

Because dopamine D2 receptors are the primary targets for antipsychotic drugs, including clozapine and quetiapine, and because some studies have found D2 receptors to be elevated in schizophrenia, we examined the mRNA of three forms of the D2 receptor, particularly the new form of the dopamine D2 receptor, D2(Longer), in post-mortem brains from patients who died with schizophrenia. Using quantitative competitive RT-PCR (reverse transcriptase-polymerase chain reaction), the D2(Longer) mRNA was higher in the frontal cortex, compared to control tissues. The mRNA concentration of D2(Long) and D2(Short) was also higher in the frontal cortex, compared to control tissues. Although most of the schizophrenia patients had received different antipsychotic drugs for varying periods of time, the mRNA of D2(Longer), as well as that for D2(Long) and D2(Short), in such medicated tissues was similar to that in a frontal cortex tissue from a patient who had reliably never received antipsychotic drugs. It is possible, therefore, that the elevation of the mRNAs for D2(Longer), D2(Long) and D2(Short) in the frontal cortex may be related to the disease of schizophrenia itself.

Schizophrenia: elevated mRNA for calcium-calmodulin-dependent protein kinase IIbeta in frontal cortex.

Because amphetamine releases two to three times more dopamine in schizophrenia patients than in control subjects, and because calcium-calmodulin-dependent protein kinase II has a key role in the enhanced action of amphetamine-induced dopamine release in rats, the synaptic content of calcium-calmodulin-dependent protein kinase IIbeta mRNA was measured (by quantitative competitive RT-PCR; reverse transcriptase-polymerase chain reaction) in seven frontal cerebral cortices of post-mortem brains from patients who had schizophrenia and in seven control tissues. The results indicate that the mRNA of this kinase is elevated in the schizophrenia frontal cortex.

New dopamine receptor, D2(Longer), with unique TG splice site, in human brain.

Brain dopamine receptor agonists alleviate the signs of Parkinson's disease, while dopamine receptor antagonists alleviate hallucinations and delusions in psychosis. The dopamine type 2 receptor (or D2) is blocked by antipsychotic drugs, including even the "atypical" drugs such as clozapine or remoxipride, in direct relation to their clinical potencies. Compared to the long form of the D2 receptor (D2(Long)), the short form (D2(Short)) may be three times more sensitive to benzamide antipsychotic drugs. Hence, it is essential to identify additional variants of dopamine receptors for which more selective antipsychotic drugs can be found. Although no family linkage has been found between the D2 receptor and schizophrenia, there can be brain region abnormalities in the RNA transcript expression of dopamine receptors. Therefore, in order to identify variant dopamine D2 receptors, we searched for mutations in the RNA transcripts for the dopamine D2 receptor in the striatum of post-mortem brains from individuals who died with psychosis, including schizophrenia. A new splice variant of the D2 receptor, D2(Longer), with a unique TG splice site, was found in one control brain and in two psychotic brains.

Rapid release of antipsychotic drugs from dopamine D2 receptors: an explanation for low receptor occupancy and early clinical relapse upon withdrawal of clozapine or quetiapine.

OBJECTIVE: In an attempt to understand the basis of early relapse after antipsychotic withdrawal, the objective of this study was to determine whether the low occupancy of dopamine D2 receptors by clozapine and by quetiapine, as seen by brain imaging, could arise from a rapid release of some of the D2-bound clozapine or quetiapine by the brain imaging compounds and by the action of a physiological concentration of dopamine. METHOD: Human cloned D2 receptors were first pre-equilibrated with the [3H]antipsychotic drug, after which raclopride, iodobenzamide, or dopamine (at the physiological concentration in the synapse) was added, and the time course of release of the [3H]antipsychotic from the D2 receptor was measured. RESULTS: Within 5 minutes, low concentrations of raclopride and iodobenzamide displaced appreciable amounts of [3H]clozapine and [3H]quetiapine from the D2 receptors but, during the course of 1 hour, did not displace any of the other antipsychotic [[3H]ligands. [3H]Clozapine and [3H]quetiapine, moreover, were displaced by dopamine (100 nM) at least 100 times faster than the other antipsychotic [3H]ligands. CONCLUSIONS: Clozapine and quetiapine are loosely bound to the D2 receptor, and the injected radioactive ligand at its peak concentration may displace some of the D2-bound antipsychotic drug, resulting in apparently low D2 occupancies. Therefore, under clinical brain imaging conditions with [11C]raclopride, D2 occupancies by clozapine and by quetiapine may be higher than currently estimated. These considerations may result in high levels of the D2 receptors being occupied by therapeutic doses of clozapine or quetiapine. The rapid release of clozapine and quetiapine from D2 receptors by endogenous dopamine may contribute to low D2 receptor occupancy and to early clinical relapse upon withdrawal of these medications.

Dopamine D2 receptor promoter polymorphism: no association with schizophrenia.

A functional polymorphism in the promoter region of the dopamine D2 receptor gene, the -141C Del allele, which may be associated with schizophrenia susceptibility, has previously been described in a Japanese sample. The present study was done in order to examine whether such an association would also be found in a North American schizophrenia patient population. However, analysis of the -141C Del allele frequency in the present group of schizophrenia patients (n = 50) and control subjects (n = 51) did not identify any significant differences. These data support the recent reports on German and British subjects that this genetic variation in the 5'-flanking region of the dopamine D2 receptor gene does not play a major role in the genetic predisposition to schizophrenia.

The human serotonin-7 receptor pseudogene: variation and chromosome location.

We report a variation of the pseudogene for the serotonin-7 receptor in human DNA. Human genomic DNA was amplified, using the polymerase chain reaction method and degenerate oligonucleotide primers for serotonin receptor-like genes. A novel gene DNA sequence of 1325 bp was found. Based on nucleotides, this gene is 88% identical to the serotonin-7 receptor coding sequence. Compared with the previously known serotonin-7 receptor pseudogene, this pseudogene has 1 nucleotide deletion and 4 nucleotide mutations. The gene is located on human chromosome 12 at 12p12.3-p13.2.

Antipsychotic drugs which elicit little or no parkinsonism bind more loosely than dopamine to brain D2 receptors, yet occupy high levels of these receptors.

This review addresses two questions. First, why does clozapine apparently occupy low levels of dopamine D2 receptors in patients, in contrast to all other antipsychotic drugs which occupy 70-80% of brain dopamine D2 receptors? Second, what is the receptor basis of action of antipsychotic drugs which elicit low levels of Parkinsonism? Antipsychotic doses of clozapine occupy between 0% and 50% of D2 receptors, as measured in patients by a variety of radioligands. It has recently been found, however, that the percent occupancy of a receptor by a drug depends on the radioligand used to measure that receptor. Based on this new finding, this review concludes that clozapine clinically occupies high levels of D2 receptors in the absence of any radioligand. This occupancy is estimated to be of the order of 70-80% in the dopamine-rich region of the human striatum, and even higher in the limbic D2-containing regions which are low in endogenous synaptic dopamine. This conclusion arises from two different approaches. One approach is to relate the reported clozapine occupancies in the human striatum with the dissociation constants of the various radioligands at the D2 receptor. This relation extrapolates to approximately 70-80% occupancy by clozapine when clozapine competes with endogenous dopamine at the D2 receptor. The second approach is to calculate the D2 occupancy of each antipsychotic drug, using the average spinal fluid concentration and the correct dissociation constant of the antipsychotic, thereby revealing that all antipsychotic drugs, including clozapine, occupy approximately 70-80% of dopamine D2 receptors in the human striatum, and possibly higher in the limbic regions. As determined by the new dissociation constants, antipsychotic drugs which elicit Parkinsonism (trifluperazine, chlorpromazine, raclopride, haloperidol, fluphenazine, risperidone) bind more tightly than dopamine to D2, while those antipsychotic drugs which elicit little or no Parkinsonism (melperone, seroquel, perlapine, clozapine, remoxipride, molindone, sulpiride, olanzapine, sertindole) bind more loosely than dopamine to D2 receptors. Compared to the tightly bound antipsychotic drugs, the more loosely bound antipsychotics generally require higher clinical doses, require fewer days for clinical adjustment, but may dissociate from the D2 receptor more rapidly and could lead to clinical relapse somewhat earlier than that found with the traditional tightly bound antipsychotic drugs.

A serotonin-4 receptor-like pseudogene in humans.

During a search for new G-protein-linked receptors for dopamine and serotonin, we found a serotonin-4 receptor-like pseudogene. This receptor-like pseudogene is intronless, contains an in-frame stop codon following transmembrane-3, and has two one-nucleotide insertions between transmembrane-5 and -6 regions which alter the reading frame. The predicted amino acid sequence of the human pseudogene is about 35% identical with that of the rat serotonin-4 receptor.

A human serotonin-7 receptor pseudogene.

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.

Dopamine D2 receptor dimers in human and rat brain.

In order to determine whether dimers of dopamine D2 receptors can occur in mammalian brain, rat and human brain striatal membranes were photolabelled with two radioactive photoaffinity compounds selective for dopamine D2 receptors, [125I]azidophenethylspiperone and [125I]-4-azido-5-iodonemonapride. It was found that [125I]azidophenethylspiperone only labelled the D2 monomer, while [125I]-4-azido-5-iodonemonapride labelled both D2 monomers and dimers, despite the fact that very high concentrations (6 nM) of both radiocompounds were used. In addition, human cloned D2 receptors were probed with a D2-specific antibody, revealing multiple bands indicating the existence of trimers, tetramers and pentamers of D2 receptors. The different D2-binding patterns of the spiperone and benzamide congeners may explain the different densities of dopamine D2 receptors found with these two radioligands in human brain positron tomography in health and disease.