A human tyrosine hydroxylase isoform associated with progressive supranuclear palsy shows altered enzymatic activity.

A novel human tyrosine hydroxylase (HTH) messenger RNA subgroup generated by alternative splicing and characterized by the absence of the third exon was recently identified. The corresponding putative protein lacks 74 amino acids including Ser31 and Ser40, two major phosphorylation sites implicated in the regulation of HTH activity. These mRNA species are detected in adrenal medulla and are overexpressed in patients suffering from progressive supranuclear palsy, a neurodegenerative disease mostly affecting catecholaminergic neurons of the basal ganglia. In the present work, an HTH protein isoform lacking exon 3 was identified in human adrenal medulla. For this purpose, an antibody was raised against the HTH exon 3. The effect of the removal of exon 3 on the enzymatic activity of HTH was studied in vitro by comparing a purified recombinant fusion protein without exon 3 (glutathione S-transferase (GST)-HTHDelta3) to the equivalent protein containing exon 3 (GST-HTH3). In initial velocity conditions, GST-HTHDelta3 has 30% of the maximal velocity of GST-HTH3. Moreover, the skipping of exon 3 results in the absence of activation of GST-HTH by heparin and increases by 10-fold the retroinhibition constant for dopamine, demonstrating the involvement of exon 3 in the regulation of HTH enzymatic activity. The identification of a variably expressed HTH isoform that lacks an exon implicated in activity regulation supports the view that HTH alternative splicing contributes to the functional diversity within the catecholaminergic system and may be implicated in some neurological diseases.

New species of human tyrosine hydroxylase mRNA are produced in variable amounts in adrenal medulla and are overexpressed in progressive supranuclear palsy.

Alternative splicing of human tyrosine hydroxylase (TH) pre-mRNA produces four mRNAs leading to four different TH isoforms and is thought to have important regulatory functions. We show that the diversity of TH mRNAs is greater than previously described in the autonomous nervous system: New splice junctions corresponding to the skipping of exon 3 were identified by amplification of cDNA synthesized from pheochromocytoma RNA. In all cases the reading frame was maintained. These species were assayed by RNase protection experiments; their abundance (4-6%) was comparable to that of the previously identified human TH-3 and -4 species in normal adrenal medulla. However, higher levels (11-34%) of these species were found in adrenal medullas of patients suffering from progressive supranuclear palsy. Whether such changes are specific to the disease or the consequences of the stress associated with this severe neurodegeneration remains to be established.

A novel rat tyrosine hydroxylase mRNA species generated by alternative splicing.

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain. A recombinant protein was produced in E. coli. Its in vitro specific activity was similar to that of the previously identified TH protein.

No evidence for linkage or association between the dopamine transporter gene and schizophrenia in a French population.

Pharmacological and clinical findings suggest that the dopamine transporter (DAT) gene may be involved in the genetic predisposition to schizophrenia. Linkage of a Taq I VNTR polymorphism in the DAT gene to schizophrenia was studied in multiplex schizophrenic families from Rouen, France (n = 10) and the Island of La Réunion (n = 21). Neither the lod score method nor nonparametric methods (the affected pedigree member method of Weeks and Lange [1988] and the sibling method of Green and Woodrow [1977]) provided any evidence for linkage. An association study, carried out within a group of 91 unrelated schizophrenic patients from Rouen and 91 matched control subjects, examined a 40 base-pair repeat polymorphism located in the 3' nontranslated end of the DAT mRNA. There was no significant difference in allelic or genotypic frequencies between the two groups. These results exclude any substantial involvement of the DAT gene in the pathogenesis of schizophrenia in the population studied.

No major role for the dopamine D2 receptor Ser-->Cys311 mutation in schizophrenia.

A new structural polymorphism (Ser311/Cys311) in the dopamine D2 receptor (DRD2) gene has recently been reported to be associated with schizophrenia, particularly in patients with a positive family history of schizophrenia (Arinimi et al., 1994). However these findings remain controversial (Asherson et al., 1994; Nanko et al., 1994; Nöthen et al., 1994; Shaikh et al., 1994). Thus we investigated the role of the Cys311 mutation in schizophrenia using both association and family studies. First, we screened for the Cys311 mutation in 113 unrelated Caucasian schizophrenics (mean age 42 +/- 0.6; 34 females and 79 males) including 25 familial cases, and 184 unrelated controls (mean age 49 +/- 0.5, 74 females and 110 males) free of any psychiatric disorders. Diagnoses were ascertained according to DSM-III criteria (Campion et al., 1994). All patients and controls were native to the area of Rouen.