A response element for the homeodomain transcription factor Ptx3 in the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines, which takes place in different types of neuronal systems and nonneuronal tissues. The transcriptional regulation of the TH gene, which is complex and highly variable among different tissues, reflects this heterogeneity. We recently isolated a homeodomain transcription factor, named Ptx3, that is uniquely expressed in the dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area, which together form the mesencephalic dopaminergic system. This strict localization and its coinciding induction of expression with the TH gene during development suggested a possible role for this transcription factor in the control of the TH gene. We report here the presence of a responsive element for Ptx3 located at position -50 to -45 of the rat TH promoter. Transient transfections using TH promoter constructs and electrophoretic mobility shift assays using Ptx3-containing nuclear extracts demonstrated that this region binds Ptx3 protein and confers a transcriptional effect on the TH gene. Depending on the cell type, the effect of Ptx3 was an eight- to 12-fold enhancement of TH promoter activity in Neuro2A neuroblastoma cells, or a 60-80% repression in nonneuronal human embryonic kidney 293 cells. Despite the close association of the Ptx3-binding site and the major cyclic AMP-response element in the TH gene, no interplay was found between Ptx3 and cyclic AMP-modulating agents. In combination with the orphan nuclear receptor Nurr1, which is required for the induction of the TH gene in mesencephalic dopaminergic neurons, the TH promoter activity to Ptx3 was enhanced in Neuro2A cells. Nurr1 alone displayed only very weak activity on the TH promoter in this cell type. The results demonstrate that the homeodomain protein Ptx3 has the potential to act on the promoter of the TH gene in a markedly cell type-dependent fashion. This suggests that Ptx3 contributes to the regulation of TH expression in mesencephalic dopaminergic neurons.

Molecular players in the development and maintenance of mesencephalic dopamine systems.

Several psychiatric diseases are considered to be neuro-developmental disorders. Amongst these are schizophrenia and autism, in which genetic and environmental components have been indicated. In these disorders intrinsic molecular mechanisms of brain development may be deranged due to genetic predispositions, or modified by external influences. Brain development is a delicate process of well-tuned cellular proliferation and differentiation of multipotent neural progenitor cells driven by spatiotemporal cues. One of the fundamental mechanisms is the interaction between external signals, e.g. growth factors, and internal regulators, e.g. transcription factors. An important transmitter system involved in behavioural and affective functions relevant for psychiatric disorders is the mesencephalic dopamine (DA) system. The mesencephalic DA system is organized in two anatomically and functionally different systems. DA neurons in the ventral tegmental area project to the mesolimbic system and are mostly related to control of behaviour. It has been implicated in drug addiction and affective disorders like dipolar disorder and schizophrenia. The dopamine system of the substantia nigra (nigro-striatal pathway) is implicated in movement control. Degeneration of this system, as in Parkinson's disease, or altered function in tardive dyskinesia have highlighted its importance in human disease. Recent findings in molecular neurobiology have provided the first clues to molecular mechanisms involved in developing and mature DA neurons. These may have clinical implications in novel therapeutic strategies.

Apolipoprotein E forms stable complexes with recombinant Alzheimer's disease beta-amyloid precursor protein.

Apolipoprotein E (apoE), a protein genetically linked to the incidence of Alzheimer's disease, forms SDS-stable complexes in vitro with beta-amyloid peptide (Abeta), the primary component of senile plaques. In the present study, we investigated whether apoE was able to bind full-length Abeta precursor protein (APP). Using a maltose-binding-protein-APP fusion protein and human very-low-density lipoprotein (VLDL), we detected an interaction of apoE with APP that was inhibited by Abeta or anti-apoE antibody. Saturation-binding experiments indicated a single binding equilibrium with an apparent 1:1 stoichiometry and a dissociation constant of 15 nM. An interaction was also observed using apoE from cerebrospinal fluid or delipidated VLDL, as well as recombinant apoE. APP.apoE complexes were SDS-stable, and their formation was not inhibited by reducing conditions; however, they were dissociated by SDS under reducing conditions. ApoE.APP complexes formed high-molecular-mass aggregates, and competition experiments suggested that amino acids 14-23 of Abeta are responsible for complex-formation. Finally, no differences were found when studying the interaction of APP with apoE3 or apoE4. Taken together, our results demonstrate that apoE may form stable complexes with the Abeta moiety of APP with characteristics similar to those of complexes formed with isolated Abeta, and suggest the intriguing possibility that apoE-APP interactions may be pathologically relevant in vivo.

Anti-brain spectrin immunoreactivity in Alzheimer's disease: degradation of spectrin in an animal model of cholinergic degeneration.

In a previous work, we described the existence of anti-brain spectrin auto antibodies in Alzheimer's disease (AD) patients (J. Neuroimmunol. 68 (1996) 39-44). In this report, we further support our previous observations, showing that sera from 9 out of 18 AD patients, but none of 14 control subjects, immunoreacted with spectrin synthesized by PC12 cells. In addition, degradation of brain spectrin was found to be greatly enhanced in the frontal cortex of rats subjected to an animal model of cholinergic degeneration. Our data suggest that spectrin degradation and generation of anti-spectrin auto antibodies may be related to the cholinergic degeneration encountered in AD.

Proteolysis of Alzheimer's disease beta-amyloid precursor protein by factor Xa.

Amyloid beta-protein is a 4-kDa peptide which originates from proteolysis of a larger protein precursor (APP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Since secreted APP inhibits factors IXa, Xa and XIa, and thrombin appears to play a role in APP secretion and proteolysis, a relationship between hemostasis system and APP metabolism seems to exist. In this work we investigate the susceptibility to proteolytic cleavage by factor Xa of a fusion construct containing full-length APP prepared in bacteria, and demonstrate that both APP695 and APP770 are substrates for this protease. Factor Xa was found to cleave APP after arginines 102, 268, 510, 573 and 601 (APP695 numeration); most of these sites appear to be common for different coagulation factors. In addition, APP incubation with factor Xa generates an array of six potentially amyloidogenic fragments. Comparative kinetic analysis of APP695 and APP770 cleavage by factor Xa suggests that Kunitz-type inhibitor-containing isoforms exert an inhibitory effect on the protease. However, this inhibition is far from complete even at a 5-fold molar excess of inhibitor. Our results raise the possibility that proteases from the coagulation cascade may contribute to APP proteolysis, and support the notion that these proteases play a role in AD pathogenesis.

Location of an epitope shared by Alzheimer's amyloid peptide and brain creatine kinase using a newly developed monoclonal antibody.

Amyloid plaques, composed mainly by a peptide termed A4-amyloid, derived by proteolytic processing from the amyloid precursor protein (APP), are a hallmark in the brain of Alzheimer's disease patients. We have prepared a collection of monoclonal antibodies as tools to study APP expression and proteolysis in different systems. One of these, 5AH10, raised against residues 9-22 of A4-peptide, was selected for its ability to recognize only A4 subpeptides having the intact APP-secretase target sequence, as well as whole recombinant APP. By using synthetic subpeptides, we have located 5AH10 epitope between amino acids 15 and 22 of A4. In addition, 5AH10 showed a strong immunoreactivity to a 47 kDa protein present in rat brain extracts, that was identified as the B (brain specific) subunit of creatine kinase by immunochemical data and direct N-terminal sequencing. The cross-reaction observed is most probably due to a high degree of sequence identity between amino acids 15 to 22 of A4 peptide and amino acids 9 to 16 of rat B creatine kinase. 5AH10 did not recognize the muscle specific isoform (M subunit) of rat creatine kinase, nor the B subunit of human and rabbit creatine kinase, suggesting that glutamine at first position of the epitope is essential for antigen recognition by 5AH10.