Peter Engels

Cloning and functional characterization of the human 5-HT2B serotonin receptor

Recently, we have reported the cloning of the rat 5‐HT2B receptor cDNA. This receptor is particularly interesting since it may be involved in diseases such as migraine. Here, we describe the isolation of a human 5‐HT2B receptor clone from a cDNA library derived from SH‐SY5Y cells. Although the receptor sequence was only 80% homologous to the rat sequence, the exon‐intron distribution was conserved between the two species. In the human body, the receptor mRNA was detected in most peripheral organs. Only low expression levels were found in the brain. After expression in HEK 293 cells, activation of the receptor stimulated the production of phosphatidylinositol. The pharmacology of this functional response correlated well with that of the rodent receptor.

Expression and regulation of human and rat phosphodiesterase type IV isogenes

Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT‐PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV‐A, ‐B, ‐C and ‐D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV‐A expression was detectable in human blood but not in rat blood, suggesting a species‐specific difference in the expression of this PDE IV isogene. PDE IV‐C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.

Parkin expression in the adult mouse brain.

Mutations in a protein designated Parkin were shown to be involved in the pathogenesis of autosomal recessive juvenile parkinsonism. Nothing is known about its regional and subcellular distribution in the mouse. In order to elucidate the Parkin mRNA and protein distribution in the adult mouse, the mouse cDNA was cloned and polyclonal antisera were generated against the N‐terminal part of mouse Parkin. The antibodies were shown to be specific using Western blot analysis, immunostaining of cells transfected with mouse Parkin and pre‐absorption tests. The Parkin protein expression profile was studied using immunohistochemistry and Western blot analysis and was compared with that of the mRNA yielded by in situ hybridization and RT‐PCR analysis. Parkin protein was widely distributed in all subdivisions of the mouse brain. Low levels were found in the telencephalon and diencephalon, while the brainstem contained a large number of cells heavily expressing Parkin. Ultrastructural analysis and double immunohistochemistry revealed that the majority of Parkin‐expressing cells were neurons, while only single glial cells exhibited immunostaining. The protein was distributed nonhomogeneously throughout the entire cytoplasm. A subpopulation of Parkin‐immunopositive cells displayed speckled immunodeposits in the nucleus. Dopaminergic cells of the substantia nigra pars compacta exhibited high levels of Parkin mRNA but no Parkin protein, while the striatum contained immunopositive profiles but no mRNA signals. Our data indicate that Parkin is neither restricted to a single functional system nor associated with a particular transmitter system. The speckled nuclear distribution of Parkin immunoreactivity strongly suggests a role for Parkin in gene expression.

Molecular cloning and functional expression in yeast of a human cAMP-specific phosphodiesterase subtype (PDE IV-C)

We have recently reported increased survival of dopaminergic substantia nigra neurons by inhibition of phosphodiesterase type IV enzymes. As a first step to unravel the involvement of PDE IV subtypes in this process, we isolated phosphodiesterase type IV cDNAs from human substantia nigra. One isolated partial cDNA clone was most homologous to the partially cloned rat and human PDE IV‐C isogene. Distribution analysis revealed that the enzyme is expressed in various tissues but not in cells of the immune system. Isolation of the full‐length human PDE IV‐C isogene cDNA and expression in a PDE‐deficient yeast strain resulted in functional complementation of the yeast heat shock response. Inhibition of the enzymatic activity by rolipram characterized this enzyme as a typical type IV phosphodiesterase.

Inhibitors of type IV phosphodiesterases reduce the toxicity of MPTP in substantia nigra neurons in vivo.

The neuropathology of Parkinsons disease is characterized by the degeneration of dopaminergic neurons in the substantia nigra. We have recently shown that the activation of protein kinase A improves the survival of dopaminergic neurons in culture and, furthermore, protects them from the dopaminergic neurotoxin, 1‐methyl‐4‐phenylpyridinium ion (MPP+) in vitro. We have now analysed the potential of phosphodiesterase inhibitors to increase cAMP levels in dopaminergic neurons, to improve their survival in culture and to protect them from the toxicity of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) in vivo. Increasing intracellular cAMP with phosphodiesterase type IV‐specific inhibitors enhanced the survival of dopaminergic neurons in culture. Inhibitors of other phosphodiesterase types were not active. In vivo, phosphodiesterase type IV inhibitors reduced the MPTP‐induced dopamine depletion in the striatum of C57BL/6 mice. Furthermore, the loss of tyrosine hydroxylase‐immunopositive neurons in the substantia nigra of these animals was diminished. After Nissl staining, a similar reduction of the MPTP‐induced loss of neurons was observed in the substantia nigra. The protective effect of protein kinase A activation did not appear to be due to the blocking of MPP+ uptake into dopaminergic neurons. This was not decreased after treatment with forskolin or 8‐(4‐chlorophenylthio)‐cAMP. Thus, protein kinase A regulates the survival and differentiation of dopaminergic substantia nigra neurons in vivo, implicating a therapeutic potential for substances which regulate cAMP turnover in these neurons.

Influence of Different Promoters on the Expression Pattern of Mutated Human α-Synuclein in Transgenic Mice

Two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein α-synuclein (asyn) are associated with rare, dominantly inherited forms of Parkinson’s disease (PD) and its accumulation in Lewy bodies and Lewy neurites. As an initial step in investigating the role of asyn in the pathogenesis of PD, we have generated C57BL/6 transgenic mice overexpressing the doubly mutated human asyn under the control of three different promoters; the chicken β-actin (chβactin), the mouse tyrosine hydroxylase 9.6 kb (msTH) and the mouse prion protein (msprp). In this study we compared the regional and cellular expression pattern of the transgenic protein in the brain and peripheral organs of various transgenic mouse lines. Western blot analysis and immunohistochemistry consistently showed that all three promoters successfully drive the expression of the transgene. The msprp promoter was found to give the highest level of transgene expression. All promoters directed the expression into the brain and specific neuron types. However, the promoters differed with respect to (i) the expression pattern in peripheral organs, (ii) the number and (iii) the regional distribution of expressing cells in the brain. Furthermore, remarkable line-to-line variation of expression patterns was observed in mouse lines carrying the same construct. Future studies will analyze how the variations in transgene expression affect the pathogenesis in the animals.

Distribution of 5-HT4 receptor mRNA in the rat brain

We have analyzed the brain distribution of the rat 5-HT4 receptor mRNA. A receptor specific probe was used for in situ hybridization of rat brain sections. Abundant expression of the 5-HT4 receptor mRNA was observed in the olfactory system, striatum, medial habenula and the hippocampal formation, while faint or no specific signals could be detected in most other areas of the brain. Several brain areas which display strong ligand binding do not contain mRNA, suggesting an axonal localization of the 5-HT4 receptor.

Differential distribution of rat PDE-7 mRNA in embryonic and adult rat brain

Currently not much is known about the distribution and function of the phosphodiesterase type 7 (PDE-7) enzyme. Therefore, we carried out an extensive distribution analysis of the rat and human PDE-7 byin situ hybridization as well as RT-PCR. We isolated a partial rat cDNA clone that is highly homologous to the sequence of the human PDE-7 gene. RT-PCR tissue distribution analyses revealed expression of the mRNA of the human and rat-enzymes in most of the examined tissues, like adult heart, lung, brain, and liver, as well as in several cell lines of the immune system.In situ hybridization with the rat PDE-7 showed a differential expression pattern during the late phases of the developing rat brain with higher levels of mRNA in cortical and telencephalic structures in d 16, 18 and 20 embryonic stages, whereas in adult rat brain, higher amounts of mRNA could only be detected in cerebellum and, to a lesser extent, in hippocampus and the olfactory system.