Reimar Schlingensiepen

Regional and temporal expression of sodium channel messenger RNAs in the rat brain during development

SummaryThe distribution of mRNA expression for three types of voltage gated neuronal sodium-channels was studied in the rat brain at different developmental stages (embryonal day E18, postnatal day P5 and adult). With the in-situ hybridization technique, using synthetic DNA-oligomer probes, pronounced regional and temporal variations in the expression levels of the different channel subtypes could be detected. In comparison with types I and III, sodium channel II mRNA was the most abundant subtype at all developmental stages. Maximal expression of sodium channel II mRNA was seen at P5 in virtually all parts of the grey matter, except for the cerebellum. In adult rat brain in contrast, sodium channel II mRNA levels were maximal in the granular layer of the cerebellum, whereas in all other regions expression had decreased to roughly 50% of postnatal levels. Na channel I expression was virtually absent at E18 and showed highest levels at P5, with maxima in the caudate nucleus and hippocampus. In the adult brain, expression of Na-channel I was nearly absent in the neocortex, but well detectable in the cerebellum and, at lower levels in the striatum and thalamus. Sodium channel III was mainly expressed at the embryonal stage and showed a decrease to very low levels with little regional preferences in the adult.

Developmental expression of the transcription factor zif268 in rat brain

Changes in the distribution pattern of mRNA encoding the zif268 transcription factor (also referred to as NGFI-A, Krox-24 or EGR-1) were investigated by in situ hybridization histochemistry during postnatal rat brain development. Marked changes in zif268 expression patterns were seen in particular in the cerebral cortex and the hippocampal formation during the first 3 wk. In the 1st postnatal week, zif268 mRNA levels were highest in the corpus striatum and the piriform cortex. In the neocortex, expression rose sharply in the sensorymotor area between postnatal days (PNDs) 10 and 12. In the frontal and occipital cortex, in contrast, an increase in zif268 mRNA levels was first seen on PND 14. After PND 17, levels decreased in the sensorymotor and the frontal cortex but remained high in the occipital and the piriform cortex. In the hippocampus, an initially uniform increase in expression during the 2nd week was followed by a marked dissociation in expression levels between CA1, with continuously high expression levels on the one hand, and CA3, CA4 and the dentate gyrus, with a strong decline of expression during the 3rd week, on the other hand. Our results indicate that zif268 expression displays a highly dynamic expression pattern during plastic adaptations of different cerebral subregions during postnatal development, suggesting a possible involvement in gene regulatory processes during these phases.

Changes in the expression of synapsin I and II messenger RNA during postnatal rat brain development

Synapsin Ia, Ib, IIa, and IIb are neuronal phosphoproteins, which are supposed to play a role in the short-term regulation of neurotransmitter release. Besides a high degree of homology among the four synapsin subtypes, there are structural differences in the 3′end of their coding region. Here we present the first extensive study of the expression of their gene transcripts by using in situ hybridization and northern blot analysis. Our results show regionally and temporally distinct expression patterns of synapsin Ia, Ib, IIa, and IIb, which suggests different functional properties of the four synapsin subtypes. There was no specific messenger RNA (mRNA) expression of synapsin IIb in most brain regions apart from the cerebellum, suggesting a minor functional role of this synapsin subtype. Synapsin Ia, Ib, and IIa mRNA were expressed earlier in ontogenetically older brain regions such as the piriform cortex, the thalamus, and the hippocampus and later in ontogenetically younger areas such as the neocortex and the cerebellum. Owing to the distinct expression pattern of the synapsin subtypes, we suppose that the synapsins might be essential for the underlying molecular mechanism of pattern formation and plasticity in distinct brain regions during different states of rat brain development.

The topology of the thalamo-cortical projections in the marmoset monkey (Callithrix jacchus)

SummaryThis paper addresses the question of a general topological principle of thalamo-cortical projections. In the lissencephalic primate brain of the common marmoset (Callithrix jacchus), large injections of horseradish peroxidase were made in various parts of the neocortex. These injections were placed in different animals and hemispheres along various caudo-rostral and mediolateral gradients. Labelled cells in the thalamus were plotted and the labelling-zones resulting from several injections along a medio-lateral and two caudo-rostral cortical vectors were drawn into semi-schematic thalamic maps. These composite maps reveal a topological organization of the whole thalamo-cortical projection. The thalamic representation of the caudo-rostral and mediolateral gradients indicate a rotation of the posterior relative to the anterior thalamus. An attempt is made to relate the organization of the thalamo-cortical projection to the development of the thalamus and the cortex. The cortex is divided into concentric zones around the sensory-motor and insular cortex. The thalamus is divided into corresponding projection zones. The topology of thalamo-cortical connections can then be regarded as a consequence of corresponding thalamic and cortical growth gradients. This is not only consistent with the general thalamo-cortical topology and the inversion of maps from thalamus to cortex, but also explains the continuity and overlap of thalamic projection zones in the pulvinar to widely separated cortical areas as the parietal, temporal and frontal association cortex.

Changes of synapsin I messenger RNA expression during rat brain development

Synapsin I is a synaptic phosphoprotein that is involved in the short-term regulation of neurotransmitter release. In this report we present the first extensive study of the developmental expression of its corresponding messenger ribonucleic acid (mRNA) by in situ hybridization and northern blot analysis in rat brain. Synapsin I mRNA showed pronounced differences in expression in different brain regions during postnatal development. The early expression of synapsin I mRNA in ontogenetically older regions such as the thalamus, the piriform cortex and the hippocampus coincides with the earlier maturation of these regions, in contrast to its later expression in ontogenetically younger areas such as the cerebellum and the neocortex. An intriguing expression pattern was found in the hippocampus. In all hippocampal subregions synapsin I mRNA expression increased from postnatal day (PND) 1 to 17. After PND 17, however, there was a marked dissociation between persisting high expression levels in CA3 and the dentate gyrus and a strong decline in synapsin I mRNA expression in CA1. The persistence of synapsin I in some adult rat brain regions indicates that it plays a part in synapse formation during plastic adaption in neuronal connectivities.

Differential expression of c-jun, junB, and junD in rat hippocampal slices.

THE temporal expression pattern of jun genes was studied in the hippocampal slice preparation. Slices were kept either in a physiological Ringer solution or a modified medium, substituting calcium (Ca2+) by magnesium (Mg2+). All three jun genes were expressed in a circumscribed, independent fashion with respect to distribution, intensity, and time course, c-jun, and junD were expressed strongly throughout the hippocampus with a shift from DG to CA1 to CA3. junB expression was confined to DG, and CA1, but substitution of Ca2+ by Mg2+ led to profound changes: the signal rose earlier, was prolonged, strongly enhanced, and more widespread. Our findings suggest, that changes in differential expression of jun gene transcripts are important for parallel processing of non-convergent stimuli.

The role of transforming growth factor-β in carcinogenesis

In their review, Gleave and Monia1 gave an overview of the current status and future directions of selected antisense compounds in clinical development for systemic cancer therapy. We would like to add some points to the discussion, as to the clinical potential of first-generation phosphorothioate oligodeoxynucleotides (PS-ODNs). Taking Gentas Genasense as an example, evidence of effectiveness, indicated by a marked increase in progression-free survival and responses in melanoma, was demonstrated2. The criticism of the Food and Drug Administration (FDA) during the Oncology Drugs Advisory Committee (ODAC) meeting was directed at study design deficiencies, not to the nature of the compound. Nevertheless, Genasense is still in advanced clinical trials and has recently shown activity for non-Hodgkin lymphoma3. Failure to meet the primary endpoint in melanoma could have several causes; the chemical nature of the compound is just one suggested reason. If the chemical modification was the reason for the lack of efficacy of antisense compounds in recent clinical trials as cited in the review, the logical consequence would be to target molecules such as protein kinase C α (PKCα) or BCL2 with second-generation ODNs. Instead, the compounds in current clinical development that were suggested as promising by the authors are directed against other targets. Consequently, the change in targets makes the comparison between the ODN-generations regarding anti-tumour efficacy impossible. In our opinion, the choice of target is most important for successful antisense therapy. The ideal drug candidate should drive tumour progression and should not, as is the case for PKCα, have redundant pathways. The crucial role in cancer of transforming growth factor β (TGFβ) has recently been the topic of numerous publications4–10 and conferences devoted to the TGFβ superfamily (American Association for Cancer Research, 2003, Keystone Symposium, 2005 and Federation of American Societies for Experimental Biology, 2005). So, we chose a target with pleiotropic effects — TGFβ overexpression in advanced tumours has been shown to correlate with tumour-induced immunosuppression, invasiveness and angiogenesis, and is associated with malignant progression. Blocking TGFβ therefore constitutes a multimodal antitumour approach. AP 12009 is a PS-ODN, referred to in the review as a first-generation PS-ODN, for the targeted downregulation of TGFβ2. Although the authors argue that the first-generation ODNs were the reason for current lack of clinical success, including insufficient potency, low stability and increased side effects compared to second-generation ODNs, we could show an excellent safety profile of this first-generation drug in both animal systemic toxicological studies11 and phase I/II studies in high-grade glioma patients with local application to circumvent the blood–brain barrier12–14 (P. Hau and K.J., personal communication). Furthermore, anti-tumour activity in phase I/II studies demonstrated TGFβ2 suppression with AP 12009 to be a promising therapeutic approach. A phase I/II study for the intravenous treatment of patients with other solid tumours is ongoing. The future therapeutic success of antisense compounds will depend, as is the case with any targeted therapy, on the careful selection of optimal targets, dosing, schedules and clinical trial design. A comparison of clinical efficacy of antisense compounds with different chemical modifications for the same target is neither given nor suggested for future studies in the review. So, the judgment on the therapeutic usefulness of different chemical modifications of antisense compounds as implied in the review cannot be made based on comparing the results of one chemical modification with a particular sequence against a particular target (for example ISIS 3521/Affinitak against PKCα ) with the results of another chemistry with a different sequence against a different target gene (for example OGX-011 against clusterin).

Targeted Downregulation of TGF-β2 with AP 12009 in Tumor Therapy

Albeit recent progress in new cancer therapies there is a high unmet medical need for the treatment of aggressive cancer types such as malignant glioma, pancreatic carcinoma, malignant melanoma, or colorectal carcinoma.