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Sponges (phylum Porifera) represent the phylogenetically oldest metazoan animals. Recently, from the marine sponge Geodia cydonium a first cDNA encoding a putative integrin receptor molecule was isolated. In the present study basic functional experiments have been conducted to test the hypothesis that in sponges integrin polypeptides also function as adhesion molecules and as outside-in signaling molecules. The sponge Suberites domuncula has been used for the experiments because from this sponge only has a cell culture been established. Here we report that aggregation factor (AF)-mediated cell-cell adhesion is blocked by the RGDS peptide which is known to interact with b integrin. Both RGDS and AF were found to stimulate DNA synthesis within 24 h. The b subunit of the integrin receptor was cloned from S. domuncula; the estimated 91-kDa molecule comprises the characteristic signatures. Evolutionary conservation of the b integrin was assessed by comparison with corresponding b integrin subunits from evolutionary higher metazoan taxa. Addition of RGDS or of AF to isolated cells of S. domuncula causes a rapid (within 1±2 min) increase in the intracellular Ca 2+ concentration which is further augmented in the presence of Ca 2+ . Furthermore, incubation of the cells with RGDS or AF causes an activation of the GTP-binding protein Ras. In addition it is shown that after a prolonged incubation of the cells with RGDS and AF the expression of the genes coding for Ras and for calmodulin is upregulated. These results suggest that the integrin receptor functions in the sponge system not only as adhesion molecule but also as a molecule involved in outside-in signaling.Keywords: Suberites domuncula, integrin, calcium, Ras, calmodulin, signal transduction, evolution, RGD(S), aggregation factor. Multicellularity in eukaryotes developed several times in evolution. Striking examples are the transition of unicellular to multicellular species within the volvocine algae (reviewed in [1]) which happened only 50±75 million years ago [2] while that of unicellular to multicellular animals dates back to 600 million years ago [3]. Until recently it remained unsolved if the evolution to multicellularity in Metazoa occurred only once (monophyly) or several times (polyphyly). Studies concentrating on those molecules which are considered to be characteristic for metazoan cells revealed that protein sequences, deduced from cDNAs obtained from the lowest metazoan phylum, the Porifera (sponges), are highly similar to those found in other metazoan phyla.Metazoan cells interact with each other differently than unicellular eukaryotes, especially with respect to (a) G-protein linked transmembrane receptors, (b) transmembrane adhesion molecules and (c) ligand-gated ion channels. Sequence data studies revealed that sponges ± the experiments have been performed mainly with the marine species Geodia cydonium and Suberites domuncula (reviewed in [4]) ± are provided with the G-protein linked transmembrane receptor, the metabotropic glutamate receptor [4a], ...

Sponges (phylum Porifera) represent the phylogenetically oldest metazoan phylum. These animals have complex cell adhesion and powerful immune systems which allow the formation of a distinct body plan. Consequently, an apoptotic machinery has to be predicted that allows sponges to eliminate unwanted cells accumulating during development. With the marine sponge Geodia cydonium, it is shown that allografts of these animals undergo apoptosis as demonstrated by apoptotic DNA fragmentation. Extracts from allografts contain an enzymic activity characteristic for caspases; as substrate to determine the cleavage activity, Ac-DEVD-AMC was applied. cDNAs encoding predicted caspase-3-related proteins were isolated; they comprise the characteristic structure known from caspases of other metazoan phyla. The two cDNAs are assumed to originate from one gene by alternative splicing; the longer form comprises a caspase recruitment domain (CARD), whereas the shorter one is missing CARD. The expression of sponge caspase genes is up-regulated during allograft rejection. In vivo incubation experiments with Ac-DEVD-CHO (a caspase-3 inhibitor) showed a reduction of apoptotic DNA fragmentation, whereas Ac-LEHD-CHO (an inhibitor of caspase-9) caused no effect. It is concluded, that for the establishment of the metazoan body plan, both the adhesion molecules and the apoptotic molecules (described here) were essential prerequisites.

Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell-cell and cell-matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs.

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To date, no conclusive evidence has been presented for the existence of neuronal-like elements in Porifera (sponges). In the present study, isolated cells from the marine sponge Geodia cydonium are shown to react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium [Ca2+]i. This effect can also be observed when the compounds L-quisqualic acid (L-QA) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP-4) are used. The effect of L-QA and L-AP-4, both agonists for metabotropic glutamate receptors (mGluRs), can be abolished by the antagonist of group I mGluRs, (RS)-alpha-methyl-4-carboxyphenylglycine. These data suggest that sponge cells contain an mGluR-like protein. A cDNA encoding rat mGluR subtype 1 has been used to identify the complete nucleotide sequence of G. cydonium cDNA coding for a 528-amino-acid-long protein (59 kDa) that displays marked overall similarity to mGluRs and to gamma-aminobutyric acid B receptors. The deduced sponge polypeptide, termed putative mGlu/GABA-like receptor, displays the highest similarity to the two families of metabotropic receptors within the transmembrane segment. The N-terminal part of the sponge sequence shows similarity to mGluR4 and mGluR5. These findings suggest that the earliest evolutionary metazoan phylum, the Porifera, possesses a sophisticated intercellular communication and signaling system, as seen in the neuronal network of higher Metazoa.

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Effective drugs are not available to protect against ß-amyloid peptide (Aß)-induced neurotoxicity. Cortical neurons from rat embryos were treated with the toxic fragment Aß25-35 at 1 p~Min the presence or absence of flupirtine, a triaminopyridine, successfully applied clinically as a nonopiate analgesic drug. Five days later 1 ‚uM Aß25-35 caused reduction of cell viability to 31.1%. Preincubation of cells with flupirtine (1 or 5~ig/ ml) resulted in a significant increase of the percentage of viable cells (74.6 and 65.4%, respectively). During incubation with Aß25-35 the neurons undergo apoptosis as determined by appearance of the characteristic stepladder-like DNA fragmentation pattern and by the TUNEL technique. Aß25-35-induced DNA fragmentation could be abolished by preincubation ofthe cells with 1 1ig/ml flupirtine. Incubation with Aß25-~35reduces the intraneuronal level of GSH from 21.4 to 7.4 nmol/10 6 cells. This depletion could be partially prevented by preincubation of the cells with flupirtine. Thus, flupirtine may be adequate for the treatment of the neuronal loss in Alzheimer's disease (where Aß accumulates in senile plaques) and probably other neurological diseases such as amyotrophic lateral sclerosis.