Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila.

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

Xenobiotic-induced activation of human aryl hydrocarbon receptor target genes in Drosophila is mediated by the epigenetic chromatin modifiers.

Aryl hydrocarbon receptor (AHR) is the key transcription factor that controls animal development and various adaptive processes. The AHR's target genes are involved in biodegradation of endogenous and exogenous toxins, regulation of immune response, organogenesis, and neurogenesis. Ligand binding is important for the activation of the AHR signaling pathway. Invertebrate AHR homologs are activated by endogenous ligands whereas vertebrate AHR can be activated by both endogenous and exogenous ligands (xenobiotics). Several studies using mammalian cultured cells have demonstrated that transcription of the AHR target genes can be activated by exogenous AHR ligands, but little is known about the effects of AHR in a living organism. Here, we examined the effects of human AHR and its ligands using transgenic Drosophila lines with an inducible human AhR gene. We found that exogenous AHR ligands can increase as well as decrease the transcription levels of the AHR target genes, including genes that control proliferation, motility, polarization, and programmed cell death. This suggests that AHR activation may affect the expression of gene networks that could be critical for cancer progression and metastasis. Importantly, we found that AHR target genes are also controlled by the enzymes that modify chromatin structure, in particular components of the epigenetic Polycomb Repressive complexes 1 and 2. Since exogenous AHR ligands (alternatively - xenobiotics) and small molecule inhibitors of epigenetic modifiers are often used as pharmaceutical anticancer drugs, our findings may have significant implications in designing new combinations of therapeutic treatments for oncological diseases.

Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

Availability of Pre- and Pro-regions of Transgenic GDNF Affects the Ability to Induce Axonal Sprout Growth.

Plasmids containing four GFP-tagged isoforms of the human GDNF gene, with both pre- and pro-regions (pre-pro- GDNF), with the pre- (pre-GDNF) or the pro-region (pro-GDNF) alone, and without the pre- and pro-regions (mGDNF), were used to transfect HEK293 cells (human embryonic kidney cell line). The effect of the transgenic products on the growth of processes was studied in the spinal ganglia of 14-day rat embryos. Media conditioned by the transgenic cells were used to culture explants and dissociated cells of embryonic dorsal root ganglia attached to the bottom of the plate. Medium conditioned by gfp-transgenic HEK293 cells was used as the control. Spinal ganglia explants and dissociated cells cultured in a medium supplemented with recombinant GDNF (recGDNF) as well as in conditioned media containing the pre-GDNF and mGDNF products demonstrated active growth of processes immunopositive for neuronal marker beta-3-tubulin as early as on culture day 4. The ganglia and cells cultured in control medium and media conditioned by cells transgenic for pro-GDNF had no or very few processes even after 10 days of culture.

Functional analysis of Drosophila HSP70 promoter with different HSE numbers in human cells.

The activation of genetic constructs including the Drosophila hsp70 promoter with four and eight HSE sequences in the regulatory region has been described in human cells. The promoter was shown to be induced at lower temperatures compared to the human hsp70 promoter. The promoter activity increased after a 60-min heat shock already at 38 °C in human cells. The promoter activation was observed 24 h after heat shock for the constructs with eight HSEs, while those with four HSEs required 48 h. After transplantation of in vitro heat-shocked transfected cells, the promoter activity could be maintained for 3 days with a gradual decline. The promoter activation was confirmed in vivo without preliminary heat shock in mouse ischemic brain foci. Controlled expression of the Gdnf gene under a Drosophila hsp70 promoter was demonstrated. This promoter with four and eight HSE sequences in the regulatory region can be proposed as a regulated promoter in genetic therapeutic systems.

Cloning and developmental expression of MARK/Par-1/MELK-related protein kinase xMAK-V in Xenopus laevis.

MAK-V/Hunk is a recently identified MARK/Par-1-related mammalian protein kinase. Although the precise function of this protein kinase is yet to be established, available data suggest its involvement in animals' development and in the physiology of the nervous system. Here we report characterization of a cDNA encoding Xenopus laevis orthologue of MAK-V/Hunk protein kinase, xMAK-V. The in silico analysis also revealed MAK-V/Hunk orthologues in the fish Fugu rubripes and primitive chordate Ciona intestinalis but not in invertebrate species such as Drosophila melanogaster and Caenorhabditis elegans, suggesting that MAK-V/Hunk is a chordate-specific protein kinase. The expression of xmak-v in X. laevis embryos was analyzed using whole-mount in situ hybridization. Expression of xmak-v has been detected in all developmental stages studied including maternal expression in unfertilized eggs. The xmak-v mRNA has a predominant occurrence on the animal hemisphere of the egg, and this pattern of expression is sustained throughout cleavage and blastula stages. At the gastrula stage xmak-v expression is restricted to the ectoderm. In the later stage embryos xmak-v is expressed over the entire embryonic surface including the open neural plate at stage 15 and also in neural tube at stage 22. At tadpole stage xmak-v expression is strong in embryonic epidermis, nervous system and sensory organs, and is also obvious in perisomitic mesoderm and brachial arches.