Different levels of expression of the clock protein PER and the glial marker REPO in ensheathing and astrocyte-like glia of the distal medulla of Drosophila optic lobe

Circadian plasticity of the visual system of Drosophila melanogaster depends on functioning of both the neuronal and glial oscillators. The clock function of the former is already quite well-recognized. The latter, however, is much less known and documented. In this study we focus on the glial oscillators that reside in the distal part of the second visual neuropil, medulla (dMnGl), in vicinity of the PIGMENT-DISPERSING FACTOR (PDF) releasing terminals of the circadian clock ventral Lateral Neurons (LNvs). We reveal the heterogeneity of the dMnGl, which express the clock protein PERIOD (PER) and the pan-glial marker REVERSED POLARITY (REPO) at higher (P1) or lower (P2) levels. We show that the cells with stronger expression of PER display also stronger expression of REPO, and that the number of REPO-P1 cells is bigger during the day than during the night. Using a combination of genetic markers and immunofluorescent labeling with anti PER and REPO Abs, we have established that the P1 and P2 cells can be associated with two different types of the dMnGl, the ensheathing (EnGl), and the astrocyte-like glia (ALGl). Surprisingly, the EnGl belong to the P1 cells, whereas the ALGl, previously reported to play the main role in the circadian rhythms, display the characteristics of the P2 cells (express very low level of PER and low level of REPO). Next to the EnGl and ALGl we have also observed another type of cells in the distal medulla that express PER and REPO, although at very low levels. Based on their morphology we have identified them as the T1 interneurons. Our study reveals the complexity of the distal medulla circadian network, which appears to consist of different types of glial and neuronal peripheral clocks, displaying molecular oscillations of higher (EnGl) and lower (ALGl and T1) amplitudes

Mottled mice and non-mammalian models of Menkes disease

Menkes disease is a multi-systemic copper metabolism disorder caused by mutations in the X-linked ATP7A gene and characterised by progressive neurodegeneration and severe connective tissue defects. The ATP7A protein is a Copper (Cu)-transporting ATPase expressed in all tissues and plays a critical role in the maintenance of copper homeostasis in cells of the whole body. ATP7A participates in copper absorption in the small intestine and in copper transport to the CNS across the blood-brain-barrier and blood–cerebrospinal fluid- barrier. Cu is essential for synaptogenesis and axonal development. In cells, ATP7A participates in the incorporation of copper into Cu-dependent enzymes during the course of its maturation in the secretory pathway. There is a high degree of homology (>80% ) between the human ATP7A and murine Atp7a genes. Mice with mutations in the Atp7a gene, called mottled mutants, are well-established and excellent models of Menkes disease. Mottled mutants closely recapitulate the Menkes phenotype and are invaluable for studying Cu-metabolism. They provide useful models for exploring and testing new forms of therapy in Menkes disease. Recently, non-mammalian models of Menkes disease, Drosophila melanogaster and Danio rerio mutants were used in experiments which would be technically difficult to carry out in mammals

The crucial involvement of retinoid X receptors in DDE neurotoxicity

Dichlorodiphenyldichloroethylene (DDE) is a primary environmental and metabolic degradation product of the pesticide dichlorodiphenyltrichloroethane (DDT). It is one of the most toxic compounds belonging to organochlorines. DDE has never been commercially produced; however, the parent pesticide DDT is still used in some developing countries for disease-vector control of malaria. DDT and DDE remain in the environment because these chemicals are resistant to degradation and bioaccumulate in the food chain. Little is known, however, about DDE toxicity during the early stages of neural development. The results of the present study demonstrate that DDE induced a caspase-3-dependent apoptosis and caused the global DNA hypomethylation in mouse embryonic neuronal cells. This study also provided evidence for DDE-isomer-non-specific alterations of retinoid X receptor α (RXRα)- and retinoid X receptor β (RXRβ)-mediated intracellular signaling, including changes in the levels of the receptor mRNAs and changes in the protein levels of the receptors. DDE-induced stimulation of RXRα and RXRβ was verified using selective antagonist and specific siRNAs. Co-localization of RXRα and RXRβ was demonstrated using confocal microscopy. The apoptotic action of DDE was supported at the cellular level through Hoechst 33342 and calcein AM staining experiments. In conclusion, the results of the present study demonstrated that the stimulation of RXRα- and RXRβ-mediated intracellular signaling plays an important role in the propagation of DDE-induced apoptosis during early stages of neural development

External and circadian inputs modulate synaptic protein expression in the visual system of Drosophila melanogaster

In the visual system of Drosophila melanogaster the retina photoreceptors form tetrad synapses with the first order interneurons, amacrine cells and glial cells in the first optic neuropil (lamina), in order to transmit photic and visual information to the brain. Using the specific antibodies against synaptic proteins; Bruchpilot (BRP), Synapsin (SYN), and Disc Large (DLG), the synapses in the distal lamina were specifically labeled. Then their abundance was measured as immunofluorescence intensity in flies held in light/dark (LD 12:12), constant darkness (DD), and after locomotor and light stimulation. Moreover, the levels of proteins (SYN and DLG), and mRNAs of the brp, syn, and dlg genes, were measured in the fly's head and brain, respectively. In the head we did not detect SYN and DLG oscillations. We found, however, that in the lamina, DLG oscillates in LD 12:12 and DD but SYN cycles only in DD. The abundance of all synaptic proteins was also changed in the lamina after locomotor and light stimulation. One hour locomotor stimulations at different time points in LD 12:12 affected the pattern of the daily rhythm of synaptic proteins. In turn, light stimulations in DD increased the level of all proteins studied. In the case of SYN, however, this effect was observed only after a short light pulse (15 min). In contrast to proteins studied in the lamina, the mRNA of brp, syn, and dlg genes in the brain was not cycling in LD 12:12 and DD, except the mRNA of dlg in LD 12:12. Our earlier results and obtained in the present study showed that the abundance of BRP, SYN and DLG in the distal lamina, at the tetrad synapses, is regulated by light and a circadian clock while locomotor stimulation affects their daily pattern of expression. The observed changes in the level of synaptic markers reflect the circadian plasticity of tetrad synapses regulated by the circadian clock and external inputs, both specific and unspecific for the visual system

Molecular mechanism of the circadian clock - how organisms count time

Rytmy okołodobowe są powszechne w przyrodzie, a ich występowanie stwierdzono niemal u wszystkich organizmów. Wspomniane rytmy przejawiają się zarówno w zachowaniu jak również metabolizmie oraz wielu ścieżkach fizjologicznych organizmu. Rytmy okołodobowe są kontrolowane przez specjalny mechanizm molekularnego zegara, opartego na działaniu pętli sprzężeń zwrotnych regulowanych poprzez białka o charakterze czynników transkrypcyjnych. Przez ostatnie lata mechanizm ten był intensywnie badany u bakterii, grzybów i roślin, oraz przede wszystkim u zwierząt bezkręgowych (Drosophila melanogaster) i ssaków. Molekularny mechanizm endogennego oscylatora różni się u Eukariota i Prokaryota, co sugeruje jego niezależne powstanie u tych grup organizmów. Pomimo znaczących różnic w regulacji mechanizmu zegara okołodobowego, wspólną cechą jest fakt, iż czynniki o charakterze aktywatorów inicjują transkrypcję czynników o charakterze inhibitorów, które zwrotnie hamują ekspresję wspomnianych aktywatorów. Kolejny cykl rozpoczyna się w momencie, gdy poziom czynników hamujących jest na tyle niski, iż możliwa jest ponowna transkrypcja czynników aktywujących. Taki samonapędzający się oscylator jest synchronizowany do zewnętrznych warunków środowiska dzięki istnieniu dróg wejściowych, które mogą odbierać informacje o warunkach świetlnych, temperaturze itp. Ponadto, białka wchodzące w skład molekularnego oscylatora odpowiedzialne są nie tylko za regulację poziomu białek zegara, ale wpływają na ekspresję innych genów, tak zwanych genów kontrolowanych przez zegar. Produkty białkowe tych genów stanowią element dróg wyjściowych zegara, które kontrolują wiele procesów fizjologicznych jak i behawior. W prezentowanej pracy został omówiony molekularny mechanizm zegara okołodobowego zwierząt modelowych, takich jak Synechococcus elongatus, Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster oraz Mus musculus.Almost all organisms exhibit circadian rhythms in behavior, metabolism and physiology. All these rhythms are controlled by a clock mechanism, which is composed of transcriptional feedback loops. Molecular composition and regulation of endogenous oscillators responsible for circadian rhythms, from cyanobacteria to humans, have been extensively investigated over past years. The molecular mechanism of the circadian clock is different between eukaryotes and prokaryotes, suggesting its independent origin. However, a common feature of the clock is that positive factors in the feedback loops activate the transcription of negative factors, which feedback to inhibit expression of positive factors. When the level of negative factors is low, the positive factors can start the next cycle of transcription. The core clock is synchronized to the environment by means of input pathways which can detect external cues such as light, temperature and other. Clock proteins are not only self-regulated molecules but can also influence expression of other genes (clock-controlled genes). These genes are part of an output pathway which controls many behavioral and physiological pathways. In this paper, the circadian clock mechanisms in different model organism (Synechococcus elongatus, Neurospora crassa, Arabidopsis thaliana, fruit fly and mouse) are reviewed

Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells

Molecular mechanisms of the circadian clock : how organisms count time

Rytmy okołodobowe są powszechne w przyrodzie, a ich występowanie stwierdzono niemal u wszystkich organizmów. Wspomniane rytmy przejawiają się zarówno w zachowaniu jak również metabolizmie oraz wielu ścieżkach fizjologicznych organizmu. Rytmy okołodobowe są kontrolowane przez specjalny mechanizm molekularnego zegara, opartego na działaniu pętli sprzężeń zwrotnych regulowanych poprzez białka o charakterze czynników transkrypcyjnych. Przez ostatnie lata mechanizm ten był intensywnie badany u bakterii, grzybów i roślin, oraz przede wszystkim u zwierząt bezkręgowych (Drosophila melanogaster) i ssaków. Molekularny mechanizm endogennego oscylatora różni się u Eukariota i Prokaryota, co sugeruje jego niezależne powstanie u tych grup organizmów. Pomimo znaczących różnic w regulacji mechanizmu zegara okołodobowego, wspólną cechą jest fakt, iż czynniki o charakterze aktywatorów inicjują transkrypcję czynników o charakterze inhibitorów, które zwrotnie hamują ekspresję wspomnianych aktywatorów. Kolejny cykl rozpoczyna się w momencie, gdy poziom czynników hamujących jest na tyle niski, iż możliwa jest ponowna transkrypcja czynników aktywujących. Taki samonapędzający się oscylator jest synchronizowany do zewnętrznych warunków środowiska dzięki istnieniu dróg wejściowych, które mogą odbierać informacje o warunkach świetlnych, temperaturze itp. Ponadto, białka wchodzące w skład molekularnego oscylatora odpowiedzialne są nie tylko za regulację poziomu białek zegara, ale wpływają na ekspresję innych genów, tak zwanych genów kontrolowanych przez zegar. Produkty białkowe tych genów stanowią element dróg wyjściowych zegara, które kontrolują wiele procesów fizjologicznych jak i behawior. W prezentowanej pracy został omówiony molekularny mechanizm zegara okołodobowego zwierząt modelowych, takich jak Synechococcus elongatus, Neurospora crassa, Arabidopsis thaliana, Drosophila melanogaster oraz Mus musculus.Almost all organisms exhibit circadian rhythms in behavior, metabolism and physiology. All these rhythms are controlled by a clock mechanism, which is composed of transcriptional feedback loops. Molecular composition and regulation of endogenous oscillators responsible for circadian rhythms, from cyanobacteria to humans, have been extensively investigated over past years. The molecular mechanism of the circadian clock is different between eukaryotes and prokaryotes, suggesting its independent origin. However, a common feature of the clock is that positive factors in the feedback loops activate the transcription of negative factors, which feedback to inhibit expression of positive factors. When the level of negative factors is low, the positive factors can start the next cycle of transcription. The core clock is synchronized to the environment by means of input pathways which can detect external cues such as light, temperature and other. Clock proteins are not only self-regulated molecules but can also influence expression of other genes (clock-controlled genes). These genes are part of an output pathway which controls many behavioral and physiological pathways. In this paper, the circadian clock mechanisms in different model organism (Synechococcus elongatus, Neurospora crassa, Arabidopsis thaliana, fruit fly and mouse) are reviewed