Endurance Training Increases the Running Performance of Untrained Men without Changing the Mitochondrial Volume Density in the Gastrocnemius Muscle.

The activity and quantity of mitochondrial proteins and the mitochondrial volume density (MitoVD) are higher in trained muscles; however, the underlying mechanisms remain unclear. Our goal was to determine if 20 weeks' endurance training simultaneously increases running performance, the amount and activity of mitochondrial proteins, and MitoVD in the gastrocnemius muscle in humans. Eight healthy, untrained young men completed a 20-week moderate-intensity running training program. The training increased the mean speed of a 1500 m run by 14.0% (p = 0.008) and the running speed at 85% of maximal heart rate by 9.6% (p = 0.008). In the gastrocnemius muscle, training significantly increased mitochondrial dynamics markers, i.e., peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) by 23%, mitochondrial transcription factor A (TFAM) by 29%, optic artrophy-1 (OPA1) by 31% and mitochondrial fission factor (MFF) by 44%, and voltage-dependent anion channel 1 (VDAC1) by 30%. Furthermore, training increased the amount and maximal activity of citrate synthase (CS) by 10% and 65%, respectively, and the amount and maximal activity of cytochrome c oxidase (COX) by 57% and 42%, respectively, but had no effect on the total MitoVD in the gastrocnemius muscle. We concluded that not MitoVD per se, but mitochondrial COX activity (reflecting oxidative phosphorylation activity), should be regarded as a biomarker of muscle adaptation to endurance training in beginner runners.

Mesencephalic Astrocyte-Derived Neurotrophic Factor Regulates Morphology of Pigment-Dispersing Factor-Positive Clock Neurons and Circadian Neuronal Plasticity in Drosophila melanogaster.

Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is one of a few neurotrophic factors described in Drosophila melanogaster (DmMANF) but its function is still poorly characterized. In the present study we found that DmMANF is expressed in different clusters of clock neurons. In particular, the PDF-positive large (l-LNv) and small (s-LNv) ventral lateral neurons, the CRYPTOCHROME-positive dorsal lateral neurons (LNd), the group 1 dorsal neurons posterior (DN1p) and different tim-positive cells in the fly's visual system. Importantly, DmMANF expression in the ventral lateral neurons is not controlled by the clock nor it affects its molecular mechanism. However, silencing DmMANF expression in clock neurons affects the rhythm of locomotor activity in light:dark and constant darkness conditions. Such phenotypes correlate with abnormal morphology of the dorsal projections of the s-LNv and with reduced arborizations of the l-LNv in the medulla of the optic lobe. Additionally, we show that DmMANF is important for normal morphology of the L2 interneurons in the visual system and for the circadian rhythm in the topology of their dendritic tree. Our results indicate that DmMANF is important not only for the development of neurites but also for maintaining circadian plasticity of neurons.

Glial expression of DmMANF is required for the regulation of activity, sleep and circadian rhythms in the visual system of Drosophila melanogaster.

DmMANF, Drosophila melanogaster mesencephalic astrocyte-derived neurotrophic factor (DmMANF) is an evolutionarily conserved orthologue of mammalian MANF. This neurotrophic factor exerts many functions in the Drosophila brain, particularly those dependent on glial cells. As we found in our earlier study, downregulation of DmMANF in glia induces degeneration of glial cells in the first optic neuropil (lamina) where DmMANF abundance is especially high. In the present study, we observed that changes in the level of DmMANF in two types of glia, astrocyte-like glia (AlGl) and ensheathing glia (EnGl), affect activity and sleep of flies. Interestingly, a proper level of DmMANF in AlGl seems to be important in guiding processes of pigment dispersing factor (PDF)-expressing clock neurons. This is supported by our finding that DmMANF overexpression in AlGl leads to structural changes in the architecture of the PDF-positive arborization in the brain. Finally, we detected that DmMANF also affects rhythms in glia themselves, as circadian oscillations in expression of the catalytic α subunit of the sodium pump in the lamina epithelial glia were abolished after DmMANF silencing. DmMANF expressed in AlGl and EnGl seems to affect the activity of neurons leading to changes in behaviour.

On Variations in the Level of PER in Glial Clocks of Drosophila Optic Lobe and Its Negative Regulation by PDF Signaling.

We show that the level of the core protein of the circadian clock Period (PER) expressed by glial peripheral oscillators depends on their location in the Drosophila optic lobe. It appears to be controlled by the ventral lateral neurons (LNvs) that release the circadian neurotransmitter Pigment Dispersing Factor (PDF). We demonstrate that glial cells of the distal medulla neuropil (dMnGl) that lie in the vicinity of the PDF-releasing terminals of the LNvs possess receptors for PDF (PDFRs) and express PER at significantly higher level than other types of glia. Surprisingly, the amplitude of PER molecular oscillations in dMnGl is increased twofold in PDF-free environment, that is in Pdf0 mutants. The Pdf0 mutants also reveal an increased level of glia-specific protein REPO in dMnGl. The photoreceptors of the compound eye (R-cells) of the PDF-null flies, on the other hand, exhibit de-synchrony of PER molecular oscillations, which manifests itself as increased variability of PER-specific immunofluorescence among the R-cells. Moreover, the daily pattern of expression of the presynaptic protein Bruchpilot (BRP) in the lamina terminals of the R-cells is changed in Pdf0 mutant. Considering that PDFRs are also expressed by the marginal glia of the lamina that surround the R-cell terminals, the LNv pacemakers appear to be the likely modulators of molecular cycling in the peripheral clocks of both the glial cells and the photoreceptors of the compound eye. Consequently, some form of PDF-based coupling of the glial clocks and the photoreceptors of the eye with the central LNv pacemakers must be operational.

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.

Downregulation of DmMANF in Glial Cells Results in Neurodegeneration and Affects Sleep and Lifespan in Drosophila melanogaster.

In Drosophila melanogaster, mesencephalic astrocyte-derived neurotrophic factor (DmMANF) is an evolutionarily conserved ortholog of mammalian MANF and cerebral dopamine neurotrophic factor (CDNF), which have been shown to promote the survival of dopaminergic neurons in the brain. We observed especially high levels of DmMANF in the visual system of Drosophila, particularly in the first optic neuropil (lamina). In the lamina, DmMANF was found in glial cells (surface and epithelial glia), photoreceptors and interneurons. Interestingly, silencing of DmMANF in all neurons or specifically in photoreceptors or L2 interneurons had no impact on the structure of the visual system. However, downregulation of DmMANF in glial cells induced degeneration of the lamina. Remarkably, this degeneration in the form of holes and/or tightly packed membranes was observed only in the lamina epithelial glial cells. Those membranes seem to originate from the endoplasmic reticulum, which forms autophagosome membranes. Moreover, capitate projections, the epithelial glia invaginations into photoreceptor terminals that are involved in recycling of the photoreceptor neurotransmitter histamine, were less numerous after DmMANF silencing either in neurons or glial cells. The distribution of the alpha subunit of Na+/K+-ATPase protein in the lamina cell membranes was also changed. At the behavioral level, silencing of DmMANF either in neurons or glial cells affected the daily activity/sleep pattern, and flies showed less activity during the day but higher activity during the night than did controls. In the case of silencing in glia, the lifespan of flies was also shortened. The obtained results showed that DmMANF regulates many functions in the brain, particularly those dependent on glial cells.