The Granuphilin homolog bitesize regulates sleep and longevity in Drosophila melanogaster

In a RNAseq based screen of long-lived dilp 2-3,5 mutants, the btsz locus was identified as a potential downstream effector. The function of Granuphilin, the mammalian homolog of btsz, has been well described to mediate the exocytosis of insulin-containing granules in pancreatic beta cells via interaction with Rab27a (Yi et al. 2002, Torii et al. 2004, Gomi et al. 2005). In contrast, the function of Btsz, specifically the P1 isoform containing the highly conserved SHD domain, is barely described in Drosophila. A previous study conducted in our lab revealed, that Btsz P1 is exclusively expressed in the mushroom body (Weigelt 2018), a brain region that has been implicated in the regulation of sleep and olfactory learning (Joiner et al. 2006). Strikingly, deletion of btsz P1 resulted in a significant lifespan extension in male and female flies and increased sleep length in young male flies (Weigelt 2018). In this study we showed that deletion of btsz P1 significantly influenced sleep patterns in both, male and female flies, in line with the observed lifespan extension in both sexes. Notably, btsz P1∆ mutants also exhibited an improved sleep quality during ageing, which was accompanied by ameliorated deterioration in locomotion during ageing, indicating that btsz P1∆ mutants do not only live longer, but also show a delayed onset of a characteristic physiological decline. Since btsz P1∆ mutants phenocopy dilp 2-3,5 mutants in extended lifespan, reduced fecundity and weight, and prolonged nocturnal sleep, we wondered if Btsz P1 acts on the insulin-signalling pathway. Therefore, we performed genetic epistasis experiments and combined both mutations. The combination of both mutations, however, did not result in additive effects regarding lifespan and sleep, but rather resulted in detrimental effects. In addition, Btsz P1 did not localize in insulin-secreting neurons, suggesting that Btsz P1 influences insulin-signalling probably indirectly. To further investigate whether Btsz P1 regulates other sleep-regulating pathways, we modulated several sleep-regulating signalling-pathways pharmacologically and genetically. Sleep analysis revealed that btsz P1∆ mutants are resistant to interventions on the dopamine-signalling pathway, implying that Btsz P1 potentially acts in dopaminergic neurotransmission. Brain immunostainings revealed that Btsz P1 is not expressed in dopaminergic neurons, but in neurons expressing DopR, further supporting our sleep data. Also, analysis of the single cell expression atlas of the ageing Drosophila brain strengthened our hypothesis that Btsz P1 probably executes its function in neurons expressing DopR, since expression of btsz P1 was detected in the same neuronal clusters as DopR. Interestingly, Ca-alpha 1T, a channel that has been implicated in the regulation of sleep, colocalised with Btsz P1 in neurons expressing DopR. In addition, Ca-alpha 1T null mutants were resistant to methamphetamine treatment, reminiscent to btsz P1∆ mutants, rising the hypothesis that Btsz P1 might influence the exocytosis of Ca-alpha 1T to the transmembrane to restore-ion currencies in order to regulate the neuronal excitability

Repeat length of C9orf72-associated glycine–alanine polypeptides affects their toxicity

G4C2 hexanucleotide repeat expansions in a non-coding region of the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). G4C2 insertion length is variable, and patients can carry up to several thousand repeats. Dipeptide repeat proteins (DPRs) translated from G4C2 transcripts are thought to be a main driver of toxicity. Experiments in model organisms with relatively short DPRs have shown that arginine-rich DPRs are most toxic, while polyGlycine–Alanine (GA) DPRs cause only mild toxicity. However, GA is the most abundant DPR in patient brains, and experimental work in animals has generally relied on the use of low numbers of repeats, with DPRs often tagged for in vivo tracking. Whether repeat length or tagging affect the toxicity of GA has not been systematically assessed. Therefore, we generated Drosophila fly lines expressing GA100, GA200 or GA400 specifically in adult neurons. Consistent with previous studies, expression of GA100 and GA200 caused only mild toxicity. In contrast, neuronal expression of GA400 drastically reduced climbing ability and survival of flies, indicating that long GA DPRs can be highly toxic in vivo. This toxicity could be abolished by tagging GA400. Proteomics analysis of fly brains showed a repeat-length-dependent modulation of the brain proteome, with GA400 causing earlier and stronger changes than shorter GA proteins. PolyGA expression up-regulated proteins involved in ER to Golgi trafficking, and down-regulated proteins involved in insulin signalling. Experimental down-regulation of Tango1, a highly conserved regulator of ER-to Golgi transport, partially rescued GA400 toxicity, suggesting that misregulation of this process contributes to polyGA toxicity. Experimentally increasing insulin signaling also rescued GA toxicity. In summary, our data show that long polyGA proteins can be highly toxic in vivo, and that they may therefore contribute to ALS/FTD pathogenesis in patients

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions

Clostridioides difficile Activates Human Mucosal-Associated Invariant T Cells

Clostridioides difficile infection (CDI) causes severe inflammatory responses at the intestinal mucosa but the immunological mechanisms underlying CDI-related immunopathology are still incompletely characterized. Here we identified for the first time that both, non-toxigenic strains as well as the hypervirulent ribotypes RT027 and RT023 of Clostridioides difficile (formerly Clostridium difficile), induced an effector phenotype in mucosal-associated invariant T (MAIT) cells. MAIT cells can directly respond to bacterial infections by recognizing MR1-presented metabolites derived from the riboflavin synthesis pathway constituting a novel class of antigens. We confirmed functional riboflavin synthesis of C. difficile and found fixed bacteria capable of activating primary human MAIT cells in a dose-dependent manner. C. difficile-activated MAIT cells showed an increased and MR1-dependent expression of CD69, proinflammatory IFNγ, and the lytic granule components granzyme B and perforin. Effector protein expression was accompanied by the release of lytic granules, which, in contrast to other effector functions, was mainly induced by IL-12 and IL-18. Notably, this study revealed hypervirulent C. difficile strains to be most competent in provoking MAIT cell responses suggesting MAIT cell activation to be instrumental for the immunopathology observed in C. difficile-associated colitis. In conclusion, we provide first evidence for a link between C. difficile metabolism and innate T cell-mediated immunity in humans

Influence of L-lactate and low glucose concentrations on the metabolism and the toxin formation of Clostridioides difficile.

The virulence of Clostridioides difficile (formerly Clostridium difficile) is mainly caused by its two toxins A and B. Their formation is significantly regulated by metabolic processes. Here we investigated the influence of various sugars (glucose, fructose, mannose, trehalose), sugar derivatives (mannitol and xylitol) and L-lactate on toxin synthesis. Fructose, mannose, trehalose, mannitol and xylitol in the growth medium resulted in an up to 2.2-fold increase of secreted toxin. Low glucose concentration of 2 g/L increased the toxin concentration 1.4-fold compared to growth without glucose, while high glucose concentrations in the growth medium (5 and 10 g/L) led to up to 6.6-fold decrease in toxin formation. Transcriptomic and metabolic investigation of the low glucose effect pointed towards an inactive CcpA and Rex regulatory system. L-lactate (500 mg/L) significantly reduced extracellular toxin formation. Transcriptome analyses of the later process revealed the induction of the lactose utilization operon encoding lactate racemase (larA), electron confurcating lactate dehydrogenase (CDIF630erm_01321) and the corresponding electron transfer flavoprotein (etfAB). Metabolome analyses revealed L-lactate consumption and the formation of pyruvate. The involved electron confurcation process might be responsible for the also observed reduction of the NAD+/NADH ratio which in turn is apparently linked to reduced toxin release from the cell

Metabolic Reprogramming of Clostridioides difficile During the Stationary Phase With the Induction of Toxin Production

The obligate anaerobe, spore forming bacterium Clostridioides difficile (formerly Clostridium difficile) causes nosocomial and community acquired diarrhea often associated with antibiotic therapy. Major virulence factors of the bacterium are the two large clostridial toxins TcdA and TcdB. The production of both toxins was found strongly connected to the metabolism and the nutritional status of the growth environment. Here, we systematically investigated the changes of the gene regulatory, proteomic and metabolic networks of C. difficile 630Δerm underlying the adaptation to the non-growing state in the stationary phase. Integrated data from time-resolved transcriptome, proteome and metabolome investigations performed under defined growth conditions uncovered multiple adaptation strategies. Overall changes in the cellular processes included the downregulation of ribosome production, lipid metabolism, cold shock proteins, spermine biosynthesis, and glycolysis and in the later stages of riboflavin and coenzyme A (CoA) biosynthesis. In contrast, different chaperones, several fermentation pathways, and cysteine, serine, and pantothenate biosynthesis were found upregulated. Focusing on the Stickland amino acid fermentation and the central carbon metabolism, we discovered the ability of C. difficile to replenish its favored amino acid cysteine by a pathway starting from the glycolytic 3-phosphoglycerate via L-serine as intermediate. Following the growth course, the reductive equivalent pathways used were sequentially shifted from proline via leucine/phenylalanine to the central carbon metabolism first to butanoate fermentation and then further to lactate fermentation. The toxin production was found correlated mainly to fluxes of the central carbon metabolism. Toxin formation in the supernatant was detected when the flux changed from butanoate to lactate synthesis in the late stationary phase. The holistic view derived from the combination of transcriptome, proteome and metabolome data allowed us to uncover the major metabolic strategies that are used by the clostridial cells to maintain its cellular homeostasis and ensure survival under starvation conditions

Regional analysis of inflammation and contractile function in reperfused acute myocardial infarction by in vivo 19F cardiovascular magnetic resonance in pigs

Inflammatory cell infiltration is central to healing after acute myocardial infarction (AMI). The relation of regional inflammation to edema, infarct size (IS), microvascular obstruction (MVO), intramyocardial hemorrhage (IMH), and regional and global LV function is not clear. Here we noninvasively characterized regional inflammation and contractile function in reperfused AMI in pigs using fluorine (19F) cardiovascular magnetic resonance (CMR). Adult anesthetized pigs underwent left anterior descending coronary artery instrumentation with either 90 min occlusion (n = 17) or without occlusion (sham, n = 5). After 3 days, in surviving animals a perfluorooctyl bromide nanoemulsion was infused intravenously to label monocytes/macrophages. At day 6, in vivo 1H-CMR was performed with cine, T2 and T2* weighted imaging, T2 and T1 mapping, perfusion and late gadolinium enhancement followed by 19F-CMR. Pigs were sacrificed for subsequent ex vivo scans and histology. Edema extent was 35 ± 8% and IS was 22 ± 6% of LV mass. Six of ten surviving AMI animals displayed both MVO and IMH (3.3 ± 1.6% and 1.9 ± 0.8% of LV mass). The 19F signal, reflecting the presence and density of monocytes/macrophages, was consistently smaller than edema volume or IS and not apparent in remote areas. The 19F signal-to-noise ratio (SNR) > 8 in the infarct border zone was associated with impaired remote systolic wall thickening. A whole heart value of 19F integral (19F SNR × milliliter) > 200 was related to initial LV remodeling independently of edema, IS, MVO, and IMH. Thus, 19F-CMR quantitatively characterizes regional inflammation after AMI and its relation to edema, IS, MVO, IMH and regional and global LV function and remodeling.ISSN:0300-8428ISSN:1435-180