Patterns of faecal glucocorticoid metabolite levels in captive roan antelope (Hippotragus equinus) in relation to reproductive status and season.

Populations of roan antelope (Hippotragus equinus) in southern Africa have experienced a drastic decline over the past few decades and this situation has led to the development of intensive breeding programmes to support conservation efforts. However, little is known about related welfare aspects, including stress-related physiological biomarkers. The present study set out to establish a non-invasive method to monitor faecal glucocorticoid metabolite (fGCM) concentrations as a measure of stress and determine fGCM concentrations in relation to male reproductive activity and female reproductive status in the roan antelope. An adrenocorticotrophic hormone challenge was performed using two adult roan antelope (one male and one female) at Lapalala Wilderness Nature Reserve, South Africa, to determine the suitability of five enzyme immunoassays (EIA) for monitoring adrenocortical function in roan antelope. An 11-oxoaetiocholanolone I EIA detecting 11,17 dioxoandrostanes performed best showing 17-20 folds increases in fGCM concentrations after 12 h-17 h post-injection. The identified EIA was then used to monitor fGCM concentrations during active and non-active reproductive periods in males (n = 3), and during periods of cyclicity, gestation, and postpartum in females (n = 10). Males showed an overall 80% increase in fGCM concentrations when reproductively active and females showed a progressively significant increase in fGCM levels throughout pregnancy, with overall fGCM concentrations being 1.5 to 2.6-fold higher than the respective fGCM concentrations during periods of postpartum and cyclicity, respectively. Furthermore, fGCM concentrations remained above baseline for up to 21 days post-partum. A correlation between ecological parameters (rainfall and temperature) and fGCM concentrations revealed elevated fGCM concentrations during the dry season for males, but not females. The non-invasive method validated in this study provides a valuable tool to quantify stress-related biomarkers in roan antelope, and findings can be used to support management decisions in conservation breeding facilities.

Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson's Disease.

The aggregation of α-synuclein is a hallmark of Parkinson's disease (PD) and a variety of related neurological disorders. A number of mutations in this protein, including A30P and A53T, are associated with familial forms of the disease. Patients carrying the A30P mutation typically exhibit a similar age of onset and symptoms as sporadic PD, while those carrying the A53T mutation generally have an earlier age of onset and an accelerated progression. We report two C. elegans models of PD (PDA30P and PDA53T), which express these mutational variants in the muscle cells, and probed their behavior relative to animals expressing the wild-type protein (PDWT). PDA30P worms showed a reduced speed of movement and an increased paralysis rate, control worms, but no change in the frequency of body bends. By contrast, in PDA53T worms both speed and frequency of body bends were significantly decreased, and paralysis rate was increased. α-Synuclein was also observed to be less well localized into aggregates in PDA30P worms compared to PDA53T and PDWT worms, and amyloid-like features were evident later in the life of the animals, despite comparable levels of expression of α-synuclein. Furthermore, squalamine, a natural product currently in clinical trials for treating symptomatic aspects of PD, was found to reduce significantly the aggregation of α-synuclein and its associated toxicity in PDA53T and PDWT worms, but had less marked effects in PDA30P. In addition, using an antibody that targets the N-terminal region of α-synuclein, we observed a suppression of toxicity in PDA30P, PDA53T and PDWT worms. These results illustrate the use of these two C. elegans models in fundamental and applied PD research.

Reproductive events and respective faecal androgen metabolite concentrations in captive male roan antelope (Hippotragus equinus).

Understanding the reproductive biology of the roan antelope (Hippotragus equinus) (É. Geoffroy Saint-Hilaire, 1803) is crucial to optimise breeding success in captive breeding programmes of this threatened species. In this study, the pattern of faecal androgen metabolite (fAM) production related to reproductive events (calving or birthing, mating, gestation, and lactation), sexual behaviours as well as environmental cues were studied in captive adult male roan antelope. Faecal sample collection and behavioural observations were carried out from August 2017 to July 2018 for three reproductive males participating in a conservation breeding programme at the Lapalala Wilderness Nature Reserve in South Africa. As a prerequisite, the enzyme immunoassay used in this study was biologically validated for the species by demonstrating a significant difference between fAM concentrations in non-breeding adults, breeding adults and juvenile males. Results revealed that in adults males, the overall mean fAM levels were 73% higher during the breeding period compared to the non-breeding periods, and 85% higher when exclusively compared to the lactation/gestation periods, but only 5.3% higher when compared to the birthing period. Simultaneously, fAM concentrations were lower during the wet season compared to the dry season, increasing with a reduction in photoperiod. With the exception of courtship, frequencies of sexual behaviours monitored changed in accordance with individual mean fAM concentrations in male roan antelope, the findings suggest that androgen production varies with the occurrence of mating activity and may be influenced by photoperiod but not with rainfall.

Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

Profiling patterns of fecal 20-oxopregnane concentrations during ovarian cycles in free-ranging southern white rhinoceros (Ceratotherium simum simum).

Unlike their wild counterparts, many white rhinoceros females in captivity fail to reproduce successfully such that current captive populations are not self-sustaining. The causes of the problem are poorly understood. Variation in cycle length and long periods of acyclicity are characteristics of the majority of these non-reproducing females in captivity but it is unknown whether these characteristics are a feature of reproductively successful free-ranging females. This study therefore aimed to monitor cyclic activity in a wild population of southern white rhinoceros at Lapalala Wilderness, South Africa, by measuring the concentrations of immunoreactive fecal progestagen metabolites (fPM). Five adult females were tracked twice per week for 20 months and if located a fresh fecal sample was collected. Reproductive events and group structural dynamics were also recorded and subsequently correlated with the fPM data. The baseline concentration of fPM was 0.69±0.20µg/g DW while concentrations during pregnancy were 30-400-fold higher. The females exhibited estrous cycle lengths of 30.6±7.7 days and, based on fPM data, gestation length in one female was 502±3 days. Year-round monitoring showed no clear evidence of seasonality in ovarian activity. During cyclic luteal activity females were often seen in the presence of a dominant bull. One female stopped cycling after removal of the local dominant bull and luteal activity only returned after a new bull was introduced. This suggests that white rhinoceros females in the wild might need external stimuli from a male to ovulate. These findings indicate that the irregular cyclicity reported for white rhinoceros housed in zoos and animal parks may result from conditions in captivity and account for reduced fertility.

Tryptophan metabolism: entering the field of aging and age-related pathologies.

Aging is an important risk factor for many debilitating diseases, including cancer and neurodegeneration. In model organisms, interfering with metabolic signaling pathways, including the insulin/insulin-like growth factor (IGF) 1 (IIS) and TOR pathways, can protect against age-related pathologies and increase lifespan. Recent studies in multiple organisms have implicated tryptophan metabolism as a powerful regulator of age-related diseases and lifespan. Its high conservation throughout evolution has enabled studies that begin to dissect the contribution of individual enzymes and metabolites. Here, we focus on the emerging view of tryptophan metabolism as a pathway that integrates environmental and metabolic signals to regulate animal biology and health.

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation.

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer's diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-ß and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra L-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

A FRET sensor for non-invasive imaging of amyloid formation in vivo.

Misfolding and aggregation of amyloidogenic polypeptides lie at the root of many neurodegenerative diseases. Whilst protein aggregation can be readily studied in vitro by established biophysical techniques, direct observation of the nature and kinetics of aggregation processes taking place in vivo is much more challenging. We describe here, however, a Förster resonance energy transfer sensor that permits the aggregation kinetics of amyloidogenic proteins to be quantified in living systems by exploiting our observation that amyloid assemblies can act as energy acceptors for variants of fluorescent proteins. The observed lifetime reduction can be attributed to fluorescence energy transfer to intrinsic energy states associated with the growing amyloid species. Indeed, for a-synuclein, a protein whose aggregation is linked to Parkinson's disease, we have used this sensor to follow the kinetics of the self-association reactions taking place in vitro and in vivo and to reveal the nature of the ensuing aggregated species. Experiments were conducted in vitro, in cells in culture and in living Caenorhabditis elegans. For the latter the readout correlates directly with the appearance of a toxic phenotype. The ability to measure the appearance and development of pathogenic amyloid species in a living animal and the ability to relate such data to similar processes observed in vitro provides a powerful new tool in the study of the pathology of the family of misfolding disorders. Our study confirms the importance of the molecular environment in which aggregation reactions take place, highlighting similarities as well as differences between the processes occurring in vitro and in vivo, and their significance for defining the molecular physiology of the diseases with which they are associated.

Identification of MOAG-4/SERF as a regulator of age-related proteotoxicity.

Fibrillar protein aggregates are the major pathological hallmark of several incurable, age-related, neurodegenerative disorders. These aggregates typically contain aggregation-prone pathogenic proteins, such as amyloid-beta in Alzheimer's disease and alpha-synuclein in Parkinson's disease. It is, however, poorly understood how these aggregates are formed during cellular aging. Here we identify an evolutionarily highly conserved modifier of aggregation, MOAG-4, as a positive regulator of aggregate formation in C. elegans models for polyglutamine diseases. Inactivation of MOAG-4 suppresses the formation of compact polyglutamine aggregation intermediates that are required for aggregate formation. The role of MOAG-4 in driving aggregation extends to amyloid-beta and alpha-synuclein and is evolutionarily conserved in its human orthologs SERF1A and SERF2. MOAG-4/SERF appears to act independently from HSF-1-induced molecular chaperones, proteasomal degradation, and autophagy. Our results suggest that MOAG-4/SERF regulates age-related proteotoxicity through a previously unexplored pathway, which will open up new avenues for research on age-related, neurodegenerative diseases.

KBP interacts with SCG10, linking Goldberg-Shprintzen syndrome to microtubule dynamics and neuronal differentiation.

Goldberg-Shprintzen syndrome (GOSHS) is a rare clinical disorder characterized by central and enteric nervous system defects. This syndrome is caused by inactivating mutations in the Kinesin Binding Protein (KBP) gene, which encodes a protein of which the precise function is largely unclear. We show that KBP expression is up-regulated during neuronal development in mouse cortical neurons. Moreover, KBP-depleted PC12 cells were defective in nerve growth factor-induced differentiation and neurite outgrowth, suggesting that KBP is required for cell differentiation and neurite development. To identify KBP interacting proteins, we performed a yeast two-hybrid screen and found that KBP binds almost exclusively to microtubule associated or related proteins, specifically SCG10 and several kinesins. We confirmed these results by validating KBP interaction with one of these proteins: SCG10, a microtubule destabilizing protein. Zebrafish studies further demonstrated an epistatic interaction between KBP and SCG10 in vivo. To investigate the possibility of direct interaction between KBP and microtubules, we undertook co-localization and in vitro binding assays, but found no evidence of direct binding. Thus, our data indicate that KBP is involved in neuronal differentiation and that the central and enteric nervous system defects seen in GOSHS are likely caused by microtubule-related defects.

Chaperone proteostasis in Parkinson's disease: stabilization of the Hsp70/alpha-synuclein complex by Hip.

The ATP-dependent protein chaperone heat-shock protein 70 (Hsp70) displays broad anti-aggregation functions and has a critical function in preventing protein misfolding pathologies. According to in vitro and in vivo models of Parkinson's disease (PD), loss of Hsp70 activity is associated with neurodegeneration and the formation of amyloid deposits of alpha-synuclein (alphaSyn), which constitute the intraneuronal inclusions in PD patients known as Lewy bodies. Here, we show that Hsp70 depletion can be a direct result of the presence of aggregation-prone polypeptides. We show a nucleotide-dependent interaction between Hsp70 and alphaSyn, which leads to the aggregation of Hsp70, in the presence of ADP along with alphaSyn. Such a co-aggregation phenomenon can be prevented in vitro by the co-chaperone Hip (ST13), and the hypothesis that it might do so also in vivo is supported by studies of a Caenorhabditis elegans model of alphaSyn aggregation. Our findings indicate that a decreased expression of Hip could facilitate depletion of Hsp70 by amyloidogenic polypeptides, impairing chaperone proteostasis and stimulating neurodegeneration.

Stem cell therapy to reduce radiation-induced normal tissue damage.

Normal tissue damage after radiotherapy is still a major problem in cancer treatment. Stem cell therapy may provide a means to reduce radiation-induced side effects and improve the quality of life of patients. This review discusses the current status in stem cell research with respect to their potential to reduce radiation toxicity. A number of different types of stem cells are being investigated for their potential to treat a variety of disorders. Their current status, localization, characterization, isolation, and potential in stem cell-based therapies are addressed. Although clinical adult stem cell research is still at an early stage, preclinical experiments show the potential these therapies may have. Based on the major advances made in this field, stem cell-based therapy has great potential to allow prevention or treatment of normal tissue damage after radiotherapy.

Thiol-disulphide oxidoreductase modules in the low-GC Gram-positive bacteria.

Disulphide bond formation catalysed by thiol-disulphide oxidoreductases (TDORs) is a universally conserved mechanism for stabilizing extracytoplasmic proteins. In Escherichia coli, disulphide bond formation requires a concerted action of distinct TDORs in thiol oxidation and subsequent quinone reduction. TDOR function in other bacteria has remained largely unexplored. Here we focus on TDORs of low-GC Gram-positive bacteria, in particular DsbA of Staphylococcus aureus and BdbA-D of Bacillus subtilis. Phylogenetic analyses reveal that the homologues DsbA and BdbD cluster in distinct groups typical for Staphylococcus and Bacillus species respectively. To compare the function of these TDORs, DsbA was produced in various bdb mutants of B. subtilis. Next, we assessed the ability of DsbA to sustain different TDOR-dependent processes, including heterologous secretion of E. coli PhoA, competence development and bacteriocin (sublancin 168) production. The results show that DsbA can function in all three processes. While BdbD needs a quinone oxidoreductase for activity, DsbA activity appears to depend on redox-active medium components. Unexpectedly, both quinone oxidoreductases of B. subtilis are sufficient to sustain production of sublancin. Moreover, DsbA can functionally replace these quinone oxidoreductases in sublancin production. Taken together, our unprecedented findings imply that TDOR systems of low-GC Gram-positive bacteria have a modular composition.