Spaceflight affects neuronal morphology and alters transcellular degradation of neuronal debris in adult Caenorhabditis elegans.

Extended space travel is a goal of government space agencies and private companies. However, spaceflight poses risks to human health, and the effects on the nervous system have to be better characterized. Here, we exploited the unique experimental advantages of the nematode Caenorhabditis elegans to explore how spaceflight affects adult neurons in vivo. We found that animals that lived 5 days of adulthood on the International Space Station exhibited hyperbranching in PVD and touch receptor neurons. We also found that, in the presence of a neuronal proteotoxic stress, spaceflight promotes a remarkable accumulation of neuronal-derived waste in the surrounding tissues, suggesting an impaired transcellular degradation of debris released from neurons. Our data reveal that spaceflight can significantly affect adult neuronal morphology and clearance of neuronal trash, highlighting the need to carefully assess the risks of long-duration spaceflight on the nervous system and to develop adequate countermeasures for safe space exploration.

Molecular Muscle Experiment: Hardware and Operational Lessons for Future Astrobiology Space Experiments.

Biology experiments in space seek to increase our understanding of what happens to life beyond Earth and how we can safely send life beyond Earth. Spaceflight is associated with many (mal)adaptations in physiology, including decline in musculoskeletal, cardiovascular, vestibular, and immune systems. Biological experiments in space are inherently challenging to implement. Development of hardware and validation of experimental conditions are critical to ensure the collection of high-quality data. The model organism Caenorhabditis elegans has been studied in space for more than 20 years to better understand spaceflight-induced (patho)physiology, particularly spaceflight-induced muscle decline. These experiments have used a variety of hardware configurations. Despite this, hardware used in the past was not available for our most recent experiment, the Molecular Muscle Experiment (MME). Therefore, we had to design and validate flight hardware for MME. MME provides a contemporary example of many of the challenges faced by researchers conducting C. elegans experiments onboard the International Space Station. Here, we describe the hardware selection and validation, in addition to the ground-based experiment scientific validation testing. These experiences and operational solutions allow others to replicate and/or improve our experimental design on future missions.

A comparison of the mitochondrial proteome and lipidome in the mouse and long-lived Pipistrelle bats.

It is accepted that smaller mammals with higher metabolic rates have shorter lifespans. The very few species that do not follow these rules can give insights into interesting differences. The recorded maximum lifespans of bats are exceptional - over 40 years, compared with the laboratory mouse of 4 years. We investigated the differences in the biochemical composition of mitochondria between bat and mouse species. We used proteomics and ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry lipidomics, to interrogate mitochondrial fractions prepared from Mus musculus and Pipistrellus pipistrellus brain and skeletal muscle. Fatty acid binding protein 3 was found at different levels in mouse and bat muscle mitochondria and its orthologues were investigated in Caenorhabditis elegans knock-downs for LBP 4, 5 and 6. In the bat, high levels of free fatty acids and N-acylethanolamine lipid species together with a significantly greater abundance of fatty acid binding protein 3 in muscle (1.8-fold, p=0.037) were found. Manipulation of fatty acid binding protein orthologues in C. elegans suggest these proteins and their role in lipid regulation are important for mitochondrial function.

Muscle strength deficiency and mitochondrial dysfunction in a muscular dystrophy model of Caenorhabditis elegans and its functional response to drugs.

Muscle strength is a key clinical parameter used to monitor the progression of human muscular dystrophies, including Duchenne and Becker muscular dystrophies. Although Caenorhabditis elegans is an established genetic model for studying the mechanisms and treatments of muscular dystrophies, analogous strength-based measurements in this disease model are lacking. Here, we describe the first demonstration of the direct measurement of muscular strength in dystrophin-deficient C. elegans mutants using a micropillar-based force measurement system called NemaFlex. We show that dys-1(eg33) mutants, but not dys-1(cx18) mutants, are significantly weaker than their wild-type counterparts in early adulthood, cannot thrash in liquid at wild-type rates, display mitochondrial network fragmentation in the body wall muscles, and have an abnormally high baseline mitochondrial respiration. Furthermore, treatment with prednisone, the standard treatment for muscular dystrophy in humans, and melatonin both improve muscular strength, thrashing rate and mitochondrial network integrity in dys-1(eg33), and prednisone treatment also returns baseline respiration to normal levels. Thus, our results demonstrate that the dys-1(eg33) strain is more clinically relevant than dys-1(cx18) for muscular dystrophy studies in C. elegans This finding, in combination with the novel NemaFlex platform, can be used as an efficient workflow for identifying candidate compounds that can improve strength in the C. elegans muscular dystrophy model. Our study also lays the foundation for further probing of the mechanism of muscle function loss in dystrophin-deficient C. elegans, leading to knowledge translatable to human muscular dystrophy.This article has an associated First Person interview with the first author of the paper.

Greater loss of mitochondrial function with ageing is associated with earlier onset of sarcopenia in C. elegans.

Sarcopenia, the age-related decline of muscle, is a significant and growing public health burden. C. elegans, a model organism for investigating the mechanisms of ageing, also displays sarcopenia, but the underlying mechanism(s) remain elusive. Here, we use C. elegans natural scaling of lifespan in response to temperature to examine the relationship between mitochondrial content, mitochondrial function, and sarcopenia. Mitochondrial content and maximal mitochondrial ATP production rates (MAPR) display an inverse relationship to lifespan, while onset of MAPR decline displays a direct relationship. Muscle mitochondrial structure, sarcomere structure, and movement decline also display a direct relationship with longevity. Notably, the decline in mitochondrial network structure occurs earlier than sarcomere decline, and correlates more strongly with loss of movement, and scales with lifespan. These results suggest that mitochondrial function is critical in the ageing process and more robustly explains the onset and progression of sarcopenia than loss of sarcomere structure.

Mouse mitochondrial lipid composition is defined by age in brain and muscle.

Functionality of the lipid rich mitochondrial organelle declines with increased age. Recent advances in lipidomic technologies allowed us to perform a global characterisation of lipid composition in two different tissue types and age ranges. Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry was used to establish and compare mitochondrial lipidomes of brain and skeletal muscle from young (4-11 weeks old) and middle age (78 weeks old) healthy mice. In middle age the brain mitochondria had reduced levels of fatty acids, particularly polyunsaturated fatty acids, while skeletal muscle mitochondria had a decreased abundance of phosphatidylethanolamine, but a pronounced increase of triglyceride levels. Reduced levels of phosphatidylethanolamines are known to decrease mitochondrial membrane fluidity and are connected with accelerated ageing. In mitochondria from skeletal muscle we propose that increased age causes a metabolic shift in the conversion of diacylglycerol so that triglycerides predominate compared with phosphatidylethanolamines. This is the first time mitochondrial lipid content in normal healthy mammalian ageing brain and muscle has been catalogued in such detail across all lipid classes. We identify distinct mitochondrial lipid signatures that change with age, revealing tissue-specific lipid pathways as possible targets to ameliorate ageing-related mitochondrial decline.

Mitochondrial proteomic profiling reveals increased carbonic anhydrase II in aging and neurodegeneration.

Carbonic anhydrase inhibitors are used to treat glaucoma and cancers. Carbonic anhydrases perform a crucial role in the conversion of carbon dioxide and water into bicarbonate and protons. However, there is little information about carbonic anhydrase isoforms during the process of ageing. Mitochondrial dysfunction is implicit in ageing brain and muscle. We have interrogated isolated mitochondrial fractions from young adult and middle aged mouse brain and skeletal muscle. We find an increase of tissue specific carbonic anhydrases in mitochondria from middle-aged brain and skeletal muscle. Mitochondrial carbonic anhydrase II was measured in the Purkinje cell degeneration (pcd5J) mouse model. In pcd5J we find mitochondrial carbonic anhydrase II is also elevated in brain from young adults undergoing a process of neurodegeneration. We show C.elegans exposed to carbonic anhydrase II have a dose related shorter lifespan suggesting that high CAII levels are in themselves life limiting. We show for the first time that the mitochondrial content of brain and skeletal tissue are exposed to significantly higher levels of active carbonic anhydrases as early as in middle-age. Carbonic anhydrases associated with mitochondria could be targeted to specifically modulate age related impairments and disease.

Mitochondrial Complex 1 Activity Measured by Spectrophotometry Is Reduced across All Brain Regions in Ageing and More Specifically in Neurodegeneration.

Mitochondrial function, in particular complex 1 of the electron transport chain (ETC), has been shown to decrease during normal ageing and in neurodegenerative disease. However, there is some debate concerning which area of the brain has the greatest complex 1 activity. It is important to identify the pattern of activity in order to be able to gauge the effect of age or disease related changes. We determined complex 1 activity spectrophotometrically in the cortex, brainstem and cerebellum of middle aged mice (70-71 weeks), a cerebellar ataxic neurodegeneration model (pcd5J) and young wild type controls. We share our updated protocol on the measurements of complex1 activity and find that mitochondrial fractions isolated from frozen tissues can be measured for robust activity. We show that complex 1 activity is clearly highest in the cortex when compared with brainstem and cerebellum (p<0.003). Cerebellum and brainstem mitochondria exhibit similar levels of complex 1 activity in wild type brains. In the aged brain we see similar levels of complex 1 activity in all three-brain regions. The specific activity of complex 1 measured in the aged cortex is significantly decreased when compared with controls (p<0.0001). Both the cerebellum and brainstem mitochondria also show significantly reduced activity with ageing (p<0.05). The mouse model of ataxia predictably has a lower complex 1 activity in the cerebellum, and although reductions are measured in the cortex and brain stem, the remaining activity is higher than in the aged brains. We present clear evidence that complex 1 activity decreases across the brain with age and much more specifically in the cerebellum of the pcd5j mouse. Mitochondrial impairment can be a region specific phenomenon in disease, but in ageing appears to affect the entire brain, abolishing the pattern of higher activity in cortical regions.

Persistent prelimbic cortex activity contributes to enhanced learned fear expression in females.

Anxiety disorders, such as post-traumatic stress, are more prevalent in women and are characterized by impaired inhibition of learned fear and medial prefrontal cortex (mPFC) dysfunction. Here we examined sex differences in fear extinction and mPFC activity in rats. Females showed more learned fear expression during extinction and its recall, but not fear conditioning. They also showed more spontaneous fear recovery and more contextual fear before extinction and its recall. Moreover, enhanced learned fear expression in females was associated with sustained prelimbic (PL) cortex activity. These results suggest that sex differences in learned fear expression may involve persistent PL activation.