A Chronic Sleep Fragmentation Model using Vibrating Orbital Rotor to Induce Cognitive Deficit and Anxiety-Like Behavior in Young Wild-Type Mice.

Sleep disturbance is generally common in populations as a chronic disease or a complained event. Chronic sleep disturbance is proposed to be closely linked to the pathogenesis of diseases, especially neurodegenerative diseases. We recently found that 2 months of sleep fragmentation initiated Alzheimer's disease (AD)-like behavioral and pathological changes in young wild-type mice. Herein, we present a standardized protocol to achieve chronic sleep fragmentation (CSF). Briefly, CSF was induced by an orbital rotor vibrating at 110 rpm and operating with a repetitive cycle of 10 s-on, 110 s-off, during light-ON phase (8:00 AM-8:00 PM) continuously for up to 2 months. Impairments of spatial learning and memory, anxiety-like but not depression-like behavior in mice as consequences of CSF modeling, were evaluated with Morris water maze (MWM), Novel object recognition (NOR), Open field test (OFT) and Forced swimming test (FST). In comparison with other sleep manipulations, this protocol minimizes the handling labors and maximizes the modeling efficiency. It produces stable phenotypes in young wild-type mice and can be potentially generated for a variety of research purposes.

Chronic sleep fragmentation shares similar pathogenesis with neurodegenerative diseases: Endosome-autophagosome-lysosome pathway dysfunction and microglia-mediated neuroinflammation.

AIMS: Insufficient sleep has been found to result in varying degrees of cognitive impairment and emotional changes. Sleep was reported probably responsible for cleaning metabolic wastes in brain by increasing extracellular bulk flow. Herein, we propose that chronic sleep insufficiency in young adult wild-type mice is also linked with dysfunction of intracellular protein degradation pathways and microglia-mediated neuroinflammation, which are potentially important mechanisms in the initiation of neurodegeneration. METHODS: We applied the chronic sleep fragmentation (CSF) model to induce chronic sleep insufficiency in wild-type mice. After 2 months of CSF, cognitive function, amyloid-ß accumulation, dysfunction of endosome-autophagosome-lysosome pathway, and microglia activation were evaluated. RESULTS: Following CSF, impairment of spatial learning and memory, and aggravated anxiety-like behavior in mice were identified by behavioral experiments. Increased intracellular amyloid-ß accumulation was observed in cortex and hippocampus. Mechanistically, CSF could significantly enhance the expression of Rab5 (early endosome marker), Rab7 (late endosome marker), as well as LC3B (autophagosome marker), and autophagy-positive regulatory factors in brain detected by immunofluorescent staining and Western blot. In addition, activation of microglia was evident by enhanced CD68, CD16/32, and CD206 levels after CSF treatment. CONCLUSIONS: Chronic sleep fragmentation could initiate pathogenetic processes similar to the early stage of neurodegeneration, including dysfunction of endosome-autophagosome-lysosome pathway and microglia-mediated neuroinflammation. Our findings further strengthen the link between chronic sleep insufficiency and the initiation of neurodegeneration even if lack of genetic predisposition.

The small GTPase MoYpt7 is required for membrane fusion in autophagy and pathogenicity of Magnaporthe oryzae.

Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.