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

Abstract

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-β 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-β 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.

Keywords: Alzheimer's disease; amyloid-β; autophagy; lysosome; microglia; sleep fragmentation.

Figure 1

The schematic figure of experimental design procedure. A, The experimental design procedure that indicates the timing of CSF model, behavioral tests, immunofluorescence, electron microscopy, and Western blot. B, Body weight curves of the CSF and control mice during the first month after the CSF model were established

Figure 2

CSF aggravated cognitive impairment and increased intracellular Aβ accumulation. A, Left, escape latency in acquisition phase; middle and right, percentage time in the platform quadrant and times of crossing the platform in probe trial. Representative tracings of two groups were shown in the upper panel. n = 10 per group, *P < .05, **P < .01, ****P < .0001. B, Left, exploration time of A1 and A2, respectively, by mice in familiar phase; right, discrimination index (DI) in the test phase. n = 10 per group, n.s. indicates no significant changes between different groups, *P < .05. C, Left, time in the central zone; right, total distance travelled by the mice. Representative tracings of two groups were shown in the upper panel. n = 10 per group, *P < .05. D, Immobility time during the last 4 min of the test. n = 10 per group, n.s. indicates no significant changes between different groups. E, Representative immunofluorescence images of intracellular Aβ accumulation in the cortex and hippocampus of CSF and control mice. Scale bar = 40 μm. Local enlarged images were presented in the white squares. Scale bar = 10 μm. F, Quantitative analysis of the number of Aβ⁺ cells was shown in the histogram, n = 5 per group, *P < .05, **P < .01

Figure 3

Intracellular endosomes and lysosomes in the cortex were dysregulated after CSF. A, Representative confocal images of coronal sections labeled by Rab5, Rab7, and Lamp1 staining in the cortex of CSF and control mice. Scale bar = 40 μm. Local enlarged images were presented in the white squares. Scale bar = 10 μm. B, Representative Western blots showing the changes in the expression of Rab5, Rab7, and Lamp1 in the cortex of CSF and control mice. C, Histograms showed the quantitative density of cells immunoreactive for Rab5, Rab7, and Lamp1. n = 5 per group, *P < .05, **P < .01. D‐F, Quantitative analysis of the Rab5, Rab7, and Lamp1 protein levels was performed. Protein expression levels were normalized to the level of β‐actin. n = 5 per group, *P < .05, **P < .01, ***P < .001

Figure 4

CSF caused enlargement and increase in intracellular lysosomes in the cortex detected by electron microscopy. A‐B, Representative electron photomicrographs of neurons from mice in the CSF and control groups. Scale bar = 5 μm. C‐D, Higher magnification images of the rectangle area in (A‐B), hollow arrows marked intracellular lysosomes. Scale bar = 2 μm

Figure 5

Intracellular autophagy process in the cortex was disordered after CSF. A, Representative confocal images of coronal sections labeled by LC3B, Beclin 1, and UVRAG staining in the cortex of CSF and control mice. Scale bar = 40 μm. Local enlarged images were presented in the white squares. Scale bar = 10 μm. B, Representative Western blots showing the changes in the expression of LC3B‐II, Beclin 1, and UVRAG in the cortex of CSF and control mice. C, Histograms showed the quantitative density of cells immunoreactive for LC3B, Beclin1, and UVRAG. n = 5 per group,*P < .05, **P < .01. D‐F, Quantitative analysis of the LC3B‐II, Beclin1, and UVRAG protein levels was performed. The protein expression levels were normalized to the level of β‐actin. n = 5 per group, *P < .05, **P < .01

Figure 6

Activation of microglia in the hippocampus was induced by CSF. A, Representative confocal images labeled with Iba1 (red) and CD68 (green) in the hippocampus of CSF and control mice. Scale bar = 40 μm. Local enlarged images were presented in the white squares. Scale bar = 10 μm. B‐C, Statistical analysis of Iba1⁺ cells and CD68⁺ cells was shown in the histograms. n = 5 per group, **P < .01

Figure 7

M1 and M2 polarized microglia in the hippocampus were activated after CSF. A, The M1 phenotype of microglia was detected by double staining for CD16/32 (green) and Iba1 (red), while the M2 phenotype was detected by double staining for CD206 (green) and Iba1 (red) in the hippocampus of CSF and control mice. Scale bar = 40 μm. Local enlarged images were presented in the white squares. Scale bar = 10 μm. B‐C, Quantitative analysis of Iba1⁺CD16/32⁺ double‐positive cells and Iba1⁺CD206⁺ double‐positive cells was shown in the histogram. n = 5 per group, **P < .01. D, Representative Western blots of CD16/32, iNOS, and CD206 in the hippocampus of CSF and control mice. E, Statistical analysis of Western blots of CD16/32, iNOS, and CD206 in the CSF and control groups. Protein expression levels were normalized to the level of β‐actin. n = 5 per group, *P < .05, **P < .01, ***P < .001

Figure 8

Schematic model of how CSF induced accumulation of endosomes, autophagosomes, and lysosomes, which led to increased intracellular Aβ accumulation. In normal conditions, the trafficking, catabolism, and elimination of APP are via the EAL pathway. First, APP is internalized into cells by endocytosis, then sorted in the early endosomes, and delivered to the late endosomes. APP can be processed by APP secretases to form Aβ in this stage. Then, the late endosomes fuse with either lysosomes or autophagosomes for lysosomal degradation. The balance of formation and clearance of Aβ is maintained by normal EAL pathway. After 2‐mo CSF, the EAL pathway is dysregulated and autophagic flux flow is impaired, leading to accumulation of abnormal vesicles in cells. It is evident by increased expression of Rab5 (early endosome marker), Rab7 (late endosome marker), Lamp1 (lysosome marker), as well as LC3B (autophagosome marker), and autophagy‐positive regulatory factors Beclin 1 and UVRAG. The accelerated APP processing in EAL pathway results in imbalance of formation and clearance of intracellular Aβ. In pathogenic process of Alzheimer's disease, overloaded intracellular Aβ can be released to extracellular space, potentially forming Aβ plaques eventually via oligomer polyermerization