Huiyang Wang,Yueyang Liu,Zhenkun Guo,Minghui Cui,Peng Pang,Jingyu Yang,Chunfu Wu
Enhancement of oligodendrocyte autophagy alleviates white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in rats
Chronic cerebral hypoperfusion,Two vessel occlusion,White matter,Cognitive impairment,Oligodendrocyte,Myelin sheath,Autophagy-lysosomal pathway,Mechanistic target of rapamycin
Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy–lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.
Enhancement of oligodendrocyte autophagy alleviates white matter injury and cognitive impairment induced by chronic cerebral hypoperfusion in rats

Cognitive impairment caused by chronic cerebral hypoperfusion (CCH) is associated with white matter injury (WMI), possibly through the alteration of autophagy. Here, the autophagy–lysosomal pathway (ALP) dysfunction in white matter (WM) and its relationship with cognitive impairment were investigated in rats subjected to two vessel occlusion (2VO). The results showed that cognitive impairment occurred by the 28th day after 2VO. Injury and autophagy activation of mature oligodendrocytes and neuronal axons sequentially occurred in WM by the 3rd day. By the 14th day, abnormal accumulation of autophagy substrate, lysosomal dysfunction, and the activation of mechanistic target of rapamycin (MTOR) pathway were observed in WM, paralleled with mature oligodendrocyte death. This indicates autophagy activation was followed by ALP dysfunction caused by autophagy inhibition or lysosomal dysfunction. To target the ALP dysfunction, enhanced autophagy by systemic rapamycin treatment or overexpression of Beclin1 (BECN1) in oligodendrocytes reduced mature oligodendrocyte death, and subsequently alleviated the WMI and cognitive impairment after CCH. These results reveal that early autophagy activation was followed by ALP dysfunction in WM after 2VO, which was associated with the aggravation of WMI and cognitive impairment. This study highlights that alleviating ALP dysfunction by enhancing oligodendrocyte autophagy has benefits for cognitive recovery after CCH.

Chronic cerebral hypoperfusion (CCH) caused by cerebrovascular pathologies has been considered as an important pathological factor for mild cognitive impairment, vascular dementia, and Alzheimer's disease1, 2, 3. It is well-known that CCH induces significant white matter injury (WMI), characterized by demyelination and loss of axonal integrity. Since oligodendrocytes are myelin-producing cells which carry out axonal myelination under normal physiological conditions and remyelination after axonal injury,, promoting the survival of oligodendrocytes may have benefits for cognitive recovery after CCH. The autophagy–lysosomal pathway (ALP) carries out lysosome-dependent self-phagocytosis of cellular components in order to maintain the metabolic balance and recycling of intracellular substances. Several studies have demonstrated that ALP also plays an important role in white matter (WM) demyelination diseases of the central nervous system (CNS). Improving the impaired autophagy flux in oligodendrocytes can improve cell viability in vanishing white matter disease and globoid cell leukodystrophy, and promote myelination in CNS myelin developmental disorders. Notably, it has been reported that the expression of microtubule-associated protein 1 light chain 3 (LC3) increases at first and then decreases, accompanied by the opposite trend in the level of soluble sequestosome 1 (SQSTM1) in WM after CCH in mice. These data indicate a possible role of autophagy in the pathophysiology of WMI though the underlying mechanism remains to be elucidated. Therefore, the abnormal ALP function in WM and its relationship with cognitive impairment were investigated in a rat model of CCH caused by two vessel occlusion (2VO). The results showed that in 2VO-operated rats, autophagy was activated in mature oligodendrocytes, followed by ALP dysfunction and neuronal axonal injury. Furthermore, these changes contributed to the aggravation of WMI and cognitive impairment. Importantly, enhancement of autophagy by either systemic treatment with rapamycin (Rapa) or oligodendrocyte-specific overexpression of Beclin1 (BECN1) markedly improved the ALP dysfunction and reduced mature oligodendrocyte death, which further alleviated WMI and delayed the progression of cognitive impairment of 2VO-operated rats. The present study highlights the important role of oligodendrocyte autophagy in regulating CCH-induced cognitive decline.

Adult male Sprague–Dawley rats, 8 weeks old and weighing 280–300 g, were obtained from Changsheng Company [SCXK (Liao) 2020-0001, Benxi, Liaoning, China]. Rats were given free access to water and food and housed in a standard experimental environment (12 h light–12 h dark cycle, 21–24 °C). All experimental procedures were designed to minimize the pain of rats and reduce the number of rats used in experiments, and carried out according to the Regulations for the Administration of Affairs Concerning Experimental Animals of China. All animal experiments were approved (Registration nos. SYPU-IACUC-C2020-6-22-206, SYPU-IACUC-C2021-6-2-203, SYPU-IACUC-C2021-8-2-202, SYPU-IACUC-C2021-9-27-206) by the Experimental Animals Ethics Committee of Shenyang Pharmaceutical University (Shenyang, China).

Rats were continuously anesthetized with isoflurane (RWD Life science Co., Ltd., Shenzhen, China) and oxygen mixture in the supine position, and the common carotid arteries were exposed via a ventral midline neck incision. The right common carotid artery was first gently separated from the vagus nerve and then ligated. The wound was sutured. One week later, the left common carotid artery was ligated in the same manner. Sham group experienced the same procedures except carotid artery ligation. All rats were randomly assigned to the sham and 2VO groups. For administration of lysosomal inhibitor, chloroquine (CQ) (Sigma, St. Louis, MO, USA) was dissolved in normal saline, and was administered to rats by intraperitoneal (i.p.) injection (20 mg/kg) once a day for 3 days before tissue collection13, 14, 15. For administration of mechanistic target of rapamycin (MTOR) inhibitor, Rapa (Yuanye Bio-Technology, Shanghai, China) was dissolved in a mixture of ethanol, polyethylene glycol-400 and Tween 80, and i.p.-injected (3 mg/kg) once a day from the onset of 2VO for 14 consecutive days,.

CBF was recorded on both sides of the cerebral cortex before and 0.5 h after right common carotid artery occlusion (RCCAO), and then before and 0.5 h after bilateral common carotid artery occlusion (BCCAO), using an RFLSI Pro+ laser speckle instrument (RWD Life Science Co., Ltd., Shenzhen, China). Throughout the measurement of CBF, rats were anesthetized with a mixture of isoflurane and oxygen and placed prone in the stereotactic frame. The skin was opened to expose the skull. The skull was thinned with a high-speed dental drill (STRONGWT, Jiangsu, China) until the blood vessels were clearly visible. During the grinding process, the skull was chilled with cold normal saline to reduce the brain injury caused by heat released from grinding. Laser speckle blood flow images were recorded and CBF was quantified in the same region of interest in both hemispheres (Supporting Information Fig. S1A).

The top of the spontaneous movement box was equipped with a box camera and infrared light source, which automatically recorded the spontaneous movement of rats in the box at the beginning of the experiment. The rats were put into the activity box gently, then the door of the box was closed, and the video recording was started. After the rats explored freely for 5 min, the video recording was stopped. The collected videos were analyzed using EthoVision XT 8.0 (Noldus Information Technology, Netherlands).

The NOR test was divided into three stages. The first stage was the adaptation stage: the rats explored freely in the experiment box without any objects. The second stage was the training stage: the rats explored the box, which contained two objects of similar shape on the same side. The third stage was the detection stage: before the experiment started, one of the two objects was replaced with a new object. Two detection stages were carried out, at 1 and 24 h after the training stage. The new objects introduced in the two detection stages were different. The rats were free to explore for 5 min in each stage, and video recording started at the same time. The collected video was analyzed by EthoVision XT 8.0 to analyze the time of the experimental animals spent exploring the different objects. The discrimination index was calculated by the ratio of the time spent exploring new objects to the time spent exploring familiar objects and the new objects in the test session.

The Y maze device consisted of three arms (named A, B and C) with the same specifications at an angle of 120°. In the experiment, rats were randomly placed in the central triangle area of the maze with their backs to the wall and were allowed to explore freely in the maze for 8 min. The total number of arm entries of each rat (the whole body except the tail of the rat entered the arm once) and the order of arm entry were recorded, and the spontaneous alternation response rate was calculated.

The MWM test device was made of a circular pool (160 cm × 60 cm) filled with black opaque water (23 ± 1 °C). The pool was divided equally into four quadrants, with a black platform (12 cm) submerged 2 cm below the surface of the water in the target quadrant. Visual cues were placed around the maze to help the rats find the hidden platform. In the navigation test, each rat was placed into the pool from four quadrants every day and was allowed to locate the hidden platform for 60 s each time. Escape latency and average velocity were recorded. During the probe test, the hidden platform was removed from the pool. The rats were placed into the pool from the quadrant opposite the original platform, and allowed to swim freely for 60 s. The number of times which the rats passed through the original region of the platform and velocity were calculated. All data were recorded and analyzed by EthoVision XT 8.0.

Animals were perfused with a modified fixative (2% paraformaldehyde–2.5% glutaraldehyde) (Damao chemical reagent factory, Tianjin, China), prepared with phosphate buffer (pH 7.4) (Beyotime, Shanghai, China). The corpus callosum (CC) was microdissected into 1 mm3 blocks. Samples were first placed in 2.5% glutaraldehyde fixation solution and then postfixed in osmium tetroxide (Zhongjingkeyi Technology Co., Ltd., Beijing, China). Afterwards, the samples were dehydrated in a graded ethanol series, embedded in araldite (Zhongjingkeyi Technology Co., Ltd.), and aggregated into a solid. Then the solid was cut into ultrathin sections (70 nm) and stained with uranyl acetate (Zhongjingkeyi Technology Co., Ltd.) and lead citrate (Zhongjingkeyi Technology Co., Ltd.). Finally, images of the sections were taken by a TEM (Chiyoda, Tokyo, Japan). The g-ratio was calculated as the diameter of the axon to the entire fiber diameter (axon diameter + total myelin thickness).

Animals were perfused with fixative (4% paraformaldehyde) in phosphate buffered saline (pH 7.4). Brain was post-fixed with 4% paraformaldehyde and cut into 40 μm vibratome sections with a vibrating microtome (Leica, Weztlar, Germany). For immunofluorescence, sections (40 μm) were rinsed with phosphate buffered saline and placed in sodium citrate buffer for antigen retrieval. The sections were blocked in 5% normal goat serum (Zhong Shan-Golden Bridge Biological Technology Co., Ltd., Beijing, China) containing 0.1% Triton X-100 (Solarbio, Beijing, China). Then the sections were incubated with the corresponding primary antibody against adenomatous polyposis coli (APC; Millipore, Darmstadt, Germany), BECN1 (Wanleibio, Shenyang, China), glial fibrillary acidic protein (GFAP; Abcam, London, UK), ionized calcium binding adapter molecule 1 (IBA1; Millipore), LC3 (Medical & Biological Laboratories, Nagoya, Japan), neurofilament 200 (NF200; Sigma), myelin basic protein (MBP; Abcam), oligodendrocyte lineage transcription factor 2 (OLIG2; Millipore) or platelet-derived growth factor receptor α (PDGFR-α; Millipore) at 4 °C overnight. After washing with phosphate buffered saline with Tween 20, the sections were incubated with a fluorescent dye-conjugated secondary antibody (Abcam). After that, the sections were incubated with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime). Fluorescence was detected with a fluorescence microscope (Nikon, Chiyoda, Tokyo). The number of positively stained cells and the integrated optical density were analyzed by Image J software (National Institutes of Health, USA).

The WM tissue was extracted and homogenized in a lysis buffer containing a mixture of protease-phosphatase inhibitors and 1% Triton X-100. The homogenate was centrifuged and the supernatant (Triton X-100-soluble fraction) and precipitate (Triton X-100-insoluble fraction) were retained. The precipitate was re-suspended in pyrolysis buffer containing 1% sodium dodecyl sulfate (SDS; Solarbio). Protein concentration was determined using a bicinchoninic acid kit (Beyotime). The supernatant or resuspended precipitate was boiled in SDS sample buffer. Samples containing an equal amount of total protein (25–30 μg) were electrophoresed in 10%–15% SDS-polyacrylamide gels at constant voltage. Then the protein was transferred to a 0.45 μm polyvinylidene fluoride membrane (Millipore), which was then sealed at room temperature for 1 h with 5% skimmed milk prepared with tris buffered saline with Tween 20. Then, the membrane was incubated overnight with the corresponding primary antibody against β-actin (ACTB; Santa Cruz Biotechnology, Dallas, TX, USA), BECN1 (Cell Signaling Technology, Boston, Massachusetts, USA; BD Biosciences, NJ, USA), cathepsin D (CTSD; Santa Cruz Biotechnology), LC3 (Medical & Biological Laboratories), lysosomal-associated membrane protein 1 (LAMP1; Abcam), lysosomal-associated membrane protein 2 (LAMP2; Sigma), MBP (Abcam), MTOR (Cell Signaling Technology), NF200 (Sigma), 70 kDa ribosomal protein S6 kinase (P70S6K; Cell Signaling Technology), p-MTOR (Cell Signaling Technology), p-P70S6K (Cell Signaling Technology), p-unc-51 like autophagy activating kinase 1 (ULK1; Cell Signaling Technology), SQSTM1 (Abcam), Ubiquitin (Santa Cruz Biotechnology), ULK1 (Cell Signaling Technology) or vacuolar protein sorting 34 (VPS34; Invitrogen, Carlsbad, CA, USA) at 4 °C. The next day, the membrane was taken out and incubated with the corresponding secondary antibody prepared with 5% milk at room temperature for 1 h. Between each procedure, the membrane was washed in tris buffered saline with Tween 20. Immune reactivity was tested using enhanced chemiluminescence (ThermoFisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. The intensity of the protein band was determined using Image J software.

For LFB, sections (40 μm) were washed with distilled water, put into LFB (Servicebio, Wuhan, China) staining solution, and sealed at 60 °C for 5 h. Then the slices were color-separated with ethanol and lithium carbonate aqueous solution, until the gray matter and WM were clearly distinguished under the microscope. The sections were dehydrated in ethanol, permeated in xylene, and finally sealed with neutral gum. The area covered by LFB staining was quantified with Image J software.

Mature oligodendrocytes (APC-positive) were stained for autophagosomes (LC3-positive) and subjected to the TUNEL assay. Immunofluorescence staining of APC and LC3 were carried out according to the procedure described in Section 2.9. After addition of the fluorescent dye-conjugated secondary antibodies, TUNEL staining was performed using an in situ cell death detection kit (Basel, Switzerland). Fluorescence was detected with a fluorescence microscope. The number of positively stained cells was analyzed by Image J software.

The AAV were purchased from OBiO Technology Company (Shanghai, China). The rat cDNA for BECN1 overexpression and the enhanced green fluorescent protein (EGFP) reporter gene were inserted into the adeno-related virus packaging vector. Packaging viruses were collected from the packaging cells and further expanded. The final recombinant AAV2/9-BECN1 overexpression cassette was pAAV-MBP-BECN1-HA-P2A-EGFP-3 × FLAG-tWPA, which expresses BECN1 and the reporter EGFP separately under control of the MBP promoter. The vector was confirmed by sequencing. AAV particles carrying this vector were used to overexpress BECN1 in oligodendrocytes. A similar vector expressing the EGFP reporter only was used as a negative control. AAV was diluted to the working concentration with artificial cerebrospinal fluid. AAV overexpressing BECN1 and EGFP sequences (labeled as BECN1 in the figures), or EGFP alone (control, labeled as EGFP in the figures), were stereotactically injected into the CC. During stereotactic injection, the rats were deeply anesthetized with isoflurane and placed prone in the stereotactic frame. A hole was drilled in the skull and 1 μL AAV (2.73 × 1012 vg/mL) or control virus (2.09 × 1012 vg/mL) was injected into both sides of the CC (AP −0.3 mm, ML ±1.0 mm, DV −3.7 mm) with a microliter syringe (Hamilton, Switzerland) at a rate of 0.25 μL/min. The left primary somatosensory barrel cortex (AP −0.3 mm, ML 5.0 mm, DV −3.0 mm) was injected with 0.2 μL of 1% (w/v) fluorescent cholera toxin B subunit (CTB) (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a microliter syringe at a rate of 0.05 μL/min. After the injection, the syringe was left in the original position for 5 min before removing it to prevent reflux.

Statistical analysis was performed via SPSS 22.0 (IBM Corp., Armonk, NY, USA), and graphs were plotted using GraphPad Prism 7.0 software (GraphPad Software, CA, USA). Data are expressed as the mean ± standard error of mean (SEM). Student's t-test was used to compare the difference between two experimental groups. Escape latency and swimming velocity in the MWM test navigation phase and body weight were analyzed by two-way analysis of variance (ANOVA) with repeated measures. Other data were analyzed with one-way ANOVA, followed by Tukey's test or LSD test to compare the differences among three or more groups. Statistical significance was set at P < 0.05.

Reduced CBF has been identified as a standard to judge successful establishment of the 2VO model in cognitive impairment and brain injury after CCH. Our data show that compared with CBF measurements before RCCAO, the left and right cerebral CBF decreased to 60%–70% after RCCAO, then the level of CBF remained stable until BCCAO, and further decreased to 40%–50% after BCCAO, confirming that the 2VO model was successfully established, (Fig. S1A–S1C). Then, the pathological tests, histological tests and behavioral tests were conducted on the 3rd, 7th, 14th, 28th and 42nd day after 2VO. Five groups of rats were subjected to 2VO or sham operation according to the schedule shown in Fig. 1A. Surgery for each group was carried out 42, 28, 14, 7, and 3 days before behavioral testing and tissue collection, respectively. The cognitive function tests included the NOR test for recognition memory, the Y maze test for working memory, and the MWM test for spatial learning and memory. The data show that compared with the sham group, both the discrimination index detected at 1 h after the training stage in the NOR test (Fig. 1B and C, Supporting Information Fig. S2C) and the spontaneous alternation rate in the Y maze test (Figs. 1D and E, S2F) were significantly decreased by the 28th and 42nd day after 2VO in rats. The escape latencies during the navigation phase of the MWM test were prolonged (Figs. 1F–H, S2G) and the number of platform crossings in the probe phase were reduced (Figs. 1I and J, S2H) by the 28th and 42nd day. No significant difference was observed between sham and 2VO groups in the distance and velocity in the locomotor activity test (Fig. S2A and S2B) and in the discrimination index detected at 24 h after the training stage in the NOR test (Fig. S2D and S2E), indicating that 2VO did not affect the motor performance and long-term memory of rats. These results indicate that cognitive impairment in 2VO-operated rats occurred by the 28th day after 2VO. Demyelination and loss of axonal integrity, which are indicators of WMI, were examined at different regions of WM (TEM, immunofluorescence and LFB staining were observed in the CC of rats. Western blotting was observed in the CC and cingulate zone of rats) (Supporting Information Fig. S3A). Significant increases in the g-ratio, which reflects the thickness of the myelin sheath was observed on the 3rd day after 2VO operation, and a significantly decreased number of myelinated axons and unmyelinated axons were observed on the 14th and 42nd day after 2VO (Fig. 2A–D). An increase in axonal diameter due to axonal swelling and a decrease in axonal number were observed on the 7th and 14th day, respectively (Fig. 2E and F). The expression of MBP in myelin sheaths was significantly decreased, followed by the reduced expression of NF200 in neuronal axons after 2VO (Fig. 2G–J). The vacuolation and disarrangement of myelin fibers in the CC of 2VO-operated rats gradually appeared with time (Fig. 2K and L). Simultaneously, the number of mature oligodendrocytes (APC-positive, OLIG2-positive) in the CC of rats was significantly decreased by the 14th day (Fig. 2M and N), while the number of oligodendrocyte precursor cells (OPCs, PDGFR-α-positive, OLIG2-positive) was increased by the 3rd day and returned to the normal level by the 14th day after 2VO, and the number of oligodendrocyte lineage cells (OLIG2-positive) remained unchanged (Figs. 2N, Fig. S3B). Moreover, the changes in microglia (IBA1-positive) and astrocyte (GFAP-positive) numbers exhibited a U-shaped profile, with a marked decrease on the 14th day (Supporting Information Fig. S4A–S4D). These results indicate that ahead of cognitive dysfunction, WMI occurred in rats by the 3rd day after 2VO operation and lasted until the end of the experimental period. WMI was paralleled with disturbance of glial cells, especially massive mature oligodendrocyte death by the 14th day after 2VO.

Next, we investigated whether autophagy is activated in specific brain cell types following 2VO. We examined the co-localization of the autophagosome marker LC3 with APC (marker for mature oligodendrocytes), NF200 (marker for neuronal axons), IBA1 (marker for microglia), and GFAP (marker for astrocytes). The results show that LC3 was mainly co-localized with APC and NF200 by the 3rd and 7th day, respectively, after 2VO in the CC of rats (Fig. 3A–C). No significant co-localization was observed with IBA1 and GFAP (Fig. S4A, S4B, S4E and S4F). The expression of LC3-II in WM of rats was significantly increased on the 3rd day, and then gradually returned to the normal level (Fig. 3D and E). Moreover, the level of the autophagy substrate soluble SQSTM1 was significantly increased on the 3rd day and decreased on the 14th day (Fig. 3D and F), while the levels of insoluble SQSTM1 (Fig. 3D and G) and Ubiquitin (Fig. 3D and H) were significantly increased on the 14th day. These data indicate that there was an abnormal accumulation of autophagy substrates following initial autophagy activation after 2VO. Furthermore, the levels of p-MTOR (S2448):MTOR (Fig. 3I and J), p-ULK1 (S757):ULK1 (Fig. 3I and K) and p-P70S6K (T389):P70S6K (Fig. 3I and L) were increased, while the levels of both VPS34 (Fig. 3I and M) and BECN1 (Fig. 3I and N) were significantly decreased around the 14th day after 2VO, suggesting that autophagy induction was inhibited. The expression levels of LAMP1 (Fig. 3O and P), LAMP2 (Fig. 3O and Q) and CTSD (Fig. 3O and R) were significantly decreased in WM of rats by the 14th day after 2VO, revealing lysosomal dysfunction. To further elucidate the changes of autophagic flux, the levels of LC3 were assayed on the 3rd day and 14th day after 2VO in the presence or absence of CQ, which inhibits intralysosomal acidification to block autophagosome–lysosome fusion (Supporting Information Fig. S5A). The results showed that CQ (20 mg/kg, i.p.) significantly increased the expression of LC3-II in the sham-operated rats, confirming the inhibitory effect of CQ on autophagic flux (Fig. S5B–S5D). The increased level of LC3-II on the 3rd day after 2VO was further enhanced by treatment with CQ (Fig. S5B and S5C), which suggests that ALP function was induced on the 3rd day after 2VO. However, on the 14th day after 2VO, the level of LC3-II was unchanged compared with the sham group, and CQ did not affect the expression of LC3-II (Fig. S5B and S5D). This suggests that impairment of autophagy induction and lysosomal dysfunction may occur concurrently in WM on the 14th day. To investigate whether the 2VO-induced abnormal change of ALP leads to the death of mature oligodendrocytes, mature oligodendrocytes (APC-positive) were co-stained for LC3 and TUNEL in WM of 2VO-treated rats. The results showed that both TUNEL and LC3 staining were co-localized with APC-positive mature oligodendrocytes, which suggests that autophagy occurred in dying mature oligodendrocytes. Moreover, compared with the sham group, the number of TUNEL-positive cells in autophagic mature oligodendrocytes (APC-positive, LC3-positive) was significantly increased by the 14th day after 2VO (Supporting Information Fig. S6A and S6B). These results suggest that ALP dysfunction resulted in the death of mature oligodendrocytes in WM on the 14th day after 2VO. All these results indicate that after initial autophagy activation, autophagy was inhibited and lysosomal function was concurrently impaired, resulting in abnormal ALP function in WM by the 14th day after 2VO in rats, which may further lead to the death of mature oligodendrocytes.

The above results show that autophagy was inhibited in WM after 2VO. Therefore, we were interested to know whether enhancing autophagy had a beneficial effect on WMI. We used continuous treatment with the MTOR inhibitor Rapa (3 mg/kg, i.p.) to stimulate autophagy for two weeks after 2VO (Fig. 4A). Rapa treatment significantly increased the expression of LC3-II (Fig. 4B and C) and decreased the levels of p-MTOR:MTOR (Fig. 4B and D), p-ULK1:ULK1 (Fig. 4B and E) and p-P70S6K:P70S6K (Fig. 4B and F) in WM in both the sham and 2VO groups on the 14th day after 2VO. Simultaneously, Rapa partially reversed the 2VO-induced reduction in expression of VPS34 (Fig. 4B and G) and BECN1 (Fig. 4B and H). These results confirm the effect of Rapa on autophagy activation. Next, we found that Rapa markedly alleviated the abnormal accumulation of autophagy substrates by reducing the 2VO-induced increase of insoluble SQSTM1 (Fig. 4I and K) and ubiquitin (Fig. 4I and L) without affecting the level of soluble SQSTM1 (Fig. 4I and J) on the 14th day after 2VO. Additionally, Rapa significantly reversed the decrease of LAMP1 (Fig. 4M and N), LAMP2 (Fig. 4M and O) and CTSD (Fig. 4M and P) expression on the 14th day following 2VO. These results suggest that Rapa alleviates lysosomal defects. Moreover, on the 14th day after 2VO, Rapa significantly increased the numbers of myelinated axons and total axons (Fig. 5A, C and E) and the expression of MBP (Fig. 5F–I), whereas the g-ratio and axonal diameter were decreased compared with the vehicle group (Fig. 5A, B and D). Rapa significantly reduced the number of TUNEL-positive cells in autophagic mature oligodendrocytes (APC-positive, LC3-positive) after 2VO (Fig. 5J and K). These results suggest that enhancement of autophagy by systemic Rapa treatment could attenuate WMI through improvement of ALP dysfunction and alleviation of lysosomal dysfunction in 2VO-operated rats.

The above results show that inhibition of autophagy in WM was accompanied by massive mature oligodendrocyte death. Thus, it is valuable to explore whether enhancement of autophagy, targeted at oligodendrocytes, could improve WMI and cognitive function after 2VO. Transduction of BECN1-overexpressing AAV into the CC of 2VO-operated rats (Fig. 6A, Supporting Information Fig. S7A) allowed oligodendrocyte-specific expression of exogenous BECN1 (EGFP-positive, BECN1-positive) and successfully increased BECN1 expression in WM (Fig. S7B–S7F). Furthermore, the results show that during the 35 days of the experiment, there was no significant difference in the body weight of rats in the different groups (Supporting Information Fig. S8A). Also, on the 28th day after 2VO, there was no significant change of the activation of microglia and astrocytes after administration of BECN1-overexpressing AAV compared with control virus (Fig. S8B–S8F). The data show that compared with control vector, overexpression of BECN1 significantly increased the level of LC3-II (Fig. 6B and C) and further decreased the level of soluble SQSTM1 after 2VO (Fig. 6B and D). The accumulation of insoluble SQSTM1 (Fig. 6B and E) and Ubiquitin (Fig. 6B and F) were also reversed by overexpression of BECN1 in 2VO-treated rats. Thus, enhancement of oligodendrocyte autophagy alleviated the ALP defects on the 14th day after 2VO. Moreover, overexpression of BECN1 significantly reversed the 2VO-induced decrease of LAMP1 (Fig. 6G and H) without affecting the levels of LAMP2 (Fig. 6G and I) and CTSD (Fig. 6G and J). These data suggest that targeted enhancement of autophagy in oligodendrocytes by overexpression of BECN1 alleviated ALP dysfunction on the 14th day in 2VO-operated rats. Moreover, overexpression of BECN1 significantly increased the expression of MBP and NF200 in WM on the 28th day after 2VO (Fig. 7A–D), and significantly prevented the 2VO-mediated reduction in the number and proportion of mature oligodendrocytes without affecting the number of OPCs or oligodendrocyte lineage cells (Fig. 7E–G, Supporting Information Fig. S9). This indicates a reversal of mature oligodendrocyte death. A fluorescent CTB, an indicator for axonal connectivity in different brain regions, was injected into the left primary somatosensory barrel cortex to retrogradely trace axonal projections. Overexpression of BECN1 significantly increased the positive cell density of CTB-labeled neurons and fluorescence intensity in CTB-injected contralateral cortex on the 28th day after 2VO (Fig. 7H–J). These results indicate that overexpression of BECN1 in oligodendrocytes improved the disruption of cortico-cortical axonal connectivity in 2VO-operated rats. These results suggest that overexpression of BECN1 in oligodendrocytes could reduce demyelination and alleviate impairment in axonal number and connectivity by reducing mature oligodendrocyte death after 2VO. Furthermore, overexpression of BECN1 in oligodendrocytes can alleviate the 2VO-induced cognitive impairment, as indicated by the increases in the discrimination index in the NOR test (Fig. 7K, Supporting Information Fig. S10C), spontaneous alternations in the Y maze test (Figs. 7L, S10D), and the number of platform crossings in the MWM (Figs. 7N and O, S10F); and the reduced escape latency in the MWM (Figs. 7M, S10E). No significant difference of the distance and velocity in the locomotor activity test (Fig. S10A and S10B) was observed between the groups. These data suggest that targeted enhancement of oligodendrocyte autophagy by overexpression of BECN1 reduced mature oligodendrocyte death, thus alleviating the WMI-related cognitive impairment on the 28th day after 2VO.

The current study showed that cognitive impairment was presented on the 28th day after 2VO in rats. Myelin sheath injury and autophagy activation in mature oligodendrocytes occurred simultaneously in WM on the 3rd day, followed by injury and autophagy activation in neuronal axons on the 7th day. On the 14th day, autophagy activation turned into ALP dysfunction caused by autophagy inhibition and lysosomal dysfunction, paralleled with massive death of mature oligodendrocytes. The study proves that ALP dysfunction in WM aggravated WMI and cognitive impairment in 2VO-operated rats. Finally, it showed that improving ALP dysfunction by enhancing oligodendrocyte autophagy may reduce mature oligodendrocyte death, thus effectively alleviating CCH-induced WMI and cognitive impairment in rats (Fig. 8). It is generally believed that cognitive impairment occurs in rats by the 14th or 28th day after 2VO, and the detection of postoperative cognitive impairment is usually conducted at 14, 28, and 42 days after 2VO,,. Therefore, we also chose these three time points to investigate postoperative behavioral changes induced by 2VO. In addition, it was reported that both neuronal soma injury and altered autophagy function occurred in gray matter at 14 days after 2VO,. Importantly, it has been shown that oligodendrocytes are more sensitive to the ischemic environment than neurons. Also, the levels of LC3 increased and the levels of the autophagy substrate soluble SQSTM1 decreased in WM from the 3rd day after CCH. Therefore, we examined the pathology and histology on the 3rd, 7th, 14th, 28th and 42nd day after 2VO to investigate the dynamic changes of autophagy and WMI of rats after 2VO. The behavioral tests were conducted on the 14th, 28th and 42nd day after 2VO to evaluate the level of cognitive impairment. Our results show that myelin sheath injury and autophagy activation in mature oligodendrocytes occurred simultaneously in WM on the 3rd day, injury and autophagy activation in neuronal axons appeared on the 7th day, and behavioral abnormalities appeared on the 28th day after 2VO. WM is mainly composed of a large number of oligodendrocytes and neuronal axons. Studies have found that oligodendrocytes in WM are more susceptible to injury than neuronal axons in ischemic stroke models such as transient focal cerebral ischemia and human subcortical white matter stroke,, and that the loss of myelinated axons is more marked than that of unmyelinated axons in ischemic neuropathy. It has been reported that increasing OPCs maturation or reducing mature oligodendrocyte death can significantly alleviate demyelination, thereby reducing WMI and improving learning and memory deficits or sensorimotor neurological function after WM lesions such as CCH or neonatal hypoxia/ischemia32, 33, 34. In the present study, we also found that oligodendrocytes injury preceded neuronal axon injury and the number of myelinated axons decreased before that of unmyelinated axons in WM of 2VO-operated rats. We show that the axonal injury could be effectively alleviated by reducing mature oligodendrocyte death. This indicates that delayed neuronal axonal injury after CCH may represent a bystander effect of impaired function and reduced number of mature oligodendrocytes. Above all, this study confirms that targeting WM, especially promoting the survival of mature oligodendrocytes, may be an effective strategy for the treatment of CCH. It is generally accepted that intervention to alleviate abnormal changes of autophagy can reduce ischemia-induced cell death35, 36, 37. Our previous study showed that there are abnormal changes of ALP function in neurons, and overexpression of neuronal-targeted TFEB can rescue the ALP dysfunction, thus alleviating the ischemic injury after focal cerebral ischemia. In the present study, we found that the number of autophagosomes was increased in mature oligodendrocytes and neuronal axons in WM on the 3rd day and 7th day after 2VO, respectively. On the 14th day, the expression of LC3-II returned to normal levels, while autophagy substrates accumulated. Meanwhile, the levels of p-MTOR, p-ULK1, and p-P70S6K were increased, while VPS34 and BECN1 were significantly decreased. These data indicate that autophagy induction was inhibited on the 14th day after 2VO. Additionally, the expression of LAMP1, LAMP2 and CTSD was significantly decreased on the 14th day after 2VO, indicating lysosomal dysfunction. All these results suggest that both autophagy inhibition and lysosomal dysfunction occurred after initial autophagy activation in WM on the 14th day after 2VO. These findings further elucidate the reason why autophagy is initially activated and then suppressed in WM, which was observed in the CCH induced by bilateral common carotid artery stenosis in mice. However, the interaction between lysosomes and autophagy is complicated. It has been shown that lysosomal positioning influences and coordinates MTOR activity, autophagosome biogenesis and autophagosome-lysosome fusion38, 39, 40. In the present study, we found that the inhibition of autophagy coexisted with lysosomal dysfunction, and therefore we could not determine the driving force leading to the abnormal change of autophagy after 2VO. However, our results show that the level of lysosomal protein LAMP2 was decreased on the 7th day after 2VO which was prior to the alteration of MTOR pathway-related proteins. This suggests that lysosomes are more likely to be the driving force that leads to the abnormal change of autophagy. Of course, to explore this hypothesis, the following issues need to be further investigated38, 39, 40: In the early stage of 2VO, does enhancing or inhibiting lysosomal function affect MTOR pathway-related proteins and the formation of autophagosomes? After 2VO, are there abnormal changes in the intracellular positioning of lysosomes, intracellular pH, and proteins involved in microtubule transport that affect MTOR activation? It is well known that Rapa, which is usually used as an autophagy agonist, can dephosphorylate ULK by inhibiting MTORC141, 42, 43. Several studies have demonstrated that Rapa can alleviate ischemic brain injury in permanent or transient focal cerebral ischemia44, 45, 46. It can also alleviate the neuronal injury in hippocampus and improve the cognitive impairment in 2VO-operated rats by increasing mitophagy. However, it is still unclear whether Rapa can improve the WMI after 2VO. Our results show that systemic administration of Rapa ameliorated ALP deficiency, leading to the alleviation of demyelination in WM of 2VO-operated rats. Notably, Rapa not only stimulated autophagy, but also promoted lysosomal function in WM of 2VO-operated rats. It has been reported that Rapa can dephosphorylate and induce nuclear localization of TFEB by inhibiting MTORC1, thereby driving the expression of CLEAR genes to promote lysosomal biogenesis,,. This may be the reason why Rapa enhanced the lysosomal function in WM of 2VO-operated rats in the present study. Oligodendrocytes maintain nerve impulse transmission between neurons in different brain regions by producing myelin sheaths. Meanwhile, activation of oligodendrocyte autophagy plays an important role in oligodendrocyte survival as well as in white matter demyelinating diseases of the CNS8, 9, 10. More importantly, our results demonstrated that oligodendrocyte injury preceded neuronal axon injury in WM, and there was an abnormal change of autophagy in mature oligodendrocytes after 2VO. Lines of evidence from previous studies indicate that enhancement of autophagy in oligodendrocytes reverses the abnormal function of autophagy, which then reduces the accumulation of toxic proteins (such as tau, β-amyloid peptides and α-synuclein) and reverses the mitochondrial dysfunction in oligodendrocytes in Alzheimer's disease, multiple sclerosis, experimental autoimmune encephalomyelitis and other CNS diseases, thus promoting oligodendrocyte survival49, 50, 51, 52, 53. It is reasonable to assume that in the present study, CCH-induced ALP dysfunction in oligodendrocytes may lead to the accumulation of toxic proteins and damaged organelles, followed by death of the oligodendrocytes. Therefore, targeting oligodendrocytes to regulate autophagy may be of benefit to alleviate WMI and cognitive decline after 2VO. In our study, Rapa was administered systemically, therefore had non-specific effects. To specifically activate autophagy in oligodendrocytes, we constructed an AAV to express BECN1 under control of the MBP promoter. The results indicate that enhancing autophagy in oligodendrocytes significantly reduced mature oligodendrocyte death and alleviated axonal injury thereby reducing WMI and delaying the progression of cognitive impairment after 2VO. However, the causal relationships among these observations are unclear, and further investigations are needed to elucidate the underlying mechanism. In addition, enhancing autophagy by overexpression of BECN1 in oligodendrocytes stimulated autophagy without affecting other proteins involved in lysosomal function except for reversing the reduction of LAMP1. It has been reported that BECN1 may interact with the lysosomal protein LAMP1 under oxidative stress in the CNS, but its direct or indirect effects on lysosomes and the underlying mechanisms need to be further studied. We therefore speculate that autophagy inhibition may be the main cause of the ALP dysfunction on the 14th day after 2VO. In addition, compared with the control treatment, long-term activation of autophagy by overexpression of BECN1 in oligodendrocytes had no significant effect on the body weight and locomotor activity of the rats. Furthermore, the activation of microglia and astrocytes, cells which are sensitive to microenvironmental changes, did not change in WM following the administration of AAV–MBP–BECN1. These data indicate that long-term activation of autophagy did not cause side-effects in both sham- and 2VO-operated rats. Of course, BECN1 also has other functions independent of autophagy, such as mediating apoptosis, endocytosis, and phagocytosis by phagocytes. Further investigations are needed to determine whether these mechanisms may be involved in BECN1-mediated improvement of WMI in the 2VO model. More importantly, the present findings identify oligodendrocyte autophagy as a promising target for neuroprotective therapy in CCH. It is still controversial whether enhancement of autophagy is beneficial or detrimental to CCH,. In our study, we found that enhancement of autophagy by systemic rapamycin treatment or overexpression of BECN1 in oligodendrocytes exerts a protective effect on WM and cognitive function after CCH. In contrast, some studies have reported that autophagy inhibition achieved the same effect,,. We summarize some possible explanations for these conflicting outcomes. First, different animal species and models may lead to different extent of ischemia, resulting in varying degrees of autophagy activation,62, 63, 64. Moderate activation of autophagy is beneficial for cell survival, while over-activated autophagy will cause serious cell injury and lead to autophagic cell death,. Second, the accumulation of autophagosomes may result from autophagy activation alone, or from lysosomal dysfunction alone, or from both. Therefore, the role of autophagy in regulating cell survival after CCH needs to be comprehensively evaluated in combination with the autophagy upstream agonists or inhibitors and lysosomal inhibitors. Third, different brain regions and cell types may contribute to varying change of autophagy,,,65, 66, 67, 68. Of course, these possibilities need to be further clarified. Since WM repair can be achieved either by reducing the death of existing mature oligodendrocytes or accelerating the differentiation of OPCs into mature oligodendrocytes, we analyzed the differentiation of OPCs following 2VO in this study. The results showed that OPCs began to proliferate from the 3rd to the 7th day after 2VO, while proliferative dysfunction occurred by the 14th day after 2VO. This was paralleled with massive death of mature oligodendrocytes, but the number of cells in the oligodendrocyte lineage remained unchanged. Importantly, overexpression of BECN1 in oligodendrocytes reversed the massive death of mature oligodendrocytes without affecting the proliferation of OPCs. This indicates that enhancement of autophagy-mediated WM repair may be due to the decreased demyelination rather than increased remyelination. However, it still remains to be clarified whether the proliferation of OPCs in the early stage after 2VO is relevant to the activation of autophagy. In the present study, we also found that the change in the numbers of microglia and astrocytes exhibited a U-shaped profile, with a marked decrease on the 14th day after 2VO, which was consistent with the results from striatum and cortex in the same rat model. The decrease in microglia on the 14th day may result from the increased anti-inflammatory environment during recovery, leading to massive death of pro-inflammatory M1 microglia. The decrease in astrocytes at this time may be due to several factors. Firstly, lower numbers of M1 will reduce the stimulation of A1 astrocyte activation; secondly, persistent hypoperfusion leads to disintegration and degeneration of astrocytes; and thirdly, injured oligodendrocytes transmit harmful substances through glial syncytium. However, the numbers of both microglia and astrocytes increased later, and this may be due to increased levels of neurotoxic M1 and A1 caused by the aggravated WMI or an increase in neuroprotective M2 and A2 during the recovery period. Detailed examination of the respective isoforms will be required to distinguish between these possibilities. Our data demonstrate that enhancement of autophagy in oligodendrocytes did not affect the number and morphology of microglia and astrocytes in WM, which suggests that oligodendrocyte-targeted autophagy played a vital role in alleviating WMI and cognitive impairment.

We identified the critical role of abnormal ALP function in WM in regulating injury after CCH. Our study show that autophagy activation occurred simultaneously with WMI in the early stage of CCH and gradually transformed into ALP defects caused by autophagy inhibition and lysosomal dysfunction. Impaired ALP function was accompanied by massive mature oligodendrocyte death, which aggravated WMI and cognitive impairment. Enhancement of oligodendrocyte autophagy to overcome the ALP deficiency significantly reduced mature oligodendrocyte death, thus alleviating the WMI and cognitive decline. This study provides the first evidence that enhancing oligodendrocyte autophagy may benefit cognitive recovery after CCH.