Background
Ischemic stroke is one of the most common brain diseases, accounting for 70% of cerebrovascular diseases [
1]. It is the fourth leading cause of death in the USA and Europe, and a major cause of adult disability [
2]. It is thus of considerable interest to investigate the cellular and molecular mechanisms of ischemic brain damage. Many progresses have been achieved, but, it remains to be a detrimental disease without an effective treatment in clinic. Thus, it is necessary to further investigate its pathological mechanisms for identification of novel intervention targets for the development of a better therapy.
Ischemic brain injury triggers a complex pathophysiological cascade, which includes impaired blood flow, hypoxia, oxidative stress, glutamate excite-toxicity, and inflammation [
1]. During an ischemic event, impaired blood flow in the brain occurs first, which results in a reduced delivery of oxygen and glucose, leading to the energy depletion and neuronal death that is largely located in the ischemic core [
3,
4]. There are glial responses to the neuronal death or damaged neurons mainly in the peri-infarct region, which include reactive astrogliosis and microglial activation.
Microglia, the resident immune cells of the brain, react quickly to ischemic brain injury with transcriptional regulation and morphological changes [
5]. Based on their transcriptional changes, microglia have been classified as M1, M2, and recently DAM (damage-associated microglia) [
6,
7]. The different types of microglia appear to have different functions during ischemic brain injury, whereas M2 microglial cells show protective effects during ischemic injury, M1 microglia appear to play a detrimental role in this event [
8,
9]. The functions of DAM in response to the ischemic injury or its relationship with M1 microglia remain to be investigated.
VPS35 (Vacuolar sorting protein 35), a critical component of retromer, is essential for selective endosome-to-Golgi retrieval of transmembrane proteins [
10]. VPS35 is a ubiquitous protein with different levels of expression in different cells of the central nervous system, including microglia. In the central nervous system, VPS35 has been shown to be involved in many key cellular physiological processes. For example, AMPA receptor mediated neurotransmission [
11], mitochondrial fusion/fission dynamics [
12], and β-amyloid (Aβ) metabolism [
13]. Pathologically, VPS35-deficiency is believed to increase the risk of neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [
14]. Mutations in VPS35 gene have been identified in patients with autosomal dominant PD [
15‐
17] or early onset AD [
18]. VPS35 loss in mouse models causes PD-like deficits as well as enhances AD-like neuropathology in Tg2576, an AD mouse models [
13,
14,
19]. Interestingly, the microglial VPS35 is decreased in the brain of AD patients [
20]. We have previously shown that microglial VPS35 knocking out in mouse model results in a selective microglial activation in the hippocampus and interferes with the neurogenesis in adult dentate gyrus [
21]. However, the function of microglial VPS35 in the cortex in response to ischemic stroke remains elusive.
Here, we report that microglial VPS35 loss regulates microglial polarization in the cortical brain after ischemic stroke. Ischemic stroke-induced injury is diminished in microglial VPS35 conditional knockout (cKO) mice. In addition, ischemic stroke-induced increase of CX3CR1 receptor levels is abolished in microglial VPS35-deficient mice, implicating CX3CR1 as a potential cargo of VPS35. Taken together, these results suggest an unrecognized function of microglial VPS35 in suppressing microglial polarization change (from pro-inflammatory to anti-inflammatory), in line with the view for pro-inflammatory microglia to play a “neurotoxic” role in ischemic brain injury.
Methods
Animals
CX3CR1
Cre-ER mice in C57BL/6 background were purchased from the Jackson Laboratory (stock number: 00790). VPS35
flox/flox:CX3CR1
Cre-ER/+ mice (termed as VPS35
CX3CR1 in this study) were generated as described previously [
21]. Mice were housed under constant 12-h light-dark cycle and fed a diet of standard rodent chow. All animal experimental procedures were approved by the Animal Subjects Committees at Case Western Reserve University and Augusta University according to US National Institutes of Health guidelines.
Photothrombotic ischemic stroke
Ischemic damage of sensorimotor cortex in mice was induced by photothrombotic ischemia as described previously [
22]. Briefly, mice (control and mutant) were anesthetized by ketamine (100 mg/kg) and intraperitoneally injected with same amount of Rose Bengal (10 mg/ml and 10 μl/g body weight). Mice with an exposed skull were then placed under the stereotaxic instrument with their heads firmly secured. Ten minutes after Rose Bengal injection, a cold light was turned on specifically on the sensorimotor cortex region for 15-min photo illumination.
We chose 10 min after Rose Bengal injection for photo illumination, because its concentration in the blood (measured by Rose Bengal-BSA binding assay) reached a peak level [
23]. To make sure same size of area exposed to the light and to prevent photo illumination of the animal by any other source of light, an opaque patch with a hole (2-mm diameter) in the center was put under the skin of the skull, and the end of illuminator fiber (with 150-W intensity) was tightly attached to the center of the patch.
Immunofluorescence staining
Immunofluorescence staining analysis was performed on 40 μm free-floating sections. Primary antibodies, including chicken anti-GFP (Aves Labs, GFP-1020), goat anti-CD206 (R&D Systems, AF2535), rabbit anti-Cleaved Caspase-3 (Cell Signaling, 9661), mouse anti-Caspase-3 (Novus Biologicals, 31A1067), mouse anti-NeuN (Millipore, MAB377), rabbit anti-Tmem119 (Abcam, ab209064), mouse anti-LPL (Abcam, ab21356), rabbit anti-CX3CR1(Abcam, ab8021), rabbit anti-GFAP (Abcam, ab7260), mouse anti-iNOS (inducible nitric oxide synthase) (Abcam, ab49999), and goat anti-Iba1 (Abcam, ab5076), were used. Corresponding secondary antibodies, including 488- and 594-conjugated secondary antibodies, were purchased from Thermo Fisher Scientific, Alexa Fluor conjugates.
All images were processed with Image J for quantification analysis. The means were calculated from 3 randomly selected microscopic fields in the ipsilateral and contralateral cortex of each section, respectively, and 3 consecutive sections were analyzed for each brain. Data are expressed as mean numbers of cells per square millimeter.
Real-time PCR
Total RNAs were isolated from ischemic brains using the RNAeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Five micrograms was used to synthesize the first strand of cDNA using the Superscript First-Strand Synthesis System for RT-PCR (Invitrogen). PCR was performed on the Opticon 2 Real-Time PCR Detection System (Bio-Rad) using corresponding primers (Table
1) and SYBR gene PCR Master Mix (Invitrogen). The cycle time values were normalized to GAPDH of the same sample. The expression levels of the mRNAs were then reported as fold changes over control.
Table 1
Primers for real-time polymerase chain reaction
CD86 | Forward: GACCGTTGTGTGTGTTCTGG Reverse: GATGAGCAGCATCACAAGGA |
CD206 | Forward:CAAGGAAGGTTGGCATTTGT Reverse: CCTTTCAGTCCTTTGCAAGC |
iNOS | Forward: CAAGCACCTTGGAAGAGGAG Reverse: AAGGCCAAACACAGCATACC |
CD32 | Forward: AATCCTGCCGTTCCTACTGATC Reverse: GTGTCACCGTGTCTTCCTTGAG |
Arg1 | Forward: TCACCTGAGCTTTGATGTCG Reverse: CTGAAAGGAGCCCTGTCTTG |
Ym1/2 | Forward: CAGGGTAATGAGTGGGTTGG Reverse: CACGGCACCTCCTAAATTGT |
IL-6 | Forward: CTCTGCAAGAGACTTCCATCCA Reverse: GACAGGTCTGTTGGGAGTGG |
IL-1β | Forward: CTGATGCAGGTCCCTATGGT Reverse: GCAGGATTTTGAGGTCCAGA |
IL-10 | Forward: CCAAGCCTTATCGGAAATGA Reverse: TCCTCACAGGGGAGAAATCG |
IL-4 | Forward: ATGATGCAGGTCCCTATGGT Reverse: GCAGGATTTTGAGGTCCAGA |
TGF-β | Forward: TGCGCTTGCAGAGATTAAAA Reverse: CGTCAAAAGACAGCCACTCA |
TNF-α | Forward: CAGGCGGTGCCTATGTCTC Reverse: CGATCACCCCGAAGTTCAGTAG |
Neurological functional tests
The two groups of mice (cKO and control) were trained for 1 week before the test. All the tests were measured before, 6 h, 1, 3, and 7 days after ischemia/stroke. Modified neurological severity score assessment, adhesive removal test, and foot fault test were performed as described previously [
24]. Neurological function assessments were performed by investigators who were blinded to the experimental groups. Neurological function was graded on a scale of 0 to 14 (normal = 0; maximal deficit = 14). The higher the score, the more serious the neurological impairment.
TTC staining and Nissl staining to assess infarct volume
The infarct volume was accessed by TTC and Nissl staining as described previously [
24]. Briefly, after anesthesia, mouse was euthanized, and its whole brain was removed. For TTC staining, the brain slice sections (750-μm thick, coronal) were incubated with 2% TTC (2,3,5-triphenyltetrazolium chloride) in PBS solution at 37 °C for 10 min, then fixed with 4% paraformaldehyde for 30 min, and washed with PBS for 5 min for 3 times. For Nissl staining, the brain sections (40-μm think, coronal) were incubated with 1% toluidine blue in PBS at 60 °C for 40 min, and then washed, dehydrated, and sealed for imaging analysis as described previously [
25].
The infarct volume in TTC-stained or Nissl-stained sections was measured by a blinded observer using National Institutes of Health Image J software. The infarct area and volume on each slice were calculated based on the following equations according to a previous publication [
26]: Infarct area = Contralateral hemisphere area − healthy area of ipsilateral hemisphere. Infarct volume = Infarct area × thickness of slice.
Statistical analysis
All data were expressed as means ± SEM. The data were statistically analyzed by two-way analysis of variance (ANOVA), followed by Tukey’s test for pairwise comparisons by using Graph Pad Prism v7.0 and Sigma Plot 13.0 software. In the case of significance, a further ad hoc two-tailed Student’s t test was applied. P value < 0.05 was considered statistically significant.
Discussion
VPS35 is known to play a key role in endosomal trafficking and cargo-selective function. Dysfunctional VPS35 increases a risk for neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD) [
36]. Stroke is also believed to be an environmental risk factor for AD [
37,
38]. While VPS35 deficiency contributes to the pathogenesis of neurodegenerative disorders [
10‐
14], its function in stroke remains largely unclear. Here, we provide evidence for microglial VPS35’s function in ischemic cortical stroke. To our surprise, microglial VPS35 loss appears to be neuroprotective in this event. In microglial VPS35 KO mice, the injury response to photothrombotic stroke is reduced, which include decreased infarct area, attenuated neuronal death and reactive astrogliosis, and better sensorimotor regulated behavior functions. Further studies of microglial response to the stroke showed reduced pro-inflammatory but increased anti-inflammatory type of microglial polarization, in the mutant mice. Thus, the pro-inflammatory cytokine expression is downregulated, but the anti-inflammatory gene expression is upregulated in the mutant mice after the stroke. Finally, stroke-induced CX3CR1 protein levels are abolished in microglial VPS35-deficient mice. Taken together, these results suggest that microglial VPS35 is likely to be necessary for stroke-induced DAM and pro-inflammatory microglial activation, and both type of microglia might be “neurotoxic” in the condition of stroke induced injury.
Microglia play key roles in the physiology and pathology of the central nervous system, including ischemic brain injury. Microglial cells are not a uniform cell population, and they respond quickly with changes in transcriptional regulation and morphology to specific environmental factors, which include tissue injury (timing and degree), brain regions (e.g., cortex vs hippocampus), and age [
39,
40]. Activated microglia/macrophages may have protective or harmful effects after ischemia injury, and these different functions may be due to different subtypes of microglia/macrophages, such as pro- or anti-inflammatory state [
41]. Pro-inflammatory microglial activation is believed to be cytotoxic phenotype, which is characterized by the production of nitric oxide, reactive oxygen species, and pro-inflammatory cytokines. In contrary, anti-inflammatory microglia are associated with the promotion of debris removal, angiogenesis, and tissue repairing [
37,
39,
40]. In line with this view are our observations of decreased iNOS
+ microglia and reduced expression of pro-inflammatory cytokines in VPS35 mutant brains after ischemic stroke.
Recent single-cell RNA-seq analysis has identified DAM, a unique type of microglia that is induced in multiple disease conditions, including stroke [
6,
29]. LPL is a marker for type 2 DAM [
6]; however, it is also reported to be higher in anti-inflammatory microglia in response to EAE (experimental allergic encephalomyelitis) [
42]. We found a decrease in LPL
+ DAM, but an increase in CD206
+ microglia (anti-inflammatory-like), in VPS35 mutant cortex after stroke (Figs.
5 and
6). Such a difference might be due to different experimental conditions (stroke vs EAE). In stroke condition, DAM may be in association with “neurotoxic” microglia (or pro-inflammatory), but under the condition of EAE, DAM, in association with beneficial microglia (or anti-inflammatory), mediate effect against EAE-induced inflammation. Further studies examining additional markers for both DAM and anti-inflammatory-microglia are necessary to test this view. Together, our results suggest associations of the reduced stroke injury in VPS35 mutant brain with decreases in iNOS
+ microglia and pro-inflammatory cytokines’ expression, and an increase in CD206
+ microglial activation. However, the molecular mechanisms underlying microglial VPS35 regulation of DAM/microglial polarization remain unclear, which need further investigation.
It is of interest to note previous reports that the loss of CX3CR1 receptors in mice has neuroprotective effects in response to ischemic brain injury [
33,
43]. In MCAO stroke model, CX3CR1-deficient mice show reductions in infarct size and neuron death [
33,
35]. In the experimental model of spinal cord injury, lack of CX3CR1 induces neurological function protection [
34]. These phenotypes are remarkable similar to the phenotypes observed in microglial VPS35-cKO mice. In addition to the reduced infarct area in response to the ischemic injury, both VPS35 and CX3CR1 mutant mice show increased anti-inflammatory, but not pro-inflammatory, microglial activation by stroke [
35] (Fig.
6) and reduced stroke-induced expressions of pro-inflammatory cytokines [
33,
40,
41] (Fig.
8). These observations suggest that like CX3CR1-deficiency, VPS35 loss in microglia appears to attenuate neuronal damage and improve recovery of function by reducing the recruitment and/or the activation of DAM or pro-inflammatory microglia and macrophages in response to stroke injury [
6,
29,
41,
43]. It is noteworthy that in contrast from stroke injury, CX3CR1 deficiency in AD animal models enhances Aβand Aβassociated brain pathology [
37,
44]; and such enhanced Aβphenotypes are also observed in VPS35-deficient mice [
13]. The similarity of phenotypes between VPS35 and CX3CR1 mutant mice leads us to wonder if CX3CR1 receptor is a cargo of VPS35/retromer in microglia. Examining CX3CR1 protein levels by Western blot analysis showed no difference between control and microglial VPS35-cKO brains, but, stroke-induced CX3CR1 protein levels were only detected in control, but not in VPS35-mutant, brains (Fig.
9a). The increase in CX3CR1 protein levels (Fig.
9a), but decrease in CX3CR1 promotor-driven GFP expression in subset of microglia (Fig.
3a), in control brains by stroke implicates that the elevation of CX3CR1 protein levels is likely due to an enrichment in populations of microglia/macrophages in the injury side of brains, as compared with that in contralateral side. The reduction in stroke-induced CX3CR1 protein levels in VPS35 cKO brain suggests a role of microglial VPS35 in stabilizing CX3CR1 proteins in response to stroke. However, this view requires further investigations.
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