The ER chaperone, BIP protects Microglial cells from ER stress-mediated Apoptosis in Hyperglycemia

doi:10.21203/rs.3.rs-2626070/v1

Download PDF

Research Article

The ER chaperone, BIP protects Microglial cells from ER stress-mediated Apoptosis in Hyperglycemia

https://doi.org/10.21203/rs.3.rs-2626070/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Background: Binding of Immunoglobulin heavy chain protein(BIP) is a major endoplasmic reticulum (ER) chaperone facilitating the assembly of newly synthesized proteins in the ER. Microglial cells vigorously respond to brain injuries and eliminate the damaged neuronal and apoptotic cells through phagocytosis in the central nervous system. However, the mechanism of BIP-mediated microglial cell function is not clear in hyperglycemia. We explored the molecular mechanism of BIP in microglial function during hyperglycemia conditions.

Methods: Hyperglycemia was induced in C57BL/6J mice by two consecutive intraperitoneal injections of streptozotocin (STZ 100/kg) and confirmed by measuring the blood glucose from day 2 to day 14. After 14 days of experimental condition, mice were sacrificed, brains were collected, and tissue lysate was prepared for ER chaperone studies. In-vitro hyperglycemia was induced by exposing HMC3 cells to 25mM glucose for 5 days and proteins involved in ER stress, apoptosis, and autophagy were analyzed. For the BIP induction, cells were treated with 25μM of BIX (BiP inducer-X) after 48 hr of hyperglycemia for 3 days.

Results: In hyperglycemia condition, the major ER chaperone BIP protein expression was dramatically reduced in HMC3 cells, which led to increased apoptosis through the activation of CHOP and mitochondrial pro-apoptotic proteins (Bax, Bad, cleaved caspase-3). The flow cytometry results also indicate that hyperglycemia-induced the apoptosis and reactive oxygen species (ROS) production. Interestingly, the BIP inducer BIX restored the apoptosis in microglia cells through the derepression of BIP expression and inhibition of ER stress.

Conclusion: These results suggest that the ER chaperone BIP is required for the microglial function and protection from apoptosis in hyperglycemia. A better understanding of the molecular mechanism and role of BIP in microglia function may contribute to the development of novel therapies for microglia dysfunction-associated neurodegenerative diseases.

ER stress

Hyperglycemia

Reactive oxygen species

Apoptosis

Autophagy

microglia function

ER chaperone

Streptozotocin

and BiP inducer-X

Binding of Immunoglobulin heavy chain Protein (BIP) is one of the major endoplasmic reticulum (ER) chaperones and is a highly conserved member of the heat shock protein family [1, 2], which plays a crucial role in protein refolding during the accumulation of unfolded or misfolded proteins in the ER [3, 4]. BIP protein expression is regulated through the binding of X-box protein (XBP1) on the ER stress-responsive element (ERSE) located in the promoter of BIP [5]. Also, serves as an ER sensor, and its expression is profoundly increased in ER stress. Protein kinase R-like ER kinase (PERK) phosphorylates the eukaryotic translation initiation factor-2 alpha (eIF2α) and activates the active transcription factor-4 (ATF4) and CHOP, which further activates the pro-apoptotic signal leading to cell death or apoptosis [6–8]. ER is the unique and dynamic compartment that is involved in the protein, lipid synthesis, and post-translation modifications of secretary proteins through glycosylation. Protein glycosylation defect causes the accumulation of unfolded or misfolded proteins in the ER leading to ER stress [9, 10]. The accumulation of unfolded protein in the ER activates the unfolded protein response (UPR) as an adaptive response to protect the cells from ER stress-mediated apoptosis [11].

Glucose is the fuel source of energy for brain physiological functions and brain cells, including neurons, astrocytes, and microglia. The brain derives energy from glucose, glutamate, lactate, and ketone bodies because these metabolites have specialized carriers that can cross the blood-brain barrier (BBB). Glucose is extremely important for ATP synthesis, neurotransmitters precursor synthesis and maintenance of ionic concentration gradients across the membrane, neuronal plasticity, learning, and memory. Dysregulation in glucose homeostasis leads to hyperglycemia or hypoglycemia, conditions associated with the impairment of brain functions [12]. Recent studies show that dysregulation of brain glucose metabolism and hyperglycemia is associated with neurodegenerative disease. Hyperglycemia and insulin resistance are risk factors that can increase the probabilities of neuronal death and stroke [13, 14]. Chronic hyperglycemia leads to diabetic neuropathy, retinopathy, and dementia, also associated with glucotoxic conditions [15, 16]. A vast amount of clinical and epidemiological studies reported that diabetes and hyperglycemia have a close association with pathological alteration of the cerebral microvasculature, which leads to cognitive deficits and an increased risk for stroke [17]. Microglia dysfunction or self-degradation is associated with enhanced neurodestructive effects, which are a major concern in diabetic brains. Microglia, a unique and motile type of immune cell in the central nervous system (CNS), play a crucial role in brain development and contributes to neural cell genesis and synaptic development [18, 19]. Microglia form the innate defensive system of the CNS and eliminate the damaged cells and dysfunctional synapses through interaction with neurons during the pathological condition [19]. They play an important role in learning-associated synaptic plasticity in the healthy adult brain and contribute to the regulation of network activity of newly born neurons and the elimination of supranumerous apoptotic neurons in the adult hippocampus [20]. Microglia have bifunctional characteristics features and their function might vary based on the activation state because they rapidly respond to chronic disease and acute insults affecting the CNS. Activated microglial cells release neurotrophic factors such as nerve growth factor (NGF), neurotrophin (NT)-4/5, transforming growth factor (TGF)-β1, glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and interleukin (IL)-3, which promote neuronal survival and differentiation [21]. Also, part of the microglia activation releasese reactive oxygen species and cytokines in acute and chronic pathological conditions. Recent studies reported that microglia could be beneficial for neurogenesis, progenitor proliferation, survival, migration, and differentiation in the early stages of Alzheimer’s disease (AD), the initial microglial activation serves a protective role in clearing amyloids [22]. When clearance fails, microglia could play a detrimental role by producing pro-inflammatory cytokines, leading to progressive neurodegeneration in the later stage of AD [23].

There is a lack of molecular and mechanistic evidence showing the effect of hyperglycemia on microglia functions. In the present study, we explored the mechanism of the ER chaperone BIP repression-mediated ER stress-induced microglia dysfunction in hyperglycemia conditions.

Cell Culture

The Human microglia clone 3 cell line (HMC3) was cultured in eagle’s minimum essential medium (EMEM) (ATCC, Manassas, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate and 1% penicillin/streptomycin at 37°C in a humidified atmosphere of 95% air/5% CO2. HMC3 cells were seeded at a density of 2×10⁵ cells in normal glucose (1 g/L, 5.5mM) and high glucose medium (4.5 g/L, 25mM), cultured for 5 days, and cells were harvested for protein expression, RNA isolation, and subcellular organelles isolation. For BIX treatment: Cells were cultured in control and hyperglycemia conditions for 48hr, after 48 hr, fresh medium was replaced with 25μM of BIX (MedChem Express LLC, NJ, USA) for 72 hr, and immunocytochemistry and Western blotting were performed as described in the method section.

Animals

We used 10 weeks old male C57BL/6J mice purchased from Jackson Laboratories (Bar Harbor, ME). Mice were maintained in a pathogen-free barrier facility under a 12-hours dark/light cycle at 21°C temperature and 40–60% humidity in the Department of Laboratory Animal Resources (DLAR), University of Toledo, Health Science Campus (HSC). All animal protocols were approved by the Institutional Animal Care and Utilization Committee (IACUC) of the University of Toledo under the National Institute of Health guidelines.

Streptozotocin-induced type-I diabetes model

Age and weight-matched mice were subdivided into two groups: normoglycemic (n=5) and hyperglycemic (n=5). Streptozotocin (Sigma-Aldrich, St. Louis, MO, USA) was prepared freshly every time before injection by dissolving in 10 mM citrate buffer (PH 4.5). The Hyperglycemic group was injected with STZ (100 mg/Kg) intraperitoneally over two consecutive days, whereas the normoglycemic group was injected with an equal volume of citrate buffer vehicle. Hyperglycemia was confirmed by measuring blood glucose levels (>300mg/dl) from two sequential tail vein samples using a Contour Next EZ glucometer (Ascenia Diabetic Care, NJ, USA).

Quantitative real-time PCR

Cells were cultured in EMEM with normal glucose 1 g/L, 5.5mM) and high glucose (4.5 g/L,25 mM) for 5 days. Total RNA was isolated using the TRIzol reagent (Life Technologies, California, USA) the RNA concentration and quality were measured using NanoDrop^TM (Thermo Fisher Scientific, MA 02451, USA). Complementary DNA (cDNA) was synthesized using the high-capacity cDNA reverse transcription kit (Thermo Scientific, USA) with 1x RT buffer, 1x random primers, 4 mM dNTP mix, 50 U/ml reverse transcriptase, and 1 μg of total RNA. The primers were designed using Primer Express® 3.0 (Applied Biosystems), and the primer sequences are listed in figure S5. Relative mRNA expressions were determined by quantitative real-time RT-PCR using SYBR Green real-time PCR master mix (Applied Biosystems) by following the manufacturer's instructions. PCR was initiated with denaturation at 95 °C for 10 min, which was followed by 40 cycles of 95 °C for 15 s, 58 °C for 60 s, and 95 °C for 15 s, and a final extension was performed at 72 °C for 5 min. Amplification was measured using an ABI 7500 instrument (Applied Biosystems). The mRNA expression levels were analyzed in triplicate, and the results were analyzed using the 2−ΔΔCT method [24]. Beta-actin was used as an endogenous control for normalization. The q-RTPCR data are presented as the mean values, and the data were analyzed using Student's t-test.

Western blotting

Cells were grown in EMEM medium with normal glucose (1g/L, 5.5mM) and high glucose (4.5 g/L, 25mM) for 5 days, washed with 1x PBS, and the cell lysate was prepared in ice-cold lysis buffer (20 mM/L Tris (pH 8.0), 137 mM/L NaCl, 1% NP40,10% glycerol) supplemented with protease inhibitors (1mM phenylmethyl sulfonyl fluoride (PMSF), 10μg/ml aprotinin, 10μg/ml leupeptin), and 3mM Na3VO4. The lysate was cleared by centrifugation and the protein concentration was determined using the Bradford assay (Bio-Rad; Hercules, CA, USA). The total protein sample (25µg) was resolved using 10 to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking for 30 min with blocking buffer (5%BSA, PBS, 0.05% Tween-20) membranes were incubated with the appropriate primary antibodies; anti-BIP (1:1000), anti-eIFα (1:1000), anti-CHOP (1:1000), anti-Bax (1:1000), anti-Bad (1:1000), anti-caspase-3 (1:1000), anti-cleaved caspase-3 (1:1000), anti-caspase-1 (1:1000), anti-cytochrome-C (1:1000), anti-Bcl2 (1:1000), calreticulin (1:1000), anti-ATG12 (1:1000), anti-LC-3(1:1000), anti-Beclin 1(1:1000) and β-actin (1:5000) at 4°C overnight. As secondary antibodies, anti-rabbit and anti-mouse HRP IgG (1:5000) were used. The proteins were visualized using an enhanced chemiluminescence detection reagent (Thermo Fisher Scientific, USA) with horseradish peroxidase-conjugated anti-rabbit or secondary antibody. Western blotting was used to determine the levels of BiP (1:1000 dilution), Cleaved caspase 3 (1:1000 dilution), caspase 3 (1:1000 dilution), and β-actin (1:3000) used as a loading control (Cell Signaling Technology, MA, USA).

Flow cytometry analysis

Cells were cultured and trypsinized (0.05%) and washed with serum-containing medium and resuspended in 0.5ml of ice-cold 1X binding buffer. 5μl of Annexine V5-FITC (fluorescein isothiocyanate-annexin-V (Thermo Fisher Scientific) was added and incubated at room temperature (18 to 24°C) for 15mins in the dark. Cells were centrifuged at 1000xg for 5mins at room temperature and the supernatant was removed and the cells were resuspended in 1X binding buffer and analyzed via Annexin binding by flow cytometry (Ex=488; Em=518 nm) using FITC signal detector (FL1) according to manufacturer instructions.

H2DCFDA- ROS staining

Cells were trypsinized (0.05%) and washed with serum-containing medium and resuspended in 1.0 ml of 1X buffer. Cells were stained with 20μM DCFDA (2',7'-dichlorodihydrofluorescein diacetate) for 30mins at 37°C in the dark, and the fluorescence intensity was measured by flow cytometry (Ex=485; Em=535nm) using a FITC signal detector (FL1) according to manufacturer instructions. The fluorescence intensity was determined by the changes as a percentage of control after background subtraction.

Immunocytochemistry

Coverslips were pre-coated with poly-D-lysine (50μg/ml) overnight and dried in a CO₂ incubator. Cells were cultured on the coverslips placed in the 6well plates containing normal glucose and high glucose medium for 5 days, washed in wash buffer (1XPBS+0.1%tween-20) and cells were fixed with 4% paraformaldehyde for 10mins at room temperature. Then cells were washed three times for 5 mins in ice-cold PBS (PH7.4) and incubated in PBS containing 0.25% TritonX-100 for 10 mins to increase the membrane permeability. Cells were incubated in PBST blocking buffer (1% BSA, 22.52 mg/ml glycine, 0.1% Tween-20) for 30 mins. Then cells were incubated in the primary antibody in a blocking buffer (1% BSA in PBST) overnight at 4°C and washed three times in PBS for 5 min. Then cells were again incubated with the secondary antibody in 1% BSA for 1 h at room temperature in the dark and washed three times with PBS for 5 min in the dark. Coverslips were removed from 6 well plates and wiped, another side of the coverslips was placed on the microscope slide and mounted with a drop of mounting medium containing DAPI staining (Life Technologies). Coverslips were sealed with nail polish to prevent drying and movement under a microscope. Imaging was performed using a fluorescence microscope at 20x magnification and fluorescence intensity.

ER chaperone BIP repression increases the ER stress

To explore the chronic effect of hyperglycemia on ER chaperone in microglia, cells were cultured in normal glucose (5.5mM) and hyperglycemia (25mM) for 5 days. We analyzed the BIP expression on day 2, day 3, day 4, and day 5. We found that the BIP expression was significantly downregulated on day 5 (Fig.1A). The BIP protein expression was significantly reduced, whereas the ER stress key marker proteins p-eIF2α and CHOP level was upregulated in hyperglycemia than control (Fig.1B, C). But there was no change in the expression of total eIF2α. Next, we analyzed the mRNA expression of genes involved in the ER stress and found BIP mRNA expression ∼ 50% reduced, and CHOP and ATF4 upregulated to ∼ 3 fold than control (Fig.1C). These results suggest that the inhibition of BIP expression induces ER stress.

ER stress increased in STZ-induced mouse model

To further validate the in-vitro BIP downregulation, we used streptozotocin (100 mg/Kg) induced type-1 diabetes mouse model. Blood glucose levels (>300mg/dl) were increased on day 1, day 7, and day 14 in STZ injected group (S4). After 14 days of experimental condition, mice were sacrificed, brains were collected, and tissue lysate was prepared for ER stress protein expression studies. Interestingly, the ER chaperone BIP was significantly downregulated, whereas p-eIF2α, CHOP, and cleaved caspase-3 expression levels were significantly upregulated in STZ group than the control group (Fig 2A, B). However, the ER chaperone calreticulin, total eIF2α, and caspase-3 protein expression levels were not changed in STZ group. These results suggest that hyperglycemia induces ER stress and apoptosis.

Hyperglycemia induced apoptosis in human microglia cells

Since we found the BIP expression was reduced and ER stress was increased in both in-vitro and in-vivo models. This finding prompted us to explore whether BIP repression induces apoptosis in microglia during hyperglycemia. Astonishingly, the pro-apoptotic markers Bax and Bad expression were significantly increased, and mitochondria-mediated apoptosis marker cytochrome-C and cleaved caspase-3 was also increased and no significant change was observed in anti-apoptosis marker Bcl2, and total caspase-3 expression in hyperglycemia (Fig. 3A). Next, we tested the expression of proteins involved in autophagy hence apoptosis was increased in hyperglycemia. In hyperglycemia, LC3-BII expression was significantly reduced and no significant change was detected in the expression of Atg12 and Beclin-1(Fig. 3A). Further, to validate our findings more comprehensively, we analyzed the apoptosis using annexin-V5-FITC staining and ROS production using DCFDA staining. The flow- cytometry data shows that hyperglycemia-induced 44.30% apoptotic cells than control (Fig. 3B, S2). Also, the DCFDA staining result shows dramatically increased ROS production in hyperglycemia (Fig. 3C,S3). These results suggest that hyperglycemia induces apoptosis in microglia cells.

BIX mitigated the ER stress through derepression of BIP expression

ER stress and apoptosis was increased in microglia cells when cultured in hyperglycemia. Therefore, we speculated that the apoptosis could be increased through the downregulation of ER chaperone BIP. To validate and prove the hypothesis comprehensively, we used BiP inducer-X (BIX) to derepress the BIP expression. HMC3 cells were cultured in 25mM glucose for 5 days. At day 2, cell were treated with 25μM BIX and cultured until day 5 and immunocytochemistry for the BIP and western blot for proteins involved in the ER stress was performed. As expected, we found significantly reduced fluorescence of BIP protein expression in hyperglycemia than the control and interestingly, BIP protein expression was restored in BIX-treated cells (Fig. 4A).

The BIP protein expression was significantly increased in BIX-treated cells than untreated hyperglycemia control but its expression was not induced as equal to normoglycemia control. And subsequently, the CHOP, p-eIF2α, and total eIF2α expression were reduced in BIX-treated cells than in control (Fig. 4B). These results suggest that hyperglycemia increased the ER stress through repression of the ER chaperone BIP and alleviated the ER stress through derepression of BIP protein expression when cells were treated with BIX.

BIX protects microglia cells from the apoptosis

The BIP protein expression was enhanced and ER stress was reduced in BIX-treated cells. Therefore, we further investigated whether the inhibition of ER stress through derepression of BIP protein expression could reduce apoptosis in microglia cells. We evaluated the expression of proteins involved in apoptosis in BIX-treated cells. Interestingly, the pro-apoptotic marker proteins including Bax, Bad, cytochrome-C, and caspase-1, cleaved caspase-3 expression levels were significantly reduced, and caspase-3 expression was not significantly changed in BIX-treated cells (Fig. 5A). Furthermore, we investigated whether the autophagy process could play a role on the protection of microglia cells from apoptosis or ER stress. Unfortunately, the BIX-treated cells did not show increased LC3-BII in hyperglycemia than control (Fig.5B), suggesting that BIX does not alter the autophagy through the induction of BIP in hyperglycemia. These results suggest that BIX protects microglia cells from apoptosis through derepression of the BIP protein expression and inhibition of ER stress.

In the present study, we reported that hyperglycemia reduced the BIP protein expression and subsequently increased ER stress in microglia cells partly due to a defect in protein folding and accumulation of unfolded or misfolded proteins in the ER.

Numerous studies suggested that inhibition of ER stress is most productive for neurodegenerative disease. The activation of UPR is an ER adaptive mechanism to protect the cells but prolonged UPR activation is also detrimental and causes cell death. The UPR adaptive mechanism comprises three principal signaling pathways, inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). In addition, the UPR adaptive mechanism is mediated by molecular chaperones and luminal enzymes, including the BIP/GRP78, GRP94, calreticulin, and lectin calnexin, disulfide isomerase (PDI) Erp57, thiol-disulfide oxidoreductase [25]. Previous studies have shown that BIP protein expression was increased in ER stress and caused more than 70% cell death during glucose deprivation [26]. In contrast, the BIP protein expression was reduced significantly, and p-eif2α protein expression and ER stress were increased in hyperglycemia conditions. It is possibly due to reduced expression of BIP or disassociation of BIP from the PERK protein in hyperglycemia. The disassociation of BIP with PERK or reduced expression could increase the phosphorylation of eIF2α through PERK [27]. The mRNA expression of ATF4 was also increased in hyperglycemia due to PERK-mediated increased phosphorylation of eIF2α, the ATF4 transcription factor that activates the expression of CHOP. The CHOP mRNA and protein expression were profoundly increased in hyperglycemia which is due to the increased p-eIF2α mediated derepression of ATF4, through the transcription of genes involved in apoptosis [28].

The in-vivo streptozotocin-induced diabetes mouse model also revealed that BIP repression induced the ER stress through the activation of CHOP and hyperphosphorylation of eIF2α. Previous studies have also shown that the knockdown of BIP increased the phosphorylation of eIF2α, and increased CHOP expression in both in-vitro and in-vivo models [29]. Inhibition or suppression of BIP could induce the compensatory mechanism by the upregulation of ER chaperones. However, there were no compensatory ER chaperone expressions detected (calreticulin) when BIP protein expression was reduced in hyperglycemia.

Caspases are critical mediators of apoptosis in mammalian cells. The Bcl2 is an anti-apoptotic protein that plays an important role in cell proliferation. In hyperglycemia, the Bcl2 protein expression was not significantly changed whereas the apoptosis marker proteins, cleaved caspase-3, Bax, Bad, and cytochrome-C expression levels were increased. It is possibly due to the activation of ER resident protein caspase-12, which activates the cytoplasmic caspase-3 via TNFα during the ER stress [30]. Increased caspase-3 activation alters the mitochondrial membrane depolarization leading to mitochondrial dysfunction [31]. Recent studies also suggested that microglial cell function is a prerequisite for appropriate brain function and important for the maintenance of neuronal circuitry, and dysfunction of microglia leads to abnormal behavior or neural dysfunction [32]. The flow cytometry results also revealed that hyperglycemia-induced 43% of apoptosis in microglia cells. The previous study also revealed the ER-resident protein caspase-12-dependent caspase-3 activation causes apoptosis in ER stress [33]. Activation of UPR is an adaptive mechanism to protect the cells from the stress-induced apoptosis or cell death. A moderate level of UPR activation may alleviate cellular dysfunction and increase the chances of survival; however, when the ER stress is prolonged or UPR fails to restore ER homeostasis leads to cause cell death or apoptosis [5].

In hyperglycemia, the increased expression of mitochondrial apoptosis markers suggests that ER stress-mediated mitochondrial apoptosis is induced. Apoptotic cell death occurs primarily through intrinsic and extrinsic signaling pathways and often involves ER and mitochondrial membrane crosstalk pathway-mediated apoptosis in ER stress [34, 35]. Previous studies have identified numerous crosstalk signaling mechanisms between ER mitochondria-mediated apoptosis [36]. A recent study showed that ER stress might be primarily related to mitochondria-regulated apoptosis [35]. ER stress can activate extrinsic and intrinsic apoptotic pathways through different molecular mechanisms. Our results show that Bax, Bad, and cleaved caspase-3 levels are profoundly increased and BIP was inhibited at day 5. It is due to derepression of CHOP mediated caspase-3 activation leading to apoptosis. Taken together, the ER stress-mediated apoptosis pathway is activated in chronic hyperglycemia (5 days), implying that inhibition of the ER stress response reduces apoptosis and protects microglia cells. Constitutive production of ROS is a main inducer of apoptosis. Cells can produce endogenous antioxidants, such as glutathione and superoxide dismutase, to prevent ROS production and protect the cells from stress and apoptosis [37]. We evaluated whether the Nrf2-dependent redox homeostatic balance could protect the microglia in hyperglycemia. The Nrf2 expression was increased in the nuclear fraction, and there was no significant change detected in total cell lysate (S1). However, the ROS levels were increased and apoptosis was not inhibited. It might possibly be due to prolonged chronic ER stress and the ratio of ER stress could be greater than the redox balance. Nrf2 is the transcription factor, which regulates the transcription of genes involved in antioxidant synthesis [38].

Interestingly, the BIP protein expression was upregulated and ultimately, ER stress was reduced through the repression of p-eIF2α and CHOP expression in BIX-treated hyperglycemia cells. Subsequently, the proteins involved in the apoptosis, Bax, Bad, cleaved caspase-3, caspase-1, and cytochrome-C levels were significantly reduced. This further led to the inhibition of apoptosis in microglia cells during hyperglycemia conditions. The LC3-BII expression was not changed in BIX-treated cells when compared with untreated hyperglycemia control, suggesting that BIX is protecting the microglia cells not through autophagy in hyperglycemia. Recent study has also shown that BIX protects the neuronal cells from ER stress in-vitro neuroblastoma cells [39, 40]. But there was a lack of evidence of whether the microglia cells mediated neuronal protection is replicated in in-vivo stroke mouse model. Understanding microglia function is complex; a current decade of studies shows that activated microglia plays a crucial role in eliminating damaged neuron cells and secreting neurotrophic factors, which is required for neurogenesis during the pathological condition. Microglial dysfunction results in behavioral deficits, indicating that microglia are essential for proper brain function. This defines a new role for microglia beyond being a mere pathologic sensor. For the first time, we described the BIP signaling mediated microglial cell protection from the ER stress in hyperglycemia, thus opening new therapeutic strategies for neurological disease and stroke. There were several limitations to this study that should be addressed. First, although we demonstrated the BIP expression was downregulated in STZ-induced mouse brain but additional systematic experiments will be required to show in primary microglia cells from STZ-induced mouse. Second, we reported that BIP repression-mediated ER stress is restored in-vitro after BIX treatment but this needs to be proved in-vivo mouse model and overexpression of BIP/HSPA5 gene to HMC3 cells in hyperglycemia is required for solid confirmation. Thus, future studies are needed to acquire more thorough information on using inhibitors for microglia proliferation in mouse models to evaluate microglia function in diabetes conditions.

In conclusion, our results suggest that BIX, a selective inducer of BIP, inhibits the apoptosis induced by ER stress in hyperglycemia. Hence, drugs that selectively induce BIP may activate defense mechanisms against ER stress, resulting in a protective effect. Such drugs may therefore have potential as new therapeutic interventions against neurological disease and microglia dysfunctions mediated complications in diabetes conditions.

BIP: Binding of Immunoglobulin heavy chain protein, CNS: Central nervous system, ROS: ER: Endoplasmic reticulum, STZ: Streptozotocin, BIX: BiP inducer-X, XBP1: Binding of X-box protein, ERSE: Endoplasmic reticulum stress responsive element, eIF2α: Eukaryotic translation initiation factor-2 alpha, ATF4: The active transcription factor-4, CHOP: C/EBP homologous protein, C-Caspase-3: Cleaved caspase-3, UPR: Unfolded protein response, BBB: Blood-brain barrier, cDNA: Complementary DNA, PCR: Polymerase chain reaction, SDS-PAGE: Polyacrylamide gel electrophoresis, PVDF: Polyvinylidene difluoride, ICC: Immunocytochemistry, AD: Alzheimer’s disease, PERK: Protein kinase R-like ER kinase, ANOVA: Analysis of variance, Annexin V-FITC: Fluorescein isothiocyanate annexin V, DAPI: 4, 6-diamidino-2-phenylindole, DCFDA: 2’-7’-Dichlorodihydrofluorescein diacetate.

Acknowledgments

We thank Dr. Katherine A. Wall, Ph.D, Professor and Chair, Department of Medicinal and Biological Chemistry, and Giselle Soares, University of Toledo, for helping Flowcytometry.

Author contribution: WJ and ZAS designed the experimental plan; WJ performed the experiments and wrote the first draft of the manuscript. GAB carried out the animal experiments and WJ analyzed the datasets. MM carried out Western blotting of in vitro experiments. ZAS corrected the draft and approved the final version.

Funding: The study was supported by grants from the National Institute of Neurological Disorders and Stroke of the National Institutes of Health #R01NS112642 to ZAS.

Availability of Data and Materials

The datasets used in the current study are available with corresponding author and will be shared with any appropriately qualifed investigator upon reasonable request.

Consent for publication

Not applicable

Declaration of competing interest

The authors declare no competing interests

Ethics Approval

All animal protocols were approved by the Institutional Animal Care and Utilization Committee (IACUC) of the University of Toledo, Ohio, USA, under the National Institute of Health guidelines The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, Toledo, Ohio USA

Antonisamy William James, Ghaith A. Bahader, Mohammad Albassan

Corresponding Author

Zahoor A. Shah, Ph.D., Department of Medicinal and Biological Chemistry, University of Toledo, 3000 Arlington Avenue, Toledo, Ohio, 43614. Phone: 419-383-1587. Email: [email protected]

Lee A S. Mammalian stress response: induction of the glucose-regulated protein family. Curr Opin Cell Biol.1992; 4, 267–273.
Lee A S. The glucose-regulated proteins: stress induction and clinical applications. Trends Biochem Sci. 2001; 26, 504–510.
Kuznetsov G, Chen L B, Nigam S K. Multiple molecular chaperones complex with misfolded large oligomeric glycoproteins in the endoplasmic reticulum. J Biol Chem.1997; 272, 3057–3063.
Klausner, R D, Sitia R. Protein degradation in the endoplasmic reticulum. Cell. 1990 ;62, 611–614.
Raghubir R, Nakka VP, Mehta SL. Endoplasmic reticulum stress in brain damage. Methods Enzymol. 2011; 489:259-275.
Marciniak SJ, Garcia-Bonilla L, Hu J, Harding HP, Ron D. Activation-dependent substrate recruitment by the eukaryotic translation initiation factor 2 kinase PERK. J Cell Biol. 2006; 172: 201–209.
Harding HP, Zhang Y, Zeng H, Novoa I, Lu PD, Calfon M. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol Cell. 2003; 11: 619–633.
Marciniak SJ, Yun CY, Oyadomari S, Novoa I, Zhang Y, Jungreis R. CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum. Genes Dev. 2004; 18: 3066–3077.
James A W, Gowsalya R and Nachiappan V. Dolichyl pyrophosphate phosphatase-mediated N-glycosylation defect dysregulates lipid homeostasis in Saccharomyces cerevisiae. Biochim Biophys Acta. 2016; 1861, 1705–1718.
William James A, Ravi C, Srinivasan M, Nachiappan V. Crosstalk between protein N-glycosylation and lipid metabolism in Saccharomyces cerevisiae. Sci rep. 2019; 9; 9(1):14485.
Helenius A. How N-linked oligosaccharides affect glycoprotein folding in the endoplasmic reticulum. Mol Biol Cell. 1994; 5, 253–265.
Seaquist E R. The impact of diabetes on cerebral structure and function. Psychosom Med. 2015; 77 (6), 616–621.
Pugazhenthi, S, Qin L, & Reddy P H. Common neurodegenerative pathways in obesity, diabetes, and Alzheimer’s disease. Biochim Biophys Acta. 2017; 1863, 1037–1045.
Sridhar G R, Lakshmi G, & Nagamani G. Emerging links between type 2 diabetes and Alzheimer’s disease. World J Diabetes. 2015; 6,744–751.
Lee H J , Seo HI, Cha HY, Yang Y J, Kwon S H, Yang S J. Diabetes and Alzheimer’s disease: mechanisms and nutritional aspects. Clin Nutr Res. 2018; 7 (4), 229–240.
Chatterjee S, Mudher A. Alzheimer’s disease and type 2 diabetes: a critical assessment of the shared pathological traits. Front Neurosci. 2018; 12, 383.
Hardigan T, Ward R, Ergul A. Cerebrovascular complications of diabetes: focus on cognitive dysfunction.Clin Sci (Lond). 2016; 1;130(20):1807-22.
Kettenmann H, Hanisch U K, Noda M , and Verkhratsky A. Physiology of microglia. Physiol Rev. 2011; 91, 461–553.
Fan Z, Brooks DJ, Okello A, Edison P. An early and late peak in microglial activation in Alzheimer's disease trajectory. Brain. 2017; 140(3):792-803.
Sierra A, Encinas J M, Deudero J J, Chancey J H, Enikolopov G, Overstreet-Wadiche L S, Tsirka S E, and Maletic-Savatic M. Microglia shape adult hippocampal neurogenesis through apoptosis-coupled phagocytosis. Cell Stem Cell. 2010; 7, 483–495.
Nakajima K, Tohyama Y, Maeda S, Kohsaka S, and Kurihara T. Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons. Neurochem Int. 2007; 50, 807–820.
Hamelin L, Lagarde J, Dorothée G, et al. Early and protective microglial activation in Alzheimer's disease: a prospective study using 18F-DPA-714 PET imaging. Brain. 2016; 139 (4):1252-1264.
Fan Z, Brooks DJ, Okello A, Edison P. An early and late peak in microglial activation in Alzheimer's disease trajectory. Brain. 2017; 140 (3):792-803.
Livak KJ, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C (T)) method. Methods. 2001;25,402–408
Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress induced apoptosis. EMBO Rep. 2006; 7:880–885
Selene G D C, Karla H F, Ignacio C A, Lourdes M. Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7- and calpain-mediated caspase-12 activation. Apoptosis. 2014; 19(3):414-27.
Harding H P, Zhang Y, Ron D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature. 1999; 21;397(6716):271-4.
Szegezdi E, Logue SE, Gorman AM, Samali A. Mediators of endoplasmic reticulum stress induced apoptosis. EMBO Rep. 2006; 7:880–885.
Rangel DF, Dubeau L, Park R, Chan P, Ha DP, Pulido MA, Mullen DJ, Vorobyova I, Zhou B, Borok Z, Offringa IA, Lee AS. Endoplasmic reticulum chaperone GRP78/BiP is critical for mutant Kras-driven lung tumorigenesis. Oncogene. 2021; 40(20):3624-3632.
Junichi H, Taiichi K, Manabu T, Akiko H, Kazunori I, Masaya T. Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett. 2004; 4:357(2):127-30.
Edaena B R , Mauricio O A, María Cristina López-Méndez G D M , Agustín HG, Julio Moran. Caspase-3 Activation Correlates With the Initial Mitochondrial Membrane Depolarization in Neonatal Cerebellar Granule Neurons. Front Cell Dev Biol. 2020; 2;8:544.
Derecki N C , Cronk J C, Lu Z, Xu E, Abbott S B, Guyenet PG, and Kipnis J. Wild-type microglia arrest pathology in a mouse model of Rett syndrome. Nature. 2012; 484, 105–109.
Jin M, Yang YW, Cheng WP, Lu JK, Hou SY, Dong XH, Liu SY. Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury. Neural Regen Res. 2015; 10:1830-1835
Sun GZ, Gao FF, Zhao ZM, Sun H, Xu W, Wu LW, He YC. Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injury. Neural Regen Res. 2016; 11:1260-1266.
Sophonnithiprasert T, Mahabusarakam W, Nakamura Y, Watanapokasin R. Goniothalamin induces mitochondria-mediated apoptosis associated with endoplasmic reticulum stress-induced activation of JNK in HeLa cells. Oncol Lett. 2017; 13:119-128.
Burton GJ, Yung HW, Murray AJ. Mitochondrial - Endoplasmic reticulum interactions in the trophoblast: Stress and senescence. Placenta.2017; 52:146-155.
Hiebert JB, Shen Q, Thimmesch AR, Pierce JD. Traumatic brain injury and mitochondrial dysfunction. Am J Med Sci. 2015;350:132-138.
Farina M, Vieira LE, Buttari B, Profumo E, Saso L. The Nrf2 Pathway in Ischemic Stroke: A Review. Molecules. 2021; 18; 26 (16):5001.
Kudo T, Kanemoto S, Hara H, Morimoto N, Morihara T, Kimura R, Tabira T, Imaizumi K, Takeda M. A molecular chaperone inducer protects neurons from ER stress. Cell Death Differ. 2008;15(2):364-75.
Oidaa Y, Izutaa H, Oyagia H, Shimazawaa M, Kudob T, Imaizumic K, Hara H. Induction of BiP, an ER-resident protein, prevents the neuronal death induced by transient forebrain ischemia in gerbil. Brain Res. 2008;7;1208:217-24.

No competing interests reported.

SupplementaryfigS1.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

The ER chaperone, BIP protects Microglial cells from ER stress-mediated Apoptosis in Hyperglycemia

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1