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. Author manuscript; available in PMC: 2026 Jul 15.
Published in final edited form as: Brain Res. 2025 Apr 16;1859:149647. doi: 10.1016/j.brainres.2025.149647

Effects of Mitochondrial O-GlcNAcylation in Pericytes after Mechanical Injury

Ji Hyun Park 1,*, Dong Bin Back 1, Shuzhen Guo 1, Masayoshi Tanaka 1, Hajime Takase 1, Michael J Whalen 2, Ken Arai 1, Kazuhide Hayakawa 1,*, Eng H Lo 1,*
PMCID: PMC12302007  NIHMSID: NIHMS2077352  PMID: 40250747

Abstract

Damage to vascular cells comprise an important part of traumatic brain injury (TBI) but the underlying pathophysiology remains to be fully elucidated. Here, we investigate the loss of O-Linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) and mitochondrial disruption in vascular pericytes as a candidate mechanism. In mouse models in vivo, TBI rapidly induces vascular oxidative stress and down-regulates mitochondrial O-GlcNAcylation. In pericytes but not brain endothelial cultures in vitro, mechanical stretch injury down-regulates mitochondrial O-GlcNAcylation. This is accompanied by disruptions in mitochondrial dynamics, comprising a decrease in mitochondrial fusion and an increase in mitochondrial fission proteins. Pharmacologic rescue of endogenous mitochondrial O-GlcNAcylation with an O-GlcNAcase inhibitor Thiamet-G or addition of exogenous O-GlcNAc-enhanced extracellular mitochondria ameliorates the mitochondrial disruption in pericytes damaged by mechanical injury. Finally, in a pericyte-endothelial co-culture model, mechanical injury increased trans-cellular permeability; adding Thiamet-G or O-GlcNAc-enhanced extracellular mitochondria rescued trans-cellular permeability following mechanical injury. These proof-of-concept findings suggest that mitochondrial O-GlcNAcylation in pericytes may represent a novel therapeutic target for ameliorating oxidative stress and vascular damage after mechanical injury following TBI.

Keywords: traumatic brain injury, oxidative stress, vascular pericyte, O-GlcNAcylation, mitochondrial dynamics

Introduction

Traumatic brain injury (TBI) is a heterogeneous condition that triggers multiple pathomechanisms, including both primary and secondary events (McKee et al., 2015; Ng and Lee, 2019). The primary insult results from direct mechanical forces, while secondary events comprise a complex mix of responses in neural, glial and vascular cells that leads to further brain damage. The original focus in TBI was based on neuronal cell death (Akamatsu and Hanafy, 2020) and gliosis (Amlerova et al., 2024; Nespoli et al., 2024). It is now increasingly recognized that vascular injury is also important (Das et al., 2022; Logsdon et al., 2015; Salehi et al., 2017). However, the molecular mechanisms underlying vascular responses after TBI remain to be fully elucidated.

Oxidative stress and mitochondrial disruption are central to TBI pathophysiology, and these responses are thought to represent upstream events that lead to multiple downstream cell death pathways in high energy-consuming neurons (Fischer et al., 2016). In addition to neurons, vascular cells should also be vulnerable to mitochondrial perturbations since metabolic requirements are high for the maintenance of key vascular properties such as the blood-brain barrier (BBB) (Salnikova et al., 2021; Wang et al., 2023). BBB leakage and subsequent edema and hemorrhage are major events after TBI (Wu et al., 2020). Furthermore, vascular injury may augment the infiltration of immune cells and amplify secondary inflammation (Wang and Chen, 2023). Is it possible that mitochondrial disruptions also serve as an early upstream event in the vascular compartment following TBI? And if so, are there ways to restore mitochondrial function and mitigate this early injury?

It has been suggested that O-GlcNAcylation is widely distributed in mitochondrial proteins, where it plays a key role in regulating mitochondrial homeostasis, oxidative stress response, energy production, membrane potential, and motility (Qiu et al., 2024; Tan et al., 2017). O-GlcNAcylation is a reversible post-translational modification in which O-linked β-N-acetylglucosamine (O-GlcNAc) is added to the serine and/or threonine residues (Lee et al., 2021). This modification is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase, with the addition and removal of O-GlcNAc moieties occurring rapidly in response to cellular metabolic changes (Ong et al., 2018). An imbalance in O-GlcNAcylation can disrupt the delicate balance between mitochondrial fusion and fission, leading to mitochondrial dysfunction (Qiu et al., 2024). Recently, O-GlcNAcylation has been demonstrated to be crucial for maintaining mitochondrial function and dynamics after stroke (Park et al., 2020; Park et al., 2023). In this study, we explore the hypothesis that in the context of TBI, O-GlcNAcylation mechanisms play a key role in mediating early pericyte responses after mechanical injury.

Results

Vascular Oxidative Stress and Mitochondrial O-GlcNAcylation after TBI

First, we asked whether signs of vascular injury can be detected early. Mice were subjected to mild traumatic brain injury (mTBI) using a closed head injury model without craniotomy. This model induces minor neurological and cognitive deficits without causing discrete tissue tearing and cavitation (Luo et al., 2017; Siebold et al., 2018). At 1 hour after TBI, brain microvessels were isolated (Fig. 1A-B). Western blot analysis demonstrated a rapid increase in 4-hydroxynonenal (4-HNE), a marker for oxidative stress in the injured brain microvessels (Fig. 1C). Since mitochondrial O-GlcNAcylation has been linked to oxidative stress and mitochondrial function after injury (Qiu et al., 2024; Xue et al., 2024), we isolated brain mitochondria and assessed O-GlcNAcylation by western blot (Fig. 1D). Mitochondrial O-GlcNAcylation was significantly down-regulated in the injured brains at 1 hour post-injury (Fig. 1E).

Figure 1. Vascular oxidative stress and mitochondrial O-GlcNAcylation after mTBI.

Figure 1.

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(A) Schematic representation of mTBI and brain microvessel isolation. (B) Immunostaining with vascular-associated proteins on isolated brain microvessels. Isolated brain microvessels were stained with vascular markers, CD13 (green, pericyte) and CD31 (red, endothelial cell). Scale bar, 25 μm. (C) Oxidative stress marker, 4-HNE, was increased in microvessels at 1 hour post-injury (n=3, p=0.006). (D) Schematic representation of mTBI and brain mitochondria isolation. (E) O-GlcNAcylation was reduced in brain mitochondria following mTBI injury (n=3, p=0.043). Sh: sham, T: TBI. All values are mean +/− SEM. *P<0.05, **P<0.01.

Mechanical Injury Induces Mitochondrial Dysfunction and a Reduction in O-GlcNAcylation in Pericytes

To explore the cellular basis for the link between reduced O-GlcNAcylation and mitochondrial dysfunction, we utilized a standard in vitro model of mechanical injury. Human brain vascular pericytes or human brain endothelial cells were cultured on bioflex plates and subjected to air pressure pulses, which deform the flexible membranes and then stretch and damage the cells. Following this mechanical stretch injury, mitochondria were isolated and analyzed by western blot (Fig. 2A). Mechanical injury rapidly down-regulated mitochondrial O-GlcNAcylation in pericytes at 1 hour post-injury, a phenomenon not observed in endothelial cells (Fig. 2B). Mechanical injury also induced rapid alterations in mitochondrial network dynamics and membrane potential, as observed by tetramethylrhodamine (TMRM) staining, along with elevated cellular oxidative stress as early as 1 hour post-injury (Fig. 2C). Importantly, no significant changes in cell viability were observed, consistent with an early upstream role for these events in the present model of mechanical injury that does not induce immediate cell death (Fig. 2D).

Figure 2. Oxidative stress, mitochondrial dysfunction in pericytes after mechanical stretch.

Figure 2.

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(A) Schematic representation of mechanical cell injury and mitochondria isolation. (B) After mechanical cell stretch of pericytes (PC) or endothelial cells (EC), mitochondrial O-GlcNAcylation was accessed by WB. Pericytes showed a reduction in O-GlcNAcylation at 1 hour post-stretch (n=3, p=0.002), while no such reduction was observed in endothelial cells. (C) At 1 hour post-stretch, mitochondrial network morphology and membrane potential (assessed by TMRM staining) were disrupted, and oxidative stress was elevated. EC: endothelial cell. Scale bar, 25 μm (TMRM); 50 μm (ROS). (D) Mechanical stretch didn’t affect to cell viability. Str: stretch. All values are mean +/− SEM. **P<0.01.

Disruption of Mitochondrial Dynamics After Mechanical Injury

To better understand the molecular mechanisms of mitochondrial dysfunction induced by stretch injury, we analyzed proteins involved in mitochondrial dynamics in pericyte cultures that were subjected to mechanical injury. At 1 hour post-injury, we observed a down-regulation of mitochondrial fusion-related proteins, including Mitofusin 2 (MFN2) and Optic Atrophy Protein 1 (OPA1), while mitochondrial fission proteins, such as Mitochondrial Fission Factor (MFF) and Dynamin-Related Protein 1 (DRP1), were up-regulated (Fig. 3A-B). Immunostaining of MFN2 revealed an altered pattern of expression following mechanical injury (Fig. 3C). These results indicate that mechanical injury disrupts mitochondrial dynamics, promoting mitochondrial fission and impairing fusion.

Figure 3. Alterations in mitochondrial dynamics after cell stretch.

Figure 3.

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(A, B) Proteins related to mitochondrial dynamics were assessed at 1 hour post-stretch. There was a decrease in mitochondrial fusion-related proteins (n=3, MFN2, p=0.004; OPA1, p=0.012) and an increase in mitochondrial fission proteins (n=3, MFF, p=0.008; DRP1, p=0.002). (C) Representative staining of MFN2 in pericytes at 1 hour post-stretch Scale bar, 25 μm. All values are mean +/− SEM. *P<0.05, **P<0.01.

O-GlcNAcylation Induction Ameliorates Mitochondrial Dynamics Induced by Stretch Injury

Next, we asked whether enhancing mitochondrial O-GlcNAcylation could mitigate mitochondrial dysfunction. Treatment with Thiamet-G (TMG), an O-GlcNAcase (OGA) inhibitor, effectively increased levels of mitochondrial O-GlcNAcylation in pericytes that were subjected to mechanical injury (Fig. 4A-B). Importantly, TMG treatment restored mitochondrial dynamics, normalizing the stretch-induced alterations in mitochondrial fusion and fission proteins (Fig. 4C-D). As an alternative approach, we explored whether these therapeutic responses could also be obtained by treating pericytes with extracellular mitochondria. Mitochondria were isolated from HEK293 cells, modified with O-GlcNAcylation (Fig. 4E) and then added to pericytes before mechanical injury (Fig. 4F). Compared to non-modified mitochondria (mt-Con), O-GlcNAcylated mitochondria (mt-OGN) up-regulated the mitochondrial fusion protein MFN2 after mechanical injury (Fig. 4G-H). Taken together, these data suggest that enhancing mitochondrial O-GlcNAcylation can help restore mitochondrial dynamics in pericytes damaged by mechanical injury.

Figure 4. Enhancing mitochondrial O-GlcNAc normalizes mitochondrial dynamics.

Figure 4.

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(A) Schematic representation of TMG treatment and stretch injury. (B) Western blot confirmed elevated mitochondrial O-GlcNAcylation in pericytes following treatment with TMG. (C, D) Proteins related to mitochondrial dynamics were assessed by western blot in TMG treated cells. TMG treatment normalized the stretch-induced alterations in mitochondrial dynamics (D, n=3, C vs Str, p=0.001; Str vs Str/TMG, p=0.018). (E) Mitochondria isolated from HEK293 cells were O-GlcNAcylated in vitro by incubating with O-GlcNAc transferase (OGT) and Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Western blot confirmed O-GlcNAc modification of mitochondria. (F) Schematic representation of mitochondria treatment and stretch injury. (G, H) Treatment with mt-OGN up-regulated the mitochondrial fusion protein MFN2 following mechanical stretch injury (H, n=3, Str vs Str/mt-OGN, p=0.002). All values are mean +/− SEM. *P<0.05, **P<0.01.

O-GlcNAcylation Ameliorates the Disruption of Cellular Permeability Induced by Stretch Injury

Finally, we asked whether O-GlcNAcylation-mediated mitochondrial protection in pericytes can rescue vascular functional integrity. Pericytes were co-cultured with brain endothelial cells and then tracers were used to measure trans-cellular permeability, an indirect in vitro surrogate marker for vascular barrier function (Fig. 5A). After mechanical stretch, trans-cellular permeability was significantly increased, consistent with loss of barrier function after vascular injury (Fig. 5B). Treating these cells with either TMG to increase endogenous O-GlcNAcylation or adding O-GlcNAcylated extracellular mitochondria significantly prevented the increase in trans-cellular permeability (Fig. 5B). Taken together, these findings suggest that enhancing mitochondrial O-GlcNAcylation through either pharmacological inhibition of OGA or adding exogenous mitochondria can help normalize mitochondrial dynamics and protect the functional integrity of vascular cells (Fig. 5C).

Figure 5. Enhancing mitochondrial O-GlcNAcylation preserves cell permeability following stretch injury.

Figure 5.

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(A) The cell permeability assay following mechanical stretch injury. (B) The increase in cell permeability induced by mechanical stretch was normalized by treatment with TMG or mt-OGN (n=4, C vs Str, p=0.001; Str vs Str/TMG, p=0.006; Str vs Str/mt-OGN, p=0.032). (C) A schematic diagram suggesting that mitochondrial O-GlcNAcylation may protect against TBI-induced mitochondrial and microvascular damage. All values are mean +/− SEM. *P<0.05, **P<0.01.

Discussion

The pathophysiology of TBI is complex and multifactorial. Beyond events in the neuronal compartment per se (e.g. neuron cell death, axonal damage, synaptic and dendritic loss), TBI can also induce significant vascular damage that contributes to BBB leakage, hemorrhage, edema, neuroinflammation and secondary neurodegeneration (Das et al., 2022; Logsdon et al., 2015; Ng and Lee, 2019). Our study demonstrates that pericyte responses may play an early role. Mechanical injury in pericytes trigger a rapid reduction of mitochondrial O-GlcNAcylation thus leading to disruptions in mitochondrial dynamics. These findings raise the possibility that up-regulating mitochondrial O-GlcNAcylation may provide novel therapeutic approaches for protecting pericytes and rescuing vascular function in TBI. Mitochondrial integrity is highly vulnerable to cellular stress and injury (Zong et al., 2024). Restoring mitochondrial integrity and function is a promising approach for treating a wide spectrum of CNS disorders (Borcherding and Brestoff, 2023; Zong et al., 2024). Often, the targets involved boosting mitochondrial metabolism (Wu et al., 2016; Zhao et al., 2024), modifying the mitochondrial unfolded protein response (Nguyen et al., 2023; Sorrentino et al., 2017), scavenging free radicals (Rossman et al., 2018; Zhao et al., 2021) or blocking cell death caspases (Li et al., 2022; Lin et al., 2022). However, many of these targets tend to occur further downstream in the pathogenic cascade. The present study may be consistent with an emerging idea that targeting initial reversible responses—such as O-GlcNAcylation —offers a promising strategy to prevent subsequent irreversible mitochondrial disruption (Dontaine et al., 2022).

However, there are several important caveats and considerations. First, while our findings implicate pericytes in the early vascular response to TBI, it remains to be determined whether, when, and how other cell types may also contribute to the observed vascular pathogenesis. In our experiments, we observed an increase in O-GlcNAcylation in endothelial cells at 24 hour post-stretch (Supplmentary Fig. 1), which could be involved in a compensatory mechanism to maintain mitochondrial function. Cell-type specific differences in O-GlcNAcylation responses has been previously reported in cell culture models of diabetic retinopathy, where pericytes exhibited greater sensitivity compared to endothelial cells or astrocytes, leading to early pericyte loss (Gurel et al., 2013; Gurel et al., 2014; Gurel and Sheibani, 2018). However, the mechanisms underlying this selective sensitivity remain unclear. Future studies should explore the molecular basis of pericyte- and other cell type-specific responses, and how interactions between pericytes and other cell types (endothelium, astrocytic endfeet, smooth muscle cells) may connect with the hypothesized role of O-GlcNAcylation and mitochondrial dynamics after TBI.

Second, our studies use pharmacologic inhibition or exogenous mitochondria to support the therapeutic potential of O-GlcNAcylation in pericytes. However, other “off-target” effects must be carefully considered, particularly in vivo. For example, TMG may also modulate microglial activity, contributing to neuroprotection (He et al., 2017). In our study, exogenous mitochondria more effectively increased the mitochondrial fusion protein MFN2 compared to TMG, but their efficacy in reducing trans-cellular permeability was comparable under our experimental conditions. Comparing therapeutic efficacy of these two approaches and further dissecting underlying mechanisms would be valuable. Additionally in our study, the pharmacological approaches were applied to a mixed culture of pericytes and endothelial cells in the trans-cellular permeability assay, therefore the drugs may affect both cell types.

Third, our interventions may have other effects besides renormalizing the homeostatic balance in fusion and fission. O-GlcNAcylated mitochondria have been reported to generate fewer immune responses and damage-associated molecular patterns (DAMPs) when administered to neurons (Park et al., 2023; Sacoman et al., 2017). Further identification of O-GlcNAcylated mitochondrial proteins could help elucidate the mechanisms and refine strategies for therapeutic approaches.

Fourth, our experiments use pericyte cultures to dissect molecular mechanisms and cell biology. However, it is important to recognize that trans-cellular permeability in cell culture models is not the same as blood-brain barrier function within an intact brain. In vivo studies are warranted to ask whether these mechanisms and targets can be applied for protecting vascular function with in vivo TBI models, including the rescue of the blood-brain barrier as well as cerebral blood flow. Importantly, it should be interesting to ask how ameliorating mitochondrial and vascular injury in these early times translate into neurological outcomes several days after injury in clinically-relevant in vivo models of TBI.

Finally, while we focused on early responses and interventions to establish the proof-of-concept, the therapeutic window for enhancing mitochondrial O-GlcNAcylation requires further investigation. Since O-GlcNAcylation is decreased as early as 1 hour post-injury, and given that mitochondrial internalization or TMG treatment typically requires several hours to become fully effective, we prioritized pre-treatment in this initial proof-of-concept study. Pre-treatment with TMG or O-GlcNAcylated extracellular mitochondria worked, but the impact of post-treatment protocols should be explored and may offer more clinically relevant therapeutic options following TBI.

In conclusion, our proof-of-concept study uncovers a novel role for pericytes in the pathogenesis of early vascular damage after mechanical injury and identifies mitochondrial O-GlcNAcylation as a potential therapeutic target for mitigating vascular dysfunction and potentially preventing further secondary damage in TBI.

Materials and Methods

Animals

C57BL6 mice were obtained from Jackson Laboratory. They were given food and water ad libitum and were housed in pathogen-free facilities with 12-h day and night cycles. All experiments were performed following an institutionally approved protocol in accordance with National Institutes of Health guidelines and with the United States Public Health Service’s Policy on Human Care and Use of Laboratory Animals. Our methods also included randomization, blinding and statistical criteria consistent with ARRIVE guidelines (Animals in Research: Reporting In vivo Experiments).

TBI induction

The trauma protocol was approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee and complied with the NIH Guide for the Care and Use of Laboratory Animals. Male C57BL6 mice (3 months old), weighing 26 to 29 g, were used. Mice were anesthetized with 2% isoflurane (Anaquest, Memphis, TN) in 70% N2O and 30% O2 using a Fluotec 3 vaporizer (Colonial Medical Amherst, NH) and positioned in a stereotaxic frame. Anesthesia was maintained with 1.8% isoflurane. Mice were shaved and subjected to controlled cortical impact on the left parieto-temporal cortex using a pneumatic cylinder with a 3-mm flat-tip impounder, using a velocity of 5 m/s, a depth of 1 mm and a 150-ms impact duration. The mice were returned to their cages to recover from anesthesia.

Brain microvessel isolation

After the cardiac perfusion, mouse brains were isolated and meninges were removed by rolling the brains on blotting paper. Cortices were dissected in cold phosphate-buffered saline (PBS) and homogenized using a Dounce grinder and centrifuged at 1,000 g for 5 min at 4 °C. The pellet was resuspended in a 18 % (wt/vol) 70-kDa dextran solution and centrifuged at 1,700g for 20 min at 4 °C. The microvessel pellets were resuspended in PBS and passed through 40-μm cell strainer. After being washed with cold PBS, lysis buffer was added to the microvessels for protein preparation.

Cell culture

Human brain vascular pericytes were purchased from ScienCell (#1200) and cultured in pericyte medium containing 2% Fetal Bovine Serum (FBS) and pericyte growth supplement (ScienCell). Dishes were coated using poly-l-lysine hydrobromide (Sigma-Aldrich). A primary human brain microvascular endothelial cell line (CSC cells) was purchased from Cell Systems Corporation (Kirkland, WA). Cells from passages 6 to 12 were grown in Endothelial Basal Medium (EBM, Millipore) and supplemented with endothelial cell growth supplements including 5% FBS.

Stretch injury and treatment

Cells were seeded on amino-coated silicone-based deformable membrane BioFlex culture plates (BF-3001A, Flexcell International, Hillsborough, NC) and incubated for 24 h in growth media to allow for adherence (37 °C, 5% CO2), after which a stretch-injury was administered as previously described (Shaughness and Byrnes, 2021). Briefly, a stretch-injury was produced using the Cell Injury Controller II (CIC II), which utilizes nitrogen gas to pressurize a BIOFLEX® well individually. The CIC II regulates the duration and pressure of gas delivered to the well via a rubber tube that feeds through a plastic circular well adaptor plug that attaches to the top of the well, creating a hermetically sealed system. Upon discharging the gas, the well becomes pressurized and a bi-axial deformation of the flexible membrane results in the cultured cells experiencing a stretch injury. All stretch-injury experiments used parameters with 25–35 psi regulator pressure for a duration of 50 mSec, resulting in a 2–3 psi pressurization of the well that created a mild-moderate degree of injury. TMG (1 μM, Tocris, 4390) or mt-OGN (10 μg/well) were administered 16 h before the stretch injury.

Mitochondria isolation and O-GlcNAc modification

Mitochondria are isolated using the Mitochondria Isolation Kit (ThermoFisher, 89874; 89801) according to the manufacturer’s protocol. Briefly, mouse brains or cells were homogenized/harvested and centrifuged at 700 × g for 10 min at 4 °C. The supernatant is collected and centrifuged at 12,000 × g for 15 min at 4 °C. The resulting mitochondria pellet is collected for subsequent western blot analysis or modification. In order to modify mitochondria with O-GlcNAc, UDP-GlcNAc (0.5 mM, Sigma, U4375) and recombinant O-GlcNAc transferase (OGT, 0.5 μg, R&D systems, 8446-GT-010) were co-incubated with isolated mitochondria for 30 min at 37 °C.

Trans-cellular permeability assay

Changes in trans-cellular permeability were measured using a modified version of a previously described method (Rosas-Hernandez et al., 2018). Briefly, biotinylated-fibronectin (Cytoskeleton, FNR03) was diluted in PBS to a working concentration of 0.04 mg/mL. The BioFlex culture plates (BF-3001C, collagen type 1-coated, Flexcell International, Hillsborough, NC) were incubated with the biotinylated-fibronectin overnight and the excess removed by 2 consecutive washes with PBS. Pericytes and endothelial cells (CSC) were seeded onto the coated plates at a 1:10 ratio. After reaching full confluency, cells were treated with TMG or mt-OGN for 16 h and subsequuently subjected to a stretch-injury. 1 h after the injury, the media was replaced with FITC-avidin (ThermoScientific, A821) in PBS and incubated for 15 min, followed by 3 consecutive washes with PBS. The membranes were then removed using a scalpel, placed into 6-well culture plates and covered with 2 mL of PBS. Fluorescence of the FITC-avidin bound to the membrane was measured using a microplate reader at 485 nm with a reference wavelength of 530 nm. Background readings from plates coated with non-biotinylated-fibronectin were subtracted.

Western blotting

Tissue samples and cells were lysed in Protein Extraction Solution (Cell Signaling, 9803). Samples were heated with equal volumes of SDS sample buffer (Invitrogen, LC2676) at 95 °C for 5 min, then each sample was loaded onto 4–20% Tris–glycine gels. After electrophoresis and transfer to nitrocellulose membranes (Thermo Fisher, IB23001), the membranes were blocked in TBS containing 0.1% Tween 20 and 5% skim milk (LabScientific). We used antibodies specific for O-Linked N-Acetylglucosamine (1:2,000, Abcam, ab2739), 4-HNE (1:2,000, abcam, ab46545), TOM40 (1:2,000, ThermoFisher, 18409–1-AP), MFN2 (1:2,000, Cell Signaling, 11925), OPA1 (1:2,000, Cell Signaling, 80471), MFF (1:2,000, Cell Signaling, 84580), DRP1 (1:2,000, Cell Signaling, 8570), β-actin (1:3,000, Sigma-Aldrich, A5441). Membranes were then incubated with peroxidase-conjugated secondary antibodies and visualized by enhanced chemiluminescence (Thermo scientific, 34580).

Immunostaining/Immunofluorescence analysis

Mouse brain microvessels or cells were fixed for 20 min in 4% paraformaldehyde followed by permeabilization with 0.1 % triton X-100 for 3 min and stained with primary antibodies overnight at 4 °C. The primary antibody information is as follows. Anti-CD13 antibody (1:200, BD bioscience, 558744), anti-CD31 antibody (1:200, BD bioscience, 551262), anti-MFN2 antibody (1:200, Cell Signaling, 11925). After washing off the primary antibody with tris-buffered saline (TBS), samples were then incubated with secondary antibodies (1:300, Jackson Immunoresearch Laboratories) for 1 h at room temperature. The samples were covered with mounting medium (Fisher scientific; Vector Laboratories) and coverslips were placed. Immunostaining was analyzed with a light/fluorescence microscope (Nikon ECLIPSE Ti-S). To detect functional mitochondria in cells, TMRM (Invitrogen, T668) was added to growth medium at a final concentration of 100 nM and incubated for 30 min at 37 °C. For oxidative stress detection, CellROX Green (Invitrogen, C10444) was administered at a final concentration of 5 μM for 30 min at 37 °C. Nuclei were stained with Hoechst 33342 (R&D systems, 5117) for 5 min. Cells were washed with PBS, and analyzed with a fluorescence microscope.

Statistical analysis

All of the experiments were randomized and performed in a blinded manner. Quantification of protein band intensity obtained by western blot was analyzed using ImageJ (http://rsb.info.nih.gov/ij/) and normalized to the density of the actin or TOM40 band. GraphPad Prism 10 was used for overall statistical analysis in this study. Results were expressed as mean ± SEM. When only two groups were compared, unpaired t-test (two-tailed) was used. Multiple comparisons were evaluated by Tukey’s test after one-way or two-way ANOVA. P < 0.05 was considered to be statistically significant.

Supplementary Material

1

Supplementary Figure 1. Changes in O-GlcNAcylation in endothelial cells after mechanical stretch

Highlights.

  • TBI rapidly induces vascular oxidative stress and decreases mitochondrial O-GlcNAcylation in a mouse model of mild TBI.

  • In vitro mechanical injury reduces mitochondrial O-GlcNAcylation and disrupts mitochondrial dynamics in pericytes.

  • Enhancing O-GlcNAcylation with Thiamet-G or O-GlcNAc-modified mitochondria restores mitochondrial dynamics in pericytes.

  • O-GlcNAcylation enhancement reduces stretch-induced transcellular permeability in pericyte-endothelial co-cultures.

  • Mitochondrial O-GlcNAcylation may serve as a novel therapeutic target to preserving vascular function after TBI.

Footnotes

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Supplementary Materials

1

Supplementary Figure 1. Changes in O-GlcNAcylation in endothelial cells after mechanical stretch

NIHMS2077352-supplement-1.pptx (97.4KB, pptx)

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