A Novel Role for BRIP1/FANCJ in Neuronal Cells Health and in Resolving Oxidative Stress-Induced DNA Lesions

Cell lines, culture method, and reagents

Human neuroblastoma cell line SHSY5Y and mouse neuronal hippocampal cell line HT22 were purchased from ATCC, Manassas, VA. Both the cell lines were cultured in Dulbecco’s modified eagle medium (Corning, Manassas, VA), supplemented with 10%fetal bovine serum (Omega Scientific Inc, Tarzana, CA) and 1%penicillin-streptomycin (50 U/mL, 50μg/mL, Invitrogen, Eugene, OR). H₂O₂ (Cat No# 216763) and L-Glutamic acid monosodium salt hydrate (Cat No# G1626) was purchased from Sigma, St. Louis, MO. To initiate differentiation, SHSY5Y cells were treated with retinoic acid (10μM) (Sigma-Aldrich, St. Louis, MO, USA) for 7 days. Media was replenished after 3 days of culture.

Animals

WT mice (C57BL6/SJL) were purchased from The Jackson Laboratory (Bar Harbor, ME). All animals were housed under air-conditioned rooms at a constant temperature of 22°C with a 12 h light/dark cycle and given access to water and food ad libitum. The animal study was approved by Texas Tech University Health Sciences Center-Institutional animal care and use committee (TTUHSC-IACUC).

Protein expression by western blot

Cells were placed on ice and washed with ice-cold PBS and lysed using cytoskeletal buffer (10 mM PIPES at pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA, 0.1 mM ATP, 0.1%Triton X-100 freshly supplemented with 1 mM dithiothreitol, 1x protease, and phosphatase inhibitors with EDTA). Bradford reagent was used to estimate protein concentration, and the proteins were normalized using cytoskeletal buffer with 6X Laemmli buffer and heated at 100°C for 15 min. The proteins were resolved on gradient polyacrylamide gels and then transferred onto nitrocellulose membrane using Biorad Trans-Blot Turbo system. The membranes were blocked using a 2.5%blocking grade blocker (BioRad, USA) in 1X TBST and incubated with the primary antibody overnight on a rocking platform at 4°C. Primary antibodies include BRIP1 (Cat No: B1310, Sigma) or (Cat No: NBP131883, Novus Biologicals), γH2AX (Cat No: 05-636, Millipore), GAPDH (Cat No: SC-32233, Santa Cruz), BRCA1 (Cat No: SC-6954, Santa Cruz), FANCD2 (Cat No: SC-20022, Santa Cruz), Parkin (Cat No: 4211, Cell Signaling), Pink1 (Cat No: 6946, Cell Signaling), and LC3 A/B (Cat No: 12741, Cell Signaling). Membranes were then washed three times with 1X TBST, and a secondary antibody was added and incubated further for 1 h. The membranes were again washed three times with 1X TBST and exposed to Western lightning plus ECL (Perklin Elmer, USA), and developed in a dark room with Konica Minolta equipment.

Immunofluorescence

Cells were seeded into fluorodish (World Precision Instruments) containing growth medium and incubated overnight for adherence. After overnight treatment with H₂O₂ or water, cells were fixed with 4%formaldehyde for 10 min at room temperature, and then permeabilized using 0.2%Triton X-100 in PBS, for 3 min and blocked using 10%goat serum in PBS for 40 min. After three washes with PBS, cells were incubated overnight at 4°C with anti-DNA-RNA Hybrid [S9.6] antibody (Cat no# ENH001, Kerafast) in PBS, followed by incubating with fluorescent secondary antibodies (Molecular Probes, Eugene, OR) for 2 h at room temperature. Cells were mounted with Vectashield, and analyzed for R-loop formation, using Nikon Eclipse TE confocal microscope.

Immunohistochemistry

Human and mouse brain sections were stained for the expression of BRIP1 by immunohistochemistry. Mice frozen tissue sections were fixed in 4%paraformaldehyde diluted in PBS for 10 min followed by permeabilization with 0.1%Triton-X100 in PBS. Paraffin-embedded human brain sections were deparaffinized and antigen retrieved using sodium citrate buffer. Both the mice and human sections were washed in PBS, quenched endogenous peroxidase, blocked, and incubated with BRIP1 (Cat No: NBP131883, Novus Biologicals) primary antibody overnight. The next day, sections were washed and incubated with biotinylated secondary antibody, and then with conjugated HRP streptavidin and then stained with DAB chromogen, and tissues were counterstained with hematoxylin. Stained sections were analyzed by Zeiss Axioscope microscope.

Transmission electron microscopy

To count the mitochondrial number and length we used transmission electron microscopy. EUFA FJ +ve and FJ -ve cells were fixed in 2%Paraformaldehyde/2.5%Glutaraldehyde in 0.1 M Sodium Cacodylate Buffer, 7.4 pH (Cat no: 1596001, Electron Microscopy Sciences). Samples were post-fixed with 1%osmium tetroxide and embedded. Ultrathin sections were post-stained with uranyl acetate and lead citrate and examined in electron microscopy at 60 kV on a Philips Morgagni transmission electron microscope equipped with a CCD, and images were collected.

MitoSOX assay

After indicated treatments (H₂O₂ or LGA), MitoSOX assays were performed accordingly to the manufacturer’s instructions (Cat No: M36008, ThermoFisher). Approximately 0.5×10⁶ cells were aliquot into micro-centrifuge tubes in a final volume of 0.5 ml PBS and then added with 1μl of 2.5 mM MitoSOX to tubes (the final concentration of MitoSOX is 5μM). Cells were incubated in a shaking, 37°C water bath for 20 min. After incubation, cells were washed twice with PBS, re-suspended in 0.5 ml of 37°C-prewarmed PBS, and analyzed by flow cytometry.

CellROX® Green staining for reactive oxygen species (ROS)

BRIP1 proficient (siControl) and knockdown (siBRIP1) cells were seeded onto fluorodish image plates, and they were treated with 10 mM LGA overnight. These cells were stained with 5μM CellROX® green reagent (Cat No: 10444, ThermoFisher) and Hoechst 33342 (Cat No: R37605, ThermoFisher) dye in complete media at 37°C for 30 min. Cells were washed with PBS, images were captured using Nikon confocal microscope at 20X magnification, and ROS intensities were calculated using Image J software in at least 50 randomly chosen cells.

MitoTracker™ Red CMXRos staining

Control and BRIP1 siRNAs transfected cells were treated with 10 mM LGA or vehicle overnight and incubated with 200 nM MitoTracker™ Red CMXRos reagent (Cat No: M7512, ThermoFisher) and Hoechst 33342 (Cat No: R37605, ThermoFisher) in complete media at 37°C for 30 min. The cells were then washed with PBS and imaged using Nikon confocal microscope at 60X magnification.

Seahorse oxygen consumption rate (OCR)

The XFe96 Extracellular Flux Analyzer and Cell Mito Stress Test Kit (Cat No: 103708-100, Seahorse Agilent Technologies) were used to examine the effects of BRIP1 on mitochondrial respiration rate in control and BRIP1 knockdown HT22 cells in normal and excitotoxicity induced conditions. Briefly, after siRNAs transfection, HT22 cells (1×10⁴ cells/well) were incubated in an XP-96-well plate in a CO2 incubator at 37°C for overnight. After 48 h of indicated siRNAs transfections, cells were treated with 10 mM LGA for 24 h. Subsequently, the cells were washed three times with Assay Medium (10 mM glucose, 1 mM pyruvate, 2 mM glutamine, and pH 7.4), and incubated in 180μl of Assay Medium in a non-CO2 incubator at 37°C for an additional 30 min and analyzed using the XFe Extracellular Flux Analyzer. OCR was measured under basal conditions or in the presence of 1.5μM oligomycin, 0.5μM fluoro-carbonyl cyanide phenylhydrazone, and 0.5μM rotenone/antimycin A.

Analysis of DNA replication status in cells

To examine the DNA replication status in undifferentiated and terminally differentiated neuronal cells, SHSY5Y cells were treated with 10μM retinoic acid (RA) or DMSO for 7 days. These cells were pulse-labeled with 10μM BrdU (Cat No: B5002, Sigma) for 30 min, washed and fixed with ethanol. Fixed cells were denatured using 2N HCL and incubated with FITC conjugated anti-BrdU antibody (Cat No: 347583, BD) and analyzed by flow cytometry.

RNA isolation and real-time RT-PCR

A Purelink RNA isolation kit from Ambion was used to isolate total RNA from mice brains, and RNA was reverse-transcribed using an Invitrogen high-capacity cDNA reverse transcription kit, following the manufacturer’s protocol. The primers from Bio-Rad were used for amplification of Brip1 and Gapdh. SYBR green dye (ABI) was used to measure PCR product amplification and fluorescence was monitored using a QuantStudio 12K Flex detection system. For each amplicon, a melting curve study was performed. Quantitation was done using the 2–ΔΔCt technique using Gapdh as an endogenous control.

COMET assay

Comet assays were performed under alkaline conditions using CometAssay Kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions. In brief, cells were collected after indicated treatments and mixed with 1 percent low melting point agarose (1 : 10 v/v), and they were added to comet slides. Cells were lysed for 30 min in a lysis solution, and the slides were electrophoresed in a horizontal electrophoresis system. Comets were fixed in 70%ethanol and stained with SYBR green dye and images were captured using a Zeiss Axioscope microscope. The ImageJ comet program was used to demarcate the “head” and “tail” areas of each comet from fluorescence photographs. The area of the comet tail was measured, and the data from three different experiments were presented.

Presto blue cytotoxicity assay

The Presto Blue™ cell viability assay (Invitrogen, Carlsbad, USA) was performed to examine the cytotoxicity of LGA treatment in control and BRIP1 siRNAs transfected HT22 and SHSY5Y cells. Approximately 5000 cells/well were seeded and incubated in a CO₂ incubator at 37°C for overnight and they were exposed to different concentrations of LGA for 48 h. These cells were incubated with media containing 10%Presto Blue™ reagent for additional 3 h and analyzed using a microplate reader.

Statistical analysis

Data are presented as mean±standard deviation of mean. The statistical significance of the differences between groups was calculated using the unpaired Student t-test.

BRIP1 is expressed in mouse and human brains, and its levels diminish during aging

BRIP1 as a DNA repair helicase resolves abnormal DNA structures formed during vital cellular processes such as DNA replication, gene transcription, chromatin remodeling. Thus, germline mutations in BRIP1 or BRIP1 deficiency causes genomic instability syndrome FA and early onset of several cancers. However, its expression during aging has not been studied, particularly in the brain and neuronal cells, as they are one of the metabolically most active cells and exposed to several reactive metabolites. To examine the expression of BRIP1 in brains, we performed immunohistochemical analysis of mouse brains using an antibody specific to mouse BRIP1. Analysis of immunohistochemical images from mouse brains revealed BRIP1 is expressed in most parts of the mouse brain, particularly its expression was prominent in the cerebral cortex, piriform area, dentate gyrus (DG), CA1, and CA3 regions (Fig. 1A and Supplementary Figure 1A). It was interesting to note that BRIP1 intensities were remarkably high in the DG regions of the brains compared to all other parts (Supplementary Figure 1A). DG has been implicated in adult neurogenesis and is known to be a critical component of the hippocampal circuit involved in episodic and spatial memory. To confirm the expression of BRIP1 in neuronal cells, we evaluated BRIP1 in co-immunofluorescence studies with NeuN, a neuronal marker. Consistent with the immunohistochemical data, BRIP1 is highly expressed in the DG area and neuronal cells, as indicated by the NeuN co-immunostaining (Fig. 1B). To test whether BRIP1 is expressed in human brains, we examined BRIP1 expression in normal human adult hippocampus brain sections (Cat No: NBP2-77762, Novus Bio). The immunohistochemical staining with human BRIP1 antibody showed expression of BRIP1 in hippocampal sections of the human brain (Fig. 1C). This is also consistent with The Human Protein Atlas, where BRIP1 is highly expressed in different parts of the brain, including the hippocampus (Supplementary Figure 1B).

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Fig. 1

BRIP1 is expressed in mouse and human brains, and its expression diminishes during aging. A) Immunohistochemical analysis of BRIP1 in mouse brain tissue sections. Scale bar represents 1000μm. B) Co-immunofluorescence staining of BRIP1 and NeuN (neuronal marker) in mouse dentate gyrus. Scale bar represents 100μm. C) Immunohistochemical staining for BRIP1 expression in normal human adult hippocampal brain section. Scale bar represents 100μm. D) Western blot analysis of BRIP1 and γH2AX expression in brains isolated from different age groups of mice. E) Densitometry analysis for BRIP1 and γH2AX expression in brains isolated from different age groups of mice, and representative standard deviations presented as error bars. F) qRT-PCR analysis of Brip1 gene expression levels in different age groups of mice brains. Gapdh was used as endogenous control and fold differences with standard deviations were presented (^*p < 0.05).

Moreover, accumulation of DNA damage and aberrant regulation of DNA repair genes have been observed with the aging process and onset of neurodegenerative diseases [16]. To examine BRIP1 expression pattern during aging, we have collected brains of mice at different age groups, extracted proteins and RNA, and evaluated for BRIP1 protein and transcript levels. Consistent with the previous studies, γH2AX, a marker for DNA DSBs were significantly upregulated with the aging in mouse brains. As indicated in Fig. 1D, in 2-month-old mouse brains, very little or undetectable levels of γH2AX were observed. However, brains of 12- and 20-month-old mice showed elevated levels of γH2AX. In contrast, expression of BRIP1 protein is higher in the 2-month-old mice (n = 5) group than in the 12-month-old (n = 4) and 20-month-old (n = 5) mice groups (Fig. 1D, E). Similarly, Brip1 transcript levels were also decreased in 12-month-old (n = 5) and 20-month-old (n = 5) mice brains compared to 2-month-old (n = 5) mice (Fig. 1F). Together these results confirm the expression of BRIP1 in both mouse and human brains, indicates it may have important functions, particularly in DG neuronal cells, and its expression regulated during aging in mouse brains.

BRIP1 is expressed in both undifferentiated and differentiated neuronal cells and is necessary to resolve DNA-RNA hybrid (R-loop) structures

As its name implies, BRIP1 interacts with the BRCA1 and facilitates in eliciting DNA damage responses and repair of DNA lesions. In BRIP1 deficient cells, stability of BRCA1 and other DNA repair proteins were affected and fails to activate efficient DDR in response to agents that cause replication blockade and DSB [17]. To examine whether BRIP1 shows similar effects in terminally differentiated neuronal cells, first we confirmed the expression of BRIP1 and BRCA1 in cycling and after terminally differentiated SHSY5Y-human neuroblastoma cells. For terminal differentiation, we cultured SHSY5Y cells in a medium containing 10μM RA for 7 days and confirmed their differentiation by observing the formation of neurites outgrowth (Fig. 2A). Additionally, we also labeled cycling and RA-treated SHSY5Y cells with BrdU, a marker for DNA replication or synthesis for 30 min and analyzed their cell cycle and replication status by flow cytometry. Consistent with their differentiation, RA treated cells showed little or no BrdU labeling compared to SHSY5Y cells that are cycling (Supplementary Figure 2). Next, we evaluated the expression of BRIP1 and its interaction partners BRCA1 and FANCD2 proteins in both differentiated and undifferentiated SHSY5Y cells. Our data shows an expression of these proteins in both undifferentiated and differentiated SHSY5Y cells; however, their levels were diminished in differentiated cells compared to undifferentiated cells (Fig. 2B). These are consistent that expression of FA-BRCA genes is induced during S and G2 phases, as they are more vulnerable to DNA damage.

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Fig. 2

BRIP1 is expressed in both undifferentiated and terminally differentiated neuronal cells. A) Representative cells images for neurite outgrowth in differentiated and undifferentiated SHSY5Y cells. B) Western blot analysis of DNA repair proteins expression in differentiated and undifferentiated SHSY5Y cells. C) Western blot analysis of DNA repair proteins expression in BRIP1 proficient and deficient terminally differentiated SHSY5Y cells. R-loop formation in BRIP1 proficient and deficient (D) HT22 and (E) SHSY5Y cells treated with H₂O₂.

Although the importance of these FA-BRCA DNA repair pathway is well studied in cycling cells, their possible roles in non-dividing and fully differentiated neuronal cells remain less defined. Our previous studies showed BRIP1 protein is critical for the stability of BRCA1 and FANCD2/FANCI proteins, as it shields them from proteasome-mediated degradation [17]. To test the influence of BRIP1 in differentiated neuronal cells, we evaluated the levels of BRCA1 and FANCD2 proteins in differentiated SHSY5Y cells after transfecting them with control and BRIP1 siRNAs. Consistent with our previous studies, BRIP1 deficiency caused significant downregulation of key DNA repair proteins BRCA1 and FANCD2 in differentiated neuronal cells (Fig. 2C).

FA-BRCA DNA repair proteins are known to form a complex and facilitates the resolution of abnormal DNA structures such as DNA-RNA hybrids or R-loops formed due to pausing of transcriptional complexes [18]. Additionally, deficiency in these proteins also causes accumulation of R-loops, DNA damage, and genomic instability. To test the BRIP1-deficiency and formation of R-loops in neuronal cells, we knocked down BRIP1 in mouse primary hippocampal (HT22) neuronal cells and exposed them to vehicle or 100μM H₂O₂ for 24 h and evaluated the formation of R-loops by staining these cells with R-loop specific antibody S9.6. In control siRNAs (siControl) transfected cells no detectable levels of nuclear staining for R-loop antibody. However, H₂O₂ treatment caused the formation of nuclear R-loops. Interestingly, BRIP1 deficient HT22 cells (siBRIP1) showed increased accumulation of nuclear R-loops compared to siControl cells (Fig. 2D). Furthermore, H₂O₂ treatment caused significantly increased accumulation of R-loops in BRIP1 deficient cells compared to BRIP1 proficient HT22 cells treated with H₂O₂ (Fig. 2D). To further confirm these results, we performed similar experiments in human neuroblastoma cell line SHSY5Y in the presence and absence of 25μM H₂O₂. Consistent with the HT22 cells data, H₂O₂ treatment increased R-loop formation compared to untreated SHSY5Y cells; and BRIP1 deficiency alone caused increased accumulation of R-loops and their levels were increased significantly in response to H₂O₂ compared to siControl and siControl cells treated with H₂O₂ respectively (Fig. 2E). Together, these results indicate a role for BRIP1 in facilitating the resolution of OS-induced R-loops in neuronal cells.

BRIP1 protects neuronal cells from LGA-induced ROS, DNA damage, and excitotoxicity

L-glutamic acid (LGA)-induced excitotoxicity contributes to several central nervous system disorders and neurodegeneration. Glutamate-induced excitotoxicity involves an abnormal Ca^2 + influx and then leads to increased ROS generation, mitochondrial dysfunction, increased DNA damage, and subsequent neuronal cell death [19]. BRIP1 has been shown to detect oxidative base damage in either strand of duplex DNA and is activated by single-stranded DNA binding protein or replication protein A (RPA) [20]. To examine the influence of BRIP1 status in LGA induced ROS formation in neuronal cells, HT22, and SHSY5Y cells were transfected with control (siCon) or BRIP1 siRNAs (siBRIP1) and treated with 15μM (HT22 cells) and 100μM LGA (SHSY5Y cells) overnight and then analyzed for ROS using CellROX® Green staining. The corrected total cell fluorescence was calculated from at least 50 individual cells for each from three independent studies using Image-J software. As expected, LGA treatment-induced cellular ROS in both HT22 (Supplementary Figure 3A, B) and SHSY5Y (Supplementary Figure 3C, D) cells. Interestingly, in both HT22 and SHSY5Y cells, BRIP1 deficiency caused significantly increased levels of ROS accumulation in response to LGA treatment compared to the control cells treated with LGA.

Additionally, LGA-induced ROS can damage nuclear DNA, and if unrepaired these lesions can cause neuronal cell death [21]. We evaluated the role of BRIP1 in the repair of LGA-induced DNA damage in neuronal cells using alkaline COMET assays, which can measure both single and DSBs. SHSY5Y cells were transfected with control and BRIP1 siRNAs and exposed to LGA, and cells were electrophoresed in alkaline conditions. COMET tails were measured from at least 50 cells for each data point using OpenComet ImageJ plugin. Consistent with the increased cellular ROS levels, LGA treatment caused nuclear DNA lesions in both BRIP1 proficient and deficient cells compared to untreated cells as evidenced by the increased COMET tail area. Interestingly, BRIP1 deficiency caused significantly increased DNA damage compared to BRIP1 proficient cells treated with LGA (Fig. 3A, B), indicating that BRIP1 is required for the repair of DNA damage caused by excitotoxins such as LGA.

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Fig. 3

BRIP1 protects neuronal cells from LGA-induced excitotoxicity. A) Representative images of comet assays from control and BRIP1 knockdown SHSY5Y cells treated with LGA for 24 h. B) Comet tail areas were measured from more than 50 cells for each from three independent experiments, and representative standard deviations were presented as error bars. C) Representative images showing LGA-induced excitotoxicity in siControl and siBRIP1 HT22 cells. Survival assays showing LGA concentration-dependent toxicities to BRIP1 proficient and deficient (D) HT22 and (E) SHSY5Y cells. (^*p < 0.05), (^***p < 0.001) and (^****p < 0.0001).

Increased generation of DNA lesions and failure to repair them timely can lead to cell death. We further evaluated the influence of BRIP1 on cell viabilities of HT22 and SHSY5Y cells in response to different concentrations of LGA using PrestoBlue® cell viability reagent. As shown in Fig. 3C, BRIP1 deficient cells showed apoptotic phenotype compared to BRIP1 proficient HT22 cells in response to LGA treatment. Consistent with the increased DNA damage, BRIP1 deficient HT22, and SHSY5Y cells were more vulnerable to LGA-mediated excitotoxicity when compared to their respective LGA-treated control cells (Fig. 3D, E). Although it is not clear by which mechanism BRIP1 suppresses the formation of cellular ROS induced by LGA, our results demonstrate BRIP1 deficiency causes a significant increase in cellular ROS in neuronal cells and implicates an important role for BRIP1 in the protection of neuronal cells from LGA-induced OS, DNA damage, and excitotoxicity.

LGA and H₂O₂ causes increased mitochondrial ROS, structural abnormalities, and functional defects in BRIP1 deficient neuronal cells

Increased generation of cellular ROS in response to LGA can alter mitochondrial membrane potential and increase mitochondrial ROS [22]. Moreover, BRIP1 is an iron-sulfur motif-containing DNA helicase; its functions may depend on mitochondrial iron-sulfur cluster biogenesis. Along these lines, a recent study showed an important role for FANCG, a component of FA core complex localizes to mitochondria and protects mitochondria from OS [23]. Additionally, FANCG deficient cells exhibit mitochondrial instability, attributed to transcriptional downregulation of mitochondrial iron-sulfur cluster biogenesis protein frataxin, which may affect iron-sulfur cluster containing BRIP1 helicase function [23]. Based on these observations, we evaluated the role of BRIP1 in LGA-induced mitochondrial OS using the MitoSox assay. After overnight treatment with 15μM and 100μM LGA to HT22 and SHSY5Y cells respectively, they were stained with MitoSox dye and analyzed by flow cytometry to evaluate mitochondrial ROS. LGA treatment caused an increased generation of mitochondrial ROS in BRIP1 proficient HT22 and SHSY5Y cells compared to their respective untreated cells (Fig. 4A, B). Remarkably, in both the neuronal cell lines, BRIP1 deficiency caused increased mitochondrial ROS at basally and in response to LGA treatment compared to their respective BRIP1 proficient cells (Fig. 4A, B). Additionally, FANCJ/BRIP1patient derived cell lines that are BRIP1-mutant or deficient cells (EUFA BRIP1-Ve) and their isogenic cells complemented with wild-type BRIP1 (EUFA BRIP1 +ve) were treated with 100μM H₂O₂ and evaluated for mitochondrial ROS. Similar to neuronal cell lines, EUFA BRIP1-ve cells showed increased mitochondrial ROS compared to EUFA BRIP1 +ve cells, both basally and in response to H₂O₂ treatment (Fig. 4C).

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Fig. 4

BRIP1 protects neuronal cells from LGA and H₂O₂ induced mitochondrial ROS. Flow cytometry analysis of mitochondrial ROS using MitoSOX staining in (A) HT22 cells treated with LGA, (B) SHSY5Y cells treated with LGA, and (C) BRIP1 mutant (EUFA BRIP1 –ve), and wild-type BRIP1 complemented (EUFA BRIP1 +ve) cells treated with H₂O₂. (^*p < 0.05), (^**p < 0.01) and (^****p < 0.0001).

Mitochondrial morphology is altered in the absence of BRIP1

Several studies demonstrated accumulation of nuclear DNA damage and cellular ROS affects mitochondrial structural integrity and function [24]. To investigate the role of BRIP1 on mitochondrial structural integrity and function in normal and in glutamate-induced excitotoxicity conditions, first, we evaluated the mitochondrial morphology using MitoTrackerH Red CMXRos dye. BRIP1 proficient HT22 and SHSY5Y cells showed a normal reticular mitochondrial network. As predicted, LGA treatment caused an increase in disrupted mitochondrial reticularity in both control and BRIP1 deficient HT22 and SHSY5Y cells (Fig. 5A–D). Quantitative analysis revealed that the number of cells with decreased mitochondrial reticularity significantly increased in BRIP1 deficient HT22 and SHSY5Y cells compared to their respective BRIP1 proficient in response to LGA treatment (Fig. 5A–D). These results suggest an important role for BRIP1 in maintaining a mitochondrial dynamic balance in normal metabolic conditions and during exposure to agents, like glutamate-induced excitotoxicity.

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Fig. 5

BRIP1 is important for mitochondrial integrity. Immunofluorescence analysis of mitochondrial morphology using MitoTracker™ Red CMXRos reagent in (A) HT22 and (C) SHSY5Y cells. Quantitative analysis of the percentage of cells with distorted mitochondria in control and LGA treated (B) HT22 and (D) SHYSY5Y cells. E) Representative images of transmission electron microscope image of EUFA BRIP1 +ve and EUFA BRIP1 –ve cells showing mitochondria. Scale bar represents 200 nm. F) Quantitative analysis of mitochondrial size and number in EUFA BRIP1 +ve and EUFA BRIP1 –ve cells. G) Western blot analysis of mitophagy proteins in BRIP1 proficient and deficient SHSY5Y cells in the presence and absence of LGA. (^*p < 0.05), (^***p < 0.001), and (^****p < 0.0001).

To further evaluate the role of BRIP1 on mitochondrial structural integrity, we used FANCJ/BRIP1 mutant patient-derived cell line (EUFA BRIP1-Ve) and their isogenic cells complemented with wild-type BRIP1 (EUFA BRIP1 +ve) and examined using a transmission electron microscope. We have counted the number of mitochondria and measured their size/shape in over 50 cells each of EUFA BRIP1-Ve and EUFA BRIP1 +ve cells. Interestingly, BRIP1-breakdeficient cells (EUFA BRIP1-Ve) showed significantly increased mitochondrial number and decreased mitochondrial size compared to EUFA BRIP1 +ve cells (Fig. 5E, F). This indicates BRIP1 deficient EUFA BRIP1-Ve cells may be undergoing chronic OS, which resulted in mitochondrial abnormalities, and complementing with wild-type BRIP1 corrected these defects in mitochondria.

Increased OS causes mitochondrial abnormalities and affects their function leading to an imbalance in mitochondrial fission and fusion, and increased mitophagy [25]. Several studies demonstrated activation of Parkin, PINK1, and LC3A/B in LGA-mediated excitotoxicity as a surrogate for mitophagy and age-related neuropathological conditions [26]. In this connection, we further evaluated the expression of mitophagy markers in BRIP1 proficient and deficient HT22 cells with and without exposure to LGA. Consistent with the previous studies, LGA treatment induced the expression of Parkin, PINK1, and LC3B (Fig. 5G). Interestingly, LGA treatment caused a remarkable increase in Parkin, PINK1, and LC3B expression in BRIP1-deficient cells (Fig. 5G). These results indicate an important role for BRIP1 in protecting mitochondria in neuronal cells in response to LGA-induced excitotoxicity.

To further evaluate the influence of BRIP1 in mitochondrial function/respiration, we used Seahorse assay and measured the OCR. First, we compared OCR between control and BRIP1 knocked down HT22 cells for any differences. As shown in Fig. 6A and 6B, transient knockdown of BRIP1 in these cells has no significant difference in basal and spare respiratory capacity of mitochondria, suggesting that transient knockdown of BRIP1 in these cells might not interfere with mitochondrial function. However, in the presence of LGA, BRIP1-deficient cells exhibited significantly decreased basal and spare respiratory capacity (Fig. 6C, D), suggesting an important role for BRIP1 in maintaining mitochondrial function during stress conditions. Collectively, our results suggest a critical role for BRIP1 in mitochondrial structural and functional integrity, particularly during chronic events such as aging and acute events like neurotransmitter-induced excitotoxicity.

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Fig. 6

A) Analysis of oxygen consumption rate using SeaHorse bioanalyzer in BRIP1 proficient and deficient HT22 cells. B) Histogram represents the oxygen consumption rate for the basal and spare respiratory capacity in HT22 cells. C) Analysis of oxygen consumption rate SeaHorse bioanalyzer in control and LGA treated HT22 cells. D) Histogram represents the oxygen consumption rate measurement for the basal and spare respiratory capacity in HT22 cells. (^*p < 0.05) and (^**p < 0.01).