Mutant NOTCH3 ECD Triggers Defects in Mitochondrial Function and Mitophagy in CADASIL Cell Models

Abstract

Background:

Cerebral autosomal-dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL) is an inherited small-vessel disease that affects the white matter of the brain. Recent studies have confirmed that the deposition of NOTCH3^ECD is the main pathological basis of CADASIL; however, whether different mutations present the same pathological characteristics remains to be further studied. Some studies have found that mitochondrial dysfunction is related to CADASIL; however, the specific effects of NOTCH3^ECD on mitochondrial remain to be determined.

Objective:

We aimed to explore the role of mitochondrial dysfunction in CADASIL.

Methods:

We established transgenic human embryonic kidney-293T cell models (involving alterations in cysteine and non-cysteine residues) via lentiviral transfection. Mitochondrial function and structure were assessed using flow cytometry and transmission electron microscopy, respectively. Mitophagy was assessed using western blotting and immunofluorescence.

Results:

We demonstrated that NOTCH3^ECD deposition affects mitochondrial morphology and function, and that its protein levels are significantly correlated with mitochondrial quality and can directly bind to mitochondria. Moreover, NOTCH3^ECD deposition promoted the induction of autophagy and mitophagy. However, these processes were impaired, leading to abnormal mitochondrial accumulation.

Conclusions:

This study revealed a common pathological feature of NOTCH3^ECD deposition caused by different NOTCH3 mutations and provided new insights into the role of NOTCH3^ECD in mitochondrial dysfunction and mitophagy.

Keywords

Alzheimer’s disease autophagy CADASIL mitochondrial dysfunction mitophagy

INTRODUCTION

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a genetically inherited disease caused by mutations in the NOTCH3 gene on chromosome 19 and is the most common monogenic cause of vascular dementia, stroke, and small vessel disease.1 –3

NOTCH3 encodes transmembrane protein receptors that are primarily expressed in arterial smooth muscle cells and pericytes.4,5, 4,5NOTCH3 mutations usually cause the amount of cysteine to affect the extracellular domain (ECD) structure of NOTCH3 receptor, thereby promoting its polymerization and aggregation into granular osmiophilic material (GOM) particles deposited around the basement membrane of blood vessels, which is a classic pathological change of CADASIL.6,7, 6,7 More than 300 mutations have been reported, most of which are cysteine alteration mutations. 8 Interestingly, an increasing number of pathogenic non-cysteine mutations have been detected recently.9 –14 Furthermore, the clinical characteristics of these two mutations are similar.15,16, 15,16 However, whether different mutations share the same pathological changes, such as NOTCH3^ECD aggregation, requires further study. Furthermore, it remains to be elucidated whether the abnormal aggregation of NOTCH3^ECD causes damage to cellular structure andfunction.

Extensive loss of the medial arterial smooth muscle in patients with CADASIL is another key pathological change, 17 which ultimately results in small blood vessel damage, white matter damage, dementia, and other neurological impairments. 18 Vascular smooth muscle cells (SMC) play an important role in the organization of the arterial wall and the maintenance of vascular tension. Once their cellular structure changes, it will lead to changes in the structure and function of cerebral blood vessels, which is the cytopathological basis of cerebral arteriosclerosis, vascular occlusion, microhemorrhage, and other vascular diseases. 19 However, the exact relationship between NOTCH3^ECD deposition and cell damage remains unclear. To explore the molecular mechanisms regulating the survival of SMC, several important pathogenic mechanisms have been reported, including inflammation and oxidative stress.20 –25 However, the detailed underlying molecular mechanisms require furtherinvestigation. Mitochondrial dysfunction has recently attracted the attention of several researchers. Mitochondria are basic organelles that regulate cell metabolism and energy production, 26 and play a central role in numerous cellular processes, including ATP (Adenosine Triphosphate) generation, calcium ion regulation, and reactive oxygen species (ROS) production. 27 Dysfunctional mitochondria undergo mitochondrial fragmentation and membrane depolarization, generating large amounts of ROS, which in turn leads to a series of pathological states. 28 Therefore, mitochondrial dysfunction is considered to be one of the characteristics of neurodegenerative diseases. 29 In CADASIL, mitochondrial dysfunction is manifested as faulty electron transport chain performance, damaged DNA, and aberrant mitochondrial morphology. 30 These mitochondrial abnormalities are not only closely related to cell death, but also may be a key factor in the pathophysiology of CADASIL. Therefore, in-depth investigation of the role of mitochondrial dysfunction in the pathogenesis of CADASIL is of great significance for the development of new therapeutic strategies.

Mitochondrial homeostasis is controlled by the balance between genesis and clearance via mitophagy (a defense mechanism).31,32, 31,32 Damaged and dysfunctional mitochondria are selectively cleared to maintain mitochondrial quality and cellular physiological functions.33,34, 33,34 The most common mitophagy pathway is PINK1/parkin receptor-mediated, followed by Bcl-2 interacting protein-3-mediated, FUN14 domain-containing 1-mediated, and lipid-mediated. 34

This study first constructed two types of NOTCH3 mutation cell models to verify whether abnormal NOTCH3^ECD protein deposition is a general pathological change associated with different pathogenic mutations. We explored whether abnormal NOTCH3^ECD deposition was associated with cell injury, including mitochondrial structure and function changes. Finally, we discuss the phenotypes of autophagy and mitophagy and the related mechanisms in these cells. Thus, our results provides a new idea for understanding the pathological mechanism of CADASIL.

MATERIALS AND METHODS

Cell culture and lentiviral transduction

Through lentiviral transduction, CADASIL cell models were created. Lentivases, including NOTCH3^WT and NOTCH3^ECD mutants, and a viral vector (sequences provided in Supplementary Table 1) were bought from BrainVTA Co., LTD (Wuhan, China). Human embryonic kidney (HEK) 293T cells (Shanghai, China) were used as tool cells to construct CADASIL cell models. The cells were treated with puromycin (3μg/mL, Sigma, USA) for 72 h to screen them. DMEM growth medium (Servicebio, G4511, China) was supplemented with 10% fetal bovine serum (VivaCell, Co4001, China) and 100 : 1 Penicillin-Streptomycin liquid (solarbio, P1400, China) for stable inducible cell lines. The cells were maintained at 37°C, 5% CO2.

Antibodies and reagent

In this study, western blot was performed using the following antibodies: anti-NOTCH3 (Abnovas, H00004854-M02, USA, 1 : 1000), anti-LC3 (Proteintech, 14600-1-AP, 1 : 2000), anti-p62 (Proteintech, 18420-1-AP, 1 : 2000), anti-ATG5 (Proteintech, 10181-2-AP, 1 : 2000), anti-VDAC1 (Proteintech, 55259-1-AP, 1 : 500), anti-PINK1 (Proteintech, 23274-1-AP, 1 : 1000), anti-parkin (Proteintech, 66674-1-Ig, 1 : 1000), anti-TOMM20 (Proteintech, 11802-1-AP, 1 : 2000), and anti-translocase of the inner mitochondrial membrane complex subunit 23 (TIMM23; Proteintech, 11123-1-AP, 1 : 2000). As sample loading controls, β-actin (beyotime, AF5003, 1 : 2000) and Glyceraldehyde-3-phosphate dehydrogenase (servicebio, gb15004, 1 : 2000) were employed. For immunofluorescence, anti-NOTCH3 (Merck, MABC594, 1 : 300), anti-LC3 (Proteintech, 14600-1-AP, 1 : 300), anti-parkin (Proteintech, 66674-1-Ig, 1 : 200), anti-TOMM20 (Proteintech, 11802-1-AP, 1 : 300), Alexa488-labeled or Alexa594-labeled goat anti-mouse secondary antibody (Invitrogen, A-11001, A-11005, 1 : 400 diluted) and Alexa488-labeled and Alexa594-labeled goat anti-rabbit secondary antibody (Invitrogen, A-11008, A-11012, 1 : 400) diluted were used.

Protein extraction and SDS– PAGE analysis

Fractionated mitochondria and HEK 293T cells were homogenized in 1×RIPA buffer (Beyotime, R0728), adding protease and phosphatase inhibitors (MCE, HY-K0010, HY-K0023). After 15-min centrifugation at 13,300 rpm and 4°C, total protein content was measured using the bicinchoninic acid protein assay (Thermo, 23227, USA). SDS-PAGE sample loading buffer was combined with protein lysates in a 4 : 1 ratio, boiled for 10 min, separated using 4-12% SDS-PAGE, and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% skim milk for 1 h at room temperature (RT), followed by overnight incubation with primary antibodies. Relevant secondary antibodies were incubated for 1 h at RT. Enhanced chemiluminescence was used to visualize protein bands. The Bio-Rad ChemiDocTM Touch Imaging System was used to image blots, finding the best imaging time for chemiluminescent detection using the signal accumulation mode.

Mitochondrial protein extraction

Cell mitochondria were isolated using the Qproteome Mitochondria Isolation Kit (Qiagen, 37612, Germany). The mitochondria were lysed with mitochondrial lysate, then centrifuged, and the resulting supernatant was then isolated and preserved. After measuring the concentration of mitochondrial protein, loading buffer is added to supernatant and boiled for 10 min. Finally, the samples are stored in a refrigerator at –80°C for later use.

Immunofluorescence

Cells were treated and fixed by the application of 4% paraformaldehyde (solarbio, P1110, China) for 30 min at RT. The cells were then permeabilized for 100 min at RT using 0.3% Triton X-100 and blocked with 5% bovine serum albumin (BSA) for 1 h at RT. The cells were then incubated overnight with primary antibodies at 4°C. On the second day, the cells were incubated with fluorochrome-labeled secondary antibodies for 1 h at room temperature. DAPI was used to stain the nuclei for 5 min. The Immunofluorescence was observed using an SP5 confocal microscope (DMD108; Leica) or an Olympus Model BX53 fluorescence microscope (Olympus, Tokyo, Japan).

Flow cytometric analysis

A JC-1 assay kit (Servicebio, G1515) was used to measure the mitochondrial membrane potential (MMP). Additionally, a ROS assay kit (Beyotime, S0033S) was used to measure the levels of ROS. All experiments were conducted in accordance with the manufacturer’s guidelines. A fluorescence-activated cell sorting (FACS) flow cytometer (BD FACSAriaTM Fusion, USA) was used to evaluate stained cells (>10,000). The experimental data was analyzed with FlowJo software.

Transmission electron microscopy

Cells were fixed with TEM fixative (Servicebio, G1102), washed with 0.1 M phosphate buffer (PB; pH 7.4), pre-embedded in agarose, post-fixed with OsO4, dehydrated, and embedded in epoxy resin. Sections were contrasted with lead citrate and uranyl acetate and examined with TEM and Olympus SIS MORADA camera.

Oxygen consumption

Oxygen consumption was measured using Extracellular Oxygen Consumption Rate Assay Kit (Elabscience, E-BC-F068) according to the manufacturer’s instructions.

Statistical analyses

GraphPad Prism version 9 and SPSS Statistic 20.0 software was used to analyze the data. One-way ANOVA was used to investigate groups of more than two variables, followed by Dunnett’s post hoc test. Significant differences are expressed as: (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001), ns means no significant difference.

RESULTS

NOTCH3^ECD mutations led to abnormal NOTCH3^ECD deposition

NOTCH3^ECD deposition is a classic pathological change in CADASIL. Different types of NOTCH3^ECD mutations, including cysteine and non-cysteine alterations, have been reported. To investigate whether abnormal NOTCH3^ECD deposition is associated with different types of mutations, we established stably transduced HEK 293T cell lines expressing normal NOTCH3^WT and NOTCH3 mutations involving alterations of cysteine residues (R133C, R110C, and R587C), alterations of non-cysteine residues (V237M, R75P, R75Q, and R1175W), and a void vector (NC). The selection of non-cysteine mutation points was based on the pathogenicity guidelines for non-cysteine-altering mutations proposed by Muiño. 16

Compared with that of the WT and NC groups, the NOTCH3^ECD protein level was significantly increased in both the NOTCH3 cysteine alteration and NOTCH3 non-cysteine alteration mutation groups (Fig. 1a– b). However, no significant differences were observed between the cysteine and non-cysteine alteration groups. To confirm this, we used immunofluorescence staining to detect NOTCH3^ECD deposition (Fig. 1c– d). This was consistent with the results of western blot tests, indicating that NOTCH3^ECD mutations led to abnormal NOTCH3^ECD deposition. These data also suggest that abnormal NOTCH3^ECD deposition may be a common core pathological change, regardless of the type of NOTCH3^ECD mutation. Overall, regardless of the type of mutation, abnormal NOTCH3^ECD may contribute to the deposition of NOTCH3^ECD.

Fig. 1

NOTCH3^ECD accumulation in cerebral autosomal-dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL) model cells. a) Immunoblot analysis of NOTCH3^ECD expression in CADASIL model cells. b) Quantification of NOTCH3^ECD protein levels (N = 5, mean±standard error mean [SEM]). c) Immunofluorescence analysis of NOTCH3^ECD in cysteine-altered (upper panel) and non-cysteine mutant cells (lower panel). Scale Bar: 20μm. d) Quantification of NOTCH3^ECD average density in c (N = 3–5). Each bar represents the mean±SEM. Data are evaluated by one-way ANOVA followed by Dunnetts post hoc test.

Abnormal NOTCH3^ECD deposition correlated with mitochondrial dysfunction

Previous study showed that mitochondrial imbalance plays an important role in pathological changes in CADASIL. Thus, to further investigate how toxic NOTCH3^ECD deposition results in cell dysfunction, we examined mitochondrial function. We stained the cells in situ and observed that the control group predominantly exhibited red fluorescence, indicating a higher intracellular mitochondrial membrane potential due to the presence of JC-1 in a polymerized form in the mitochondrial matrix. Conversely, the mutant group displayed a high proportion of green fluorescence, signifying a decrease or loss of mitochondrial membrane potential, as JC-1 was present in the cytoplasm as a monomer. We observed a decrease in mitochondrial membrane potential in the mutant group (Fig. 2a) was observed. In addition, JC-1 was detected using flow cytometry, and the proportion of healthy cells in the mutant group decreased significantly (Fig. 2b, c). Thus, compared with that in the WT control group, NOTCH3 mutant cells showed a decrease in MMP, which was determined by uptake of JC-1. Moreover, using ROS generation tests, we found that mitochondria in the NOTCH3 mutant group produced more superoxide than those in the WT and NC groups (Fig. 2d). We then measured the OCR to evaluate mitochondrial respiratory function. We found that mutant cells significantly inhibited mitochondrial respiration (Fig. 2e). Therefore, we believe that mitochondrial function is impaired in NOTCH3^ECD mutant cells.

Fig. 2

HEK-293T cells overexpressing NOTCH3^ECD display impaired mitochondrial function. a) The mitochondrial membrane potential of the cells was detected using the JC-1 probe. Cyanide 3-chlorophenylhydrazine carbonate (CCCP) was used as the positive control. The normal mitochondrial membrane potential (MMP) was red, the JC-1 dimer (JC-1 aggregation), the depolarized membrane potential, and the JC-1 monomer was green. The fluorescent color changed from red to green, indicating a decrease in MMP. Scale bar: 50μm. b,c) Flow cytometry analysis and quantification of MMP in overexpressing NOTCH3^ECD cells. Normal MMP is shown in the JC-1 dimer (JC-1 aggregate), whereas depolarized membrane potential is shown in the JC-1 monomer. Data represent mean±SEM for three replicates. d) Flow cytometry to detect reactive oxygen species (ROS) accumulation in NOTCH3^ECD-overexpressing cells. e) The relative Oxygen consumption rate (OCR) in every group. Data are shown as mean±SEM of values from three independent experiments. Data are evaluated by one-way ANOVA followed by Dunnetts post hoc test.

Next, we examined the morphology and structure of the mitochondria using TEM. Compared with that in the control groups, the NOTCH3^ECD mutation groups showed severe mitochondrial shrinkage or vacuolation, and the mitochondrial cristae were significantly altered. Moreover, using the four classified categories of mitochondrial morphology, we conducted a comprehensive analysis of structural alterations in mitochondria. 35 The specific classifications are as follows: Class I: mitochondria maintain their normal oval shape, the mitochondrial matrix is uniform, the ridge structure is dense and evenly distributed, and the whole presents a relatively dark electron density; class II: mitochondrial ridge structure rupture, resulting in a decrease in matrix density, presenting an uneven electron density; class III: vacuolation of mitochondria, ridge marginalization, disordered structure, lack of obvious structural integrity; and class IV: mitochondria swelling, even the integrity of the cell membrane is destroyed, manifested as a significant expansion of the mitochondrial shape (Fig. 3a, b). In the control group of cells, we observed that approximately 80% of the mitochondria belonged to the Class I, maintaining a healthy and normal structure, and class II accounted for 20%. However, in the NOTCH3^ECD mutant group of cells, the proportion of healthy mitochondria (class I and II) decreased sharply, while the proportion of unhealthy mitochondria (categories III and IV) increased significantly (Fig. 3c). Together, these results demonstrate that NOTCH3^ECD mutation accelerates mitochondrial injury, including structural and functional imbalances.

Fig. 3

HEK-293T cells overexpressing NOTCH3^ECD display impaired mitochondrial structure. a) Electron microscopy ultrastructure of HEK-293T cells stably transfected with NOTCH3^ECD. Scale bars correspond to 2 or 0.5μm. b,c) Representative images of mitochondria classes I, II, III, and IV and their quantitative distribution in control and CADASIL model cells. The quantification was done in at least five different fields (>40 mitochondria). D) Correlation analysis between the relative amount of NOTCH3^ECD and the proportion of “unhealthy” mitochondria (III + IV).

Co-localization of mutant NOTCH3^ECD deposits with mitochondria

Based on these results, we hypothesized that NOTCH3^ECD was the main cause of mitochondrial dysfunction. To verify the relationship between NOTCH3^ECD deposition and mitochondrial structure and function, we performed a correlation analysis. We found that increased NOTCH3^ECD deposition was associated with the number of severely unhealthy mitochondria (Fig. 3d). There was a significant positive correlation between the level of NOTCH3^ECD deposition and the number of unhealthy mitochondria (III + IV). Next, we examined whether abnormal NOTCH3^ECD was the core cause of mitochondrial dysfunction. To further verify the relationship between NOTCH3^ECD and mitochondria, we examined their positional relationship between NOTCH3^ECD and mitochondria using immunofluorescence. Interestingly, we found that NOTCH3^ECD displays a strong co-localization with mitochondria in both the control group and the mutant group. (Fig. 4a, b). In addition, we extracted mitochondrial proteins from the cells to detect NOTCH3^ECD levels and found that NOTCH3^ECD levels were elevated in the mutant group (Fig. 4c, d). These data suggest that abnormal NOTCH3^ECD may contribute to mitochondrial dysfunction.

Fig. 4

Co-localization of mutant NOTCH3^ECD deposits with mitochondria. a) Immunofluorescence analysis of the co-localization of NOTCH3^ECD and translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20) in NOTCH3^ECD-overexpressing cells. Scale Bar: 10μm. b) Graphs show the Pearson’s R values using whole cells in Image J. (n = 6– 7 fields). c) Immunoblotting analysis of NOTCH3^ECD in NOTCH3^ECD-overexpressing cells. d) Graphs show the relative amount of NOTCH3^ECD in mitochondrial proteins (N = 3– 5, mean±SEM). Data are evaluated by one-way ANOVA followed by Dunnetts post hoc test.

Abnormal NOTCH3^ECD deposition enhanced basal autophagy

Selective removal of mitochondria is facilitated by a specialized form of autophagy called mitophagy. Hence, this mechanism plays a key role in mitochondrial quality control, assisting in the removal of poorly functioning mitochondria. Considering that the results of the previous study indicated that NOTCH3 mutation results in mitochondrial dysfunction, we decided to investigate whether or not this mutation causes an induction of mitophagy in a CADASIL cell model.

The activation of general autophagy is a necessary step before autophagic clearance of damaged mitochondria. Therefore, we analyzed basal autophagy in CADASIL cells using western blot analysis of the autophagy substrate sequestosome 1 (p62/SQSTM1), autophagy-related gene 5 and LC3B-II, which indicates the number of autophagosomes. It was observed that NOTCH3 mutant cells exhibited elevated amounts of both LC3-II and SQSTM1 proteins (Fig. 5a– c). Moreover, we also detected the expression of beclin-1 and observed an increase in it (Supplementary Figure 1). Consistently, the NOTCH3 mutation model cells displayed an accumulation of autophagy vesicles and lysosomes under TEM, confirming that the NOTCH3 mutation increased endogenous LC3 puncta levels (Fig. 5d, e). In order to evaluate the basal flux of autophagy, including lysosomal degradation, we observed that LC3-II puncta increased significantly in cells treated with chloroquine (CQ) for 24 h compared to the WT group cells. Elevated levels of LC3B-II may indicate autophagy-induced enhancement or blockade of downstream lysosomal degradation (Fig. 5f, g).

Fig. 5

NOTCH3^ECD enhances autophagy in cerebral autosomal-dominant arteriopathy with subcortical infarction and leukoencephalopathy (CADASIL) model cell lines. a) Immunoblotting analysis of microtubule-associated protein 1A/1B-light chain 3 (LC3) B, autophagy related 5 (ATG5), and p62 expression in CADASIL model cells. b,c) Quantification of the LC3, p62 and ATG5 protein levels in a (N = 3–4, mean±SEM). d) Representative images of autophagy-lysosome transmission electron microscopy in CADASIL model cells. e) Quantification of autophagic vesicles in d. Data represent mean±SEM with five replicates. Quantification was performed in at least five different fields (>40 mitochondria). f) Immunofluorescence analysis of LC3 in cysteine altered mutant cells (upper panel) and non-cysteine mutant cells (lower panel) treated with 10 m chloroquine (CQ) for 24 h. Scale Bar: 10μm. g) Quantification of LC3 puncta numbers in f. Data are evaluated by one-way ANOVA followed by Dunnetts post hoc test.

Abnormal NOTCH3^ECD deposition activated mitophagy via the PINK1-parkin pathway

The process of mitophagy, which includes eliminating malfunctioning mitochondria, is a type of quality control mechanism for mitochondria. As the above results confirmed mitochondrial dysfunction and autophagy defects due to NOTCH3 mutations, we investigated whether NOTCH3 mutations induced mitophagy in a CADASIL cell model. In order to ascertain whether autophagosomes had consumed the mitochondria, we first quantified the level of of LC3-II in the mitochondrial fraction isolated from NOTCH3 mutant and WT cells. Compared with that in cytoplasmic proteins, the main form of LC3 in mitochondrial proteins is LC3-II, whereas the main form of LC3 in cytoplasmic proteins is LC3-I, indicating that mitochondria are targeted by LC3-II(Fig. 6a).

Fig. 6

Abnormal NOTCH3^ECD deposition stimulates mitophagy by inducing mitochondrial parkin translocation in CADASIL model cells. a) Immunoblotting analysis of LC3 in the cytosolic (Cyto) and mitochondrial (Mito) fractions in cysteine altered mutant cells and non-cysteine mutant cells. b) Immunoblotting analysis of parkin and phosphatase and tensin homolog-induced kinase 1 (PINK1) protein levels in NOTCH3^ECD-expressing cells. c) Quantification of PINK1/Parkin protein levels in b (N = 3–4, mean±SEM]. d) Immunoblotting of the mitochondrial outer membrane protein translocase of the outer mitochondrial membrane complex subunit 20 (TOMM20) and inner membrane protein translocase of the inner mitochondrial membrane complex subunit 23 (TIMM23) in NOTCH3^ECD-expressing cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control for the expression of TOMM20 and TIMM23. Representative images were obtained from three independent experiments. e) Quantification of TOMM20 and TIMM23 protein levels in d (N = 3– 4, mean±SEM). f) Immunofluorescence analysis of the expression of endogenous parkin and TOMM20 in NOTCH3^ECD-overexpressing cells. Scale Bar: 10μm. Data are evaluated by one-way ANOVA followed by Dunnetts post hoc test.

Mitophagy passthy in mammalian cells is mediated a signaling pathway known as PINK1/parkin, which plays a crucial role in biological processes. Thus, we examined the levels of PINK1 and Parkin in NOTCH3 mutant cells. We observed that the levels of parkin with PINK1 were significantly enhanced in NOTCH3 mutant cells compared with those in control cells (Fig. 6b, c). However, the mitochondrial outer membrane protein TOMM20 and inner membrane protein TIMM23 levels were not significantly elevated in the CADASIL cell model. Considering the previously mentioned increase in p62 protein levels and the abnormal lysosomal degradation, this observation may be due to abnormal lysosomal degradation during mitophagy (Fig. 6d, e). We examined the recruitment of damaged mitochondria by autophagy to identify the processes that can block the autophagic transport of mitochondria to lysosomes.34,36, 34,36 The translocation of parkin to mitochondria was successfully detected in mutant cells by immunofluorescence analysis. This translocation phenomenon is regarded as one of the markers of mitophagy. We found that the parkin protein is widely distributed in NOTCH3 mutant cells but less co-localized with mitochondria. (Fig. 6f). Our data suggested that the mitophagy pathway mediated by PINK1/parkin was activated; however, this was hindered or impaired in the presence of other factors. These data suggest that mitophagy is impaired in NOTCH3 mutants.

Mitophagy was impaired in cells overexpressing NOTCH3^ECD

To ascertain if autophagosome-lysosome fusion failure is the cause of impaired autophagic flux, a tandem Ad-mCherry-GFP-LC3B was used. In this experiment, cherry red fluorescence was consistently visible, while GFP green fluorescence was rapidly diminished in acidic environments (Fig. 7d). As shown in Fig. 7a and 7b the NOTCH3 mutant cells exhibited a significant increase in the number of LC3B puncta, with a large proportion of puncta exhibiting red fluorescent (RFP+/GFP–) signaling (autolysosomes). However, compared with that of the control group, a greater proportion of spots exhibited yellow fluorescent (RFP+/GFP+) signaling (autophagosomes). In addition, after CQ treatment, NOTCH3 mutant cells showed a substantial proportion of these puncta exhibiting yellow fluorescent (RFP+/GFP+) signaling (autophagosomes; Fig. 3c). Therefore, NOTCH3 mutations result in impaired or deficient lysosomal function in the CADASIL cell model.

Fig. 7

Blocked autophagy flow in cells overexpressing NOTCH3^ECD. a) Immunofluorescence analysis of NOTCH3^ECD-overexpressing cells transiently transfected with tandem Ad-mCherry-GFP-LC3B. b) followed by treatment with 10μM chloroquine for another 24 h. Scale Bar: 10μm. c) Quantification of the ratio of autolysosome (AL, red puncta) versus autophagosome (AP, yellow puncta). d) Schematic diagram of autophagy flow.

DISCUSSION

CADASIL is the most common single-gene inherited cerebrovascular disease and has received extensive attention as a small vessel disease model in recent years. 1 With increased confirmed cases, our understanding of the complex pathological mechanisms has strengthen. Recent studies pointed to mitochondrial abnormalities in the cells of patients with CADASIL, suggesting that NOTCH3 gene mutations may be associated with mitochondrial dysfunction.27,30,37 , 27,30,37 However, the mechanisms through which NOTCH3 mutations contribute to mitochondrial dysfunction remain unclear. This study revealed a novel mechanism underlying NOTCH3 mutations in CADASIL. We provided evidence that NOTCH3 mutations lead to abnormal NOTCH3^ECD deposition and mitochondrial dysfunction. However, mitophagy cannot reverse this process, leading to the accumulation of dysfunctional mitochondria, which ultimately affects cell survival. Our findings provide new insights into the pathological mechanisms underlying CADASIL.

As one of the most important organelles in the cell, mitochondria play a crucial role in maintaining the normal physiological functions of the cell. However, dysfunctional mitochondrial function can have serious consequences for cell survival and function, particularly in neurodegenerative diseases. Mitochondrial dysfunction not only leads to a lack of energy supply but also causes imbalances in the intracellular environment, including oxidative stress, mitophagy, and mitochondria-induced apoptosis, all of which are key links in the development of diseases. In a study on the hereditary vascular disease CADASIL, recent findings further strengthened the central role of mitochondrial dysfunction in the pathophysiology of this disease. Studies have found that the activity of mitochondrial respiratory complex I is significantly reduced in muscle biopsy samples from patients with CADASIL.38,39, 38,39 Impairment of the respiratory complex I is a key component of the mitochondrial respiratory chain, and its function directly affects the ability of mitochondria to produce energy. In addition, studies have found that the mitochondrial DNA content in the peripheral blood of patients with CADASIL is significantly increased. Abnormal increases in mitochondrial DNA may be a mechanism by which cells compensate for mitochondrial dysfunction and may also be a feature of disease progression. 27 In terms of mitochondrial number and morphology, vascular smooth muscle cells in patients with CADASIL exhibit substantial abnormalities, including increased mitochondrial number, morphological irregularities, and structural abnormalities within the mitochondria 30 . In this study, we observed an increase in NOTCH3^ECD deposition in the cells of the mutant group. This suggests that mutations in NOTCH3, whether cysteine or non-cysteine, can lead to NOTCH3^ECD deposition.37,40, 37,40 We observed significant mitochondrial dysfunction in cells that overexpressed the mutation NOTCH3^ECD. The results of the correlation analysis showed that increased NOTCH3^ECD deposition was closely associated with severe mitochondrial dysfunction and a significant positive correlation with the number of unhealthy mitochondria. In addition, we found significant differences in the levels of NOTCH3 in the mitochondrial proteins. Simultaneously, we observed a strong colocalization phenomenon between NOTCH3^ECD and mitochondria, but the Pearson colocalization correlation coefficient (R-value) between the control and mutant groups was insignificant. This suggests a correlation between NOTCH3^ECD and mitochondria and that the toxic effect of NOTCH3^ECD mutations on mitochondria may be the main cause of mitochondrial dysfunction. At present, we cannot determine whether NOTCH3^ECD directly interacts with mitochondria. Future studies are needed to explore further specific interaction mechanism between NOTCH3^ECD and mitochondria and how this interaction affects mitochondrial function and cell health.

Autophagy and mitophagy are key cellular responses to mitochondrial dysfunction. 9 Inside the cell, damaged mitochondria are selectively cleared by mitophagy to maintain mitochondrial quality and quantity of mitochondria. 41 This process requires the involvement of lysosomes and acidic organelles with a pH range of 4.5– 5, which play crucial roles in intracellular waste disposal. 42 Previous studies showed that NOTCH3^ECD is degraded in a lysosome-dependent manner. 43 In this study, we found that autophagy and mitophagy were activated; we even observed that mitochondria were encapsulated by lysosomes, which is direct evidence of mitophagy. However, we found an accumulation of p62/SQSTM1 and autophagosome. Collectively, these results indicate inadequate or impaired lysosomal function, ultimately leading to the accumulation of p62/SQSTM1 and autophagosomes, impaired mitochondria, and, ultimately, the accumulation of harmful substances. In addition, this result is also supported by the absence of a significant decrease in the levels of the inner and outer membrane proteins TIMM23 and TOMM20, which is also consistent with previous studies showing that the average number of abnormal mitochondria in the CADASIL model cells was higher than in the control group cells; 30 muscle biopsies in CADASIL patients showed abnormal mitochondrial accumulation again consistent with this result.40,44, 40,44

Extensive loss of medial smooth muscle cells is a key pathogenic characteristic of CADASIL patients. 17 The ability of blood vessels to contract is greatly impacted by this alteration, which results in aberrant cerebral vascular hemodynamics. Patients may get migraines, psychosocial problems, gradual impairment of cognition, and even ischemic stroke. Autophagy and mitophagy play crucial roles in regulating myoblast differentiation, maintaining signaling pathways associated with mitochondrial networks, and responding to mitochondrial oxidative stress. 45 Studies have shown that mitochondrial dysfunction can trigger a complex set of responses, while under mild stress conditions, cells are often able to clear mild mitochondrial damage by activating mitophagy.34,45, 34,45 However, in patients with CADASIL, genetic mutations may impair mitophagy function, leading to dysfunctional mitochondria that are impossible to eliminate. This, in turn, continuously generates toxic substances like ROS, damaging cells continuously and ultimately resulting in apoptosis.23,46–48 , 23,46–48 Simultaneously, the insufficient or impaired lysosomal function not only affects mitophagy, leading to the accumulation of mitochondrial dysfunction but may also be the main cause of cellular aging or apoptosis.49,50, 49,50 In patients with CADASIL, lysosomal insufficiency may also exacerbate the abnormal deposition of NOTCH3^ECD, forming a vicious circle that further exacerbates cellular dysfunction. Decreased lysosomal function also promotes the deposition of NOTCH3^ECD, thus forming a vicious cycle that exacerbates cellular dysfunction.

Although our research has made progress, it has some limitations. First, as diseased brain tissue was unavailable, relevant studies on diseased tissues could not be conducted. Second, our research is limited to the cellular level and does not involve in vivo experiments. The next step is to explore this disease using animal models to understand the mechanisms underlying this disease more comprehensively and provide a solid scientific basis for future therapeutic strategies.

Our results suggest that the abnormalities in mutant NOTCH3^ECD are core pathological alterations that lead to cellular dysfunction. NOTCH3^ECD deposition led to defects in mitochondrial morphology and function, and mitophagy is one of the main pathways activated in response to this stress. However, owing to inadequate or defective lysosomal function, damaged mitochondria accumulate and produce harmful substances, forming a vicious circle that may eventually lead to cell death. Therefore, our findings provide a new perspective for understanding the pathological mechanisms of CADASIL and may identify potential targets for the development of therapeutic strategies targeting CADASIL. These findings strengthen our understanding of how mutations in NOTCH3 affect mitochondrial function and help uncover the molecular mechanisms of CADASIL, providing an important scientific basis for future clinical research and treatment.

AUTHOR CONTRUBUTIONS

Wan Wang (Conceptualization; Methodology; Software; Writing – original draft); Zhenping Gong (Data curation; Software; Writing – original draft); Yadan Wang (Data curation; Visualization); Ying Zhao (Data curation; Visualization); Yaru Lu (Writing – review & editing); Ruihua Sun (Writing – review & editing); Haohan Zhang (Writing – review & editing); Junkui Shang (Conceptualization; Project administration; Supervision); Jiewen Zhang (Conceptualization; Project administration; Supervision).

Footnotes

ACKNOWLEDGMENTS

We express our gratitude to all participants for their involvement in the study, as well as to all other contributors for their efforts and support.

FUNDING

This research was supported by the Key Research and Development Program of Henan Province, China (241111313500).

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

DATA AVAILABILITY

The data supporting the findings of this study are available within the article and/or its supplementary material.

The supplementary material is available in the electronic version of this article: .

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Mutant NOTCH3 ECD Triggers Defects in Mitochondrial Function and Mitophagy in CADASIL Cell Models

Abstract

Background:

Objective:

Methods:

Results:

Conclusions:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Cell culture and lentiviral transduction

Antibodies and reagent

Protein extraction and SDS– PAGE analysis

Mitochondrial protein extraction

Immunofluorescence

Flow cytometric analysis

Transmission electron microscopy

Oxygen consumption

Statistical analyses

RESULTS

NOTCH3 ECD mutations led to abnormal NOTCH3 ECD deposition

Abnormal NOTCH3 ECD deposition correlated with mitochondrial dysfunction

Co-localization of mutant NOTCH3 ECD deposits with mitochondria

Abnormal NOTCH3 ECD deposition enhanced basal autophagy

Abnormal NOTCH3 ECD deposition activated mitophagy via the PINK1-parkin pathway

Mitophagy was impaired in cells overexpressing NOTCH3 ECD

DISCUSSION

AUTHOR CONTRUBUTIONS

Footnotes

ACKNOWLEDGMENTS

FUNDING

CONFLICT OF INTEREST

DATA AVAILABILITY

References

NOTCH3^ECD mutations led to abnormal NOTCH3^ECD deposition

Abnormal NOTCH3^ECD deposition correlated with mitochondrial dysfunction

Co-localization of mutant NOTCH3^ECD deposits with mitochondria

Abnormal NOTCH3^ECD deposition enhanced basal autophagy

Abnormal NOTCH3^ECD deposition activated mitophagy via the PINK1-parkin pathway

Mitophagy was impaired in cells overexpressing NOTCH3^ECD