A comprehensive functional investigation of the human translocator protein 18 kDa (TSPO) in a novel human neuronal cell knockout model: from molecule to depression

doi:10.21203/rs.3.rs-4024118/v1

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A comprehensive functional investigation of the human translocator protein 18 kDa (TSPO) in a novel human neuronal cell knockout model: from molecule to depression

https://doi.org/10.21203/rs.3.rs-4024118/v1

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The translocator protein 18 kDa (TSPO) is a multifunctional outer mitochondrial membrane protein associated with various aspects of mitochondrial physiology and multiple roles in health and disease. Here, we aimed to analyze the impact of TSPO on the regulation of mitochondrial and cellular function in a human neuronal cell model. We used the CRISPR/Cas9 technology and generated TSPO knockout (KO) and control (CTRL) variants of human induced pluripotent stem cells (hiPSCs). In a multimodal phenotyping approach, we investigated cellular and mitochondrial function in neural progenitor cells (NPCs), astrocytes, and neurons differentiated from hiPSC CTRL and TSPO KO cell lines. Our analysis revealed reduced mitochondrial respiration and glycolysis, altered Ca²⁺ levels in the cytosol and mitochondrial matrix, a depolarized MMP, and increased levels of reactive oxygen species, as well as a reduced cell size. Notably, TSPO deficiency was accompanied by reduced expression of the voltage-dependent anion channel (VDAC). We also observed a reduced TSPO and VDAC expression in cells derived from patients suffering from major depression (MDD). Considering the modulatory function of TSPO and the similar functional phenotype of cells derived from patients with depression, we discuss a role of TSPO in the aetiology or pathology of MDD. Taken together, these findings indicate the impairment of mitochondrial function in TSPO KO cells, contributing to the understanding of the multifaceted role of TSPO and setting the stage for further investigations to unravel the underlying mechanisms and its involvement in various physiological and pathological processes.

Biological sciences/Neuroscience/Molecular neuroscience

Biological sciences/Neuroscience/Cellular neuroscience

The translocator protein 18 kDa (TSPO) is a highly conserved multifunctional protein residing in the outer membrane of mitochondria (outer mitochondrial membrane, OMM) ¹.

Originally described as peripheral-type benzodiazepine receptor (PBR), it was renamed translocator protein to reflect its suspected role in the transport of cholesterol into the mitochondria in the de novo generation of steroids. In addition to its role in cholesterol transport and steroid synthesis ^{2, 3, 4, 5}, TSPO has been shown to be involved in the regulation of various cellular and mitochondrial functions, such as mitochondrial bioenergetics (the oxidative phosphorylation system, OXPHOS, and the provision of ATP) ^{6, 7, 8}, the beta-oxidation of fatty acids ⁹, the production of ROS ¹⁰, and Ca²⁺ homeostasis ^{7, 8, 11}. Moreover, TSPO is involved in regulating cellular downstream processes such as proliferation, survival, and apoptosis ^{12, 13, 14} and plays multiple roles in health and disease, ranging from neurodegeneration and neoplasia to psychiatric disorders ^{15, 16}.

TSPO is expressed to varying degrees in multiple tissues, with the highest levels of expression found in steroidogenic tissues and relatively low expression in the central nervous system. In the brain, TSPO can be detected predominantly in microglia and reactive astrocytes ¹⁷, where its expression is upregulated in the context of inflammation, neurodegeneration, and malignant neoplasia ^{12, 15}. Steroids (especially neuroactive steroids) are important modulators of stress and stress-related disorders. At the subcellular level, steroid synthesis originates in the mitochondria, which places these organelles and the TSPO-associated functions at the center of stress-related disorders ^{18, 19, 20}.

In the literature, differing roles and functions of TSPO and TSPO ligands in various species and tissues have been reported ^{21, 22, 23}. The diverse functions of TSPO are likely mediated by a TSPO-containing protein complex, whose composition of interacting protein partners could be adapted to meet specific needs in various tissues and physiological or pathophysiological conditions. Among the many candidates, VDAC, a versatile channel for all kinds of ions and metabolites, seems to be one of the most important interaction partners ^{24, 25}.

To investigate the effect of TSPO expression or deficiency in the human neural system, we used CRISPR/Cas9 to generate TSPO-deficient induced pluripotent stem cells (iPSCs) reprogrammed from dermal fibroblasts obtained from a healthy donor. Furthermore, we differentiated the TSPO-deficient or TSPO-expressing iPSCs into neural progenitors, neurons, and astrocytes and performed a comprehensive analysis of mitochondrial function in the respective lines. We assessed the steroid synthesis, mitochondrial respiration (OXPHOS), the glycolysis, the mitochondrial membrane potential, the cytosolic and mitochondrial Ca²⁺ and reactive oxygen species (ROS) levels, the expression of inflammation- and mitochondrial dynamic-associated genes, the general mitochondrial content, and the cell size. Our findings point to a central role of TSPO in the regulation of mitochondrial and cellular function, underlining the significance of this protein in physiological and pathophysiological states. Confirming that alterations in mitochondrial functions are also evident in a human cell model of depression ^{26, 27, 28}, we extended our studies to fibroblasts of patients with depression and found reduced expression of both TSPO and VDAC in MDD fibroblasts, highlighting a possible causal link between TSPO and depression.

Fibroblasts and human induced pluripotent stem cells

The fibroblast cell lines derived from 16 MDD patients and their sex- and age-matched controls ²⁸ were used in this study. The hiPSC line from healthy fibroblasts was generated prior to this study using non-integrating episomal plasmid vectors encoding KLF4, SOX2, L-MYC, LIN28, OCT3/4, and p53DD (pCXB-EBNS, pCE-hsk, pCE-hUL, pCE-hOCT3/4, and pCE-mp53DD (Addgene plasmid #41857, #41814, #41855, #41813, and #41856, gifted by Shinya Yamanaka)) using the Amaxa Nucleofactor (Lonza). Undifferentiated human hiPSCs were cultured and maintained in mTeSR Plus medium (StemCell) supplemented with 50 µg/mL gentamycin on Matrigel-coated plates (8.7 µg/cm²) under standard conditions (37°C, 5% CO2).

Generation of TSPO knockout in iPSC using CRISPR/Cas9 genome editing

CRISPR/Cas9-TSPO sgRNA plasmids were prepared according to a previously published protocol ²⁹. Two independent sgRNA-targeting sequences, #116: 5`-TCCCGCTTTGTCCACGGCGAGGG-3`, and #126: 5`-TCCACGGCGAGGGTCTCCGCTGG-3` within exon 2 of the human TSPO gene were selected using the bioinformatics tool CRISPOR (http://crispor.tefor.net/). The DNA sequences were separately cloned into the modified pSpCas9 (BB)-2A-Puro (PX459) V2.0 vector (#62988, Addgene), kindly provided by Emmanuel Nivet ³⁰), using BbsI restriction sites. Constructs were transfected into iPSC cells by nucleofection using human stem cell nucleofector kit 2 (Lonza), and the puromycin-resistant single clones were picked and expanded. Homozygous TSPO ^−/− clones, were identified by immunoblotting using an anti-TSPO antibody. To determine whether the individual clones were homozygous for TSPO ^−/−, genomic DNA spanning exon 2 was PCR-amplified using the following primers: TSPO-ex2F: 5´-CTGGAAATGCGTTCACTCAG-3` and TSPO-ex2R: 5`- GCCTGGAGAAGACCCTCTGT-3`, and the PCR product was cloned into the pGEM-T cloning vector (Promega). Single bacterial colonies of each TSPO-KO clone were subsequently sequenced. The top five potential off-target sites, which were selected using the CRISPOR web tool were likewise PCR amplified, subcloned, and sequenced using specific primers (Supp. Table 1)

Table 1

Generation of PAX6/SOX2 positive hiPSC-derived NPC. Numbers represent proportion [%] of SOX2 and PAX6 positive cells in the analysed cultures.
Cell line	SOX2 positive [%]	PAX6 positive [%]
CTRL1	89.7	93.0
CTRL2	91.6	95.2
CTRL3	88.8	91.1
KO1	88.7	94.7
KO2	93.8	95.9
KO3	87.1	93.2

Differentiation of iPSCs into neural progenitor cells (NPCs), neurons and astrocytes

NPCs were derived from iPSCs following a monolayer culture method based on a previously published protocol ³¹. For neuronal differentiation, dissociated NPCs from passages 5 to 12 were plated onto laminin-coated µ-dishes (Ibidi) at a density of 1 to 1.5x10⁵ cells/cm² and differentiated for 21 days in BrainPhys neuronal medium (StemCell) supplemented with 1% B27 Plus, 0.5% GlutaMax, 0.5% non-essential amino acids, 0.5% Culture One, 200 nM ascorbic acid, 20 ng/mL BDNF, 20 ng/mL GDNF, 1 mM dibutyryl-cAMP, 50 U/mL Penicillin, and 50 µg/mL streptomycin, with half medium change twice per week. Additionally, 4 µg/mL laminin was freshly added each time the medium was changed.

iPSC-derived NPCs were differentiated into astrocytes, according to a previously described protocol ³². Briefly, NPCs were plated at 30,000 cells/cm² on Matrigel-coated plates (8.7 µg/cm²) and grown for 30 days in astrocyte medium (ScienCell). Astrocytes between 31 and 60 days of differentiation were used in this study.

Western blotting

To block de novo protein biosynthesis and thus determine protein half-life, iPSC-derived NPCs were treated with 100 µg/mL of the translational inhibitor cycloheximide (CHX) for the indicated time points. Whole-cell protein samples were prepared in RIPA buffer, separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Blotted proteins were detected using anti-TSPO (ab109497), anti-VDAC1 (ab186321) antibodies, both from Abcam and anti-LC3B (#3868S, Cell Signalling). Anti-Beta 1 tubulin (clone E7, from DSHB) and anti-GAPDH (sc-47724, Santa Cruz Biotechnology) antibodies were used as loading controls. Densitometric analysis was performed using ImageJ software (version 2.9.2).

Immunofluorescence

For immunocytochemistry, cells grown on either Geltrex-, Matrigel-, or laminin-coated glass coverslips (12 mm) or µ-dishes (Ibidi) were fixed in 4% PFA. Cells were permeabilised and blocked using a blocking solution containing 1x PBS, 0.5% Triton X-100, and 10% normal goat serum. Antigen retrieval was performed prior to staining of TSPO and VDAC1 by steaming coverslips in citrate buffer, pH 6. Cells were incubated overnight at 4 ^oC with following primary antibodies: anti-ALDHL1 (ab87117; 1:500), EAAT1 (ab416-1001; 1:200), anti-MAP2 (ab5392; 1:500), anti-NeuN (ab177487; 1:1000), anti-TSPO (ab109497; 1:500), anti-SYP (ab52636; 1:250), anti-VDAC1 (ab186321; 1:500), anti-VGLUT1 (ab180188; 1:250), all from Abcam. Anti-GFAP (C53893; 1:400) and anti-S100β (S2532; 1:1000) were purchased from Sigma, and anti-PSD95 (K28/43; 1:250) from NeuroMab. After three washing steps, cells were incubated for 1 hour with corresponding secondary antibodies conjugated with Alexa Fluor 488, Cy3, Cy5, and Hoechst to stain nuclei. Cells were mounted on glass slides with DAKO fluorescent mounting medium and examined using a Zeiss Axio Observer Z.1 microscope.

Fluorescent live-cell imaging

Live cell imaging experiments were conducted as described in ²⁶. Briefly, all recordings were taken using an inverted Zeiss Axio Observer Z.1 microscope and detected using an AxioCam MRm CCD camera (Zeiss). Illumination control and image acquisition were performed using a Lambda DG-4 high-speed wavelength switcher (Sutter Instruments) and the ZEN 2012 imaging software. Analysis of the measurements in the regions of interest, manually drawn over selected cells in the visual field, was performed using ImageJ (version 2.9.2). The macros used for background subtraction and analysis are provided upon request.

To analyse mitochondrial membrane potential (MMP) in NPCs and neurons the ratiometric fluorophore JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolylcarbocyanineiodide) was used. In astrocytes, the fluorescent dyes TMRE (tetramethylrhodamine, ethyl ester) in non-quenching mode and MitoTracker® Green (MTG) were used.

Cytosolic and mitochondrial Ca²⁺ levels were measured using Fura2-AM and Rhod-2AM. Approximately 1.8x10⁶ NPCs and 2x10⁵ astrocytes were seeded onto Matrigel-coated 25 mm glass coverslips and incubated overnight in a humidified incubator at 37°C and 5% CO2. The following day, the cells were simultaneously loaded with Fura-2/AM and Rhod-2/AM at a final concentration of 2 µM in OptiMEM. Neurons were likewise stained and measured on day 21 of differentiation.

Quantitative real-time PCR and mtDNA copy number analysis

For gene expression analysis, total RNA was extracted using RNA Plus Kit (Macherey-Nagel) according to the manufacturer’s instructions. First strand cDNA synthesis from 1 µg of total RNA was performed with QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR was performed on a RotorGeneQ (Qiagen) machine using the Takyon SYBR mastermix (Eurogentec), and specific intron-spanning primers, listed in Supp. Table 2. Measurements were performed in technical duplicates with a minimum of three biological replicates. The results were analysed using RotorGeneQ software V2.3 (Qiagen) by applying the ΔΔCt method for relative quantification.

To quantify the mtDNA copy number, primers for the mitochondrial encoded tRNA leucine 1 (mt-TL1) and the nuclear gene Beta-2 microtubulin (B2M) were used. Instead of total RNA, genomic DNA was used, and the following equation for quantification of mtDNA content relative to the diploid nuclear DNA was applied: mtDNA copy number = 2 x E^(-∆∆Ct).

Mitochondrial respirometry

A total of 80,000 NPCs/well or 30,000 astrocytes/well were grown on a Matrigel-coated XFp miniplate (Agilent). The sensor cartridges were prepared according to the manufacturer´s recommendations. To test oxidative phosphorylation, the sensor plate was loaded with oligomycin (1 µM), FCCP (1 µM for NPCs and 2 µM for astrocytes), and a combination of rotenone (0.5 µM) and antimycin A (0.5 µM). For measuring the glycolytic rate by calculating and subtracting mitochondrial-produced acidification the mitochondrial inhibitors rotenone and antimycin A were used in a final concentration of 0.5 µM each as well as 50 mM 2-deoxy-D-glucose (2-DG) as a glycolytic inhibitor.

The total ATP production rate was measured by obtaining both OCR and ECAR, simultaneously under basal conditions and after serial addition of 1.5 µM oligomycin followed by 0.5 µM of each rotenone and antimycin A. For the normalisation of respiration data, cells were stained with Hoechst and nuclei were counted using an ImageJ macro (provided on request).

Total ATP content quantification

For quantification of whole-cell ATP content, the CellTiter-Glo® Luminescent Cell Viability Kit (Promega) based on an ATP-dependent luciferase reaction was used. ATP was measured in cell pellets of 1x10⁶ NPCs or 1x10⁵ astrocytes according to the manufacturer´s instructions. ATP concentrations were normalised to µg/mL protein using a BCA assay (Thermo Fisher Scientific).

Scanning Electron Microscopy (SEM)

A detailed procedure has been described previously ³³. Briefly, iPS neurons grown on glass slides were viewed both in high-vacuum (high-vac) SEM mode. Cells were fixed for 30 min at room temperature with 2.5% glutaraldehyde (Serva) in 0.1 M Soerensen buffer, pH 7.4 (Merck) and aqua bidest. After treatment with the critical point dryer Balzers CPD 030 (BAL-TEC AG, Leica), and positioning on stubs via Leit-Tabs (Science Services), cells were sputtered with platinum twice for 30 s at 30 mA at a distance of 50 mm (BAL-TEC SCD 005, BAL-TEC AG, Leica). Cells were kept in the exicator and investigated under dry conditions with a FEI Quanta 400 FEG in high-vac mode (4.0 kV, Spot 3, WD ≈ 6 mm, ≤ 10 − 4 Torr, tilt 30°).

Pregnenolone quantification

Pregnenolone quantification was performed using an enzyme-linked immunosorbent assay (ELISA), adapted from a previously published protocol ³⁴. Briefly, pregnenolone concentration was measured in the supernatant from 1.5x10⁵ astrocytes/well of a Matrigel-coated 24-well plate. To quantify pregnenolone accumulation, the supernatant was collected after 3 h of incubation at 37°C in ELISA buffer (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mM MgSO₄, 10 mM glucose, 10 mM (HEPES)-NaOH, pH 7.4, and 0.1% BSA) containing inhibitors of pregnenolone metabolism trilostane (25 µM) and SU10603 (10 µM) using pregnenolone ELISA (LDN) according to the manufacturer’s instructions. Quantified pregnenolone concentrations were normalised to the protein concentration of each sample, as determined using the Pierce BCA Protein Assay (Thermo Fisher Scientific).

Flow cytometry analyses

Flow cytometry was performed on a BD Biosciences FACS Celesta™, which was run using the Diva software v7.0 (BD Biosciences). The cytometer was equipped with violet (405 nm), blue (488 nm) and red (640 nm) lasers. Particle size and granularity were measured using forward scatter (FSC) and side scatter (SSC) detectors. The area, height, and width parameters of the FSC and SSC detectors were used for doublet exclusion. Compensation was not required. For each sample 0.15x10⁶ to 0.3x10⁶ (astrocytes) or 0.5x10⁶ to 1x10⁶ (NPCs) cells were used. All tests were performed in at least three independent experiments. Data were acquired immediately after staining using the FACS Celesta™ flow cytometer (BD Bioscience). The FlowJo software (V10.8., Tree Star) was used for analysis.

Cytosolic and mitochondrial reactive oxygen species/oxidative stress

Cytosolic ROS levels were determined by applying 10 µM 2',7'-dichlorofluorescein diacetate (H2DCFDA) for 20 min under standard culture conditions in FACS buffer (DPBS supplemented with 2% FBS) in airtight tubes. To analyse mitochondrial ROS production, cells were washed with Hank’s Balanced Salt Solution (HBSS), stained with 5 µM MitoSOX™ Red Superoxide indicator in HBSS, and incubated in a humidified atmosphere at 37°C and 5% CO2. Flow cytometry data were acquired using the Fortessa System (BD) or Celesta System (BD). Data were analyzed using FlowJo (v10.8.1).

Mitochondrial mass

Mitochondrial content was assessed by staining with 1 µM MitoTracker® Green in RPMI 1640 supplemented with 2 mM L-glutamine for 1 h at 37°C in a cell incubator. To prevent mitochondrial depolarization 1.5 µM cyclosporin A was added to prevent mitochondrial depolarisation. After washing and resuspension in 200–300 µL FACS buffer, the cells were filtered and immediately analysed by flow cytometry.

Statistical analysis

Graphical depictions and statistical analyses were performed using GraphPad Prism 9.5.1 (GraphPad Software) for at least three independent experiments. For all experiments, two to three technical replicates were calculated and at least three biological replicates were averaged. Statistical outliers were identified using the ROUT method and removed before the analysis. The normal distribution of the data was assessed using the D’Agostino-Pearson omnibus normality test. Nonparametric data that did not follow a Gaussian distribution were analysed using the Mann-Whitney U test (MW), whereas parametric data were analysed using an independent samples t-test. The cut-off value for statistical significance was set at p ≤ 0.05. Results are presented as mean ± standard error of the mean (SEM). Asterisks in the figures represent p-values as follows: * p ≤ 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Validation of TSPO knockout in hiPSCs and off-target screening

To study the role and function of TSPO in neural cells, CRISPR/Cas9-mediated TSPO knockout variants were generated from hiPSCs reprogrammed from primary skin fibroblasts of a healthy donor. Knockouts were validated by DNA sequencing of PCR products and Western Blot analysis.

In one of the clones (KO1), the nucleofection of the cAB03 plasmid containing the sgRNA-targeting sequence #116 deleted a 17 bp fragment, which resulted in a shift in the open reading frame (ORF) after the amino acid V26 in allele 1. An additional 246 bp insertion in allele 2 generated a stop codon (*) after residue R24. A second clone (KO2) demonstrated a 26 bp deletion in allele 1 and a deletion of a 16 bp fragment in allele 2, resulting in a frameshift and generation of a non-functional TSPO gene. Electroporation of hiPSCs with the cAB03 plasmid, including the #126 targeting sequence, generated a third clone (KO3), which lost the complete exon 2 and adjacent bases (286 bp) in allele 1. The deletion of 19 bp in allele 2 led again to a shift in the ORF and a non-functional TSPO gene (Fig. 1A).

Western Blot analysis of TSPO protein expression in all three clones revealed a complete loss of TSPO in the mutated cell lines KO1, KO2, and KO3, whereas the corresponding isogenic control cell lines CTRL1, CTRL2, and CTRL3 exhibited prominent TSPO expression at 18 kDa (Fig. 1B).

Differentiation of hiPSC-derived NPCs into astrocytes

iPSCs were differentiated into NPCs and stained for the neural progenitor markers SOX2 and PAX6 ³⁵. Table 1 shows that most cells co-express both markers and could, therefore, be considered neural progenitors. Furthermore, astrocytes and induced cortical-like neurons were generated by differentiating NPCs following the protocol described by ³² and ³¹, respectively.

To identify and confirm the maturity of the astrocytes (Astro), the expression of specific markers was examined by immunostaining. Within 30 days, hiPSC-derived astrocytes were immunopositive for glial fibrillary acidic protein (GFAP), S100 calcium-binding protein B (S100β), glutamate transporter excitatory amino acid transporter 1 (EAAT-1) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1) (Fig. 1C).

The functionality of mature astrocytes was verified by the presence of spontaneous and ATP-elicited Ca²⁺ transients, as previously described for astrocytes ^{32, 36, 37, 38, 39}. The Ca²⁺ indicator Fura-2/AM was used to monitor Ca²⁺ signalling under basal conditions and in response to a pulse of extracellular ATP (100 µM) ³⁷. While some cells showed already spontaneous Ca²⁺ spikes (Fig. 1D), a single pulse of the gliotransmitter ATP produced a slow Ca²⁺ response in most cells (Fig. 1E), suggesting a network of interconnected astrocytes.

Differentiation of hiPSC-derived NPCs into neurons

To confirm the identity of hiPSC-derived neurons, the expression of specific neuronal markers was investigated using immunocytochemical analysis. Somato-dendritic microtubule-associated protein 2 (MAP-2)- and class III β-tubulin (Tuj-1)-positive neuronal cells were detected after 21 days of differentiation, labelling the neurite network. Additionally, visualisation of the post-mitotic nuclei using the neuronal marker NeuN, co-localising with the nuclear-specific dye Hoechst, indicated a highly pure neuronal culture ⁴⁰. The induced neurons displayed mature synaptic terminals, as indicated by distinct punctate labelling with presynaptic (synaptophysin, VGLUT1) and postsynaptic (PSD95) markers (Fig. 1F). The vesicular glutamate transporter (VGLUT) and the scaffolding postsynaptic density protein 95 (PSD95) serve as distinctive biomarkers for glutamatergic synapses, confirming that the cells are cortical-like glutamatergic neurons. To further visualise neuronal morphology, high-resolution electron micrographs were generated, revealing densely interconnected networks of neurites and protrusions (Fig. 1G).

Excitability is a hallmark of neuronal function and is involved in both spontaneous and evoked neurotransmission as well as synaptic control. Neuronal activity plays a crucial role in the maturation of the developing brain and is vital for processes such as neuronal migration, axonal and dendritic growth, and the formation and refinement of neuronal connectivity and synapses ³⁶. A fundamental characteristic of the nervous system ⁴¹ present also in cultured neurons is the tendency to self-organize a functional network showing spontaneous activity. As changes and oscillations in intracellular Ca²⁺ levels through voltage-dependent Ca²⁺ channels evoked by the electrical activity of individual neurons ⁴² are an important aspect of spontaneous activity, neuronal Ca²⁺ signalling was monitored using Fura-2/AM in hiPSC-derived neurons to verify their functionality (Fig. 1H). Additionally, growing hiPSC-derived neurons on high-density multielectrode arrays (HD-MEAs) and extracellular recording of electrical activity demonstrated synchronous and asynchronous patterns of neural network activity ³⁶. These preliminary experiments further confirmed successful differentiation into neurons (Fig. 1I).

Validation of TSPO knockout in NPCs, astrocytes, and neurons

TSPO knockout was validated in differentiated NPCs, astrocytes, and neurons using immunofluorescence. Anti-VDAC1 fluorescence clearly demonstrated the presence of a mitochondrial network in both the CTRL and KO cells. TSPO expression, as indicated by anti-TSPO antibody staining, was only visible in CTRL cells but was completely absent in KO cells, validating the loss of TSPO expression in mutated NPCs, astrocytes, and neurons. (Fig. 1J).

Therefore, TSPO knockout cell lines can be considered suitable tools for studying the role of TSPO in different cell types derived from induced human neural cells.

Effect of TSPO expression on steroid synthesis in hiPSC-derived astrocytes

TSPO is highly expressed in activated glial cells and has been reported to be involved in mitochondrial cholesterol transport, thus making it a key regulator of pregnenolone synthesis ^{2, 3, 14, 15, 18, 43}. To evaluate the involvement of TSPO in neurosteroidogenesis, the concentration of pregnenolone in hiPSC-derived astrocytes was investigated using ELISA. The supernatants collected from TSPO-knockout cells contained significantly lower concentrations of pregnenolone than those from CTRL cells (3.961 ± 0.18 pg/µg/mL protein, n = 17 vs. 5.144 ± 0.33 pg/µg/mL protein, n = 18, p = 0.0031) after 3 h of incubation with the enzyme inhibitors trilostane (3β-hydroxysteroid dehydrogenase, HSD3B1) and SU10603 (17-OH-steroid hydroxylase, CYP17A1), which were used to prevent further metabolism of the acutely synthesised pregnenolone (Fig. 2A). Omitting these inhibitors from the assay medium resulted in a low basal concentration of pregnenolone in both CTRL and TSPO^−/− cells. These data indicate that the hiPSC-derived astrocytes are capable of steroid production, which is regulated by the presence of TSPO.

To gain insight into the steroid-synthesising capacity of hiPSC-derived astrocytes, analysis of gene expression was performed for key steroidogenic enzymes, including cytochrome P450 (CYP11A1), HSD3B1, and CYP17A1, which are crucial for the conversion of cholesterol to pregnenolone and subsequent neurosteroid production, as well as steroidogenic acute regulatory protein (StAR), which is essential for cholesterol transport. As shown in Fig. 2B, the results demonstrate higher mRNA transcript levels of these proteins in astrocytes than in the C20 microglial cell line, which does not produce substantial amounts of pregnenolone ⁷. However, it is noteworthy that the mRNA levels observed in hiPSC-derived astrocytes were still considerably lower than those found in the steroidogenic adrenocarcinoma cell line H295-R, indicating a relatively low steroid-synthesising capacity of astrocytes. Nevertheless, these findings support the potential importance of TSPO in the regulation of neurosteroidogenesis, as evidenced by the reduced pregnenolone concentration in the TSPO-knockout cells.

Impact of TSPO expression on mitochondrial respiration

Mitochondria are crucial for the regulation of cellular bioenergetics by providing most of the cell’s energy through the oxidative phosphorylation system (OXPHOS). To investigate the functionality and performance of the OXPHOS, the oxygen consumption rate (OCR) was measured using extracellular flux analysis in CTRL and TSPO KO NPCs (Fig. 3A and B) and astrocytes (Fig. 3D and E), respectively. We found that TSPO expression significantly affected different aspects of mitochondrial respiration. KO cells of both cell types showed significantly lower basal respiration than TSPO-expressing CTRL cells (NPC: p = 0.0002; Astro: p = 0.0022). Moreover, maximal respiration in the presence of the uncoupler FCCP and the resulting spare respiratory capacity, which reflects the cell’s bioenergetic reserve and flexibility in meeting high energetic demands, were significantly reduced in TSPO-deficient cells (maximal respiration: NPC p < 0.0001; Astro p = 0.0019; spare respiratory capacity: NPC p = 0.0145; Astro p = 0.0039). Additionally, the oxygen consumption related to ATP production differed significantly between KO and CTRL neural progenitors and astrocytes (NPC p = 0.0004; Astro p = 0.003). The proton leak was also significantly lower in the knockout group than in the CTRLs (NPC p = 0.0031; Astro p = 0.0123).

To provide a more comprehensive understanding of OXPHOS activity and to study the bioenergetic core function of mitochondrial metabolism the total ATP levels of the cells were measured. Interestingly, we didn’t detect a difference in cellular ATP content, neither in TSPO KO NPCs nor astrocytes, compared with their respective controls (NPC p = 0.7834; Astro p = 0.5011) (Fig. 3C and 3F).

The altered respiratory parameters in TSPO-deficient cells highlight the significance of TSPO in regulating mitochondrial bioenergetics in neural cells. Moreover, the unaffected ATP levels suggest compensatory mechanisms in TSPO^−/− cells to maintain intracellular ATP levels, despite the potentially reduced energy production associated with decreased OXPHOS.

Role of TSPO in the modulation of the mitochondrial membrane potential

During OXPHOS, a proton gradient across the IMM contributes to the formation of the mitochondrial membrane potential (MMP), which provides valuable insights into mitochondrial function, metabolic activity, and potential imbalances arising from mitochondrial dysfunction.

To visualise the MMP in NPCs and neurons, mitochondria were labelled with the ratiometric cationic dye JC-1 (200 nM), which forms potential-dependent red fluorescent aggregates in highly energised mitochondria, whereas in response to the dissipation of the MMP, the dye molecules disaggregate into green fluorescing monomers. The MMP of hiPSC-derived astrocytes was assessed by loading the cells with a combination of the cationic dye TMRE (25 nM, non-quenching mode) and MitoTracker Green (MTG, 200 nM). TMRE also accumulates in negatively charged mitochondria to an extent that depends on the strength of the electric field. The mitochondria-specific dye MTG is nearly potential-independent and is used to normalise the TMRE fluorescence. The ratio of the fluorescence signals emitted by the two different states of JC-1 (red/green), as well as the ratio F_TMRE/F_MTG, is therefore a measure of the strength of the MMP ⁴⁴.

TSPO deficiency in hiPSC-derived neural progenitors and astrocytes decreased the fluorescence ratio from 1.08 ± 0.01 and 0.49 ± 0.005 to 0.86 ± 0.01 and 0.47 ± 0.006, respectively (p < 0.0001) (Fig. 3G and H). Given that the mitochondria residing in the soma or neurites of the neurons appeared at different focal planes, images of JC-1 fluorescence of the relevant structures were captured separately. The TSPO-knockout neurons showed again a significantly reduced JC-1 ratio (p < 0.0001), especially in the neurites, thereby indicating a less hyperpolarised membrane potential in the mitochondria devoid of TSPO (Soma 0.88 ± 0.23 vs. 0.45 ± 0.008; Neurites 12.07 ± 0.23 vs. 7.85 ± 0.16) (Fig. 3I). Overall, knocking out TSPO resulted in a robust and significant decline in the mitochondrial membrane potential across all examined cell types. This again indicates that the overall function of the ETC, i.e., the translocation of protons, and the parallel backflow of protons through the ATP synthase, which determines the mitochondrial membrane potential, is diminished.

Involvement of TSPO in Ca²⁺ Homeostasis

Mitochondria play a critical role in Ca²⁺ homeostasis, thereby regulating numerous signalling pathways, enzyme function, and cellular metabolism. Driven by their negative membrane potential, mitochondria attract and accumulate calcium ions within the matrix, thereby directly influencing and regulating cellular Ca²⁺ dynamics. Owing to their capability to store or release calcium ions, mitochondria serve as calcium buffers with both beneficial and detrimental effects. The MMP, along with Ca²⁺-conducting channels (e.g., voltage-dependent anion channel, VDAC) and the activity of Ca²⁺-transporting proteins in the IMM (e.g., mitochondrial calcium uniporter, MCU), influence the extent of mitochondrial Ca²⁺ uptake.

Using Ca²⁺-sensitive dyes, the cytosolic and mitochondrial Ca²⁺ levels were investigated. Cytosolic Ca²⁺ concentrations were measured by loading the cells with the ratiometric dye Fura-2/AM, whereas Rhod-2/AM was used to assess mitochondrial Ca²⁺ levels. Consistent within all analysed cell types, a significantly reduced Fura-2 ratio (F_340nm/F_380nm) was observed in the TSPO^−/− cell lines compared to the respective CTRL cells, showing decreased basal cytosolic Ca²⁺ levels (p < 0.0001) (Fig. 3J). The Rhod-2 fluorescence intensity showed a significant increase amongst all KO cell lines (p < 0.0001) which is indicative of altered Ca²⁺ flux or active transport processes (Fig. 3J).

According to current hypotheses, TSPO plays a crucial role in coordinating and regulating the activity and expression of proteins within a multimeric complex. As a highly interactive protein, TSPO may function as a molecular hub of VDAC-containing protein complexes, thereby contributing to the maintenance of intracellular Ca²⁺ homeostasis.

Western Blot analyses with an anti-VDAC1 antibody showed that TSPO deficiency in hiPSC-derived NPCs and astrocytes was accompanied by a 33.5 ± 4.4% and 44.8 ± 4.2% reduction in VDAC1 protein expression, respectively, compared to the respective control (p < 0.0001, Mann-Whitney U test), as shown in Fig. 4A. This finding supports the idea of functional and structural interactions between these two proteins. This altered VDAC1 expression suggests that changes in TSPO expression have direct or indirect effects on key physiological aspects of mitochondrial function.

To gain insight into the underlying mechanisms, analysis of VDAC1 mRNA expression and protein stability was performed. qRT-PCR analysis indicated that loss of TSPO expression had no effect on VDAC1 transcription (Fig. 4B).

To assess the stability of the endogenous VDAC1 protein, a cycloheximide (CHX) chase protein degradation assay was conducted. Therefore, neural progenitors were incubated with 100 µg/mL CHX, a well-known inhibitor of eukaryotic translation, for a maximum of 24 hours to evaluate VDAC1 protein levels at different time points after CHX treatment. VDAC1 protein expression was quantified as a percentage of the initial VDAC1 protein level (0 h of CHX treatment) and was normalized to BTUB1 expression. Our findings indicate that the amount of VDAC1 protein in CTRL NPCs declined to 67.4 ± 3.8% of the initial value after 24 h, while the KO cells showed higher protein degradation (55.9 ± 5.6% of initial value), as shown in Fig. 4C.

Impact of TSPO on cellular bioenergetics and glycolysis

ATP production via the glycolytic pathway is an alternative way for the provision of energy. To measure the glycolytic flux, the proton efflux rates (PER) of the TSPO KO and CTRL groups were analysed. Using the Agilent Seahorse Glycolytic Rate Assay, TSPO^−/− neural progenitors (Fig. 5A), and astrocytes (Fig. 5B) showed significantly lower basal glycolysis (glycoPER) than CTRL cells (NPC p < 0.0001; Astro p = 0.0023). According to basal glycolysis, TSPO-deficient cells also had significantly reduced compensatory glycolysis (NPC p < 0.0001; Astro p = 0.0002). In addition, the basal PER and post-2-DG acidification rate of KO cells were also statistically significant different from those of CTRL cells (basal PER: NPC p < 0.0001; Astro p < 0.001; post-2-DG acidification: NPC p = 0.0005; Astro p = 0.00238). Conversely, the CTRL and KO groups did not differ significantly in their basal mitoOCR/glycoPER ratios (NPC p = 0.31; Astro p = 0.297).

Collectively, TSPO KO cells demonstrated lower basal respiration, spare respiratory capacity, glycolysis, and compensatory glycolysis in comparison with CTRL cells, but a similar mitoOCR/glycoPER ratio, indicating a lower metabolic flux in TSPO^−/− cells.

The ATP production rate is an effective means of characterising cellular metabolism. Using the Agilent Seahorse XFp Real-Time ATP Rate Assay, the total ATP production rates as well as the fractions of ATP produced through mitochondrial OXPHOS and glycolysis of hiPSC-derived neural progenitors and astrocytes were analysed. As shown in Fig. 5C and D and consistent with the Seahorse Mito Stress Test, the mitochondrial-derived ATP production rate (basal mitoATP production rate) was significantly decreased in the TSPO^−/− group of both cell types (NPC p = 0.0035; Astro p < 0.0001). Regarding the glycolysis-specific ATP rate fraction, only TSPO deficiency in NPCs resulted in a significant reduction compared to that in the control cells (NPC p < 0.0001). The contribution of glycolysis to the total ATP production rate was not altered in KO and CTRL astrocytes (Astro p = 0.17).

However, consistent with the unchanged basal mitoOCR/glycoPER ratio, the overall metabolic flux, represented by the total ATP production rate, was lower in both TSPO-deficient NPCs and astrocytes than in TSPO-expressing controls (NPC p < 0.0001; Astro p = 0.002). The XF ATP Rate Index, a measure for detecting changes or differences in metabolic phenotypes, showed a significant decrease in TSPO-KO astrocytes, indicating a less oxidative phenotype compared to CTRL cells (Astro p = 0.0051). As both mitochondrial and glycolytic ATP rates were decreased in TSPO-deficient NPCs, the XF ATP Rate Index remained unchanged (NPC p = 0.22). Taken together, these findings demonstrate the impact of TSPO expression on the bioenergetic capacity of cells and support the regulatory role of TSPO in cellular and mitochondrial energy homeostasis.

TSPO-deficient Neural Progenitors and astrocytes show oxidative stress

Mitochondria exert a crucial role in maintaining the cell’s redox homeostasis. Efficient functioning of the electron transport chain coupled with mitochondrial ATP production is associated with reactive oxygen species (ROS), generated primarily at complexes I and III of the ETC. When maintained at low or moderate concentrations, ROS are involved in intracellular signalling. However, an imbalance between the production and detoxification of reactive oxygen species causes oxidative stress, leading to molecular and cellular damage. Cellular redox was analysed by flow cytometry using the fluorescent dyes DCFDA and MitoSOX™ Red to detect global cytosolic hydrogen peroxide (H₂O₂), peroxyl radicals (HO₂), and mitochondrial superoxide, respectively. As shown in Fig. 6, both cytosolic and mitochondrial ROS were increased in TSPO-knockout neural progenitors (cytosolic ROS p = 0.0028; mitochondrial ROS p = 0.013) (Fig. 6A) and astrocytes (cytosolic ROS p = 0.031; mitochondrial ROS p = 0.046) (Fig. 6B) compared with the respective controls, indicating oxidative stress and an imbalanced redox state of the cell.

Effect of TSPO loss on mtDNA copy number, mitochondrial content and cell size

Mitochondria possess their own genetic material, circular mtDNA molecules, which encode some rRNA, tRNA, and essential components of the mitochondrial ETC. The dynamic equilibrium between mtDNA degradation and synthesis, which does not coincide with the cell cycle, determines the mtDNA copy number, ranging from 10³ to 10⁴ copies in different cells. The quantity and quality of mtDNA directly affect mitochondrial function, with mtDNA copy number serving as an indicator of mitochondrial integrity. Levels of mtDNA copy number are correlated with energy reserves, oxidative stress, and changes in MMP. Thus, altered mtDNA copy number can disrupt mitochondrial energy metabolism and contribute to diverse pathologies.

To evaluate whether the observed differences in the mitochondrial membrane potential and respiratory activity are related to lower mitochondrial content, the mtDNA copy number was determined in relation to the diploid nuclear genome in TSPO^−/− and CTRL cells. The relative mtDNA content was quantified using quantitative polymerase chain reaction (qRT-PCR) targeting the mitochondrial-encoded gene tRNA leucine 1 (mt-TL1) and the nuclear-encoded single-copy gene beta-2-microglobulin (B2M).

Consistent with the observation of altered bioenergetic properties and oxidative stress, TSPO-deficient NPCs, astrocytes, and neurons contained significantly less mtDNA compared to their controls (NPC p < 0.0001; Astro p = 0.017; Neuron p < 0.0001) (Fig. 7A).

Reduced mitochondrial mass is an adaptive response to decreased mtDNA copy number. Consequently, the mitochondrial content was determined by flow cytometry using the fluorescent dye MitoTracker Green. However, MTG fluorescence did not differ significantly between KO and CTRL cells (NPC p = 0.26; Astro p = 0.67) (Fig. 7B), indicating no effect of TSPO expression on the mitochondrial mass in our cells.

Interestingly, consistent among all cell types examined, cells lacking TSPO protein exhibited a considerably reduced soma size (p < 0.0001), as determined by pixel counting of the fluorescent area during Fura-2 imaging (Fig. 7C).

Impact of TSPO on mitochondrial dynamics and morphology

Dynamic fusion and fission processes help to adapt the cellular mitochondrial network to cellular demands and external stressors. In addition, mitophagy selectively eliminates damaged or dysfunctional mitochondria, thereby preventing detrimental effects on cellular health.

To investigate the impact of TSPO deficiency on mitochondrial dynamics, the mRNA expression levels of key fusion proteins, mitofusin 1 (MFN1) and optic atrophy protein 1 (OPA1), and fission protein dynamin-related protein 1 (DRP1) were analysed in hiPSC-derived astrocytes. Figure 7D shows that TSPO deficiency significantly reduced MFN1 mRNA levels (p = 0.035), whereas OPA1 and DRP1 transcripts remained unchanged. Additionally, the gene expression of mitochondrial transcription factor A (TFAM), a crucial activator of mitochondrial transcription and regulator of mtDNA copy number, was examined. Consistent with the decreased mtDNA copy number, TFAM mRNA expression was significantly reduced in astrocytes lacking TSPO protein (p = 0.05) (Fig. 7E). Furthermore, the role of TSPO in mitophagy was examined by gene and protein expression analyses. While PTEN-induced putative protein kinase 1 (PINK1) transcripts were not significantly different between TSPO^−/− and CTRL cells, parkin (PARK2) mRNA levels were considerably lower in TSPO-deficient astrocytes (p = 0.004) (Fig. 7F). Surprisingly, LC3B-II protein expression, the cleaved lipidated form of LC3B, which is tightly associated with autophagosomal membranes and indicates the degree of autophagic activation, was upregulated in TSPO-deficient NPCs and astrocytes. Western Blot analyses with an anti-LC3B antibody showed that TSPO deficiency in hiPSC-derived NPCs and astrocytes led to an increase of 42.1 ± 10.3% and 49.9 ± 14.3% in LC3B-II protein expression, compared to the respective control (NPC p = 0.007; Astro p = 0.0066), as shown in Fig. 7G. These findings suggest a role for TSPO in modulating mitochondrial dynamics, mitophagy, and the maintenance of the mitochondrial genome.

As mitochondria morphology is regulated by the dynamic interplay between fusion and fission, ensuring mitochondrial network integrity and function, electron micrographs of hiPSC-derived astrocytes were quantified to investigate the influence of TSPO expression on mitochondrial morphology. Mitochondria devoid of TSPO were significantly smaller, as indicated by decreased surface area (p < 0.0001) and perimeter (p < 0.0001). Additionally, the sphericity of the mitochondria was significantly increased (p = 0.03) in TSPO KO astrocytes, indicating a rounder, less polymorphic shape, as depicted in Fig. 7H and I.

TSPO expression in Major Depressive Disorder

TSPO has been implicated in neurodegenerative and neuroinflammatory processes, as presumed by the observation of increased TSPO expression in various neuropathologies such as Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, and major depressive disorder (MDD) ^{12, 15}. Although the molecular pathomechanisms underlying MDD are still not fully understood, accumulating evidence suggests a link between mitochondrial dysfunction, bioenergetic imbalance, and the aetiology and pathology of depression ^{26, 27, 28, 45, 46, 47}. Using a human cell model of depression, our group found mitochondrial function to be altered in a way similar to what we observed in TSPO-deficient cells ^{27, 28}. This possible connection prompted us to analyse the protein expression of TSPO and VDAC1 in cells from patients with MDD. Therefore, we used fibroblast cell lines derived from 16 patients with MDD and their sex- and age-matched controls. Interestingly, we found that the levels of both TSPO (Fig. 8A) and VDAC1 (Fig. 8B) proteins were significantly reduced (TSPO 76.03 ± 4.94%, p = 0.0004; VDAC1 76.48 ± 3.92% p = 0.0028) in patients with MDD, pointing towards a potential involvement of TSPO in the development or cause of mitochondrial dysfunction and manifestation of MDD.

In the present study, the effect of TSPO on cellular and mitochondrial homeostasis was investigated using a novel CRISPR/Cas9-mediated TSPO knockout model in human iPSCs. As the physiological significance of TSPO seems to be tissue- and cell type-specific, TSPO^−/− and CTRL hiPSCs were differentiated into neural progenitor cells, astrocytes, and neurons to elucidate metabolic changes related to neurosteroid synthesis, cellular and mitochondrial bioenergetics, VDAC1 protein expression, redox and calcium homeostasis, and mitochondrial dynamics as well as morphology in the presence and absence of TSPO across different cell types.

Role of TSPO in neurosteroidogenesis

TSPO has long been considered essential for mitochondrial cholesterol import, because of its localisation in the OMM, the specificity of the structural C-terminal CRAC domain with high affinity for cholesterol, and its particularly high expression in steroid-synthesising tissues. Studies using biochemical, pharmacological, and genetic experimental approaches have provided convincing evidence for the important role of TSPO in steroid production ^{3, 43, 48, 49, 50, 51}. However, recent in vivo and in vitro TSPO knockout studies have refuted the role of TSPO in steroidogenesis ^{52, 53, 54}, therefore, its specific role in neurosteroid synthesis is still a matter of debate ⁵⁵.

We found a significant decrease in pregnenolone levels in TSPO-deficient astrocytes, supporting an important role of TSPO in neurosteroid synthesis. However, the amount of detected pregnenolone was considerably low, ranging between 2.5 and 7.2 pg/µg/mL protein. These low levels indicate a low steroid-synthesising capacity of hiPSC-derived astrocytes, similar to what we found in human C20 microglia cells ⁷.

Neurosteroidogenesis occurs in both neurons and glia, although the expression of steroidogenic enzymes in the CNS is specific to certain cell types and regions ⁵⁶. Recent results from a TSPO knockdown study in murine microglial BV-2 cells support a TSPO-related de novo neurosteroid synthesis pathway as well as a direct and TSPO-mediated effect of TSPO ligands on neurosteroidogenesis ¹⁴. In contrast, a study using human C20 microglial cells reported low pregnenolone synthesis, independent of TSPO expression, which could not be stimulated by specific TSPO ligands or dibutyryl-cAMP ⁷. A more recent study not only confirmed the steroidogenic role of human microglia but also highlighted the explicit involvement of TSPO in this process ³⁴. Moreover, Angeloni et al. also suggested a role of TSPO in cholesterol trafficking and neurosteroid synthesis in human microglia, since TSPO silencing resulted in impaired cholesterol homeostasis, leading to excessive cholesterol accumulation ⁵⁷. Lin et al. demonstrated the ability of human microglia and astrocytes to produce pregnenolone despite low levels of P450scc mRNA and no detectable protein expression. They further provided evidence for a CYP11A1-independent pathway for pregnenolone synthesis and proposed the involvement of an alternative CYP450 isoform, CYP450 1B1, responsible for producing pregnenolone in the human CNS ⁵⁸.

Although TSPO is highly expressed in microglia and astrocytes, it is less abundant in neurons. Single-cell transcriptomics and TSPO-antibody immunolabelling have confirmed low neuronal TSPO expression in rodent and human brains ^{18, 59, 60}, which could be upregulated by neuronal activity ⁶⁰.

Taken together, in contrast to the periphery, where TSPO is not essential for maintaining baseline steroidogenesis, except during aging or in response to stress ¹⁸, these findings suggest a crucial role for TSPO in steroid production in the brain.

Impact of TSPO on mitochondrial and cellular respiration

TSPO-deficient NPCs and astrocytes showed reduced mitochondrial respiration, as indicated by lower basal and maximal respiration rates. While the cellular ATP contents were not different, the mitochondrial and total ATP production rates were significantly reduced in TSPO^−/− NPCs and astrocytes, indicating a decreased metabolic flux. Testing the glycolytic activity of TSPO KO cells which could compensate for decreased OXPHOS and maintain total intracellular ATP levels revealed a reduced basal and compensatory glycolysis in TSPO-deficient NPCs and astrocytes.

In the human C20 microglial cell line, basal and maximal respiration, as well as ATP-related oxygen consumption, were significantly reduced in TSPO-knockout cells compared to wild-type cells. Lentiviral overexpression of TSPO was able to rescue the impaired mitochondrial respiration in C20 knockout cells ⁷. Similarly, TSPO deficiency in primary murine microglia restrained mitochondrial OXPHOS, as indicated by lower basal and maximal OCR and subsequent ATP production, resulting in reduced mitochondrial ATP levels ^{61, 62}. In a lentiviral TSPO knockdown model in the rodent microglial cell line BV-2, mitochondrial respiration was unchanged compared with that in SCR control cells ¹⁴.

Reduced mitochondrial respiration may be attributed to diminished mitochondrial respiratory chain activity or the limited availability of substrates or enzymes. If glucose or fatty acid substrates are restricted or enzymes involved in their metabolic breakdown would be affected, the levels of NADH/H⁺ and FADH₂ reduction equivalents would decrease. Moreover, the transport of these reduction equivalents into the mitochondria may be a limiting factor for ETC function ⁶³.

The voltage-dependent anion channel VDAC, is a crucial regulator of multiple mitochondrial functions. As the major pore of the OMM, VDAC1 facilitates the translocation of respiratory chain substrates, such as ADP, ATP, NAD⁺, and NADH. Furthermore, VDAC is involved in the regulation of metabolite diffusion, including glucose, pyruvate, glutamate, succinate, and citrate and cations, such as Ca²⁺ and Mg^{2+ 64}. The association between TSPO and VDAC has been extensively studied, suggesting their involvement in an insufficiently characterised protein complex that exerts a joint influence on physiological functions.

TSPO deficiency was accompanied by reduced VDAC1 protein expression in hiPSC-derived NPCs and astrocytes. While the gene expression level was unchanged, protein stability assays using cycloheximide revealed higher protein degradation in TSPO^−/− NPCs. This negative correlation between TSPO and VDAC expression has also been shown in a TSPO-knockout model of human C20 microglial cells ^{7, 8}. Moreover, decreased VDAC expression in glioblastoma cancer cells resulted in a decline in TSPO expression ⁶⁵, whereas patients with bipolar disorder showed highly increased expression levels of both TSPO and VDAC in their PBMCs ⁶⁶, further demonstrating the mutual dependence of TSPO and VDAC expression.

A compromised MMP, e.g., during oxygen deprivation or caused by damaged mitochondrial respiratory proteins, would reverse the enzymatic activity of the ATP synthase. Under such conditions, the enzyme would function as a proton-motive ATPase, thereby consuming ATP and pumping protons out of the mitochondrial matrix to sustain MMP. However, inhibitor factor 1 (IF₁) can mitigate ATP hydrolysis by binding to the F₁F_o complex and inhibiting ATPase activity without affecting ATP synthesis during OXPHOS ⁶⁷. A putative IF₁ activity could explain such a scenario with MMP depolarisation and unchanged ATP levels in the hiPSC-derived TSPO-deficient cells. However, KO cells also exhibited reduced ATP-related OCR and lower total ATP production rates, suggesting a lower ATP demand and turnover. The phosphocreatine (PCr) shuttle is used to meet immediate energy requirements ⁶⁸. Mitochondrial creatine kinase, along with cytosolic isoenzymes, produces highly diffusible phosphocreatine for the temporal and spatial buffering of ATP levels to maintain cellular energy homeostasis ⁶⁹. Interestingly, mitochondrial creatine kinase, has been identified in the brain TSPO interactome network ⁶¹. Additionally, the coordinated actions of AMP deaminase (AMPD), AMP-activated protein kinase (AMPK), and adenylate kinase (AK) contribute to energy regulation. AMPK serves as an energy-charge sensor that modulates AMPD activity. AMPD converts AMP to IMP to maintain a higher energy charge and to facilitate the forward AK reaction, which generates ATP and AMP from two ADP molecules ⁶⁸.

In summary, the reduced mitochondrial and cellular respiration observed in TSPO-deficient cells can be caused by various factors, including diminished ETC activity, restricted substrate or enzyme availability, malfunctioning of ETC complexes, and improper functioning of the TSPO-VDAC interactome. Further investigations are needed to fully understand the mechanisms underlying the regulatory role of TSPO in cellular and mitochondrial respiration.

Mitochondrial membrane potential and Ca and redox homeostasis

A more negative or polarised MMP implies an increase in the coupling efficiency of OXPHOS, whereas a less negative or depolarised MMP suggests a decrease in efficiency or may result from rate limitations in the ETC or substrate oxidation ⁷⁰. Consistent across all analysed cell types in our study, TSPO deficiency resulted in prominent depolarisation of MMP. This is in line with several studies reporting that inhibition of TSPO expression causes depolarisation of the mitochondria. In murine BV-2 microglial cells, RNA interference-mediated TSPO knockdown resulted in lower MMP ^{14, 62}. Similarly, mitochondrial depolarisation has been observed in primary microglia isolated from genetically modified TSPO-knockout mice ^{61, 62}. Moreover, CRISPR/Cas9-mediated knockout of TSPO in human C20 microglia caused a robust reduction in MMP ⁷. A comprehensive study on the impact of TSPO on key mitochondrial functions reported similar results. TSPO-knockout mouse glioma cells also showed lower MMP ⁷¹. In contrast, downregulation of TSPO expression in canine C35 epithelial cells resulted in hyperpolarised mitochondria ¹¹, whereas TSPO deletion in murine MA-10 Leydig cells did not affect the MMP ⁹. However, Fan et al. reported a significantly decreased MMP in TSPO-deficient MA-10 Leydig cells ⁷². The authors attributed the depolarisation of MMP to the lack of regulation of VDAC/tubulin interaction caused by the loss of TSPO expression ⁷².

Consistently, all TSPO-knockout cells showed decreased cytosolic and increased mitochondrial Ca²⁺ levels. This is in line with a previous study that reported an ATP-induced increase in [Ca²⁺]_m and a decrease in [Ca²⁺]_c in TSPO-knockdown cells and reversed effects in TSPO-overexpressing cells from different species, suggesting a conserved mechanism of mitochondrial Ca²⁺ homeostasis. This mechanism involves TSPO, which limits the transport efficiency of Ca²⁺ ions into the mitochondria and controls mitochondrial Ca²⁺ uptake through VDAC ¹¹. In contrast, TSPO loss in human C20 microglia resulted in increased cytosolic Ca²⁺ levels ⁷. However, [Ca²⁺]_m was also increased in microglia devoid of TSPO ⁸.

Alterations in cellular Ca²⁺ signalling cascades, such as altered Ca²⁺ release from intracellular stores, impaired Ca²⁺ sensing and buffering mechanisms, and dysregulation of Ca²⁺-transporting proteins or channels, can affect mitochondrial function and compromise OXPHOS. Although Ca²⁺ is required for ATP synthesis, excess influx of Ca²⁺ into the mitochondrial matrix can lead to increased ROS formation, which eventually induces MMP loss and may trigger mPTP opening, ultimately resulting in apoptosis ⁷³. While low levels of ROS serve crucial signalling functions, excessive ROS accumulation leads to oxidative stress and molecular damage. Flow cytometry analysis of hiPSC-derived NPCs and astrocytes revealed a significant increase in cytosolic ROS and mitochondrial superoxide levels in TSPO-deficient cells. Similarly, TSPO-knockout murine glioma cells and TSPO-knockdown human microglial cells displayed significantly higher cytosolic ROS levels than wild-type cells ^{57, 71}.

Furthermore, Gatliff et al. reported that dysregulation of mitochondrial Ca²⁺ signalling and the subsequent parallel increase in intracellular [Ca²⁺]_c in TSPO-overexpressing cells resulted in elevated mitochondrial ROS levels following the activation of Ca²⁺-dependent NADPH oxidase ¹¹.

Involvement of TSPO in mitochondrial dynamics and mitophagy

In accordance with the observed impaired bioenergetic status, mtDNA copy numbers were significantly reduced in TSPO-knockout cells across all analysed cell types. Consistently, human TSPO^−/− C20 microglial cells contained significantly less mtDNA than control cells ⁸. In contrast, Yao et al. demonstrated that although TSPO knockdown in BV-2 microglia resulted in decreased MMP and OXPHOS, the mtDNA copy number increased in addition to a higher amount of released mtDNA, indicating IMM damage ⁶².

Nuclear-encoded mitochondrial transcription factor A (TFAM) serves as a key activator of both transcription and replication and plays a crucial role in regulating mtDNA copy number ⁷⁴. TSPO-deficient hiPSC-derived astrocytes exhibited significantly lower levels of TFAM transcripts. TFAM knockdown cell models with reduced mtDNA copy numbers showed downregulation of mitochondrial transcription, lower respiratory enzyme activity, and reduced expression of mtDNA-coded complex proteins involved in OXPHOS ⁷⁵. However, Gatliff et al. reported no effect of TSPO on TFAM gene expression ¹⁰.

TSPO-knockout hiPSC-derived astrocytes contained significantly lower MFN1 mRNA levels than CTRL cells, whereas OPA1 and DRP1 transcripts were unaltered. TSPO-deficient mouse glioma cells also showed a marked reduction in MFN1 levels and no change in DRP1 protein expression. However, the expression of mitochondrial fission proteins MFF and FIS1 as well as of the fusion proteins MFN2 and OPA was increased in TSPO-deficient cells ⁷¹. In contrast, Gatliff et al. did not find a difference in the expression pattern of MFN1 and MFN2 in TSPO-knockdown or TSPO-overexpressing cells ¹⁰.

Although only the mitochondrial fusion gene MFN1 was affected in TSPO-knockout astrocytes, mitochondria devoid of TSPO protein were significantly smaller and rounder. TSPO-silenced mitochondria from hepatocellular carcinoma cells were also found to have smaller mitochondria with fewer cristae ⁷⁶. Similarly, TSPO knockout in mouse GL261 glioma cells resulted in mitochondrial fragmentation, whereas wild-type cells contained more fused or elongated mitochondria ⁷¹. In contrast, analysis of mitochondrial morphology in human SH-SY5Y and canine CF35 cells revealed a more elongated and branched mitochondrial network in TSPO-knockdown cells ^{11, 77}.

LC3B-II protein expression, which is tightly associated with the autophagosomal membrane and indicates the degree of autophagic activation, was upregulated in TSPO-deficient NPCs and astrocytes. Gatliff et al. proposed an inhibitory function of TSPO via VDAC1 by limiting parkin-dependent ubiquitination. TSPO, VDAC1, and parkin may collectively serve as molecular platforms for regulating the autophagosome-mediated removal of mitochondria, depending on the homeostatic expression ratio of these proteins ¹⁰. Thus, reduced VDAC expression caused by TSPO deficiency may lead to increased autophagy through ubiquitination and degradation of mitochondrial proteins. It has been observed that cells expressing only low levels of TSPO show upregulation of mitochondrial ubiquitination and increased mitophagy, which is associated with a depolarisation of the MMP ^{10, 11}. Treatment with the mitochondrial uncoupler FCCP, which is commonly used to activate PINK1/parkin-mediated mitophagy upon MMP depolarisation, further increased the mitophagy index. In the same line of evidence, TSPO overexpression reduced mitophagy ⁷⁷.

It has been demonstrated that pro-mitophagy stimuli and MMP depolarisation cause the externalization of the IMM phospholipid cardiolipin to the mitochondrial surface ^{78, 79}. The autophagy protein LC3 contains cardiolipin-binding sites, which are important for the engulfment of mitochondria by the autophagic system ⁷⁸. Thus, the mitochondrial depolarisation observed in the iPSC-derived TSPO-knockout model may trigger cardiolipin redistribution, which acts as an “eat-me” signal for the elimination of damaged mitochondria, further increasing mitophagy.

Therefore, our findings indicate that mitochondrial dysfunction induced by TSPO depletion can trigger the elimination of malfunctioning mitochondria, linking TSPO function to mitochondrial dynamics, mtDNA maintenance, and mitophagy.

TSPO-knockout NPCs, astrocytes, and neurons not only displayed impaired mitochondrial function but also exhibited a significant reduction in cellular size, which was also recently reported for human TSPO-deficient and -silenced C20 microglial cells ^{8, 57}. Typically, cellular organelles and protein content scale isometrically with cell size. However, mitochondrial content and functional scaling are uncoupled. Thus, mitochondrial activity, i.e., MMP and OXPHOS, is highest in cells of intermediate size. This nonlinearity of mitochondrial functionality results in an optimal cell size to maximise cellular fitness and proliferation capacity ⁸⁰. It has been shown that maintaining optimal cell size and scaling of mitochondrial functions depends on the normal morphology and dynamics of the mitochondria. Moreover, the mevalonate pathway, which synthesizes plasma membrane components including cholesterol, affects mitochondrial dynamics and functionality scaling ⁸⁰. According to a study by Mourier et al., mitofusin knockout leads to decreased protein and metabolite levels in the mevalonate pathway ⁸¹. Given the reduced MFN1 gene expression and apparent increased mitochondrial fragmentation observed in TSPO^−/− cells, TSPO-deficient cells appear to lose the ability to maintain their optimal cell size. However, the involvement of impaired mevalonate metabolism requires further investigation.

Possible Role for TSPO in mitochondrial dysfunction in depression

Mitochondrial function has emerged as an important factor in the pathophysiology of major depressive disorder (MDD). Reduced bioenergetic capability, oxidative stress, and impaired mitochondrial function render cells vulnerable to stress and may contribute to the development of MDD and other psychiatric disorders ^{46, 82, 83}. Malfunctioning mitochondria-related effects are not limited to neuronal cells, but have been reported in peripheral non-neuronal cells, such as fibroblasts^{28, 47}, muscle ⁸⁴, PBMCs ⁴⁵, and platelets ^{85, 86} of depressed patients, highlighting the systemic nature of mitochondrial dysfunction in MDD.

TSPO has already been linked to anxiety and depression disorders, making it a potential diagnostic and therapeutic target. Moreover, TSPO expression is lower in MDD patients receiving antidepressant medication than in unmedicated patients ⁸⁷, and TSPO binding is greater in patients with chronologically advanced MDD and a long duration of untreated depression ⁸⁸.

Our data demonstrate a significant decrease in the expression levels of both TSPO and the TSPO-interacting protein VDAC1 in fibroblasts obtained from depressed patients. Given the fact that TSPO deficiency in hiPSC-derived neural cells was accompanied by reduced VDAC1 protein levels and led to severe mitochondrial dysfunction, altered TSPO and VDAC expression may affect mitochondrial function, neurotransmitter signalling, and neurosteroidogenesis, thereby contributing to the aetiology of MDD pathogenesis. Remarkably, the impact of TSPO deficiency on mitochondrial bioenergetics closely resembles parameters of mitochondrial function observed in a human cell model of depression ^{26, 27, 28}.

The fact that TSPO loss impaired cellular energy metabolism, as evidenced by altered Ca²⁺ homeostasis, reduced OXPHOS, and depolarisation of MMP, is in favour of the hypothesis that TSPO might be a promising treatment target for psychiatric disorders. This is also supported by the role of TSPO in the regulation of neurosteroid synthesis. Neurosteroids are important modulators of the GABAergic and glutamatergic systems which are important targets in the treatment of anxiety and stress-related disorders ^{15, 20, 89}. However, correlation does not imply causation and a deeper understanding of the mechanisms behind mitochondrial function and the disruption of mitochondrial bioenergetics in MDD in relation to TSPO expression and function is needed.

The comprehensive functional investigation of our human neuronal cell model revealed severe mitochondrial dysfunction in TSPO-deficient cells, which encompassed various mutually dependent aspects of mitochondrial biology. The reduced pregnenolone synthesis observed in TSPO-knockout astrocytes confirms TSPO’s contribution to the production of neurosteroids, such as progesterone and allopregnanolone, which are important modulators of neuronal activity and neurotransmission, necessary for normal brain function. The impaired cellular and mitochondrial bioenergetics in TSPO-deficient cells indicate the importance of TSPO in sustaining the energy-producing capacity of mitochondria. Reduced OXPHOS and glycolysis suggest disruptions in the metabolic processes necessary for cellular energy supply. The altered mitochondrial morphology and dynamics observed in TSPO^−/− astrocytes suggest the involvement of TSPO in regulating the dynamic structural integrity and functional organisation of mitochondria, which is important for preserving mitochondrial health and functionality. Moreover, increased mitophagy and a subsequent decrease in mtDNA copy number in TSPO-KO cells indicate a disturbance in the normal turnover and maintenance of mitochondria. This suggests that TSPO may be involved in modulating the removal of damaged mitochondria, thereby contributing to mitochondrial quality control. Overall, these findings highlight the intricate effects of TSPO on the maintenance of mitochondrial integrity and function.

The precise molecular mechanisms underlying the effects of TSPO, and the context-dependent nature of its action remain widely elusive. However, the interaction of TSPO with other mitochondrial and cellular proteins, forming complex molecular networks that regulate various processes, appears to be crucial for its function. The composition of these functional complexes remains a subject of ongoing research. VDAC is the major pore in the OMM and plays a crucial role in mitochondrial homeostasis itself. Reduced VDAC expression in TSPO-knockout cells suggests a potential interaction between TSPO and VDAC. This positive correlation may contribute to the mechanism of action of TSPO in mitochondrial physiology.

The observed decrease in TSPO and VDAC protein expression in a human cell model of depression raises intriguing possibilities regarding the role of TSPO in the pathophysiology of stress-related disorders and depression. Further research on the function of TSPO and its physiologically relevant interaction partners will contribute to a deeper understanding of its role in maintaining cellular homeostasis. Moreover, these investigations may uncover new therapeutic targets for mitochondria-related disorders.

All participants gave written informed consent, and all study procedures were approved by the ethics committee of the University of Regensburg (ref: 13-101-0271).

Conflicts of Interest:

The authors declare no conflict of interest.

Funding

The work has been supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under project number 422182557 to C.H.W. and S.B. within the framework of the FOR2858.

Author Contributions

SB and CHW, conceived and designed the experiments. SB, TJ, AD, DM, VMM and CHW performed the experiments and/or contributed to data acquisition. SB, VMM and CHW analyzed and interpreted the data. SB and CHW wrote the original manuscript. CHW and VMM critically revised and edited the

Acknowledgements

The authors would like to thank Richard Warth for providing access to the seahorse device. We are grateful to Marianne Federlin and Matthias Widbiller for their expertise and support with the scanning electron microscopy of induced neurons.

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A comprehensive functional investigation of the human translocator protein 18 kDa (TSPO) in a novel human neuronal cell knockout model: from molecule to depression

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Abstract

Figures

Introduction

Materials and Methods

Fibroblasts and human induced pluripotent stem cells

Generation of TSPO knockout in iPSC using CRISPR/Cas9 genome editing

Differentiation of iPSCs into neural progenitor cells (NPCs), neurons and astrocytes

Western blotting

Immunofluorescence

Fluorescent live-cell imaging

Quantitative real-time PCR and mtDNA copy number analysis

Mitochondrial respirometry

Total ATP content quantification

Scanning Electron Microscopy (SEM)

Pregnenolone quantification

Flow cytometry analyses

Cytosolic and mitochondrial reactive oxygen species/oxidative stress

Mitochondrial mass

Statistical analysis

Results

Validation of TSPO knockout in hiPSCs and off-target screening

Differentiation of hiPSC-derived NPCs into astrocytes

Differentiation of hiPSC-derived NPCs into neurons

Validation of TSPO knockout in NPCs, astrocytes, and neurons

Effect of TSPO expression on steroid synthesis in hiPSC-derived astrocytes

Impact of TSPO expression on mitochondrial respiration

Role of TSPO in the modulation of the mitochondrial membrane potential

Involvement of TSPO in Ca2+ Homeostasis

Impact of TSPO on cellular bioenergetics and glycolysis

TSPO-deficient Neural Progenitors and astrocytes show oxidative stress

Effect of TSPO loss on mtDNA copy number, mitochondrial content and cell size

Impact of TSPO on mitochondrial dynamics and morphology

TSPO expression in Major Depressive Disorder

Discussion

Role of TSPO in neurosteroidogenesis

Impact of TSPO on mitochondrial and cellular respiration

Mitochondrial membrane potential and Ca and redox homeostasis

Involvement of TSPO in mitochondrial dynamics and mitophagy

Possible Role for TSPO in mitochondrial dysfunction in depression

Summary & Conclusion

Declarations

Conflicts of Interest:

Funding

Author Contributions

Acknowledgements

References

Additional Declarations

Supplementary Files

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Involvement of TSPO in Ca²⁺ Homeostasis