Human fibroblasts from sporadic Alzheimer's disease (AD) patients show mitochondrial alterations and lysosome dysfunction.

Mitophagy is a mechanism that maintains mitochondrial integrity and homeostasis and is thought to promote longevity and reduce the risk of age-related neurodegenerative diseases, including Alzheimer's disease (AD). Here, we investigate the abundance of mitochondrial reactive oxygen species (ROS), mitochondrial function, and mitophagy in primary fibroblasts from patients with sporadic AD (sAD) and normal healthy controls. The results show increased levels of mitochondrial ROS, changes in mitochondrial morphology, altered bioenergetic properties, and defects in autophagy, mitophagy, and lysosome-mediated degradation pathways in sAD fibroblasts relative to control fibroblasts. Interestingly, lysosome abundance and the staining of lysosomal markers remained high, while the capacity of lysosome-dependent degradation was lower in sAD fibroblasts than in controls fibroblasts. Nicotinamide riboside supplementation decreased mitochondrial ROS, while capacity for lysosomal degradation remained unchanged in sAD fibroblasts relative to healthy control fibroblasts. These findings provide insight into molecular mechanisms involving the dysregulation of lysosome and autophagy/mitophagy pathways that may contribute significantly to clinical signs and pathological features of sAD.

Glycolysis inhibition affects proliferation and cytotoxicity of Vγ9Vδ2 T cells expanded for adoptive cell therapy.

BACKGROUND AIMS: Vγ9Vδ2 T cells are under investigation as alternative effector cells for adoptive cell therapy (ACT) in cancer. Despite promising in vitro results, anti-tumor efficacies in early clinical studies have been lower than expected, which could be ascribed to the complex interplay of tumor and immune cell metabolism competing for the same nutrients in the tumor microenvironment. METHODS: To contribute to the scarce knowledge regarding gamma delta T-cell metabolism, we investigated the metabolic phenotype of 25-day-expanded Vγ9Vδ2 T cells and how it is intertwined with functionality. RESULTS: We found that Vγ9Vδ2 T cells displayed a quiescent metabolism, utilizing both glycolysis and oxidative phosphorylation (OXPHOS) for energy production, as measured in Seahorse assays. Upon T-cell receptor activation, both pathways were upregulated, and inhibition with metabolic inhibitors showed that Vγ9Vδ2 T cells were dependent on glycolysis and the pentose phosphate pathway for proliferation. The dependency on glucose for proliferation was confirmed in glucose-free conditions. Cytotoxicity against malignant melanoma was reduced by glycolysis inhibition but not OXPHOS inhibition. CONCLUSIONS: These findings lay the groundwork for further studies on manipulation of Vγ9Vδ2 T-cell metabolism for improved ACT outcome.

Nucleotide metabolism in the regulation of tumor microenvironment and immune cell function.

Nucleotide metabolism plays a crucial role in the regulation of the tumor microenvironment (TME) and immune cell function. In the TME, limited availability of nucleotide precursors due to increased consumption by tumor cells and T cells affects both tumor development and immune function. Metabolic reprogramming in tumor cells favors pathways supporting growth and proliferation, including nucleotide synthesis. Additionally, extracellular nucleotides, such as ATP and adenosine, exhibit dual roles in modulating immune function and tumor cell survival. ATP stimulates antitumor immunity by activating purinergic receptors, while adenosine acts as a potent immunosuppressor. Targeting nucleotide metabolism in the TME holds immense promise for cancer therapy. Understanding the intricate relationship between nucleotide metabolism, the TME, and immune responses will pave the way for innovative therapeutic interventions.

Rev1 deficiency induces a metabolic shift in MEFs that can be manipulated by the NAD+ precursor nicotinamide riboside.

Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1-/- mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1-/- MEFs can be partially reversed by NR supplementation, which also protects the mitochondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs.

Involvement of Mitochondrial Dysfunction in FOXG1 Syndrome.

FOXG1 (Forkhead box g1) syndrome is a neurodevelopmental disorder caused by a defective transcription factor, FOXG1, important for normal brain development and function. As FOXG1 syndrome and mitochondrial disorders have shared symptoms and FOXG1 regulates mitochondrial function, we investigated whether defective FOXG1 leads to mitochondrial dysfunction in five individuals with FOXG1 variants compared to controls (n = 6). We observed a significant decrease in mitochondrial content and adenosine triphosphate (ATP) levels and morphological changes in mitochondrial network in the fibroblasts of affected individuals, indicating involvement of mitochondrial dysfunction in FOXG1 syndrome pathogenesis. Further investigations are warranted to elucidate how FOXG1 deficiency impairs mitochondrial homeostasis.

Partial inhibition of mitochondrial-linked pyrimidine synthesis increases tumorigenic potential and lysosome accumulation.

The correlation between mitochondrial function and oncogenesis is complex and is not fully understood. Here we determine the importance of mitochondrial-linked pyrimidine synthesis for the aggressiveness of cancer cells. The enzyme dihydroorotate dehydrogenase (DHODH) links oxidative phosphorylation to de novo synthesis of pyrimidines. We demonstrate that an inhibition of DHODH results in a respiration-independent significant increase of anchorage-independent growth but does not affect DNA repair ability. Instead, we show an autophagy-independent increase of lysosomes. The results of this study suggest that inhibition of mitochondrial-linked pyrimidine synthesis in cancer cells results in a more aggressive tumor phenotype.

Mitochondrial-Linked De Novo Pyrimidine Biosynthesis Dictates Human T-Cell Proliferation but Not Expression of Effector Molecules.

T-cell activation upon antigen stimulation is essential for the continuation of the adaptive immune response. Impairment of mitochondrial oxidative phosphorylation is a well-known disruptor of T-cell activation. Dihydroorotate dehydrogenase (DHODH) is a component of the de novo synthesis of pyrimidines, the activity of which depends on functional oxidative phosphorylation. Under circumstances of an inhibited oxidative phosphorylation, DHODH becomes rate-limiting. Inhibition of DHODH is known to block clonal expansion and expression of effector molecules of activated T cells. However, this effect has been suggested to be caused by downstream impairment of oxidative phosphorylation rather than a lower rate of pyrimidine synthesis. In this study, we successfully inhibit the DHODH of T cells with no residual effect on oxidative phosphorylation and demonstrate a dose-dependent inhibition of proliferation of activated CD3+ T cells. This block is fully rescued when uridine is supplemented. Inhibition of DHODH does not alter expression of effector molecules but results in decreased intracellular levels of deoxypyrimidines without decreasing cell viability. Our results clearly demonstrate the DHODH and mitochondrial linked pyrimidine synthesis as an independent and important cytostatic regulator of activated T cells.

Real-time Monitoring of Mitochondrial Respiration in Cytokine-differentiated Human Primary T Cells.

During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is indispensable for T cell activation, and the survival of memory T cells is dependent on mitochondrial remodeling. Consequently, this affects the long-term clinical outcome of cancer immunotherapies. Changes in T cell quality are often studied by flow cytometry using well-known surface markers and not directly by their metabolic state. This is an optimized protocol for measuring real-time mitochondrial respiration of primary human T cells using an Extracellular Flux Analyzer and the cytokines IL-2 and IL-15, which differently affect T cell metabolism. It is shown that the metabolic state of T cells can clearly be distinguished by measuring the oxygen consumption when inhibiting key complexes in the metabolic pathway and that the accuracy of these measurements is highly dependent on optimal inhibitor concentration and inhibitor injection strategy. This standardized protocol will help implement mitochondrial respiration as a standard for T cell fitness in monitoring and studying cancer immunotherapies.

Atorvastatin impairs liver mitochondrial function in obese Göttingen Minipigs but heart and skeletal muscle are not affected.

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

Bloom syndrome DNA helicase deficiency is associated with oxidative stress and mitochondrial network changes.

Bloom Syndrome (BS; OMIM #210900; ORPHA #125) is a rare genetic disorder that is associated with growth deficits, compromised immune system, insulin resistance, genome instability and extraordinary predisposition to cancer. Most efforts thus far have focused on understanding the role of the Bloom syndrome DNA helicase BLM as a recombination factor in maintaining genome stability and suppressing cancer. Here, we observed increased levels of reactive oxygen species (ROS) and DNA base damage in BLM-deficient cells, as well as oxidative-stress-dependent reduction in DNA replication speed. BLM-deficient cells exhibited increased mitochondrial mass, upregulation of mitochondrial transcription factor A (TFAM), higher ATP levels and increased respiratory reserve capacity. Cyclin B1, which acts in complex with cyclin-dependent kinase CDK1 to regulate mitotic entry and associated mitochondrial fission by phosphorylating mitochondrial fission protein Drp1, fails to be fully degraded in BLM-deficient cells and shows unscheduled expression in G1 phase cells. This failure to degrade cyclin B1 is accompanied by increased levels and persistent activation of Drp1 throughout mitosis and into G1 phase as well as mitochondrial fragmentation. This study identifies mitochondria-associated abnormalities in Bloom syndrome patient-derived and BLM-knockout cells and we discuss how these abnormalities may contribute to Bloom syndrome.

Simvastatin improves mitochondrial respiration in peripheral blood cells.

Statins are prescribed to treat hypercholesterolemia and to reduce the risk of cardiovascular disease. However, statin users frequently report myalgia, which can discourage physical activity or cause patients to discontinue statin use, negating the potential benefit of the treatment. Although a proposed mechanism responsible for Statin-Associated Myopathy (SAM) suggests a correlation with impairment of mitochondrial function, the relationship is still poorly understood. Here, we provide evidence that long-term treatment of hypercholesterolemic patients with Simvastatin at a therapeutic dose significantly display increased mitochondrial respiration in peripheral blood mononuclear cells (PBMCs), and platelets compared to untreated controls. Furthermore, the amount of superoxide is higher in mitochondria in PBMCs, and platelets from Simvastatin-treated patients than in untreated controls, and the abundance of mitochondrial superoxide, but not mitochondrial respiration trends with patient-reported myalgia. Ubiquinone (also known as coenzyme Q10) has been suggested as a potential treatment for SAM; however, an 8-week course of oral ubiquinone had no impact on mitochondrial functions or the abundance of superoxide in mitochondria from PBMCs, and platelets. These results demonstrate that long-term treatment with Simvastatin increases respiration and the production of superoxide in mitochondria of PBMCs and platelets.

Spatial Transcriptomics Reveals Genes Associated with Dysregulated Mitochondrial Functions and Stress Signaling in Alzheimer Disease.

Alzheimer disease (AD) is a devastating neurological disease associated with progressive loss of mental skills and cognitive and physical functions whose etiology is not completely understood. Here, our goal was to simultaneously uncover novel and known molecular targets in the structured layers of the hippocampus and olfactory bulbs that may contribute to early hippocampal synaptic deficits and olfactory dysfunction in AD mice. Spatially resolved transcriptomics was used to identify high-confidence genes that were differentially regulated in AD mice relative to controls. A diverse set of genes that modulate stress responses and transcription were predominant in both hippocampi and olfactory bulbs. Notably, we identify Bok, implicated in mitochondrial physiology and cell death, as a spatially downregulated gene in the hippocampus of mouse and human AD brains. In summary, we provide a rich resource of spatially differentially expressed genes, which may contribute to understanding AD pathology.

Cytoplasmic Citrate Flux Modulates the Immune Stimulatory NKG2D Ligand MICA in Cancer Cells.

Immune surveillance of cancer cells is facilitated by the Natural Killer Group 2D (NKG2D) receptor expressed by different lymphocyte subsets. It recognizes NKG2D ligands that are rarely expressed on healthy cells, but upregulated by tumorigenesis, presenting a target for immunological clearance. The molecular mechanisms responsible for NKG2D ligand regulation remain complex. Here we report that cancer cell metabolism supports constitutive surface expression of the NKG2D ligand MHC class I chain-related proteins A (MICA). Knockout of the N-glycosylation gene N-acetylglucosaminyltransferase V (MGAT5) in HEK293 cells induced altered metabolism and continuous high MICA surface expression. MGAT5 knockout cells were used to examine the association of cell metabolism and MICA expression through genetic, pharmacological and metabolic assays. Findings were verified in cancer cell lines. Cells with constitutive high MICA expression showed enhanced spare respiratory capacity and elevated mitochondrial efflux of citrate, determined by extracellular flux analysis and metabolomics. MICA expression was reduced by inhibitors of mitochondrial function, FCCP and etomoxir e.g., and depended on conversion of citrate to acetyl-CoA and oxaloacetate by ATP citrate lyase, which was also observed in several cancer cell types. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis revealed that upregulated MICA transcription was associated with an open chromatin structure at the MICA transcription start site. We identify mitochondria and cytoplasmic citrate as key regulators of constitutive MICA expression and we propose that metabolic reprogramming of certain cancer cells facilitates MICA expression and NKG2D-mediated immune recognition.

Mitochondrial Function in Gilles de la Tourette Syndrome Patients With and Without Intragenic IMMP2L Deletions.

Background: Gilles de la Tourette syndrome (GTS) is a neurodevelopmental condition characterized by motor and vocal tics. The underlying etiology remains largely unknown, and GTS is considered as a complex multifactorial disorder associated with effects of several genes in combination with environmental factors. The inner mitochondrial membrane peptidase, subunit 2 (IMMP2L) has been suggested as one of the susceptibility genes for GTS, and IMMP2L-deficient mouse and human cells show increased levels of mitochondrial oxidative stress and altered cell fate programming. Hence, a potential involvement of IMMP2L-induced mitochondrial dysfunction in GTS pathology is yet to be elucidated. To address this, we investigated mitochondrial function in a group of GTS patients with intragenic IMMP2L deletions and compared with GTS without IMMP2L deletions and healthy controls. Methods: Mitochondrial function in fibroblasts from GTS patients and non-GTS parents (with and without IMMP2L deletions) compared to healthy controls were evaluated by measuring mitochondrial superoxide production, mitochondrial membrane potential, mitochondrial mass, and mitochondrial respiration. In addition, we evaluated apoptosis and senescence. Results: None of the mitochondrial parameters assessed in this study were significantly distinctive when comparing GTS patients with and without IMMP2L deletions against healthy controls or parents with or without IMMP2L deletions, and we did not observe altered cell programming. Conclusion: This study suggests that IMMP2L deletions do not lead to a substantial general mitochondrial dysfunction in GTS fibroblasts. Assessing a large cohort of controls and patients of similar age and gender would possibly reveal small differences in mitochondrial function. However, it is possible that IMMP2L variants affect mitochondrial function during specific instances of stress stimuli or in brain regions suggested to be affected in GTS.

MERTK Acts as a Costimulatory Receptor on Human CD8+ T Cells.

The TAM family of receptor tyrosine kinases (TYRO3, AXL, and MERTK) is known to be expressed on antigen-presenting cells and function as oncogenic drivers and as inhibitors of inflammatory responses. Both human and mouse CD8+ T cells are thought to be negative for TAM receptor expression. In this study, we show that T-cell receptor (TCR)-activated human primary CD8+ T cells expressed MERTK and the ligand PROS1 from day 2 postactivation. PROS1-mediated MERTK signaling served as a late costimulatory signal, increasing proliferation and secretion of effector and memory-associated cytokines. Knockdown and inhibition studies confirmed that this costimulatory effect was mediated through MERTK. Transcriptomic and metabolic analyses of PROS1-blocked CD8+ T cells demonstrated a role of the PROS1-MERTK axis in differentiation of memory CD8+ T cells. Finally, using tumor-infiltrating lymphocytes (TIL) from melanoma patients, we show that MERTK signaling on T cells improved TIL expansion and TIL-mediated autologous cancer cell killing. We conclude that MERTK serves as a late costimulatory signal for CD8+ T cells. Identification of this costimulatory function of MERTK on human CD8+ T cells suggests caution in the development of MERTK inhibitors for hematologic or solid cancer treatment.

Mitochondrial oxidative phosphorylation capacity of cryopreserved cells.

Defects in mitochondrial oxidative phosphorylation are a feature of many human diseases. To date, determination of oxidative phosphorylation has required fresh and live sample material and therefore also access to specialized equipment and trained personnel. Cryopreservation of samples is an attractive alternative, where samples can be collected and stored in an economic and practical fashion for later bulk assays. Here, we present an accurate, reliable method for estimating mitochondrial oxidative phosphorylation capacity of cryopreserved human cells. Broad adoption of this method will allow uncomplicated collection of samples and measurements of oxidative phosphorylation.

From Powerhouse to Perpetrator-Mitochondria in Health and Disease.

In this review we discuss the interaction between metabolic stress, mitochondrial dysfunction, and genomic instability. Unrepaired DNA damage in the nucleus resulting from excess accumulation of DNA damages and stalled replication can initiate cellular signaling responses that negatively affect metabolism and mitochondrial function. On the other hand, mitochondrial pathologies can also lead to stress in the nucleus, and cause sensitivity to DNA-damaging agents. These are examples of how hallmarks of cancer and aging are connected and influenced by each other to protect humans from disease.

The inhibitors of soluble adenylate cyclase 2-OHE, KH7, and bithionol compromise mitochondrial ATP production by distinct mechanisms.

Soluble adenylate cyclase (sAC) is a non-plasma membrane-bound isoform of the adenylate cyclases signaling via the canonical second messenger, 3',5'-cyclic AMP (cAMP). sAC is involved in key physiological processes such as insulin release, sperm motility, and energy metabolism. Thus, sAC has attracted interest as a putative drug target and attempts have been made to develop selective inhibitors. Since sAC has a binding constant for its substrate, ATP, in the millimolar range, reductions in mitochondrial ATP production may be part of the mechanism-of-action of sAC inhibitors and the potential of these compounds to study the physiological outcomes of inhibition of sAC might be severely hampered by this. Here, we evaluate the effects of two commonly employed inhibitors, 2-OHE and KH7, on mitochondrial ATP production and energy metabolism. For comparison, we included a recently identified inhibitor of sAC, bithionol. Employing mitochondria isolated from mouse brain, we show that all three compounds are able to curb ATP production albeit via distinct mechanisms. Bithionol and KH7 mainly inhibit ATP production by working as a classical uncoupler whereas 2-OHE mainly works by decreasing mitochondrial respiration. These findings were corroborated by investigating energy metabolism in acute brain slices from mice. Since all three sAC inhibitors are shown to curb mitochondrial ATP production and affect energy metabolism, caution should be exercised when employed to study the physiological roles of sAC or for validating sAC as a drug target.

Author Correction: Rev1 contributes to proper mitochondrial function via the PARP-NAD+-SIRT1-PGC1α axis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

The Role of Mitochondrial Dysfunction in the Progression of Alzheimer's Disease.

The current molecular understanding of Alzheimer's disease (AD) has still not resulted in successful interventions. Mitochondrial dysfunction of the AD brain is currently emerging as a hallmark of this disease. One mitochondrial function often affected in AD is oxidative phosphorylation responsible for ATP production, but also for production of reactive oxygen species (ROS) and for the de novo synthesis of pyrimidines. This paper reviews the role of mitochondrial produced ROS and pyrimidines in the aetiology of AD and their proposed role in oxidative degeneration of macromolecules, synthesis of essential phospholipids and maintenance of mitochondrial viability in the AD brain.