Over‐expression of Nicotinamide phosphoribosyltransferase in mouse cells confers protective effect against oxidative and ER stress‐induced premature senescence

A main feature of aged organisms is the accumulation of senescent cells. Accumulated senescent cells, especially stress‐induced premature senescent cells, in aged organisms lead to the decline of the regenerative potential and function of tissues. We recently reported that the over‐expression of NAMPT, which is the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, delays replicative senescence in vitro. However, whether Nampt‐overexpressing cells are tolerant of stress‐induced premature senescence remains unknown. Here, we show that primary mouse embryonic fibroblasts derived from Nampt‐overexpressing transgenic mice (Nampt Tg‐MEF cells) possess resistance against stress‐induced premature senescence in vitro. We found that higher oxidative or endoplasmic reticulum (ER) stress is required to induce premature senescence in Nampt Tg‐MEF cells compared to wild‐type cells. Moreover, we found that Nampt Tg‐MEF cells show acute expression of unfolded protein response (UPR)‐related genes, which in turn would have helped to restore proteostasis and avoid cellular senescence. Our results demonstrate that NAMPT/NAD⁺ axis functions to protect cells not only from replicative senescence, but also from stress‐induced premature senescence in vitro. We anticipate that in vivo activation of NAMPT activity or increment of NAD⁺ would protect tissues from the accumulation of premature senescent cells, thereby maintaining healthy aging.     

Nicotinamide phosphoribosyltransferase delays cellular senescence by upregulating SIRT1 activity and antioxidant gene expression in mouse cells

Senescent cells accumulate in tissues of aged animals and deteriorate tissue functions. The elimination of senescent cells from aged mice not only attenuates progression of already established age‐related disorders, but also extends median lifespan. Nicotinamide phosphoribosyltransferase (NAMPT), the rate‐limiting enzyme in mammalian NAD⁺ salvage pathway, has shown a protective effect on cellular senescence of human primary cells. However, it still remains unclear how NAMPT has a protective impact on aging in vitro and in vivo. In this study, we found that primary mouse embryonic fibroblast (MEF) cells undergo progressive decline of NAMPT and NAD⁺ contents during serial passaging before becoming senescent. Furthermore, we showed that constitutive Nampt over‐expression increases cellular NAD⁺ content and delays cellular senescence of MEF cells in vitro. We further found that constitutive Nampt over‐expression increases SIRT1 activity, increases the expression of antioxidant genes, superoxide dismutase 2 and catalase and promotes resistance against oxidative stress. These findings suggest that Nampt over‐expression in MEF cells delays cellular senescence by the mitigation of oxidative stress via the upregulation of superoxide dismutase 2 and catalase gene expressions by SIRT1 activation.     

Expression of a single ICAM‐1 isoform on T cells is sufficient for development of experimental autoimmune encephalomyelitis

Intercellular adhesion molecule‐1 (ICAM‐1) plays an important role in leukocyte trafficking, induction of cellular immune responses, and immunological synapse formation. As a member of the immunoglobulin superfamily of adhesion proteins, ICAM‐1 is composed of repeating Ig‐like domains, a transmembrane domain, and short cytoplasmic tail that participates in intracellular signaling events. At least seven ICAM‐1 protein isoforms are generated by alternative splicing, however little is known regarding their immunobiology. We have previously shown using different lines of ICAM‐1 mutant mice (Icam1^tm1Jcgr and Icam1^tm1Bay) that expression of alternatively spliced ICAM‐1 isoforms can significantly influence the disease course during the development of EAE. In this study, we show using a newly developed transgenic mouse (CD2‐Icam1^D4del/Icam1^null) that T‐cell‐specific expression of a single ICAM‐1 isoform composed of Ig domains 1, 2, 3, and 5 can mediate the initiation and progression of EAE. Our results indicate that the ICAM‐1 isoform lacking Ig domain 4 can drive pathogenesis in demyelinating disease and may be a novel therapeutic target for treating multiple sclerosis.     

Quantification of α‐subunit isoforms of Na,K‐ATPase in rat resistance vessels

Aim: Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual α‐subunit isoforms of Na,K‐ATPase. Methods: Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K‐ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na⁺‐dependent ³²P‐phosphorylation of Na,K‐ATPase isolated from rat kidney was used for α₁ calibration and that for high‐affinity (³H)ouabain binding of Na,K‐ATPase isolated from rat brain was used for (α₂ + α₃) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform‐specific antibodies and radiolabelled secondary antibodies. The signals from adjacent α spots were used for qualitative and quantitative characterization of rat vessels. Results: A concentration of 100.7 ± 14.4 pmol (n = 11) per g wet weight of the α₁‐isoform containing Na,K‐ATPase was found in RV from 12–14‐week rats. A much lower and more unreliable content of α₂‐ and α₃‐isoforms was found. These ouabain‐sensitive isoforms seem to represent a maximum of 5–10% each compared with the ouabain‐insensitive rat α₁‐isoform. Conclusions: The isoform pattern in RV, in which the isoform with high/intermediate Na⁺‐affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain‐insensitive α₁‐isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain‐like factors in hypertension.     

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Prehensile tails are defined as having the ability to grasp objects and are commonly used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, as all opossums have a prehensile tail and tail use varies between arboreal and terrestrial forms. Expanding on our previous work in the Virginia opossum, this study tests the hypothesis that arboreal and terrestrial opossums differentially express faster versus slower MHC isoforms, respectively. MHC isoform expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of Caluromys derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immunohistochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) that are distributed (mean percentage) as 8.2% MHC‐1, 2.6% 1/2A, and 89.2% 2A/X hybrid fibers. M. domestica also expresses MHC‐1, 2A, and 2X, in addition to the 2B isoform, distributed as 17.0% MHC‐1, 1.3% 1/2A, 9.0% 2A, 75.2% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar isoform fiber types differed significantly between species (P < 0.001). Although not statistically significant, C. derbianus was observed to have larger cross‐sectional area (CSA) for each corresponding fiber type along with a greater amount of extra‐cellular matrix. An overall faster fiber type composition (and larger fibers) in the tail of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers in the caudal musculature is well suited for tail nest building/remodeling behaviors of terrestrial opossums. Anat Rec, 297:1364–1376, 2014. © 2014 Wiley Periodicals, Inc.     

Functional Analysis of Dynamin Isoforms in Drosophila Melanogaster

Dynamin and dynamin-like proteins are required for endocytosis. synaptic vesicle recycling and membrane trafficking. From the shibire locus in Drosophila melanogaster, six different isoforms of dynamin are generated by alternative splicing. However, the roles of the individual isoforms in cellular processes are unknown. To investigate functional differences among the dynamin isoforms, transgenic lines were generated that individually expressed each of 3 different isoforms under UAS^GAL4 control. The expression of the isoforms was controlled by neural promoter (elav)-driven GAL4, or by a shibire-promoter driven GAL4 transgene. Reporter gene expression indicated that the shi promoter is active during embryogenesis, and in larvae, pupae, and adults in a pattern consistent with normal dynamin expression. To assay for the ability of dynamin isoforms to function in vivo, the isoforms expressed via these GAL4 drivers were tested for the ability to rescue shibire phenotypes. When expressed at very high levels all individual isoforms tested rescued the temperature-sensitive paralytic phenotype of shl^ls2 flies; however, this rescue was partial, suggesting that no single tested isoform is sufficient for synaptic vesicle recycling in vivo. When tested for ability to rescue lethality induced by heat-pulsing larvae during development, shi- promoter driven expression of individual isoforms conferred significant resistance to heat treatment during larval development. However, all 3 isoforms were unable to rescue the lethality of shi^12-12B mutants which are severely hypomorphic (or null) for shibire function. Taken together. these observations suggest that individual shibire isoforms have specific molecular activities in vivo.     

Proinflammatory cytokine interferon‐γ and microbiome‐derived metabolites dictate epigenetic switch between forkhead box protein 3 isoforms in coeliac disease

Coeliac disease (CD) is an autoimmune enteropathy triggered by gluten and characterized by a strong T helper type 1 (Th1)/Th17 immune response in the small intestine. Regulatory T cells (T_reg) are CD4⁺CD25⁺⁺forkhead box protein 3 (FoxP3⁺) cells that regulate the immune response. Conversely to its counterpart, FoxP3 full length (FL), the alternatively spliced isoform FoxP3 Δ2, cannot properly down‐regulate the Th17‐driven immune response. As the active state of CD has been associated with impairments in T_reg cell function, we aimed at determining whether imbalances between FoxP3 isoforms may be associated with the disease. Intestinal biopsies from patients with active CD showed increased expression of FOXP3 Δ2 isoform over FL, while both isoforms were expressed similarly in non‐coeliac control subjects (HC). Conversely to what we saw in the intestine, peripheral blood mononuclear cells (PBMC) from HC subjects did not show the same balance between isoforms. We therefore hypothesized that the intestinal microenvironment may play a role in modulating alternative splicing. The proinflammatory intestinal microenvironment of active patients has been reported to be enriched in butyrate‐producing bacteria, while high concentrations of lactate have been shown to characterize the preclinical stage of the disease. We show that the combination of interferon (IFN)‐γ and butyrate triggers the balance between FoxP3 isoforms in HC subjects, while the same does not occur in CD patients. Furthermore, we report that lactate increases both isoforms in CD patients. Collectively, these findings highlight the importance of the ratio between FoxP3 isoforms in CD and, for the first time, associate the alternative splicing process mechanistically with microbial‐derived metabolites.     

Effect of cyclopiazonic acid,an inhibitor of the sarcoplasmic reticulum Ca‐ATPase,on skeletal muscles from normal and mdx mice

Aim: In this study, we investigated Ca²⁺ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. Method: Experiments were conducted on fast‐ (extensor digitorum longus, EDL) and slow‐ (soleus) twitch muscles expressing different isoforms of Ca²⁺‐ATPase, which is responsible for the uptake of Ca²⁺ by the sarcoplasmic reticulum. Results: In sarcoplasmic reticulum vesicles, the ATP‐dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca²⁺‐ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically‐skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca²⁺ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4‐week‐old mdx mice, the expression of the slow Ca²⁺‐pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca²⁺‐ATPase in mdx soleus muscles at 4‐ and 11‐weeks of development did not differ from those in age‐matched controls. Conclusion: These findings show that in dystrophic muscle, where the Ca²⁺ homeostasis was perturbed, the Ca²⁺ handling by the sarcoplasmic reticulum was altered in fast‐twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca²⁺ uptake could then contribute to an elevated concentration of Ca²⁺ in the cytosol, and also to Ca²⁺ depletion of the sarcoplasmic reticulum.     

Detection of cerebral NAD+ by in vivo 1H NMR spectroscopy

Nicotinamide adenine dinucleotide (NAD⁺) plays a central role in cellular metabolism both as a coenzyme for electron‐transfer enzymes as well as a substrate for a wide range of metabolic pathways. In the current study NAD⁺ was detected on rat brain in vivo at 11.7T by 3D localized ¹H MRS of the NAD⁺ nicotinamide protons in the 8.7–9.5 ppm spectral region. Avoiding water perturbation was critical to the detection of NAD⁺ as strong, possibly indirect cross‐relaxation between NAD⁺ and water would lead to a several‐fold reduction of the NAD⁺ intensity in the presence of water suppression. Water perturbation was minimized through the use of localization by adiabatic spin‐echo refocusing (LASER) in combination with frequency‐selective excitation. The NAD⁺ concentration in the rat cerebral cortex was determined at 296 ± 28 μm , which is in good agreement with recently published ³¹P NMR‐based results as well as results from brain extracts in vitro (355 ± 34 μm ). The T₁ relaxation time constants of the NAD⁺ nicotinamide protons as measured by inversion recovery were 280 ± 65 and 1136 ± 122 ms in the absence and presence of water inversion, respectively. This confirms the strong interaction between NAD⁺ nicotinamide and water protons as observed during water suppression. The T₂ relaxation time constants of the NAD⁺ nicotinamide protons were determined at 60 ± 13 ms after confounding effects of scalar coupling evolution were taken into account. The simplicity of the MR sequence together with the robustness of NAD⁺ signal detection and quantification makes the presented method a convenient choice for studies on NAD⁺ metabolism and function. As the method does not critically rely on magnetic field homogeneity and spectral resolution it should find immediate applications in rodents and humans even at lower magnetic fields. Copyright © 2014 John Wiley & Sons, Ltd.     

Balance of pro‐ versus anti‐angiogenic splice isoforms of vascular endothelial growth factor as a regulator of neuroblastoma growth

Neuroblastoma (NB) is the second most common extracranial tumour of childhood. Angiogenesis plays a crucial role in the growth and development of NB and vascular endothelial growth factor (VEGF), one of the most potent stimuli of angiogenesis, has been studied extensively in vitro. VEGF₁₆₅ has been shown to be the predominant angiogenic isoform expressed in NB cell lines and tumours. In this study, we investigated the anti‐angiogenic isoform of VEGF‐A, generated from distal splice site selection in the terminal exon of VEGF (VEGF₁₆₅b) and shown to be down‐regulated in epithelial malignancies. The expression of both the pro‐ (VEGF_xxx) and the anti‐angiogenic (VEGF_xxxb) isoforms was compared in a range of NB and ganglioneuroma (GN) tumours. Whereas VEGF_xxxb and VEGF_xxx were both expressed in GN, specific up‐regulation of the VEGF_xxx isoforms was seen in NB at RNA and protein levels. Highly tumourigenic NB cell lines also showed up‐regulation of the angiogenic isoforms relative to VEGF_xxxb compared to less tumourigenic cell lines, and the isoforms were differentially secreted. These results indicate that VEGF₁₆₅ is up‐regulated in NB and that there is a difference in the balance of isoform expression from anti‐angiogenic VEGF₁₆₅b to angiogenic VEGF₁₆₅. Treatment with recombinant human VEGF₁₆₅b significantly reduced the growth rate of established xenografts of SK‐N‐BE(2)‐C cells (4.24 ± 1.01 fold increase in volume) compared with those treated with saline (9.76 ± 3.58, p < 0.01). Microvascular density (MVD) was significantly decreased in rhVEGF₁₆₅b‐treated tumours (19.4 ± 1.9 vessels/mm³) in contrast to the saline‐treated tumours (45.5 ± 8.6 vessels/mm³). VEGF₁₆₅b had no significant effect on the proliferative or apoptotic activity, viability or cytotoxicity of SK‐N‐BE(2)‐C cells after 48 h. In conclusion, VEGF₁₆₅b is an effective inhibitor of NB growth. These findings provide the rationale for further investigation of VEGF₁₆₅b in NB and other paediatric malignancies. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Developmental Expression Profile of the CXCL12γ Isoform: Insights Into its Tissue‐Specific Role

The CXCL12γ chemokine arises by alternative splicing from Cxcl12, an highly conserved gene that plays pivotal, non‐redundant roles during development. The interaction of the highly cationic carboxy‐terminal (C‐ter) domain of CXCL12γ with glycosaminoglycans (GAG) critically determines the biological properties of this chemokine. Indeed, CXCL12γ isoform displays sustained in vivo recruitment of leukocytes and endothelial progenitor cells as compared to other CXCL12 isoforms. Despite the important, specific roles of CXCL12γ in vivo, the current knowledge about its distribution in embryo and adult tissues is scarce. In this study, we have characterized by both RT‐PCR and immunohistochemistry the expression profile and tissue distribution of CXCL12γ, which showed a distinct mRNA expression pattern during organogenesis that correlates with the specific expression of the CXCL12 γ protein in several tissues and cell types during development. Our results support the biological relevance of CXCL12 γ in vivo, and shed light on the specific roles that this novel isoform could play in muscle development and vascularization as well as on the regulation of essential homeostatic functions during the embryonic development. Anat Rec, 292:891–901, 2009. © 2009 Wiley‐Liss, Inc.     

Temporal expression of calcium/calmodulin-dependent adenylyl cyclase isoforms in rat articular chondrocytes: RT-PCR and immunohistochemical localization

A multitude of signalling cascades are implicated in the homeostasis of articular chondrocytes. However, the identity of these signalling pathways is not fully established. The 3, 5′‐cyclic AMP‐mediated signalling system is considered to be a prototype. Adenylyl cyclase (AC) is an effector enzyme responsible for the synthesis of cAMP. There are 10 mammalian AC isoforms and some of these are differentially regulated by calcium/calmodulin (Ca²⁺/CaM). Ca²⁺ is known to play an important role in the development and maintenance of skeletal tissues. Ca²⁺/CaM‐dependent AC isoforms and their temporal expression in articular chondrocytes in rats were identified using RT‐PCR and immunohistochemistry techniques. All Ca²⁺/CaM‐dependent AC isoforms were expressed in chondrocytes from all age groups examined. Each isoform was differentially expressed in developing and adult articular chondrocytes. Generally, expression of AC isoforms was observed to increase with age, but the increase was not uniform for all Ca²⁺/CaM‐dependent AC isoforms. Expression of Ca²⁺/CaM‐dependent AC isoforms along with other signalling molecules known to be present in articular chondrocytes indicate complicated and multifactorial signalling cascades involved in the development and homeostasis of articular cartilage. The significance of these findings in terms of articular chondrocyte physiology is discussed.     

Cardiovascular ATIP (Angiotensin receptor type 2 interacting protein) expression in mouse development

Background : ATIPs (Angiotensin receptor type 2 [AT2] interacting proteins) are described as AT2 interacting protein variants, whereas their expression and functions during development are not known yet. Results : Here, we provide a detailed expression pattern of ATIP variants during mouse development by visualizing Mtus1 promotor activity, Mtus1 RNA, and subsequent ATIP protein expression. ATIPs are strongly expressed in the developing cardiovascular system, including the vascular plexus of the yolk sac and the fetal vascular part of the placenta. Moreover, ATIP is expressed spatially distinct during eye and limb bud development, and in later stages in lung and nervous system. The three murine ATIP isoforms are expressed in a distinct manner, whereupon isoform 1 and 4 are correlated to cardiovascular, lung, and limb bud development and isoform 3 is restricted to brain and eye development. Interestingly, Mtus1 expression is not necessarily correlated to Agtr2 expression, suggesting novel but yet unknown functions for ATIP, independent of AT2 signaling. Conclusions : ATIPs seem to be mainly involved in the developmental regulation of the cardiovascular system and may act in different AT2‐dependent and ‐independent manners. Hence, these results deliver valuable information to further elucidate the different functions of ATIPs in the processes of mammalian development. Developmental Dynamics 243:699–711, 2014. © 2013 Wiley Periodicals, Inc.     

Localization and functional characterization of the human NKCC2 isoforms

Aim: Salt reabsorption across the apical membrane of cells in the thick ascending limb (TAL) of Henle is primarily mediated by the bumetanide-sensitive Na⁺/K⁺/2Cl⁻ cotransporter NKCC2. Three full-length splice variants of NKCC2 (NKCC2B, NKCC2A and NKCC2F) have been described. The NKCC2 isoforms have specific localizations and transport characteristics, as assessed for rabbit, rat and mouse. In the present study, we aimed to address the localization and transport characteristics of the human NKCC2 isoforms. Methods: RT-PCR, in situ hybridization and uptake studies in Xenopus oocytes were performed to characterize human NKCC2 isoforms. Results: All three classical NKCC2 isoforms were detected in the human kidney; in addition, we found splice variants with tandem duplicates of the variable exon 4. Contrary to rodents, in which NKCC2F is the most abundant NKCC2 isoform, NKCC2A was the dominant isoform in humans; similarly, isoform-specific in situ hybridization showed high expression levels of human NKCC2A along the TAL. Compared to NKCC2B and NKCC2F, human NKCC2A had the lowest Cl⁻ affinity as determined by ⁸⁶Rb⁺ uptake studies in oocytes. All NKCC2 isoforms were more efficiently inhibited by bumetanide than by furosemide. A sequence analysis of the amino acids encoded by exon 4 variants revealed high similarities between human and rodent NKCC2 isoforms, suggesting that differences in ion transport characteristics between species may be related to sequence variations outside the highly conserved sequence encoded by exon 4. Conclusion: The human NKCC2 is an example of how differential splicing forms the basis for a diversification of transporter protein function.     

Relationship between in vivo muscle force at different speeds of isokinetic movements and myosin isoform expression in men and women

In an attempt to explore the relationship between force production during voluntary contractions at different speeds of isokinetic movement and the myofibrillar protein isoform expression in humans, an improved isokinetic dynamometer that corrects for gravitation, controls for acceleration and deceleration, and identifies a maximum voluntary activation was used. Muscle torque recordings were compared at the same muscle length (knee angle) and the torque was calculated as the average torque at each angle over a large knee angle range (75°–25°) to reduce the influence of small torque oscillation on the calculated torque. Muscle torque at fast (240° s⁻¹) versus slow (30° s⁻¹) speeds of movement, torque normalized to muscle cross-sectional area (specific tension), and absolute torque at fast speeds of movement were measured in 34 young healthy male and female short-, middle-, and long-distance runners. The relationship between the different measures of muscle function and the expression of myosin heavy chain (MyHC) isoforms using enzyme–histochemical and electrophoretic protein separation techniques were investigated. A significant correlation between the 240° s⁻¹ vs 30° s⁻¹ torque ratio and the relative area of the type II fibers and type II MyHC isoforms were observed in both the men (r=0.74;P<0.001) and the women (r=0.81; P<0.05). Thus, the present results confirm a significant relationship between in vivo human muscle function and the MyHC isoform expression in the contracting muscle. Electronic Publication     

PARP inhibitor rucaparib induces changes in NAD levels in cells and liver tissues as assessed by MRS

Poly(adenosine diphosphate ribose) polymerases (PARPs) are multifunctional proteins which play a role in many cellular processes. Namely, PARP1 and PARP2 have been shown to be involved in DNA repair, and therefore are valid targets in cancer treatment with PARP inhibitors, such as rucaparib, currently in clinical trials. Proton magnetic resonance spectroscopy (¹H‐MRS) was used to study the impact of rucaparib in vitro and ex vivo in liver tissue from mice, via quantitative analysis of nicotinamide adenosine diphosphate (NAD⁺) spectra, to assess the potential of MRS as a biomarker of the PARP inhibitor response. SW620 (colorectal) and A2780 (ovarian) cancer cell lines, and PARP1 wild‐type (WT) and PARP1 knock‐out (KO) mice, were treated with rucaparib, temozolomide (methylating agent) or a combination of both drugs. ¹H‐MRS spectra were obtained from perchloric acid extracts of tumour cells and mouse liver. Both cell lines showed an increase in NAD⁺ levels following PARP inhibitor treatment in comparison with temozolomide treatment. Liver extracts from PARP1 WT mice showed a significant increase in NAD⁺ levels after rucaparib treatment compared with untreated mouse liver, and a significant decrease in NAD⁺ levels in the temozolomide‐treated group. The combination of rucaparib and temozolomide did not prevent the NAD⁺ depletion caused by temozolomide treatment. The ¹H‐MRS results show that NAD⁺ levels can be used as a biomarker of PARP inhibitor and methylating agent treatments, and suggest that in vivo measurement of NAD⁺ would be valuable.