Abstract
Chemotherapy-induced cognitive impairment (CICI) is often reported as a neurotoxic side effect of chemotherapy. Although CICI has emerged as a significant medical problem, meaningful treatments are not currently available due to a lack of mechanistic understanding underlying CICI pathophysiology. Using the platinum-based chemotherapy cisplatin as a model for CICI, we show here that cisplatin suppresses nicotinamide adenine dinucleotide (NAD+) levels in the adult female mouse brain in vivo and in human cortical neurons derived from induced pluripotent stem cells in vitro. Increasing NAD+ levels through nicotinamide mononucleotide (NMN) administration prevented cisplatin-induced abnormalities in neural progenitor proliferation, neuronal morphogenesis, and cognitive function without affecting tumor growth and antitumor efficacy of cisplatin. Mechanistically, cisplatin inhibited expression of the NAD+ biosynthesis rate-limiting enzyme nicotinamide phosphoribosyl transferase (Nampt). Selective restoration of Nampt expression in adult-born neurons was sufficient to prevent cisplatin-induced defects in dendrite morphogenesis and memory function. Taken together, our findings suggest that aberrant Nampt-mediated NAD+ metabolic pathways may be a key contributor in cisplatin-induced neurogenic impairments, thus causally leading to memory dysfunction. Therefore, increasing NAD+ levels could represent a promising and safe therapeutic strategy for cisplatin-related neurotoxicity.
Increasing NAD+ through NMN supplementation offers a potential therapeutic strategy to safely prevent cisplatin-induced cognitive impairments, thus providing hope for improved quality of life in cancer survivors.
Graphical Abstract:
Introduction
Chemotherapy-induced cognitive impairment (CICI) comprises adverse neurotoxic effects that consist of subtle to moderate impairments in several cognitive domains, including working memory, processing speed, and executive functioning. Although this symptomatology may be derived from exacerbated stress, anxiety, and depression stemming from cancer diagnosis, these cognitive dysregulation (also called “chemobrain” or “chemofog”) can persist for months or years following chemotherapy. Although CICI has been reported in up to 75% of patients treated with chemotherapy, a subset of survivors (15%–25%) experience measurably persistent cognitive decline, thus representing a significant public health concern (1). However, meaningful treatments are not currently available due to a lack of mechanistic understanding underlying CICI pathophysiology.
Along with the prefrontal cortex, white matter, and other brain structures, the hippocampus is a brain region important for memory formation that is significantly affected by CICI. Notably, the hippocampus is one of a few brain regions where adult-born neurons derived from neural stem/progenitor cells (NPC) are constantly generated throughout life in most mammals, including humans (2, 3). This process, called adult hippocampal neurogenesis, represents a remarkable example of brain plasticity that plays a pivotal role for healthy learning and memory function (4, 5). Emerging evidence shows that NPCs are particularly vulnerable to the neurotoxic effects of chemotherapy, which results in significant reductions of neurogenesis (6–8), and may contribute to CICI (9). However, the molecular pathways that underlie CICI are still largely unknown.
Proper brain function is highly dependent upon controlled energy metabolism. Nicotinamide adenine dinucleotide (NAD+) is an important metabolite involved in genomic stability, cell division, and cell survival. Declines in cellular NAD+ levels and its rate-limiting enzyme, nicotinamide phosphoribosyltransferase (Nampt), play a pathogenic role in age-related diseases (10, 11). Importantly, these age-related impairments are attenuated by increasing NAD+ through NAD+ precursors such as nicotinamide mononucleotide (NMN) or nicotinamide riboside. Both compounds have been reported as promising therapeutic compounds to delay aging, extend the lifespan, and improve cognition (10). Notably, chemotherapy detrimentally alters brain function similar to advanced aging as evidenced by reduced neurogenesis, increased neuroinflammation (astrocyte and microglia activation), and memory dysfunction (7, 8, 12). In particular, chemotherapy accelerates biological aging as reported in a subset of breast cancer patients (13, 14). These observations suggest that CICI and brain aging may share common pathophysiologic mechanisms mediating cognitive impairment. Therefore, we sought to determine whether chemotherapy causes cognitive dysfunction through depletion of NAD+ metabolism, while investigating if increasing NAD+ levels have beneficial therapeutic effects in preventing CICI.
Materials and Methods
Mouse husbandry
All experiments were performed on 3 to 4-month-old female C57BL/6J mice (The Jackson Laboratory) unless noted otherwise. Nampt OXf/f mice were kindly provided by Dr. Joseph Baur at the University of Pennsylvania (Philadelphia, PA) and were generated as previously described (15). To generate adult-born neuron specific overexpression of Nampt mice, Ascl1-CreERT2 mice were purchased from the Jackson Laboratory (Stock # 012882; ref. 16). Briefly, Nampt OXf/f mice were crossed with Ascl1-CreERT2 mice to produce Nampt OXf/+;Ascl1-CreERT2 (cNampt OX) and WT;Ascl1-CreERT2 mice (control). All mice were housed in standard cages under a 12-hour light/dark cycle with water and food ad libitum. The animal experiments were conducted in accordance with NIH guidance on the care and use of laboratory animals. All procedures were approved by the Mayo Clinic Institutional Animal Care and Use Committee (IACUC protocol #A00005043 and A00004190).
Cisplatin and NMN administration
To induce cognitive dysfunction in mice, we used the platinum-based compound cisplatin (Fresenius Kabi, cat. # 100351), which has been reported to accumulate in significant concentrations in patient brains (17). Cisplatin is used to treat central nervous system (CNS) cancers such as neuroblastoma, germ cell tumors, and primary CNS neoplasms. In addition, it is also a widely used compound to treat non-CNS cancers including ovarian, testicular, lung, breast, and bladder cancer. Briefly, female mice at age of 3 to 4 months were treated with daily injections of cisplatin (2.3 mg/kg/day, i.p.) or vehicle for 5 consecutive days followed by 5 days without injections. This treatment regimen (5 injection days followed by 5 noninjection days) is indicative of 1 cycle of treatment and mice were administered 3 to 4 cycles as appropriate for each experiment objective (Fig. 1A). This cisplatin regimen is known to be comparable to clinical treatment for cancer patients (18–21), and therefore widely used for rodent models of CICI as well as chemotherapy-induced neuropathic pain (CIPN; ref. 22). Specifically, this dose regimen showed antitumor efficacy, while still inducing significant neurotoxicity to dorsal root ganglion and hippocampal neurons, leading to cisplatin-induced peripheral neuropathy and cognitive impairments in mice, respectively (23).
To test the effect of NMN in cisplatin-induced cognitive impairments as presented in Fig. 2B, mice were pretreated with NMN (250 mg/kg/day, i.p.; Sigma-Aldrich, cat. # N-3501) 4 hours prior to cisplatin administration for 4 cycles. Although cisplatin-treated mice exhibited significant decreases in body weight (Supplementary Fig. S1A), this did not meet moribund criteria by institution guidelines.
Measurement of NAD+ levels
To assess NAD+ levels in the hippocampus, striatum, and cerebellum (Fig. 2A), these brain tissues were freshly extracted (∼10–25 mg/tissue sample) without undergoing perfusion with iced saline or PFA prior to brain dissection, snap frozen in dry ice, and stored at −80°C until processed. NAD+ levels were determined by using the commercial colorimetric NAD+/NADH Quantification kit (Bio Vision, cat. #K337) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a spectrometer (Molecular Devices, Spectra Max3).
Behavior analyses
Mice were transferred from the housing room to acclimate to the behavior facility holding area for 1 hours before testing. Testing equipment was sanitized with 70% ethanol between trials.
Novel Object Recognition test
Novel Object Recognition (NOR) test is a relatively fast and efficient means for testing different phases of learning and memory in rodent animals (24). The main advantage of the test is that it relies on rodents' natural proclivity for exploring novel objects and environments. NOR testing was performed across 3 consecutive days (Fig. 2C), where on day 1 mice were placed in an empty arena (40 × 40 × 40 cm chamber) to freely explore and habituate to the environment for 10 minutes and then placed back in their home cage. On day 2 (training phase), two identical objects (familiar objects) were placed at the bottom of the arena diagonally opposed to each other (e.g., one in the NW corner and one in the SE corner; Fig. 2C), and animals were placed in the arena to explore the objects for 10 minutes. On day 3 (testing phase), one of the objects was replaced by a new object (novel object), and animals were allowed to explore for 10 minutes. The time spent exploring each object was recorded by EthoVision XT 10 (Noldus, Inc.) video tracking software. The total amount of time explored on the each object with the nose was recorded and analyzed. The amount of time taken to explore the novel object in relation to the familiar object provides an index of recognition memory. As depicted in Fig. 2D, NOR results were presented as described previously (25–27).
Morris water maze
Morris water maze (MWM) is a classic behavioral procedure mostly used with rodents to study spatial learning and memory. The MWM has proven to be a robust and reliable test that is strongly correlated with hippocampal-dependent cognitive function (28, 29). The arena setting contains a round pool (120 cm diameter and 90 cm deep) filled with water mixed with non-fat dry milk power to make opaque water. A video camera (supported by EthoVision XT by Noldus, Inc video tracking program) was set up directly above the water pool to ensure the entire maze is within the camera's field of view. The maze was divided into four quadrants of equal dimensions with each designated as N, S, E, W quadrants, respectively. Each quadrant had a visible spatial cue above it to facilitate spatial memory function. A submerged escape platform (30 cm high) was placed in the center of the SE quadrant. During each testing day, the water temperature was maintained between 25°C and 28°C. As shown in Supplementary Fig. S1B, the test was conducted in 6 consecutive days consisting of visible platform days (days 1 and 6), training days (days 2–4), and a memory probe day (day 5). Each test day, mice would undergo 4 swimming trials per day for a duration of 2 minutes/trial, and interspaced with a 2- to 5-minute interval between each trial, totaling 4 trials/day. Each trial commenced following releasing each test mouse from each quadrant release point (NE, SE, SW, and NW). Mice that failed to find the location of the visible or submerged (hidden) escape platform in each trial were manually guided to the platform for 30 seconds to learn the platform location. Briefly, on day 1 for habituation, the escape platform was placed slightly above the water level with an orange flagpole to signal its location; thus, mice were able to see and learn the platform location to escape the water. On days 2 to 4, mice underwent acquisition training for spatial learning to find the hidden escape platform, which was set 1 inch below the water level, so mice were trained to rely on spatial cues to learn to find the submerged platform. The latency, which is the time spent to find the platform, was recorded for further data analysis. If mice failed to find the target in 2 minutes, their latency was recorded as 2 minutes and they were guided to the platform and allowed to stay there for 30 seconds. On day 5 for the memory probe test, the escape platform was removed to determine whether mice relied on spatial cues and learned to memorize the position of the hidden platform. The frequency of crossing the platform zone and latency to find the target platform zone was recorded for data analysis. On day 6 for visual test, the escape platform was set slightly above the water level and signaled by flag of its location. The latency to find this visible platform was recorded.
Perfusion process and immunostaining
Mice were anesthetized by a standard dosing regimen of ketamine (100 mg/kg), xylazine (10 mg/kg), and acepromazine (3 mg/kg) cocktail (i.p. injection at 0.1 mL/10 g body weight), and perfused transcardially through the left ventricle with cold 0.1 M phosphate-buffered saline at pH 7.4 followed by a phosphate-buffered solution of 4% paraformaldehyde (PFA). Brains were post-fixed with 4% PFA overnight and 24 hours later placed into 30% sucrose. Brains were sectioned and stored in antifreeze solution at −20°C. Coronal sections (40 μm in thickness) from anterior to posterior through the entire brain were prepared in serial order and processed for histologic analysis. For MCM2 and Nestin immunostaining in Fig. 3A and B, and Nampt, doublecortin (DCX), and NeuN immunostaining in Supplementary Fig. S5A and S5D, an antigen retrieval procedure was carried out using a microwave as previously described (30, 31). Briefly, citrate buffer (1.8 mmol/L citric acid and 8.2 mmol/L trisodium citrate) was preheated for 5 minutes at maximum power. Sections were then placed in hot citrate buffer and incubated for another 7 minutes. Sections were then allowed to cool at room temperature in citrate buffer for 1 hour. Immunostaining was performed with the primary antibodies followed by secondary antibodies. DAPI (Molecular Probes, cat. #D1306) was used for counterstaining. Antibodies used in this study are listed in Supplementary Table S1. Images were acquired on a Zeiss LSM 780 single-photon confocal system using a multitrack configuration.
Retrovirus production, stereotaxic surgery, and dendrite analysis
Engineered self-inactivating murine onco-retroviruses were used to express GFP selectively in proliferating neural progenitors as previously described (31). Briefly, high titers of engineered retroviruses were produced by cotransfection of retroviral vectors and vesicular stomatitis virus G (VSVG) into HEK293gp cells followed by ultra-centrifugation of viral supernatant. To visualize dendrite morphology of adult-born neurons (Fig. 3C, top left), adult female mice at 3 to 4 months of age were anesthetized, and retroviruses were stereotaxically injected into the dentate gyrus at 4 sites (0.5 μL per site at 0.25 μL/min) with the following coordinates (in mm): posterior = 2 from Bregma, lateral = ±1.6, ventral = 2.5; posterior = 3 from Bregma, lateral = ±2.6, ventral = 3.2. At 3 days post-retroviral injection when adult-born NPCs mature into post-mitotic neurons, mice were treated with cisplatin for 3 cycles followed by NMN daily treatment 7 days prior to completion of cisplatin administration (Fig. 3C; top right). One day after completion of NMN and cisplatin administration, total dendritic length and branch numbers of GFP+ adult-born neurons were analyzed. Briefly, three-dimensional (3-D) reconstructions of the dendritic processes of each GFP+ neuron were made from Z-series stacks of confocal images (Zeiss 780). The projection images were semiautomatically traced with NIH ImageJ using the Neuron J plugin. The total dendritic length and branch number of individual GFP+ neurons were subsequently analyzed (31). Summaries of total dendritic length and branch number of each individual neuron are shown in cumulative distribution plots (Fig. 3D, right). To induce Cre recombinase (Fig. 6C), mixed male and female Nampt OXf/+ (heterozygous) mice of 3 to 4 months of age were stereotaxically injected with the pCAG-GFP-IRES-Cre (Addgene plasmid #48201) retrovirus to produce overexpression of Nampt in adult-born neurons of the dentate gyrus. The pCAG-GFP (Addgene plasmid #16664) retrovirus was used as control. At 3 days post-retroviral injection, mice were treated with cisplatin for 3 cycles and sacrificed, and brains were processed 1 day after the last cisplatin injection for dendrite morphologic analysis.
Breast cancer cell lines
All breast cancer cell lines were obtained from ATCC and were provided by J.R. Hawse (Mayo Clinic) for this study. All cell lines were routinely tested for Mycoplasma using the Mycoplasma Detection Kit (SouthernBiotech, cat. #13100-01) and have been confirmed to be negative. All cells were grown in DMEM/F12 media (Gibco, cat.#11330032) supplemented with 10% FBS (HyClone, cat. #SH30071.03) and 1% penicillin–streptomycin (Gibco, cat. #15070063) and cultured at 37°C and 5% CO2. Cells were utilized at low passage and were cultured for no more than 2 months from time of thawing to experimentation.
Cell viability analysis
To test the effect of NMN on tumor growth, we performed an in vitro cell viability analysis using 9 different breast cancer cell lines and two ovarian cancer patient-derived xenografts (PDX) 3D models that has demonstrated strong correlation with clinical platinum sensitivity in patients (32).
For breast cancer cell line studies (Supplementary Fig. S4A and S4B), ∼1,000 cells/well were plated in 96-well tissue culture plates in replicates of 8 and allowed to adhere overnight. Cells were subsequently treated with vehicle control or indicated concentrations of NMB and allowed to proliferate for 7 days. Following treatment, cells were lysed and cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assays according to the manufacturer's protocol. Vehicle-treated cells were set to 100% viability, and values from NMN treatments are depicted relative to controls.
For PDX studies (Supplementary Fig. S4C), ovarian cancer ex vivo 3D culture was performed on fresh PDX tissue resected from mice. Tumors (1–1.5 g) were first minced with surgical shears and partially digested using a MACS Tumor Dissociation Kit (Miltenyi Biotec Inc., cat. #130-095-929) with gentleMACS C Tubes in an Octo Dissociator for 1 hour at 37°C. Cell aggregates were washed twice with MACS buffer and seeded on 96-well low-binding plates at 1 to 5 × 104 cells per well in DMEM with 10% FBS and 1% penicillin/streptomycin. Cells were allowed to acclimate for 24 hours before exposure to one of three conditions: cisplatin (0, 1.5, 3, 6.25, 12.5, 25, and 50 μmol/L), NMN (0, 31.25, 62.5,125, 250, 500, and 1,000 μmol/L), or both in combination. RealTime-Glo MT Cell Viability reagent (Promega, cat. #G9711) was added 72 hours after plating, and cells were imaged for viability ∼120 hours after plating.
Xenograft tumor models
To test the in vivo effects of NMN and cisplatin on tumor growth (Fig. 5), MDA-MB-231 cells (triple-negative) and MCF7 ERα+ cells (estrogen receptor-alpha) were injected orthotopically into 4- to 5-month-old CB17/Icr-Prkdcscid SCID female mice (Charles River, Strain Code: 236). Briefly, cells with a concentration of ∼1 × 106 cells in 100 μL + 100 μL of matrigel (Corning, cat. #356231) were injected into the mammary fat pads of 4- to 5-month-old CB17 SCID female mice. For mice injected with MCF7 cells, 17β-estradiol (0.54 mg/90-day release; Innovative Research of America, cat. #NE-121) pellets were implanted into the nape of the neck using a trochar to induce tumor growth. Once the average tumor volume for all animals reached approximately 50 mm3, mice were treated with 4 cycles of 5 daily i.p. injections of cisplatin (2.3 mg/kg/day) concurrently with NMN (250 mg/kg/day) or saline, followed by a 5-day rest without injections.
Statistical analysis
All statistical analyses were performed using GraphPad Prism 8. A two-tailed unpaired or paired Student t test, one or two-way ANOVA, and Dunnett or Tukey post hoc test for multiple comparisons were used, as appropriate for each experiment. For dendrite analysis in Figs. 3C and 6C, the Kolmogorov–Smirnov test was performed. Statistical significance was defined as P ≤ 0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001). P > 0.05 were defined as statistically not significant (n.s.). Two outliers on Fig. 2D (day 2) and Fig. 2G were identified via GraphPad Prism Grubbs outlier test, and these values were removed. All exact P values, detailed statistical analysis information, and animal sources, group size, and gender of the mouse models are provided in Supplementary Table S2. All experiments and data analyses were performed in a blinded fashion.
See Supplementary Information for additional methods.
Results
Cisplatin penetrates the blood–brain barrier
We established an in vivo model of CICI using female mice administered cisplatin (Fig. 1A), a platinum-based compound widely used to treat various cancers including ovarian, testicular, lung, and breast cancer. Given that cisplatin has been reported to induce cognitive dysfunction in cancer survivors (33), understanding the mechanisms of how cisplatin induces neurotoxicity will be critical for the development of therapies to prevent or treat CICI. To explore whether cisplatin permeates the brain, we performed imaging mass cytometry (IMC) to visualize platinum distribution in the hippocampus and prefrontal cortex from mice treated with cisplatin or vehicle. We have chosen the hippocampus and the prefrontal cortex because both structures are essential for cognitive function and are reported to be more vulnerable to chemotherapy in humans (34–37). As shown in Fig. 1B, we detected platinum in the brains of mice treated with cisplatin at 2 and 5 weeks following the last cisplatin injection, thus demonstrating that cisplatin penetrates the blood–brain barrier and persists long after cessation of cisplatin administration.
Preventative effects of NMN on cisplatin-induced cognitive impairments in adult female mice
Declines in NAD+ have emerged as a molecular hallmark of age-related diseases (10). Given the similarity between CICI and brain aging, we tested if cisplatin alters cellular NAD+ levels in the adult female mouse brain. We observed that NAD+ is significantly reduced by cisplatin in the hippocampus and cerebellum (Fig. 2A). This result led us to test whether increasing NAD+ levels could rescue cisplatin-induced cognitive deficits. NAD+ precursor NMN is known to increase hippocampal NAD+ levels, promote NSC proliferation (11), and improve memory function in aged mice (38). Adult female mice were then pretreated with NMN at the daily dose of 250 mg/kg i.p., followed by cisplatin treatment 4 hours later in each of 4 cycles (Fig. 2B). Body weight (BW) assessment did not find detrimental effects of NMN, although cisplatin-treated mice displayed significant decreases in BW (Supplementary Fig. S1A). We next performed NOR (Fig. 2C) testing and the MWM (Supplementary Fig. S1B), both of which assess memory and are strongly affected by hippocampal impairments. In addition, cisplatin has been reported to impair memory function in these tests (8, 39). Our NOR results showed that while there was a slight increase in mice treated with NMN alone, no significant differences were detected in time spent exploring the two familiar objects (day 2; Fig. 2D, left), indicating a lack of location exploration preference. However, when a familiar object was replaced by a novel object during test day 3, cisplatin-treated mice spent less time exploring the novel object, relative to vehicle-treated mice, indicating that cisplatin impairs memory function. Notably, when NMN was given to vehicle and cisplatin-treated mice, both groups significantly increased exploration of the novel object (Fig. 2D, right), suggesting that NMN prevents cisplatin-induced deficits in recognition memory.
To strengthen our results and avoid reliance on a single memory test such as NOR, we performed the MWM, which is a more nuanced memory assay. The MWM test showed that cisplatin-treated mice had longer latencies to find the hidden platform during acquisition training (days 2–4), suggesting that cisplatin-treated mice displayed learning impairments. However, NMN administration prior to cisplatin treatment significantly reduced escape latencies on day 4 (Fig. 2E, left). During the probe test (day 5), cisplatin-treated mice exhibited significantly fewer target platform zone crossings (Fig. 2E, right and Fig. 2F) and longer latencies to reach the target platform zone (Supplementary Fig. S1C) compared with vehicle-treated mice, indicating spatial memory dysfunction by cisplatin. Importantly, NMN in cisplatin-treated mice showed greater crossing frequencies in the target platform zone and shorter escape latencies when compared with mice given cisplatin, suggesting a neuroprotective role for NMN in cisplatin-induced spatial memory dysfunction in female mice. Notably, there were no differences in swim speed or visual function (Supplementary Fig. S1D and S1E, respectively), confirming that the effects of NMN or cisplatin are on learning and memory rather than on physical or visual function. Furthermore, we found that the observed neuroprotective effects of NMN on memory function are correlated with increased levels of NAD+ in the hippocampus and cerebellum of cisplatin-treated mice (Fig. 2G).
Neuroprotective effects of NMN on cisplatin-induced defects in neurogenesis in adult female mice in vivo
Significant reduction of hippocampal neurogenesis by cisplatin and other chemotherapeutics is a common pathologic feature that may contribute to cognitive dysfunction (6–8). As an initial step of neurogenesis, radial glia-like neural stem cells (RGL) in the subgranular zone (SGZ) produce intermediate progenitor cells (IPC), which give rise to neuroblasts in the adult mouse dentate gyrus (DG; Supplementary Fig. S2A). Following completion of behavior testing, we analyzed the effects of cisplatin and NMN on RGLs defined by coexpressing MCM2 (a proliferative marker) and nestin (a neural stem cell marker) cells with radial processes. IPCs were defined by MCM2 expression. Our quantitative analysis showed a decreased trend in RGL population in cisplatin-treated female mice, whereas this reduction was significantly restored by NMN administration (Fig. 3A). Similarly, MCM2-expressing IPCs were significantly reduced by cisplatin, which was prevented by NMN administration (Fig. 3B). No obvious sex differences were found as cisplatin induced reductions in RGL and IPC populations (Supplementary Fig. S2B). We further analyzed if cisplatin affects neuronal morphogenesis of adult-born neurons and whether NMN can reverse this abnormal process. Adult female mice were stereotaxically injected with a GFP-expressing retrovirus to visualize adult-born neuron morphology. At 3 days post-retroviral injection when adult-born NPCs mature into post-mitotic neurons, mice were treated with cisplatin for 3 cycles followed by NMN daily treatment 7 days prior to completion of cisplatin administration (Fig. 3C). We show that cisplatin significantly decreases total dendritic length and branch numbers of GFP+ adult-born neurons compared with vehicle administration. Remarkably, the cisplatin-induced reduction in dendrite length and branch number was fully recovered by NMN (Fig. 3D). Taken together, these findings suggest a therapeutic effect of NMN in cisplatin-induced impairments in NPC proliferation, neuronal morphogenesis, and memory function in adult female mice.
Neuroprotective effects of NMN in cisplatin-induced abnormality in neuronal morphogenesis in human cortical neurons derived from iPSCs in vitro
To further assess the effects of cisplatin and NMN in human neurons, we generated excitatory cortical neurons derived from human induced pluripotent stem cells (iPSC; Fig. 4A and B; Supplementary Fig. S3A). Cortical human neurons that were exposed to cisplatin displayed significant suppression of neuronal NAD+ levels at the lowest dose (0.01 μmol/L; Fig. 4C) without significantly affecting cell viability (Supplementary Fig. S3B), indicating that neuronal NAD+ reductions were not due to decreased cell numbers by cisplatin. Intriguingly, although NMN alone does not affect cell viability, pretreatment with NMN significantly prevents cisplatin-induced reduction in cell survival (Supplementary Fig. S3C). Morphologic analysis also showed that cisplatin dramatically suppressed total neurite length in human neurons in a dose-dependent fashion (Supplementary Fig. S3D). More importantly, these abnormal phenotypes were ameliorated by NMN at doses ranging from 250 to 1,000 μmol/L, with the greatest efficacy at 500 μmol/L (Fig. 4D). Notably, NMN administration without cisplatin treatment significantly promoted neurite outgrowth, suggesting that neuronal morphogenesis is highly dependent on neuronal NAD+ levels. Collectively, our results confirm a neuroprotective role for NMN in cisplatin-induced defects in cell viability and neuronal morphogenesis in human cortical neurons.
No impact of NMN on tumor growth or antitumor activity of cisplatin
As we aimed to identify NMN as a potential compound to prevent CICI, an important consideration is whether NMN inadvertently promotes tumor growth or interferes with the efficacy of cisplatin. We did not find cell viability differences in 9 breast cancer cell lines when exposed to our NMN doses (0–1,000 μmol/L NMN), indicating that NMN does not promote tumor growth in these cell lines (Supplementary Fig. S4A and S4B). Similarly, NMN does not promote tumor growth or impair cisplatin's antitumor activity in two ovarian cancer PDX 3D models (Supplementary Fig. S4C). To further test the effects of NMN in vivo, triple-negative (MDA-MB-231; Fig. 5A and B) and estrogen receptor–positive (MCF7; Fig. 5C and D) breast cancer cell lines were implanted orthotopically into adult severe combined immunodeficiency female mice followed by NMN or cisplatin administration. Although cisplatin administration reduced tumor sizes, there were no significant tumor size changes by NMN treatment in either vehicle-treated mice or cisplatin-treated mice. Our results suggest that NMN neither promotes tumor growth nor interrupts the antitumor activity of cisplatin, ensuring NMN safety at the effective dose utilized for behavior memory tests.
Genetic Nampt overexpression selectively in adult-born neurons is sufficient to prevent cisplatin-induced impairments in dendrite morphogenesis and memory function
We next asked how cisplatin suppresses cellular NAD+ levels to potentially impair adult hippocampal neurogenesis and consequent memory function. Given that Nampt is the rate-limiting enzyme in NAD+ biosynthesis (Fig. 6A), we assessed if cisplatin inhibits Nampt levels in the adult mouse hippocampus. Western blot analysis showed that Nampt expression was suppressed by cisplatin to about 50% of vehicle-treated mice (Fig. 6B). Notably, we detected reduction of Nampt expression exclusively in hippocampal neurons (Supplementary Fig. S5A). In addition, NAD+-dependent Sirtuin enzyme, Sirt2, expression was significantly reduced by cisplatin without significantly changing Sirt1 levels (Fig. 6B). Therefore, these data indicate that cisplatin reduces expression of Nampt and NAD+-dependent Sirt2 in the adult female mouse hippocampus.
Genetic deletion of Nampt or Sirt2 is known to impair NPC proliferation, synaptic plasticity, and memory function (40, 41). To determine if reduction in Nampt levels is responsible for cisplatin's aberrant effects on neurogenesis, we reasoned that sustained Nampt levels may be able to prevent cisplatin-induced neurogenic deficits. To selectively increase Nampt levels within adult-born neurons, adult floxed Nampt overexpressing mice (Nampt OXf/+ mice) were stereotaxically injected in the hippocampal DG with a coexpressing Cre recombinase and GFP-retrovirus (Cre-retrovirus) or GFP-retrovirus that does not express Cre recombinase (Control-retrovirus), followed by 3 cycles of cisplatin injections (Fig. 6C, left). Morphologic assessment at 1 day post cisplatin administration demonstrated an increase in total dendrite length of Cre-expressing adult-born neurons compared with controls (Fig. 6C, right). These results suggest that Nampt overexpression promotes adult-born neuron dendrite outgrowth in cisplatin-treated mice in a cell-autonomous manner. We then examined whether selectively increasing Nampt levels within adult-born neurons is sufficient to prevent cisplatin-induced memory impairment by generating conditional Nampt OXf/+;Ascl1-CreERT2 (cNampt OX) and WT;Ascl1-CreERT2 mice (Control; Supplementary Fig. S5B–S5E). We selected Ascl1-CreERT2 mice because the majority of Ascl1-expressing neural progenitors in the adult DG mature into adult dentate granule neurons expressing NeuN within 30 days after tamoxifen administration (16), thus minimizing confounding glial contribution to behavior function derived from progenitors. As expected, cisplatin-treated mice exhibited longer latencies to reach the hidden platform during acquisition trials, fewer platform crossings and longer latencies to reach the target platform zone during the probe test when compared with vehicle-treated controls (Fig. 6D; left, center, and right, respectively, and Supplementary Fig. S5F–S5H). In contrast, cisplatin-treated cNampt OX mice showed decreased latencies to find the hidden platform, increased platform crossings, and decreased latencies to reach the target platform zone relative to cisplatin-treated controls. Interestingly, cNampt OX mice did not show a complete recovery from cisplatin's effects. A potential explanation is that the adult-born neuron population comprises a small portion (approximately 6%) of the whole dentate granule neuron population in the adult hippocampus (42). Therefore, we expect that Nampt overexpression selectively within adult-born neurons will only partially increase NAD+ levels at the whole-tissue level, thus incompletely preventing the learning and memory impairments induced by cisplatin. Nonetheless, these results suggest that selective sustained expression of Nampt in adult-born neurons is sufficient to prevent cisplatin-induced impairment in spatial learning and memory function, demonstrating critical causative roles of Nampt and adult-born neurons that are detrimentally affected by cisplatin.
Discussion
Here, we identify that disruption in Nampt-mediated NAD+ metabolism may be a contributor in cisplatin-induced impairments on neurogenesis and memory function. Although early studies of methotrexate-induced memory impairment attributed glial dysregulation and neuroinflammation as contributors to CICI (12), our study extends a new causative role for adult-born neurons underlying cisplatin-induced memory impairment. Furthermore, we identify Nampt as an essential enzyme that mediates cisplatin-induced NAD+ loss, indicating that Nampt-NAD+ dysregulation results in CICI. Notably, the Nampt-mediated NAD+ metabolic pathway is known to play a role in cancer pathogenesis (43). However, we did not find any detrimental impact of NMN on tumor growth and cisplatin's antitumor activity, ensuring that NMN is a safe and promising compound for CICI. In addition, since other chemotherapies are associated with cognitive dysfunction (12), it is of importance to determine whether the neurotoxic effects of these drugs are mediated through dysregulated NAD+ metabolism. If this is the case, NMN could be a general therapeutic option to abate the neurotoxicity associated with other chemotherapies. Moreover, given that CICI is commonly reported among cancer patients with breast cancer, we focused our study on the ameliorative effects of NMN on cisplatin-CICI in female mice. However, whether NMN may have similar neuroprotective effects in male mice remains an open question. Nonetheless, Nampt-NAD+ metabolism is actively being developed as a pharmacotherapy against aging and age-related diseases, and NMN is currently undergoing clinical trials to improve age-related metabolic dysfunction. Therefore, our prophylactic strategies to prevent NAD+ loss using NMN supplementation during cisplatin-chemotherapy is a promising therapeutic strategy that is rapidly and safely applicable to prevent cisplatin-induced neurotoxicity and improve quality of life for cancer patients.
Authors' Disclosures
S.J. Weroha reports personal fees from Kiyatec and AstraZeneca outside the submitted work. J.A. Baur reports grants from NIDDK during the conduct of the study and grants from Metro International Biotech and Elysium Health outside the submitted work. No disclosures were reported by the other authors.
Authors' Contributions
K.H. Yoo: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. J.J. Tang: Conceptualization, data curation, formal analysis, supervision, validation, investigation, visualization, methodology, writing–review and editing. M.A. Rashid: Data curation, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. C.H. Cho: Conceptualization, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. A. Corujo-Ramirez: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. J. Choi: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. M.G. Bae: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. D. Brogren: Formal analysis, validation, investigation, visualization, methodology, writing–review and editing. J.R. Hawse: Formal analysis, validation, investigation, methodology, writing–review and editing. X. Hou: Formal analysis, validation, investigation, methodology, writing–review and editing. S.J. Weroha: Resources, data curation, supervision, validation, investigation, methodology, writing–review and editing. A. Oliveros: Resources, data curation, supervision, validation, investigation, methodology, writing–review and editing. L.A. Kirkeby: Resources, investigation, methodology, writing–review and editing. J.A. Baur: Resources, methodology, writing–review and editing. M.-H. Jang: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, methodology, writing–original draft, writing–review and editing.
Acknowledgments
The authors thank Dr. Edurado Chini, Dr. Jann Sarkaria, Dr. Nathan LeBrasseur, and Dr. Anthony Windebank for their helpful suggestions, and Dr. Zachary T. Resch at Center for Regenerative Medicine Biotrust at Mayo Clinic for generating iPSC line. This work was supported by NIH (R01CA242158 and R01AG058560), Mayo Clinic Breast Cancer SPORE (P50CA116201), Regenerative Medicine Minnesota (RMM091718 DS 005), Eagles 5th District Cancer Telethon Funds to M.H. Jang, The Mayo Clinic Post-Baccalaureate Research Education Program (R25GM075148) to A. Corujo-Ramirez, Mayo Clinic SPORE in Ovarian Cancer (CA136393) to S.J. Weroha, and the 2019 The Bosarge Family Foundation–Waun Ki Hong Scholar Award for Regenerative Cancer Medicine from the American Association for Cancer Research (19-40-60-OLIV) to A. Oliveros.
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