Jang, Mi‐Hyeon

Advances in cancer treatment by chemotherapy, radiation, and personalized targeted therapy have significantly improved cancer survivorship. Life extension from chemotherapy is arguably the most accessible non-surgical treatment strategy, but it is accompanied by neurotoxic sequelae that are detrimental to cognitive function in survivors. Using the platinum-based drug cisplatin to model chemotherapy induced cognitive impairments (CICI; also called chemobrain) in mice, we recently identified that elevated hippocampal adenosine A 2A receptor (A 2A R) levels are causally associated with CICI. Although neuroinflammation is a major pathological mechanism underlying CICI, it remains unclear whether A 2A R mediates cisplatin-induced neuroinflammation. To address this knowledge gap, we demonstrate for the first time that cisplatin increases microglial process extension and branching complexity, consistent with a primed, hyper-surveillant morphological phenotype. Remarkably, caffeine, a non-specific A 2A R antagonist and known cognitive enhancer, attenuated the primed microglial morphology caused by cisplatin. Furthermore, we show that caffeine mitigates cisplatin-induced body-weight loss, accelerates physical recovery, and protects against motor dysfunction. We also show that caffeine confers protection against cisplatin potentiated impairments in hippocampal dendritic spine density, neurogenesis, and confers modest memory benefits. Collectively, these results suggest that A 2A R-mediated microglial priming is associated with cognitive dysfunction in chemobrain and A 2A R inhibition by caffeine may represent a strategy to ameliorate the physical and cognitive impairments caused by cisplatin.

Abstract Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Mice lacking the ethanol‐sensitive equilibrative nucleoside adenosine transporter 1 (ENT1 –/– ) display enhanced goal‐directed behaviors due to decreased adenosine A 2A R function. However, the role of A 2A R signaling in the regulation of impulsivity remains unknown. Using Pavlovian conditioning, second‐order schedule discrimination, and the 5‐choice serial reaction time task (5‐CSRTT), which incorporates differential‐reward of low rate (DRL) schedules, we reveal that deficits in A 2A R function promote impulsivity. ENT1 –/– mice display exaggerated impulsive responses and faster reaction times to retrieve reward during conditioning and extinction. Interestingly, our results show that basal ERK1/2 phosphorylation is reduced in the dorsal hippocampus (dHPC) of ENT1 –/– mice and that impulsive Pavlovian conditioning significantly increases ERK1/2 phosphorylation. Subsequently, we validate that inhibition of A 2A R promotes impulsivity during Pavlovian conditioning and the 5‐CSRTT. In addition to increased ERK1/2 phosphorylation, mice demonstrating exaggerated impulsivity as a result of A 2A R inhibition exhibit enhanced expression of doublecortin and increased incorporation of BrdU within the HPC. Taken together, these findings indicate that impaired ENT1/A 2A R signaling promotes impulsivity during cue‐induced conditioning and contributes to reward seeking during extinction. More importantly, these results demonstrate that impulsive behavior is associated with aberrant hippocampal neurogenesis, which may contribute to the development of maladaptive reward‐seeking behaviors. Support or Funding Information NIH R01 AA018779

Abstract Chemotherapy-induced cognitive impairment (CICI, also termed “chemobrain”) is a major neurotoxic side effect exhibited by a wide range of chemotherapeutic agents. These side effects affect approximately 14 million cancer survivors. More importantly, CICI persists well after cessation of therapy, severely interfering with quality of life. Unfortunately, there is no treatment and novel therapies are urgently needed. Utilizing cisplatin, a platinum-based chemotherapy, to model chemobrain mice in vivo, we have established that cisplatin accelerates the brain aging process thus leading to long-term memory impairment in mice similar to what is clinically reported. We further show that cisplatin causes oxidative DNA damage, mitochondrial defects, impaired neurogenesis, synaptic defects, and increased gliosis in the adult hippocampus, a brain region critical for learning and memory. Mechanistically, our RNA-sequencing and qRT-PCR analysis revealed that cisplatin dramatically increases COX-2 (Ptgs2) expression and its major product, prostaglandin E2 (PGE2) in adult mouse brain and human cortical neurons derived from induced pluripotent stem cells (iPSCs). Similar as cisplatin treatment, the levels of COX-2 expression began to increase at 0.1 µM methotrexate treatment, which significantly increased at 1 µM in human cortical neurons, indicating that COX-2 induction is a common pathogenic mechanism mediating cognitive impairment associated with cisplatin and methotrexate in spite of the fact that these compounds have different mechanisms of action for cancer eradication. Most importantly, NS-398, a selective COX-2 inhibitor, effectively prevents cognitive deficits in mice and reduction in cell viability of human neurons associated with these chemotherapies, without promoting tumor growth or interfering with cisplatin’s anti-tumor activity. Taken together, our findings strongly suggest that COX-2 induction is a main cause of CICI, making COX-2 inhibition a therapeutic target for CICI. Given that COX-2 inhibitors are currently being tested in cancer prevention as well as age-related memory loss in clinical trials, inhibiting COX-2 may have far-reaching therapeutic effects on CICI and cancer treatment, which can be applied quickly, safely, and effectively to clinical trials, in the effort to improve cancer survivor quality of life. Citation Format: Mohammad Abdur Rashid, Jason J. Tang, Ki-Hyun Yoo, Ana Corujo-Ramirez, Sang Hoon Kim, Alfredo Oliveros, Peter Cole, John R. Hawse, Mi-Hyeon Jang. Blockade of the cyclooxygenase-2 prevents chemotherapy-induced cognitive impairments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2694.

Background: Chemotherapy-induced cognitive impairment (CICI) is a neurotoxic side effect of chemotherapy that has yet to have an effective treatment. Objective: Using cisplatin, a platinum-based chemotherapy together with excitatory cortical neurons derived from human induced pluripotent cells (iPSCs) to model of CICI, our recent study demonstrated that dysregulation of brain NAD + metabolism contributes to cisplatin-induced impairments in neurogenesis and cognitive function, which was prevented by administration of the NAD + precursor, nicotinamide mononucleotide (NMN). However, it remains unclear how cisplatin causes neurogenic dysfunction and the mechanism by which NMN prevents cisplatin-induced cognitive impairment. Given that mitochondrial dysfunction is thought to play a prominent role in age-related neurodegenerative disease and chemotherapy-induced neurotoxicity, we sought to explore if NMN prevents chemotherapy-related neurotoxicity by attenuating cisplatin-induced mitochondrial damage. Results: We demonstrate that cisplatin induces neuronal DNA damage, increases generation of mitochondrial reactive oxygen species (ROS) and decreases ATP production, all of which are indicative of oxidative DNA damage and mitochondrial functional defects. Ultrastructural analysis revealed that cisplatin caused loss of cristae membrane integrity and matrix swelling in human cortical neurons. Notably, pretreatment with NMN prevents cisplatin-induced defects in mitochondria of human cortical neurons. Conclusion: Our results suggest that increased mitochondrial oxidative stress and functional defects play key roles in cisplatin-induced neurotoxicity. Thus, NMN may be an effective therapeutic strategy to prevent cisplatin-induced deleterious effects on mitochondria, making this organelle a key factor in amelioration of cisplatin-induced cognitive impairments.

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A 2A receptor (A 2A R) and its downstream effectors, cAMP and CREB, by cisplatin in the adult mouse hippocampus, a critical brain structure for learning and memory. Notably, A 2A R inhibition by the Food and Drug Administration–approved A 2A R antagonist KW-6002 prevented cisplatin-induced impairments in neural progenitor proliferation and dendrite morphogenesis of adult-born neurons, while improving memory and anxiety-like behavior, without affecting tumor growth or cisplatin’s antitumor activity. Collectively, our study identifies A 2A R signaling as a key pathway that can be therapeutically targeted to prevent cisplatin-induced cognitive impairments.

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. Significance: 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.

e23082 Background: The hippocampus is one of two regions in the adult brain where new neurons are generated well into adulthood. Newborn neurons develop from neural stem cells, and are consequently integrated into the hippocampal circuitry to function in learning and memory. Temozolomide (TMZ) is a commonly used alkylating agent for treatment of malignant gliomas. In this study, we examine the effects of TMZ on adult neurogenesis and cognitive function in the murine model. Methods: Adult male mice (8-week-old) were injected daily with TMZ (25 mg/kg, i.p.) for 2, 2.5 and 3.5 weeks to estimate progenitor proliferation, neuronal maturation and animal behavior respectively. For progenitor proliferation, a single dose of BrdU (50mg/kg, i.p.) was injected at 2 weeks and the mice sacrificed 2 hours later. For maturation analysis, five separate doses of EdU (41.1 mg/kg, i.p.) were injected over five days starting at 2 weeks. Three weeks after first EdU injection mice were sacrificed. Both BrdU and EdU are novel thymidine analogues that label dividing cells. Immunostaining was performed on hippocampal brain slices to label progenitors at different stages of development. For behavioral analysis, daily tests measuring anxiety, memory and motor activity were started at 2 weeks into TMZ administration. Results: TMZ significantly reduced the resident radial glia-like and non-radial glia-like population (MCM2+Nestin+), as well as the overall proliferating progenitor pool (n = 2, p < 0.05). In addition, it significantly reduced the population of Type-3 neuroblasts (EdU+DCX+NeuN-) (n = 4, p < 0.05). During neuronal maturation, immature new neurons (EdU+DCX+NeuN+) and mature new neurons (EdU+DCX-NeuN+) were also reduced by TMZ (n = 4, p < 0.05). These effects of TMZ coincided with specific behavioral deficits. While TMZ had no effect on working memory and locomotor activity, mice that underwent TMZ administration showed significantly increased anxiety on the open field test (n = 7, p < 0.05). Conclusions: Our findings demonstrate TMZ significantly impairs cellular proliferation of neural stem cells and fate-committed neuroblasts, and increases anxiety with sparing of non-hippocampal-dependent memory. TMZ may thus be associated with specific deficits in hippocampal function in the adult brain, providing mechanistic insight into adverse effects that may be observed with TMZ treatment.

Abstract BUB-related 1 (BubR1) encoded by Budding Uninhibited by Benzimidazole 1B (BUB1B) is a crucial mitotic checkpoint protein ensuring proper segregation of chromosomes during mitosis. Mutations of BUB1B are responsible for mosaic variegated aneuploidy (MVA), a human congenital disorder characterized by extensive abnormalities in chromosome number. Although microcephaly is a prominent feature of MVA carrying the BUB1B mutation, how BubR1 deficiency disturbs neural progenitor proliferation and neuronal output and leads to microcephaly is unknown. Here we show that conditional loss of BubR1 in mouse cerebral cortex recapitulates microcephaly. BubR1-deficient cortex includes a strikingly reduced number of late-born, but not of early-born, neurons, although BubR1 expression is substantially reduced from an early stage. Importantly, absence of BubR1 decreases the proportion of neural progenitors in mitosis, specifically in metaphase, suggesting shortened mitosis owing to premature chromosome segregation. In the BubR1 mutant, massive apoptotic cell death, which is likely due to the compromised genomic integrity that results from aberrant mitosis, depletes progenitors and neurons during neurogenesis. There is no apparent alteration in centrosome number, spindle formation or primary cilia, suggesting that the major effect of BubR1 deficiency on neural progenitors is to impair the mitotic checkpoint. This finding highlights the importance of the mitotic checkpoint in the pathogenesis of microcephaly. Furthermore, the ependymal cell layer does not form in the conditional knockout, revealing an unrecognized role of BubR1 in assuring the integrity of the ventricular system, which may account for the presence of hydrocephalus in some patients.

AbstractWnt signaling is a well‐known molecular pathway in age‐related pathogenesis and therapy of disease. While prior studies have mainly focused on Wnt ligands or Wnt activators, the in vivo functions of naturally secreted Wnt inhibitors are not clear, especially in brain aging. Using BubR1H/H mice as a novel mouse model of accelerated aging, we report that genetic inhibition of sFRP3 restores the reduced body and brain size observed in BubR1H/H mice. Furthermore, sFRP3 inhibition ameliorates hypomyelination in the corpus callosum and rescues neural progenitor proliferation in the hippocampal dentate gyrus of BubR1H/H mice. Taken together, our study identifies sFRP3 as a new molecular factor that cooperates with BubR1 function to regulate brain development, myelination, and hippocampal neurogenesis.