Divergent Immediate and Delayed Effects of Juvenile Exposure to Doxorubicin on the Thymus in C57BL/6 Mice.

BACKGROUND: The understanding of alterations within the immune system following doxorubicin (DOX) chemotherapy, and subsequent restoration, in childhood cancer survivors remains limited. This investigation endeavors to elucidate the immediate and delayed changes in thymic immune cell populations and their phenotypes in response to clinically relevant low doses of DOX in a juvenile mouse model. METHODS: Male mice underwent a regimen of repeated low-dose DOX intraperitoneal injections at 4 mg/kg/week for three consecutive weeks. One week after the last dose of DOX, a subset of mice was euthanized to assess the immediate effects of DOX administration. A second subset of mice was euthanized five weeks after the last DOX dose to evaluate the delayed effects. Thymic samples were collected for multiparameter flow cytometry analysis to evaluate alterations in immune cell composition and phenotype. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to measure gene expression of- cytokines and senescence markers. RESULTS: One week following DOX administration, DOX treatment resulted in significant decline in thymus weight, with notable alterations in immune cell subpopulations. Reduced frequencies of mature CD3+CD4+ and CD3+CD8+ T cells were observed, along with changes in proliferation and exhaustion markers. Gene expression analysis revealed upregulation of Foxn, Pax1, Ifnγ, and Il7 alongside decreased Il6 and Il17 expression. Furthermore, Cdkn1a (p21Cip1) expression was elevated, suggesting immunosenescence. Five weeks following DOX administration, delayed effects of DOX treatment manifested in rebound increase in thymus weight and altered frequencies of CD4+ and CD8+ T cell subsets, with distinct patterns of proliferation and exhaustion observed. Notably, central memory CD4+ T cells exhibited significant decrease in frequency, while naive and effector memory CD4+ T cells showed reduced proliferation (Ki67+) and PD1 expression. Similar trends were observed in CD8+ T cell subsets, indicating selective effects of DOX on T cell differentiation and function. Although expression of thymus-related genes was normalized, p21Cip1 gene expression remained elevated. CONCLUSION: DOX treatment elicits a multifaceted influence on immune cell subsets and thymic weight. Immediate effects included thymic atrophy and reductions in mature T cell populations, while delayed effects showed rebound thymic hyperplasia and selective changes in CD4+ and CD8+ T cell subsets. Notably, both central memory and effector memory T cells exhibited reduced proliferation and exhaustion, suggesting unique impacts of DOX on immune cell function. The enduring elevation in p21Cip1 gene expression 5 weeks after DOX treatment suggests an immunosenescent phenotype. These observations collectively illuminate the formidable task of preserving immune competence and overall well-being in childhood cancer survivors subjected to DOX therapy.

Sex-related differences in delayed doxorubicin-induced cardiac dysfunction in C57BL/6 mice.

Cancer survivors may experience long-term cardiovascular complications due to chemotherapeutic drugs such as doxorubicin (DOX). The exact mechanism of delayed DOX-induced cardiotoxicity has not been fully elucidated. Sex is an important risk factor for DOX-induced cardiotoxicity. In the current study, we identified sex differences in delayed DOX-induced cardiotoxicity and determined the underlying molecular determinants of the observed sexual dimorphism. Five-week-old male and female mice were administered intraperitoneal injections of DOX (4 mg/kg/week) or saline for 6 weeks. Echocardiography was performed 5 weeks after the last dose of DOX to evaluate cardiac function. Thereafter, mice were sacrificed and gene expression of markers of apoptosis, senescence, and inflammation was measured by PCR in hearts and livers. Proteomic profiling of the heart from both sexes was conducted to determine differentially expressed proteins (DEPs). Only DOX-treated male, but not female, mice demonstrated cardiac dysfunction, cardiac atrophy, and upregulated cardiac expression of Nppb and Myh7. No sex-related differences were observed in DOX-induced expression of most apoptotic, senescence, and pro-inflammatory markers. However, the gene expression of Trp53 was significantly reduced in hearts of DOX-treated female mice only. The anti-inflammatory marker Il-10 was significantly reduced in hearts of DOX-treated male mice only, while the pro-inflammatory marker Il-1α was significantly reduced in livers of DOX-treated female mice only. Gene expression of Tnf-α was reduced in hearts of both DOX-treated male and female mice. Proteomic analysis identified several DEPs after DOX treatment in a sex-specific manner, including anti-inflammatory acute phase proteins. This is the first study to assess sex-specific proteomic changes in a mouse model of delayed DOX-induced cardiotoxicity. Our proteomic analysis identified several sexually dimorphic DEPs, many of which are associated with the anti-inflammatory marker Il-10.

Metformin mitigates SASP secretion and LPS-triggered hyper-inflammation in Doxorubicin-induced senescent endothelial cells.

Introduction: Doxorubicin (DOX), a chemotherapeutic drug, induces senescence and increases the secretion of senescence-associated secretory phenotype (SASP) in endothelial cells (ECs), which contributes to DOX-induced inflammaging. Metformin, an anti-diabetic drug, demonstrates senomorphic effects on different models of senescence. However, the effects of metformin on DOX-induced endothelial senescence have not been reported before. Senescent ECs exhibit a hyper-inflammatory response to lipopolysachharide (LPS). Therefore, in our current work, we identified the effects of metformin on DOX-induced endothelial senescence and LPS-induced hyper-inflammation in senescent ECs. Methods: ECs were treated with DOX ± metformin for 24 h followed by 72 h incubation without DOX to establish senescence. Effects of metformin on senescence markers expression, SA-ß-gal activity, and SASP secretion were assessed. To delineate the molecular mechanisms, the effects of metformin on major signaling pathways were determined. The effect of LPS ± metformin was determined by stimulating both senescent and non-senescent ECs with LPS for an additional 24 h. Results: Metformin corrected DOX-induced upregulation of senescence markers and decreased the secretion of SASP factors and adhesion molecules. These effects were associated with a significant inhibition of the JNK and NF-κB pathway. A significant hyper-inflammatory response to LPS was observed in DOX-induced senescent ECs compared to non-senescent ECs. Metformin blunted LPS-induced upregulation of pro-inflammatory SASP factors. Conclusion: Our study demonstrates that metformin mitigates DOX-induced endothelial senescence phenotype and ameliorates the hyper-inflammatory response to LPS. These findings suggest that metformin may protect against DOX-induced vascular aging and endothelial dysfunction and ameliorate infection-induced hyper-inflammation in DOX-treated cancer survivors.

Canagliflozin mitigates carfilzomib-induced endothelial apoptosis via an AMPK-dependent pathway.

Carfilzomib (CFZ) is a proteasome inhibitor approved for relapsed/refractory multiple myeloma (MM) but its clinical use is limited by cardiovascular toxicity. The mechanisms of CFZ-induced cardiovascular toxicity are not fully understood but endothelial dysfunction may be a common denominator. Here, we first characterized the direct toxic effects of CFZ on endothelial cells (HUVECs and EA.hy926 cells) and tested whether SGLT2 inhibitors, known to have cardioprotective effects, can protect against CFZ-induced toxicity. To determine the chemotherapeutic effect of CFZ in the presence of SGLT2 inhibitors, MM and lymphoma cells were treated with CFZ with or without canagliflozin. CFZ decreased cell viability and induced apoptotic cell death in endothelial cells in a concentration-dependent manner. CFZ also upregulated ICAM-1 and VCAM-1 and downregulated VEGFR-2. These effects were associated with the activation of Akt and MAPK pathways, inhibition of p70s6k, and downregulation of AMPK. Canagliflozin, but not empagliflozin or dapagliflozin, protected endothelial cells from CFZ-induced apoptosis. Mechanistically, canagliflozin abrogated CFZ-induced JNK activation and AMPK inhibition. AICAR (an AMPK activator) protected from CFZ-induced apoptosis, and compound C (an AMPK inhibitor) abrogated the protective effect of canagliflozin, strongly suggesting that AMPK mediates these effects. Canagliflozin did not interfere with the anticancer effect of CFZ in cancer cells. In conclusion, our findings demonstrate for the first time the direct toxic effects of CFZ in endothelial cells and the associated signaling changes. Canagliflozin abrogated the apoptotic effects of CFZ in endothelial cells in an AMPK-dependent mechanism, without interfering with its cytotoxicity in cancer cells.

Juvenile exposure to doxorubicin alters the cardiovascular response to adult-onset psychosocial stress in mice.

Childhood cancer survivors have a high risk for premature cardiovascular diseases, mainly due to cardiotoxic cancer treatments such as doxorubicin (DOX). Psychosocial stress is a significant cardiovascular risk factor and an enormous burden in childhood cancer survivors. Although observational studies suggest that psychosocial stress is associated with cardiovascular complications in cancer survivors, there is no translationally relevant animal model to study this interaction. We established a "two-hit" model in which juvenile mice were administered DOX (4 mg/kg/week for 3 weeks), paired to a validated model of chronic subordination stress (CSS) 5 weeks later upon reaching adulthood. Blood pressure, heart rate, and activity were monitored by radio-telemetry. At the end of CSS experiment, cardiac function was assessed by echocardiography. Cardiac fibrosis and inflammation were assessed by histopathologic analysis. Gene expressions of inflammatory and fibrotic markers were determined by PCR. Juvenile exposure to DOX followed by adult-onset CSS caused cardiac fibrosis and inflammation as evident by histopathologic findings and upregulated gene expression of multiple inflammatory and fibrotic markers. Intriguingly, juvenile exposure to DOX blunted CSS-induced hypertension but not CSS-induced tachycardia. There were no significant differences in cardiac function parameters among all groups, but juvenile exposure to DOX abrogated the hypertrophic response to CSS. In conclusion, we established a translationally relevant mouse model of juvenile DOX-induced cardiotoxicity that predisposes to adult-onset stress-induced adverse cardiac remodeling. Psychosocial stress should be taken into consideration in cardiovascular risk stratification of DOX-treated childhood cancer survivors.

EA.hy926 Cells and HUVECs Share Similar Senescence Phenotypes but Respond Differently to the Senolytic Drug ABT-263.

Doxorubicin (DOX) induces endothelial cell (EC) senescence, which contributes to endothelial dysfunction and cardiovascular complications. Senolytic drugs selectively eliminate senescent cells to ameliorate senescence-mediated pathologies. Previous studies have demonstrated differences between immortalized and primary EC models in some characteristics. However, the response of DOX-induced senescent ECs to senolytics has not been determined across these two models. In the present work, we first established a comparative characterization of DOX-induced senescence phenotypes in immortalized EA.hy926 endothelial-derived cells and primary human umbilical vein EC (HUVECs). Thereafter, we evaluated the senolytic activity of four senolytics across both ECs. Following the DOX treatment, both EA.hy926 and HUVECs shared similar senescence phenotypes characterized by upregulated senescence markers, increased SA-ß-gal activity, cell cycle arrest, and elevated expression of the senescence-associated secretory phenotype (SASP). The potentially senolytic drugs dasatinib, quercetin, and fisetin demonstrated a lack of selectivity against DOX-induced senescent EA.hy926 cells and HUVECs. However, ABT-263 (Navitoclax) selectively induced the apoptosis of DOX-induced senescent HUVECs but not EA.hy926 cells. Mechanistically, DOX-treated EA.hy926 cells and HUVECs demonstrated differential expression levels of the BCL-2 family proteins. In conclusion, both EA.hy926 cells and HUVECs demonstrate similar DOX-induced senescence phenotypes but they respond differently to ABT-263, presumably due to the different expression levels of BCL-2 family proteins.

Divergent Cardiac Effects of Angiotensin II and Isoproterenol Following Juvenile Exposure to Doxorubicin.

Hypertension is the most significant risk factor for heart failure in doxorubicin (DOX)-treated childhood cancer survivors. We previously developed a two-hit mouse model of juvenile DOX-induced latent cardiotoxicity that is exacerbated by adult-onset angiotensin II (ANGII)-induced hypertension. It is still not known how juvenile DOX-induced latent cardiotoxicity would predispose the heart to pathologic stimuli that do not cause hypertension. Our main objective is to determine the cardiac effects of ANGII (a hypertensive pathologic stimulus) and isoproterenol (ISO, a non-hypertensive pathologic stimulus) in adult mice pre-exposed to DOX as juveniles. Five-week-old male C57BL/6N mice were administered DOX (4 mg/kg/week) or saline for 3 weeks and then allowed to recover for 5 weeks. Thereafter, mice were administered either ANGII (1.4 mg/kg/day) or ISO (10 mg/kg/day) for 14 days. Juvenile exposure to DOX abrogated the hypertrophic response to both ANGII and ISO, while it failed to correct ANGII- and ISO-induced upregulation in the hypertrophic markers, ANP and BNP. ANGII, but not ISO, worsened cardiac function and exacerbated cardiac fibrosis in DOX-exposed mice as measured by echocardiography and histopathology, respectively. The adverse cardiac remodeling in the DOX/ANGII group was associated with a marked upregulation in several inflammatory and fibrotic markers and altered expression of Ace, a critical enzyme in the RAAS. In conclusion, juvenile exposure to DOX causes latent cardiotoxicity that predisposes the heart to a hypertensive pathologic stimulus (ANGII) more than a non-hypertensive stimulus (ISO), mirroring the clinical scenario of worse cardiovascular outcome in hypertensive childhood cancer survivors.

Doxorubicin Paradoxically Ameliorates Tumor-Induced Inflammation in Young Mice.

Doxorubicin (DOX) is one of the most widely used chemo-therapeutic agents in pediatric oncology. DOX elicits an inflammatory response in multiple organs, which contributes to DOX-induced adverse effects. Cancer itself causes inflammation leading to multiple pathologic conditions. The current study investigated the inflammatory response to DOX and tumors using an EL4-lymphoma, immunocompetent, juvenile mouse model. Four-week old male C57BL/6N mice were injected subcutaneously with EL4 lymphoma cells (5 × 104 cells/mouse) in the flank region, while tumor-free mice were injected with vehicle. Three days following tumor implantation, both tumor-free and tumor-bearing mice were injected intraperitoneally with either DOX (4 mg/kg/week) or saline for 3 weeks. One week after the last DOX injection, the mice were euthanized and the hearts, livers, kidneys, and serum were harvested. Gene expression and serum concentration of inflammatory markers were quantified using real-time PCR and ELISA, respectively. DOX treatment significantly suppressed tumor growth in tumor-bearing mice and caused significant cardiac atrophy in tumor-free and tumor-bearing mice. EL4 tumors elicited a strong inflammatory response in the heart, liver, and kidney. Strikingly, DOX treatment ameliorated tumor-induced inflammation paradoxical to the effect of DOX in tumor-free mice, demonstrating a widely divergent effect of DOX treatment in tumor-free versus tumor-bearing mice.

CYP1B1 as a therapeutic target in cardio-oncology.

Cardiovascular complications have been frequently reported in cancer patients and survivors, mainly because of various cardiotoxic cancer treatments. Despite the known cardiovascular toxic effects of these treatments, they are still clinically used because of their effectiveness as anti-cancer agents. In this review, we discuss the growing body of evidence suggesting that inhibition of the cytochrome P450 1B1 enzyme (CYP1B1) can be a promising therapeutic strategy that has the potential to prevent cancer treatment-induced cardiovascular complications without reducing their anti-cancer effects. CYP1B1 is an extrahepatic enzyme that is expressed in cardiovascular tissues and overexpressed in different types of cancers. A growing body of evidence is demonstrating a detrimental role of CYP1B1 in both cardiovascular diseases and cancer, via perturbed metabolism of endogenous compounds, production of carcinogenic metabolites, DNA adduct formation, and generation of reactive oxygen species (ROS). Several chemotherapeutic agents have been shown to induce CYP1B1 in cardiovascular and cancer cells, possibly via activating the Aryl hydrocarbon Receptor (AhR), ROS generation, and inflammatory cytokines. Induction of CYP1B1 is detrimental in many ways. First, it can induce or exacerbate cancer treatment-induced cardiovascular complications. Second, it may lead to significant chemo/radio-resistance, undermining both the safety and effectiveness of cancer treatments. Therefore, numerous preclinical studies demonstrate that inhibition of CYP1B1 protects against chemotherapy-induced cardiotoxicity and prevents chemo- and radio-resistance. Most of these studies have utilized phytochemicals to inhibit CYP1B1. Since phytochemicals have multiple targets, future studies are needed to discern the specific contribution of CYP1B1 to the cardioprotective and chemo/radio-sensitizing effects of these phytochemicals.

Sexual Dimorphism in Doxorubicin-induced Systemic Inflammation: Implications for Hepatic Cytochrome P450 Regulation.

Doxorubicin (DOX) is an effective chemotherapeutic agent used to treat a wide variety of malignancies. In addition to its multi-organ toxicity, DOX treatment has been shown to induce systemic inflammation in patients and experimental animals. Inflammation alters the expression of hepatic cytochrome P450 (CYP) enzymes, which play important roles in drug metabolism and DOX-induced toxicity. Significant sex differences have been reported in DOX-induced toxicity; however, sex differences in DOX-induced systemic inflammation and the potential effects on hepatic CYP expression have not been determined. In the current work, male and female C57Bl/6 mice were administered DOX (20 mg/kg by intraperitoneal injection), and groups of mice were sacrificed 24 and 72 h after DOX administration. DOX elicited a systemic inflammatory response in both male and female mice, but the inflammatory response was stronger in male mice. DOX altered the expression of hepatic CYP isoforms in a sex-dependent manner. Most notably, inhibition of Cyp2c29 and Cyp2e1 was stronger in male than in female mice, which paralleled the sex differences in systemic inflammation. Therefore, sex differences in DOX-induced systemic inflammation may lead to sexually dimorphic drug interactions, in addition to contributing to the previously reported sexual dimorphism in specific DOX-induced organ toxicity.

Leveraging the Cardio-Protective and Anticancer Properties of Resveratrol in Cardio-Oncology.

Cardio-oncology is a clinical/scientific discipline which aims to prevent and/or treat cardiovascular diseases in cancer patients. Although a large number of cancer treatments are known to cause cardiovascular toxicity, they are still widely used because they are highly effective. Unfortunately, therapeutic interventions to prevent and/or treat cancer treatment-induced cardiovascular toxicity have not been established yet. A major challenge for such interventions is to protect the cardiovascular system without compromising the therapeutic benefit of anticancer medications. Intriguingly, the polyphenolic natural compound resveratrol and its analogs have been shown in preclinical studies to protect against cancer treatment-induced cardiovascular toxicity. They have also been shown to possess significant anticancer properties on their own, and to enhance the anticancer effect of other cancer treatments. Thus, they hold significant promise to protect the cardiovascular system and fight the cancer at the same time. In this review, we will discuss the current knowledge regarding the cardio-protective and the anticancer properties of resveratrol and its analogs. Thereafter, we will discuss the challenges that face the clinical application of these agents. To conclude, we will highlight important gaps of knowledge and future research directions to accelerate the translation of these exciting preclinical findings to cancer patient care.

Sexual dimorphism of acute doxorubicin-induced nephrotoxicity in C57Bl/6 mice.

Doxorubicin (DOX) is a chemotherapeutic agent that has been reported to cause nephrotoxicity in rodent models and to a lesser degree in cancer patients. Female rodents have been shown to be protected against several features of DOX-induced nephrotoxicity. Nevertheless, the underlying mechanisms of this sexual dimorphism are not fully elucidated. Therefore, in the current study, we investigated the sex and time-dependent changes in pathological lesions as well as apoptotic and fibrotic markers in response to acute DOX-induced nephrotoxicity. We also determined the effect of acute DOX treatment on the renal expression of the sexually dimorphic enzyme, soluble epoxide hydrolase (sEH), since inhibition of sEH has been shown to protect against DOX-induced nephrotoxicity. Acute DOX-induced nephrotoxicity was induced by a single intra-peritoneal injection of 20 mg/kg DOX to male and female adult C57Bl/6 mice. The kidneys were isolated 1, 3 and 6 days after DOX administration. Histopathology assessment, gene expression of the apoptotic marker, BAX, protein expression of the fibrotic marker, transforming growth factor-ß (TGF-ß), and gene and protein expression of sEH were assessed. DOX administration caused more severe pathological lesions as well as higher induction of the apoptotic and fibrotic markers in kidneys of male than in female mice. Intriguingly, DOX inhibited sEH protein expression in kidneys of male mice sacrificed at 3 and 6 days following administration, suggesting that induction of sEH is not necessary for acute DOX-induced nephrotoxicity. However, DOX-induced inhibition of renal sEH in male mice may protect the kidney from further DOX-induced injury in a negative feedback mechanism. We also observed lower constitutive expressions of TGF-ß and sEH in the kidney of female mice which may contribute, at least in part, to sexual dimorphism of DOX-induced nephrotoxicity.

Co-administration of resveratrol with doxorubicin in young mice attenuates detrimental late-occurring cardiovascular changes.

Aims: Doxorubicin (DOX) is among the most effective chemotherapies used in paediatric cancer patients. However, the clinical utility of DOX is offset by its well-known cardiotoxicity, which often does not appear until later in life. Since hypertension significantly increases the risk of late-onset heart failure in childhood cancer survivors, we investigated whether juvenile DOX exposure impairs the ability to adapt to angiotensin II (Ang II)-induced hypertension later in life and tested a treatment that could prevent this. Methods and results: Five-week-old male mice were administered a low dose of DOX (4 mg/kg) or saline once a week for 3 weeks and then allowed to recover for 5 weeks. Following the 5-week recovery period, mice were infused with Ang II or saline for 2 weeks. In another cohort, mice were fed chow containing 0.4% resveratrol 1 week before, during, and 1 week after the DOX administrations. One week after the last DOX administration, p38 mitogen-activated protein kinase (MAPK) was activated in hearts of DOX-treated mice demonstrating molecular signs of cardiac stress; yet, there was no change in cardiac function between groups. However, DOX-treated mice failed to develop compensatory cardiac hypertrophy in response to Ang II-induced hypertension later in life. Of importance, mice receiving DOX with resveratrol co-administration displayed normalization in p38 MAPK activation in the heart and a restored capacity for cardiac hypertrophy in response to Ang II-induced hypertension. Conclusion: We have developed a juvenile mouse model of DOX-induced cardiotoxicity that displays no immediate overt physiological dysfunction; but, leads to an impaired ability of the heart to adapt to hypertension later in life. We also show that co-administration of resveratrol during DOX treatment was sufficient to normalize molecular markers of cardiotoxicity and restore the ability of the heart to undergo adaptive remodelling in response to hypertension later in life.

Sex-dependent alteration of cardiac cytochrome P450 gene expression by doxorubicin in C57Bl/6 mice.

BACKGROUND: There is inconclusive evidence about the role of sex as a risk factor for doxorubicin (DOX)-induced cardiotoxicity. Recent experimental studies have shown that adult female rats are protected against DOX-induced cardiotoxicity. However, the mechanisms of this sexual dimorphism are not fully elucidated. We have previously demonstrated that DOX alters the expression of several cytochrome P450 (CYP) enzymes in the hearts of male rats. Nevertheless, the sex-dependent effect of DOX on the expression of CYP enzymes is still not known. Therefore, in the present study, we determined the effect of acute DOX exposure on the expression of CYP genes in the hearts of both male and female C57Bl/6 mice. METHODS: Acute DOX cardiotoxicity was induced by a single intraperitoneal injection of 20 mg/kg DOX in male and female adult C57Bl/6 mice. Cardiac function was assessed 5 days after DOX exposure by trans-thoracic echocardiography. Mice were euthanized 1 day or 6 days after DOX or saline injection. Thereafter, the hearts were harvested and weighed. Heart sections were evaluated for pathological lesions. Total RNA was extracted and expression of natriuretic peptides, inflammatory and apoptotic markers, and CYP genes was measured by real-time PCR. RESULTS: Adult female C57Bl/6 mice were protected from acute DOX-induced cardiotoxicity as they show milder pathological lesions, less inflammation, and faster recovery from DOX-induced apoptosis and DOX-mediated inhibition of beta-type natriuretic peptide. Acute DOX exposure altered the gene expression of multiple CYP genes in a sex-dependent manner. In 24 h, DOX exposure caused male-specific induction of Cyp1b1 and female-specific induction of Cyp2c29 and Cyp2e1. CONCLUSIONS: Acute DOX exposure causes sex-dependent alteration of cardiac CYP gene expression. Since cardiac CYP enzymes metabolize several endogenous compounds to biologically active metabolites, sex-dependent alteration of CYP genes may play a role in the sexual dimorphism of acute DOX-induced cardiotoxicity.

Longitudinal testing of hippocampal plasticity reveals the onset and maintenance of endogenous human Aß-induced synaptic dysfunction in individual freely behaving pre-plaque transgenic rats: rapid reversal by anti-Aß agents.

Long before synaptic loss occurs in Alzheimer's disease significant harbingers of disease may be detected at the functional level. Here we examined if synaptic long-term potentiation is selectively disrupted prior to extracellular deposition of Aß in a very complete model of Alzheimer's disease amyloidosis, the McGill-R-Thy1-APP transgenic rat. Longitudinal studies in freely behaving animals revealed an age-dependent, relatively rapid-onset and persistent inhibition of long-term potentiation without a change in baseline synaptic transmission in the CA1 area of the hippocampus. Thus the ability of a standard 200 Hz conditioning protocol to induce significant NMDA receptor-dependent short- and long-term potentiation was lost at about 3.5 months of age and this deficit persisted for at least another 2-3 months, when plaques start to appear. Consistent with in vitro evidence for a causal role of a selective reduction in NMDA receptor-mediated synaptic currents, the deficit in synaptic plasticity in vivo was associated with a reduction in the synaptic burst response to the conditioning stimulation and was overcome using stronger 400 Hz stimulation. Moreover, intracerebroventricular treatment for 3 days with an N-terminally directed monoclonal anti- human Aß antibody, McSA1, transiently reversed the impairment of synaptic plasticity. Similar brief treatment with the BACE1 inhibitor LY2886721 or the γ-secretase inhibitor MRK-560 was found to have a comparable short-lived ameliorative effect when tracked in individual rats. These findings provide strong evidence that endogenously generated human Aß selectively disrupts the induction of long-term potentiation in a manner that enables potential therapeutic options to be assessed longitudinally at the pre-plaque stage of Alzheimer's disease amyloidosis.

Endogenous expression of nNOS protein in several neuronal cell lines.

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma x glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells.