Jane E. Cavanaugh

The role of ERK1, 2, and 5 in dopamine neuron survival during aging

Extracellular signal-regulated kinases (ERKs) 1, 2, and 5 have been shown to play distinct roles in proliferation, differentiation, and neuronal viability. In this study, we examined ERK1, 2, and 5 expression and activation in the substantia nigra (SN), striatum (STR), and ventral tegmental area (VTA) during aging. An age-related decrease in phosphorylated ERK5 was observed in the SN and STR, whereas an increase in total ERK1 was observed in all 3 regions. In primary cultures of the SN and VTA, inhibition of ERK5 but not ERK1 and 2 decreased dopamine neuronal viability significantly. These data suggest that ERK5 is essential for the basal survival of SN and VTA dopaminergic neurons. This is the first study to examine ERK1, 2, and 5 expression and activation in the SN, STR, and VTA during aging, and the relative roles of ERK1, 2, and 5 in basal survival of SN and VTA dopaminergic neurons. These data raise the possibility that a decline in ERK5 signaling may play a role in age-related impairments in dopaminergic function.

Dietary supplementation with resveratrol protects against striatal dopaminergic deficits produced by in utero LPS exposure

The purpose of this study was to determine the effect of dietary supplementation with the anti-inflammatory compound resveratrol in pregnant dams on lipopolysaccharide (LPS)-induced dopaminergic deficits in pups exposed to LPS in utero. Gravid female rats were fed with a resveratrol-enriched diet during gestational days 3-22.5 (E3-E22.5) and received an intraperitoneal (i.p.) injection of 1mg/kg LPS at E10.5. The striata were isolated from the pups at postnatal days 10 (P10) and P21. LPS-induced dopaminergic deficits were noted at P21, but not P10. These DA deficits at P21 were exhibited by a loss of DA and DA metabolite [3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA)] levels and tyrosine hydroxylase (TH) expression in the striatum. The LPS-induced loss of DA, DA metabolites, and TH expression were attenuated in the striata of pups from the dams fed with the resveratrol-supplemented diet. These data suggest that a resveratrol-supplemented diet may restore homeostasis of the striatal DA neuronal system following disruption by LPS.

Identification of benzimidazole-based inhibitors of the mitogen activated kinase-5 signaling pathway

The MEK-signaling pathways are complex but critical signaling cascades that correlate an extracellular signaling event with internal cell processes. To date at least seven MEK isozymes have been identified. MEK5, in particular, is upregulated in multiple forms of tumors. Analysis of the EGF-induced MEK5 signaling cascade in cultured HEK cells has identified compounds that can inhibit MEK5 phosphorylation of ERK5; observed biological activity is dependent on chemical variation.

Loss of motor coordination in an aging mouse model

With age, there is an increase in motor deficits that leads to an increased incidence of slips and falls. As the elderly population continues to grow, there is a need for aging models and research that focus on behavioral deficits that occur with normal, non-diseased aging. The present study was designed to examine the appropriateness of C57Bl/6 male mice as aging animal models using the challenging beam and cylinder tests to measure motor coordination and spontaneous activity, respectively. Using young (2-4 mo), middle-aged (10-12 mo), and aged (22-24 mo) mice, we observed that aged C57Bl/6 male mice make more errors on the challenging beam task and take fewer hind limb steps as compared to young and middle-aged mice. Body weight and food intake were also measured to determine if these parameters were confounding factors in the interpretation of the behavioral data. Increases in body weight and food consumption were not observed in the oldest group that made the most errors. Together these data indicate that aged C57BL/6 mice display age-related motor deficits similar to those seen in humans and are an appropriate model of motor deficits that occur with age.

ERK1, 2, and 5 expression and activation in dopaminergic brain regions during postnatal development.

Degeneration and dysfunctioning of dopaminergic neurons in the midbrain have been associated with serious neurodegenerative and neuropsychiatric disorders. Elucidating the underlying neurobiology of these neurons during early postnatal development may provide important information regarding the etiology of these disorders. Cellular signaling pathways have been shown to regulate postnatal neuronal development. Among several signaling pathways, extracellular‐regulated mitogen kinases (ERK) 1, 2, and 5 have been shown to be crucial for the survival and function of dopaminergic neurons. In this study, the basal expression and activation of ERK1, 2, and 5 were studied during postnatal development in regions rich in DA cells and terminals. In the striatum (STR) and ventral mesencephalon regions of the substantia nigra (SN) and ventral tegmental area (VTA), ERK5 expression and activation were high during early postnatal days and declined with aging. Interestingly, sharp increases in phosphorylated or activated ERK1 and ERK2 were observed at postnatal day (PND) 7 in the SN and VTA. In contrast, in the STR, the levels of phosphorylated ERK1 and 2 were significantly higher at PND0 than at any other PND examined. Overall, the understanding of alterations in ERK signaling in regions rich in DA cells and DA terminals during postnatal neuronal development may provide information about their role in regulation of dopamine neuronal development which may ultimately provide insight into the underlying mechanisms of dopamine neurodegeneration.

Oncogenic signaling of MEK5-ERK5

Mitogen-activated protein kinases (MAPKs) regulate diverse cellular processes including proliferation, cell survival, differentiation, and apoptosis. While conventional MAPK constituents have well-defined roles in oncogenesis, the MEK5 pathway has only recently emerged in cancer research. In this review, we consider the MEK5 signaling cascade, focusing specifically on its involvement in drug resistance and regulation of aggressive cancer phenotypes. Moreover, we explore the role of MEK5/ERK5 in tumorigenesis and metastatic progression, discussing the discrepancies in preclinical studies and assessing its viability as a therapeutic target for anti-cancer agents.

Structure–activity relationships of benzimidazole-based selective inhibitors of the mitogen activated kinase-5 signaling pathway

In a prior communication we identified a novel class of benzimidazole-based inhibitors of EGF-induced phosphorylation of ERK5. In this paper we examine the biological activity of several 1-isopropyl-4-amino-6-ether linked benzimidazole-based compounds for their ability to selectively inhibit EGF-mediated ERK5 phosphorylation; potential utility of variation at the 6-position was indicated by the initial structural feature survey. Modification of EGF-induced formation of pERK1/2 and pERK5 in HEK293 cells were analyzed by Western blot analysis. Subsequent analysis of selected compounds in a high-throughput multiple kinase scan and the NCI 60-cell-line screen is presented.

Abstract 2242: The MAPK and PI3K signaling pathways in breast cancer: Crosstalk mechanisms and the effect on cell proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The purpose of the current study is to assess crosstalk between the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3-kinase (PI3K) pathways in breast cancer. More specifically, the goal is to investigate the interaction between the extracellular signal regulated kinases 1/2 and 5 (ERK1/2 and ERK5) and Akt and how these kinases act in concert to promote sustained proliferation in a breast cancer model. The MAPK and PI3K pathways mediate numerous cellular processes, including differentiation, growth, proliferation, and survival. To date, most related studies have focused on assessment of the crosstalk mechanisms between ERK1/2 and Akt, while the interplay of ERK5 has remained largely unstudied. Breast cancer patients with increased levels of ERK5 have been found to have a poorer prognosis than patients with lower levels, warranting further investigation of this kinase. The interaction between ERK5 and ERK1/2, as well as between ERK5 and Akt, and how these signaling mechanisms act in conjunction to promote cell proliferation in breast cancer cells remains to be elucidated. The BT-474 (ER+/PR+/HER2+) and MDA-MB-231 (ER-/PR-/HER2-) human breast cancer cell lines were utilized to examine ERK1/2, ERK5 and Akt activation following treatment with ERK and Akt inhibitors or siRNA for these kinases, respectively. Western blot analysis was performed to determine expression and activation of the kinases. An MTT assay was used to assess changes in cell proliferation following kinase inhibition for 12, 24, 48, 72 and 96 hours. In vivo studies utilizing the transgenic MMTV-neu mouse model were also performed. In the BT-474 cell line, inhibition of Akt resulted in increased ERK1/2 and ERK5 expression and activation. Interestingly, the increases in both total and phosphorylated ERK1/2 and ERK5 following Akt-inhibition were dose-dependent, meriting further study. Excised mammary tumors from our animal model demonstrated an age-dependent increase in both ERK1/2 and ERK5. Notably, an age-dependent decrease in Akt was also observed, supporting the hypothesis that crosstalk between these pathways may exist. Future studies involving dual kinase inhibition are planned to further assess crosstalk and proliferation. The data obtained from these studies indicate the presence of crosstalk between the ERK1/2, ERK5 and Akt pathways. A better understanding of this crosstalk may lead to patient-specific drug regimens to decrease the cytotoxic effects of chemotherapeutics and improve patient mortality. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2242. doi:1538-7445.AM2012-2242

Breaking Down Barriers to Pharmacy Graduate Education: The Report of the 2017-2018 Research and Graduate Affairs Committee

EXECUTIVE SUMMARY The 2017-2018 Research and Graduate Affairs Committee (RGAC) was given three charges aimed at helping academic pharmacy address barriers that must be overcome by both students and schools to attract, retain, and support the development of a diverse, well-rounded, and successful graduate student population. These charges were (1) identifying teaching methodologies, tools and opportunities that graduate programs can introduce into curriculum to overcome barriers to success of today’s and tomorrow’s learners; (2) developing a strategy for achieving member support of the 2016-2017 recommended graduate competencies by identifying gaps in and existing examples of courses or opportunities that achieve competency-based pharmacy graduate education; and (3) identifying potential strategies to address identified barriers to pursuing graduate education, especially among under-represented student populations. This report describes attitudes toward and opportunities related to competency-based education in graduation education in colleges and schools of pharmacy, identifies types of tools schools could use to enhance training towards the competency framework developed by the 2016-2017 RGAC, particularly with regards to the so-called power skills, and outlines a role for AACP in facilitating this training. This report also considers a number of barriers, both perceived and real, that potential students encounter when considering graduate training and suggests strategies to understand the impact of and mitigate these barriers. To strengthen competency-based graduate education, the RGAC puts forth two recommendations that AACP develop a toolkit supporting the training of power skills and that AACP should develop or curate programs or tools to support the use of individual development plans (IDPs). The RGAC also puts forth a suggestion to schools that IDPs be implemented for all students. In considering the barriers to pursuing graduate education, the Committee proposes one policy statement that AACP supports the training and development of an increasingly diverse population of researchers at pharmacy schools through active efforts to promote M.S. and Ph.D. education along with Pharm.D. education. Additionally, the Committee provides recommendations that AACP should expand its efforts in career tracking of graduate students to include collection and/or analysis of data that could inform the Academy’s understanding of barriers to pursuing graduate education in pharmacy schools, the AACP Office of Institutional Research and Effectiveness should expand upon graduate program data described in the annual Profile of Pharmacy Students report, and finally that AACP should include graduate programs in efforts to increase diversity of students at pharmacy schools.

Resveratrol and pinostilbene confer neuroprotection against aging-related deficits through an ERK1/2-dependent mechanism

Age-related declines in motor function may be due, in part, to an increase in oxidative stress in the aging brain leading to dopamine (DA) neuronal cell death. In this study, we examined the neuroprotective effects of natural antioxidants resveratrol and pinostilbene against age-related DAergic cell death and motor dysfunction using SH-SY5Y neuroblastoma cells and young, middle-aged, and old male C57BL/6 mice. Resveratrol and pinostilbene protected SH-SY5Y cells from a DA-induced decrease in cell viability. Dietary supplementation with resveratrol and pinostilbene inhibited the decline of motor function observed with age. While DA and its metabolites (DOPAC and HVA), dopamine transporter, and tyrosine hydroxylase levels remain unchanged during aging or treatment, resveratrol and pinostilbene increased ERK1/2 activation in vitro and in vivo in an age-dependent manner. Inhibition of ERK1/2 in SH-SY5Y cells decreased the protective effects of both compounds. These data suggest that resveratrol and pinostilbene alleviate age-related motor decline via the promotion of DA neuronal survival and activation of the ERK1/2 pathways.