Francis M. Amara

TGF-β1 is increased in a transgenic mouse model of familial Alzheimer's disease and causes neuronal apoptosis

Alzheimers disease (AD) is a neurodegenerative disorder, due to excess amyloid-beta peptide (Abeta). TGF-beta1 and beta-catenin signaling pathways have been separately implicated in modulating Abeta-neurotoxicity. However, the underlying mechanisms remain unclear. Here, we report that TGF-beta1 and nuclear Smad7 and beta-catenin levels were markedly upregulated in cortical brain regions of the TgCRND8 mice, a mouse model of familial Alzheimers disease. Coimmunoprecipitation of cortical brain tissue lysates revealed an interaction between Smad7 and beta-catenin. This interaction which was significantly enhanced in the TgCRND8 mice was also associated with an increase in TCF/LEF DNA-shift binding activity. TCF/LEF reporter gene activity was significantly increased in mouse primary cortical neuronal cultures (MCN) from the TgCRND8 mice, compared to controls. Interestingly, exposure of MCN to Abeta(1-42) led to an increase in TGF-beta1 and nuclear levels of both beta-catenin and Smad7. Furthermore, addition of TGF-beta1 to the MCN caused an increase in apoptosis and Smad7 levels. When Smad7 or beta-catenin levels were reduced by siRNA, TGF-beta1-induced apoptosis was suppressed, indicating that both Smad7 and beta-catenin are required for TGF-beta1-induced neurotoxicity. Since Abeta(1-42)-induced TGF-beta1, we suggest that TGF-beta1 may amplify Abeta(1-42)-mediated neurodegeneration in AD via Smad7 and beta-catenin interaction and nuclear localization.

Lovastatin protects human neurons against Aβ-induced toxicity and causes activation of β-catenin–TCF/LEF signaling

Abstract Alzheimers disease (AD) is characterized by cognitive decline due to excess amyloid beta peptide (Aβ), neurofibrillary tangles, and neuronal loss. Aβ promotes neuronal apoptosis in AD by activating glycogen synthase kinase-3β (GSK-3β), leading to degradation of β-catenin and inactivation of Wnt signaling. β-Catenin interacts with the T-cell factor (TCF)/Lymphoid enhancer factor (LEF)-nuclear complex to mediate Wnt signaling and cell survival. Statins are associated with decreased prevalence of AD. Lovastatin has been shown to decrease the production of Aβ and to promote neuronal survival. The mechanisms of how statins promote neuronal survival are unclear. We propose that the neuroprotective effect of lovastatin may be due to inactivation of GSK-3β activity, resulting in induction of Wnt signaling. Here, we report that lovastatin prevented Aβ-induced apoptosis in human SK-NSH cells. This was accompanied by reduction in active GSK-3β, and increased nuclear translocation of β-catenin, TCF-3, and LEF-1. Lovastatin treatment induced an increase in TCF/LEF–chloramphenicol acetyl transferase (CAT) gene reporter activity. More importantly, β-catenin and TCF were required for the neuroprotective function of lovastatin. Our results suggest that lovastatin protects neuronal cells from Aβ-induced apoptosis and causes reduction in GSK-3β activity, resulting in activation of Wnt signaling.

Anti-apoptotic wild-type Alzheimer amyloid precursor protein signaling involves the p38 mitogen-activated protein kinase/MEF2 pathway

Alzheimer amyloid precursor protein (APP) effectively protects against apoptosis in neuronal cells under stress, but the mechanisms of this anti-apoptotic effect remain largely unknown. Transcription factors act as critical molecular switches in promoting neuronal survival. The myocyte enhancer factor-2 (MEF2) is a transcription factor, and is known to be necessary for neurogenesis and activity-dependent neuronal survival. This study examined the possible role of MEF2 in the anti-apoptotic signaling pathways activated by APP. We report that expression of wild-type human APP (hAPPwt) but not familial Alzheimers disease mutant APP (FAD-hAPPmut) in APP-deficient rat B103 cells led to a significant increase in the level of phosphorylated MEF2. This differential phosphorylation was dependent on enhanced activation of p38 mitogen-activated protein kinase (p38 MAPK). Also, expression of hAPPwt mediated an increase in MEF2 DNA binding affinity that correlated with p38 MAPK-dependent trans-activation of a MEF2-responsive reporter gene. Furthermore, over-expression of dominant negative MEF2 in hAPPwt-expressing cells enhanced staurosporine-induced apoptosis, in contrast MEF2wt enhanced the capacity of hAPPwt to confer resistance to apoptosis. Thus, MEF2 plays a critical role in APP-mediated signaling pathways that inhibit neuronal apoptosis. A model of anti-apoptotic APP signaling is proposed where APP mediates p38 MAPK-dependent phosphorylation and activation of MEF2. Once activated MEF2 regulates neuronal survival by stimulation of MEF2-dependent gene transcriptions. Alteration of this function by mutations in APP and aberrant APP processing could contribute to neuronal degeneration seen in AD.

Defining a Novel cis-Element in the 3′-Untranslated Region of Mammalian Ribonucleotide Reductase Component R2 mRNA cis-trans-INTERACTIONS AND MESSAGE STABILITY

Mammalian ribonucleotide reductase is a highly regulated activity essential for DNA synthesis and repair. The 3′-untranslated region (3′-UTR) of mammalian ribonucleotide reductase R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3′-UTR contains regulatory information for R2 mRNA turnover. Using 3′-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3′-UTR a novel 9-nucleotide cis-element, 5′-UCGUGUGCU-3′, which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G → A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for ribonucleotide reductase gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.

The role of transforming growth factor beta signaling in messenger RNA stability

Transforming growth factor beta (TGF-β) is a biologically multipotent regulatory protein implicated in functions that include the regulation of cellular growth, differentiation, extracellular matrix formation, and wound healing. It also plays a role in the pathologies of Alzheimers disease, cancer and autoimmune disorders. TGF-β modulates gene expression by affecting transcriptional activation and mRNA turnover rate. Steady-state mRNA levels depend on both the transcriptional activity and mRNA half-life. The stability of mRNA can be modified by the binding of trans-acting factors to cis-elements on the message. These can protect the mRNA from cleavage by RNAses, or they may promote mRNA cleavage. Changes in mRNA stability can lead to changes in the proteome and subsequently in cellular metabolism. The SMAD family of proteins has been implicated in the transduction of the TGF-β signal, where they regulate transcriptional activity. This review attempts to provide new insights into the role played by TGF-β in the regulation of mRNA turnover.

Myogenic signaling by lithium in cardiomyoblasts is Akt independent but requires activation of the β-catenin-Tcf/Lef pathway

Rat H9c2 cardiomyoblasts can proliferate and maintain an undifferentiated state in the presence of serum. These cardiomyoblasts have been used as a cellular model to study myogenic differentiation after serum withdrawal. Here, we examined the effects of lithium, a known inhibitor of glycogen synthase kinase-3beta and activator of Wnt pathway in myogenic differentiation. We show that in the presence of serum, lithium induced the differentiation of H9c2 cells as measured by multinucleated myotube formation and expression of the muscle-specific proteins, myogenin and skeletal alpha-actin. This differentiation was preceded by nuclear accumulation of beta-catenin, which was associated with increased Tcf/Lef-dependent transcription. We also observed that lithium mediated the activation of phosphatidylinositol 3-kinase (PI3-kinase) and its downstream target Akt. Inhibition of PI3-kinase by LY294002 and over-expression of dominant-negative PI3-kinase caused a marked reduction in beta-catenin levels. This inhibition was associated with decreased beta-catenin-Tcf/Lef-dependent transcription, lack of multinucleated myotube formation, and expression of the muscle-specific proteins. In contrast, expression of dominant-negative Akt failed to inhibit the effects of lithium. We conclude that the capacity of lithium to overcome the inhibitory effects of serum and to induce the differentiation of H9c2 cardiomyoblasts is mediated, in part, by the stabilization and nuclear translocation of beta-catenin in a PI3-kinase-dependent but Akt-independent manner. Once activated, beta-catenin then interacts with the Lef/Tcf complex to regulate expression of myogenic-inducing genes.

Theories and Practical Steps for Delivering Effective Lectures

Several approaches to teaching, such as problem-based learning, team-based learning and case method teachings are increasingly being adopted. However, the lecture format is still the most widely used approach to teaching, especially for a large class size. Nevertheless, traditional lectures or didactic lectures are considered ineffective in affecting learning outcomes of knowledge retention, student satisfaction, synthesis and elaboration of knowledge. Consequently, new strategies to transform didactic lectures into effective lectures, and to facilitate deeper learning are emerging due to recent advances in our understanding of the cognitive sciences on learning and memory. These advances can be applied to teaching to diverse learners and across different settings in the health professions education. They can also be used as a guide to faculty development, organization of lectures and curriculum development. In this article, we reviewed selected principles of learning and memory to determine those that are most critical to improving didactic lectures, guiding instructors to effective teaching, and deeper student learning. Our analyses of these principles have produced key practical steps that are essential to enhancing lectures, making them interactive and effective. Further, these steps can be adopted across different learning environments of health education.

Neuroprotective Role of Statins in Alzheimer`s Disease: Anti-Apoptotic Signaling~!2009-02-06~!2010-04-04~!2010-06-22~!

Alzheimers disease (AD), a severe form of senile dementia is a neurodegenerative disorder. One of the most well characterized hallmarks of AD are extra-neuronal aggregates of amyloid-beta peptide (A� ), known as amyloid plaques. Recent epidemiological studies suggest a link between statin intake, and a lowered incidence of AD. Statins are 3-hydroxy-3-methylglutaryl co-enzyme reductase (HMG) inhibitors, which are one of the most commonly prescribed drug groups used to lower serum cholesterol levels in patients with heart disease. Some of the pleiotropic effects of statins which are gaining attention are its ability to reduce Aproduction and deposition, inhibit caspase-3 mediated apoptosis, and demonstrate anti-inflammatory properties by reducing interleukin-6 (IL-6) levels. The molecular mechanisms respon- sible for the pleiotropic effects of statins in promoting neuronal survival are not fully understood. Our own research has shown that statins promote anti-apoptotic responses against A� -neurotoxicity through � -catenin-TCF/LEF signaling how- ever, other anti-apoptotic statin mediated signaling pathways may also be involved. This review will describe AD patho- genesis, Aproduction, and the role of statins in mitigating these effects.

A Canadian Medical School in Partnership with an Inner City School Division and Community Organization to Promote Interest in Science to Aboriginal and is advantaged Youth: Plugging the First Leakage in the Medical Pipeline

The Biomedical Youth Program and the Inner City Science Centre are the main components of a partnership between the Faculty of Medicine, University of Manitoba, Winnipeg School Division, and the Winnipeg Foundation. This partnership serves the inner city elementary schools that have a predominant population of aboriginal youth. It aims to promote diversity in health professions education, which have underrepresentation of aboriginal and inner city youth. Although aboriginal people represent 4% of the Canadian population, they make up less than 0.25% of the physician workforce. The partnership has three main goals: to provide professional development for teachers; mentor students to build their knowledge, motivation, and confidence in science; and to promote teachers’, students’, and parents’ interests and excitement in the sciences and health professions careers. The partnership’s activities include workshops for teachers to teach science from a lecture-based format into a problem-based curriculum and to develop their leadership skills in order to positively influence science education. Since 2006 approximately 4035 elementary pupils, of whom 43% are aboriginal, have participated in the activities. Evaluations provide evidence of the activities’ effectiveness in promoting teachers’ and students’ interest in science and the health professions, and significant gain in students’ achievements. 75% of the aboriginal participants are now in high schools, and 46% of them have gone on to participate in science fairs, with a success rate of 37% medal awards. 27% of the teachers have been recognized as science consultants, and appointed as site facilitators. These facilitators are now providing workshops for other teachers. Thus, enhancing science education early on in elementary school rather than in high school is likely to stop the leakage in the medical pipeline.