Amalia M. Issa

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimers disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β‐peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ‐related peptides, under acute conditions, potently inhibit K+‐evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ‐mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high‐affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ1–40 (10−8M) potently inhibited veratridine‐evoked endogenous ACh release from rat hippocampal slices and also decreased the K+‐evoked release potentiated by the nitric oxide‐generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10−14 to 10−6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10−6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.

ETHICAL PERSPECTIVES ON PHARMACOGENOMIC PROFILING IN THE DRUG DEVELOPMENT PROCESS

Pharmacogenomics, which is a field that encompasses the study of genetic polymorphisms that underlie individual differences in drug response, is rapidly advancing. The potential for the widespread use of pharmacogenomics in the drug development process merits an examination of its fundamental impact on clinical-trial design and practice. This article provides a critical analysis of some of the issues that pertain to pharmacogenomics in the drug development process. In particular, four areas will be discussed: clinical-trial design; subject stratification; some new social risks; and economic concerns. Recommendations are offered for addressing the issues that are discussed and anticipating the regulatory needs for pharmacogenomics-based trials.

The Efficacy of Omega–3 Fatty Acids on Cognitive Function in Aging and Dementia: A Systematic Review

We systematically reviewed the published literature on the effects of omega–3 fatty acids on measures of cognitive function in normal aging, incidence and treatment of dementia. Computerized databases were searched for published literature to identify potentially relevant studies with the intent to conduct a meta-analysis. We screened 5,865 titles, reviewed 497 studies of which 49 underwent a detailed review, and found 5 studies that pertained to our objectives. We included controlled clinical trials and observational studies, including prospective cohort, case-control, and case series designs; we excluded case reports. We had no language restrictions. We abstracted data on the effects of omega–3 fatty acids and on study design, relevant outcomes, study population, source, type, amount, and duration of omega–3 fatty acid consumption, and parameters of methodological quality. A single cohort study has assessed the effects of omega–3 fatty acids on cognitive function with normal aging and found no association for fish or total omega–3 consumption. In four studies that assessed the effects of omega–3 fatty acids on incidence and treatment of dementia, a trend in favor of omega–3 fatty acids (fish and total omega–3 consumption) toward reducing risk of dementia and improving cognitive function was reported. The available data are insufficient to draw strong conclusions about the effects of omega–3 fatty acids on cognitive function in normal aging or on the incidence or treatment of dementia. However, limited evidence suggests a possible association between omega–3 fatty acids and reduced risk of dementia.

Ethical considerations in clinical pharmacogenomics research.

In recent years there have been unprecedented advances in our understanding of the involvement of genetic polymorphisms in the response to drug therapies. Polymorphisms have been identified that lead to variable patient responses to several medications including cardiovascular, psychiatric, anti-infective and analgesic therapies. The potential for the development of customized, genotype-based therapies is scientifically and clinically attractive. However, these developments, although bearing scientific promise, raise ethical concerns for the conduct of research with human subjects, particularly with respect to confidentiality, risk-benefit analysis, DNA-banking and pharmacoeconomic issues. This article discusses some of the ethical considerations that are related to the use of pharmacogenomics in clinical research protocols.

Cost effectiveness of gene expression profiling for early stage breast cancer: a decision-analytic model.

Gene expression profiling (GEP) is being used increasingly for risk stratification to identify women with lymph node‐negative, estrogen receptor‐positive, early stage breast cancer who are most likely to benefit from adjuvant chemotherapy. The authors of this report evaluated the cost effectiveness of recurrence score‐guided treatment using 2 commercially available GEP tests, Oncotype DX (Genomic Health, Redwood City, Calif) and MammaPrint (Agendia Inc., Irvine, Calif), from a third‐party payers perspective.

Drug Withdrawals in the United States: A Systematic Review of the Evidence and Analysis of Trends

There have been a number of highly publicized safety-based drug withdrawals in the United States in recent years. We conducted a review of drugs withdrawn since 1993 and examined trends in drug withdrawals. Our objective was to determine the frequency and characteristics of withdrawn drugs and trends since 1993, and to discuss the implications of the findings. We found that a mean of 1.5 drugs per year have been withdrawn since 1993, and that the number of withdrawals has not increased over time. However, some recent drug withdrawals have impacted large numbers of people. The rate of withdrawals alone is not an adequate measure of the status of drug safety in the US, and there is a serious dearth of data that can be used to examine the impact of drug withdrawals. Although drug withdrawals are an important issue to address, drug safety policies need to be developed within the broader context of drug safety and effectiveness. A comprehensive approach will be needed to address the improvement of drug safety. We propose improvements to the evidence base to increase drug safety and assess how new scientific evidence can be incorporated into drug safety efforts.

Assessing patient readiness for the clinical adoption of personalized medicine.

Background/Aims: Although pharmacogenomics-based diagnostics and therapeutics are increasingly being translated into personalized medicine applications, relatively little evidence exists about how novel pharmacogenomics-based technologies will be accepted and adopted by patients. It is important to understand the characteristics of genomic diagnostics and targeted therapeutics that might impact utilization or serve as barriers to adoption of these novel technologies in order to formulate appropriate policies and procedures. The objective of this study was to investigate patients’ understanding and knowledge of personalized medicine and the process of decision-making regarding pharmacogenomics testing and targeted therapeutics and to better understand how patients value receiving pharmacogenomics-based care. Methods: We conducted 4 focus groups with 8–10 individuals in each group with patients recruited from out-patient clinics at The Methodist Hospital in Houston, Tex., USA. Results: The use of genomic diagnostics and targeted therapeutics to facilitate personalized medicine has considerable support from patients. However, our data revealed that participants were concerned with issues surrounding privacy and confidentiality of genetic test results, particularly with respect to access of information by insurers, with potential costs of testing and issues related to accuracy of test results. Questions regarding willingness to pay revealed that patients would be more willing to pay out-of-pocket if the disease associated with pharmacogenomic testing for treatment was perceived to be high risk (e.g., colorectal cancer) versus a chronic condition that was perceived as lower risk (e.g., high cholesterol). Conclusion: As the personalized medicine approach is increasingly incorporated into health care, understanding patients’ needs and their readiness to adopt these novel technologies will become progressively more important for the development of appropriate health policies.

Effects of the Phosphatase Inhibitors Calyculin A and Okadaic Acid on Acetylcholine Synthesis and Content of Rat Hippocampal Formation

Abstract: The biochemical mechanisms involved in the regulation of acetylcholine (ACh) turnover are poorly understood. In the experiments reported here, we examined whether inhibition of the serine/threonine phosphatases 1 and 2A by calyculin A or okadaic acid alters ACh synthesis by rat hippocampal preparations. With hippocampal slices, calyculin A (50 nM) and okadaic acid (50 nM) reduced significantly (p < 0.01) the synthesis of [3H]ACh from [3H]choline. Both calyculin A and okadaic acid produced significant depletion of endogenous tissue ACh in a concentration‐dependent manner (p < 0.01). This depletion was not the result of a drug‐induced increase of spontaneous ACh release, which was not changed significantly (p > 0.7) by either drug. Choline acetyltransferase (ChAT) activity from tissue exposed to calyculin A or okadaic acid was reduced in a concentration‐dependent manner (p < 0.05), but these phosphatase inhibitors did not act directly on ChAT in vitro; i.e., enzymatic activity was not altered significantly (p > 0.4) in the presence of calyculin A or okadaic acid. Both high‐affinity and low‐affinity [3H]choline uptake by hippocampal synaptosomes were reduced significantly in a concentration‐dependent manner in the presence of calyculin A or okadaic acid; these agents reduced Vmax values for high‐ and low‐affinity choline uptake (p < 0.01) with no significant change in Km values (p > 0.1), indicating a noncompetitive inhibition. Taken together, these data suggest that phosphatase activity plays a role in presynaptic central cholinergic nerve terminal function, in particular in the modulation of ACh synthesis.

Apolipoprotein E genotyping for pharmacogenetic purposes in Alzheimer's disease: emerging ethical issues.

Objective: To present some of the ethical concerns pertaining to the anticipated use of apolipoprotein E genotyping in selecting therapy in Alzheimers disease. Method: We review studies that support the utility of apolipoprotein E (APO E) genotyping for predicting drug responsiveness along with the published consensus statements and position papers related to APO E genotyping. Ethical issues pertaining to the use of APO E genotyping for pharmacogenetic purposes have not yet been fully explored, and these are discussed. Results: This paper explores the bioethics surrounding the use of APO E genotyping for pharmacogenetic purposes. The rapidly increasing pace of clinical trials in Alheimers disease necessitates a critical examination of the evolving ethical issues. A framework for establishing guidelines is suggested. Conclusions: Clinical research trials for Alzheimers disease with a genotyping component will increasingly be influenced by and benefit from a serious analysis of the ethics emerging alongside the scientific and clinical advances.

Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation

Abstract : The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P3 fraction, but cytoplasmic ACh contained in the S3 fraction was not significantly affected. The P3 fraction is known to be heterogeneous ; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E‐F and G‐H) were not so affected. K+‐evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction Ds vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [3H]ACh from [3H]choline, the ratio of [3H]ACh in the S3 to P3 fractions was not much changed by exposure of tissue to calyculin A or okadaic acid ; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.