Blood biomarkers for memory: toward early detection of risk for Alzheimer disease, pharmacogenomics, and repurposed drugs.

Short-term memory dysfunction is a key early feature of Alzheimer's disease (AD). Psychiatric patients may be at higher risk for memory dysfunction and subsequent AD due to the negative effects of stress and depression on the brain. We carried out longitudinal within-subject studies in male and female psychiatric patients to discover blood gene expression biomarkers that track short term memory as measured by the retention measure in the Hopkins Verbal Learning Test. These biomarkers were subsequently prioritized with a convergent functional genomics approach using previous evidence in the field implicating them in AD. The top candidate biomarkers were then tested in an independent cohort for ability to predict state short-term memory, and trait future positive neuropsychological testing for cognitive impairment. The best overall evidence was for a series of new, as well as some previously known genes, which are now newly shown to have functional evidence in humans as blood biomarkers: RAB7A, NPC2, TGFB1, GAP43, ARSB, PER1, GUSB, and MAPT. Additional top blood biomarkers include GSK3B, PTGS2, APOE, BACE1, PSEN1, and TREM2, well known genes implicated in AD by previous brain and genetic studies, in humans and animal models, which serve as reassuring de facto positive controls for our whole-genome gene expression discovery approach. Biological pathway analyses implicate LXR/RXR activation, neuroinflammation, atherosclerosis signaling, and amyloid processing. Co-directionality of expression data provide new mechanistic insights that are consistent with a compensatory/scarring scenario for brain pathological changes. A majority of top biomarkers also have evidence for involvement in other psychiatric disorders, particularly stress, providing a molecular basis for clinical co-morbidity and for stress as an early precipitant/risk factor. Some of them are modulated by existing drugs, such as antidepressants, lithium and omega-3 fatty acids. Other drug and nutraceutical leads were identified through bioinformatic drug repurposing analyses (such as pioglitazone, levonorgestrel, salsolidine, ginkgolide A, and icariin). Our work contributes to the overall pathophysiological understanding of memory disorders and AD. It also opens new avenues for precision medicine- diagnostics (assement of risk) as well as early treatment (pharmacogenomically informed, personalized, and preventive).

Antibiofilm and anti-quorum sensing activities of eugenol and linalool from Ocimum tenuiflorum against Pseudomonas aeruginosa biofilm.

AIMS: The aim of this study is to determine the ability of two bioactive compounds, namely, eugenol and linalool, purified from leaves of Ocimum tenuiflorum for eradication of biofilm produced by Pseudomonas aeruginosa. METHODS AND RESULTS: The phytoextract of O. tenuiflorum (KT), a common ethno-botanical plant of India, was purified through high-performance liquid chromatography and was analysed using ultraviolet (UV) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Eugenol and linalool were found to be the most active amongst all phytocompounds present in phytoextract and showed a significant reduction in the viability of sessile cells of P. aeruginosa and the minimum revival after withdrawal of phyto-challenge. They could bring about notable reduction in the protein and carbohydrate content of exopolysaccharide of biofilm. Eugenol and linalool could affect the synthesis of quorum sensing (QS) proteins like LasA and LasB as well as virulence factors such as pyocyanin, and rhamnolipids, which seriously hamper the formation of biofilm. The biofilm framework was extremely affected by the phytocompounds through the reduction of protein and carbohydrate content of extracellular polymeric substance (EPS). Another interesting found out was that they brought about maximum inhibition to the genomic DNA and RNA content. The studies were supported by in silico interaction between eugenol and linalool with the QS proteins. The antibiofilm efficacies of eugenol, linalool and phytoextract (KT) were further confirmed by microscopic studies with scanning electron microscopy (SEM), atomic force microscopy and fluorescence confocal microscopy microscopic studies. CONCLUSIONS: The phytocompounds are proved to be more effective than conventional antibiotics in inhibiting the biofilm forming sessile cells and can be used as a replacement for antibiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: Pure eugenol extracted from common basil leaves can be used as a safe substitute for common antibiotic for treatment of chronic infections caused by P. aeruginosa. It will be cost effective, devoid of notable side effects and will not generate antibiotic resistance in host body.

Novel roles of amyloid-beta precursor protein metabolites in fragile X syndrome and autism.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and is associated with up to 5% of autism cases. Several promising drugs are in preclinical testing for FXS; however, bench-to-bedside plans for the clinic are severely limited due to lack of validated biomarkers and outcome measures. Published work from our laboratories has demonstrated altered levels of amyloid-beta (Aß) precursor protein (APP) and its metabolites in FXS and idiopathic autism. Westmark and colleagues have focused on ß-secretase (amyloidogenic) processing and the accumulation of Aß peptides in adult FXS models, whereas Lahiri and Sokol have studied α-secretase (non-amyloidogenic or anabolic) processing and altered levels of sAPPα and Aß in pediatric autism and FXS. Thus, our groups have hypothesized a pivotal role for these Alzheimer's disease (AD)-related proteins in the neurodevelopmental disorders of FXS and autism. In this review, we discuss the contribution of APP metabolites to FXS and autism pathogenesis as well as the potential use of these metabolites as blood-based biomarkers and therapeutic targets. Our future focus is to identify key underlying mechanisms through which APP metabolites contribute to FXS and autism condition-to-disease pathology. Positive outcomes will support utilizing APP metabolites as blood-based biomarkers in clinical trials as well as testing drugs that modulate APP processing as potential disease therapeutics. Our studies to understand the role of APP metabolites in developmental conditions such as FXS and autism are a quantum leap for the neuroscience field, which has traditionally restricted any role of APP to AD and aging.

Obstructive sleep apnea induced bilateral tonic- clonic seizure of unknown origin: A case series of a novel association.

Background: Obstructive sleep apnea is a highly prevalent disorder, characterized by recurrent events of upper airway obstruction during sleep and associated with recurrent cycles of desaturation and re-oxygenation, sympathetic hyperactivity, and intra-thoracic pressure fluctuations, resulting in fragmentation of sleep and subsequent daytime fatigue with excessive sleepiness. Obstructive sleep apnea-induced bilateral tonic-clonic seizures are unheard of. We aimed to report 3 patients with previously undiagnosed obstructive sleep apnea who presented to the emergency department with new onset bilateral tonic-clonic seizure without any evidential neurological or metabolic cause. Methods: Patient data were obtained from medical records from the Department of Internal Medicine, IPGMER and SSKM Hospital, Kolkata, and Belle Vue Clinic, Kolkata, India. Results: Three male patients (67, 58, and 44 years old) presented with bilateral tonic-clonic seizure disorder without any underlying cause of seizures after rigorous investigations except for moderate to severe obstructive sleep apnea on polysomnography. All 2 patients were seizure-free after being treated with levetiracetam, chronic continuous positive airway pressure therapy in 2, and only continuous positive airway pressure in the other. The patients remained seizure-free on continuous positive airway pressure, even when levetiracetam was withdrawn, suggesting obstructive sleep apnea's causality in their new-onset acute seizures. Conclusion: Although further investigation is required to clarify this association, underlying obstructive sleep apnea should be ruled out in patients with a first-ever bilateral tonic-clonic seizure. Whether or not continuous positive airway pressure alone could effectively treat hypoxia and deranged cortical excitability, which may lead to seizures in cases with longstanding obstructive sleep apnea, is yet to be explored.

Introducción: La apnea obstructiva del sueño es una enfermedad con una alta prevalencia que se caracteriza por episodios recurrentes de obstrucción de las vías respiratorias altas durante el sueño, lo que conlleva ciclos repetidos de hipoxia y reoxigenación, hiperactividad simpática y fluctuaciones en la presión intratorácica. Todos estos procesos dan lugar a una fragmentación del sueño, lo que provoca fatiga diurna y somnolencia excesiva. Las crisis tónico-clónicas bilaterales inducidas por apnea obstructiva del sueño son poco conocidas. Presentamos los casos de tres pacientes con apnea obstructiva del sueño sin diagnosticar previamente que acudieron a urgencias por crisis tónico-clónicas de nueva aparición sin causa neurológica o metabólica aparente. Métodos: Los datos de nuestros pacientes se recogieron de los historiales médicos del servicio de Medicina Interna del Institute of Post-Graduate Medical Education and Research and Seth Sukhlal Karnani Memorial Hospital y de la Belle Vue Clinic, ambos en Kolkata (India). Resultados: Tres pacientes varones de 67, 58 y 44 años de edad presentaron convulsiones tónico-clónicas bilaterales sin causa identificada tras examen riguroso, exceptuando una apnea obstructiva del sueño de gravedad moderada a grave observada en la polisomnografía. Los tres pacientes recibieron tratamiento con levetiracetam durante el ingreso; al alta, se pautó tratamiento crónico con presión positiva continua de las vías respiratorias más levetiracetam en dos pacientes, y en el tercero solo presión positiva continua de las vías respiratorias. Ninguno presentó nuevas crisis tras la retirada de levetiracetam, lo que sugiere que la causa de las convulsiones era la apnea obstructiva del sueño. Conclusión: Aunque es necesario realizar más estudios para aclarar esta asociación, debemos descartar la apnea obstructiva del sueño en pacientes con crisis tónico-clónicas bilaterales de nueva aparición. Queda aún por determinar si la presión positiva continua podría tratar de forma efectiva la hipoxia y las alteraciones en la excitabilidad cortical, que podrían provocar crisis en casos de apnea obstructiva del sueño de larga evolución.

Identification of blood biomarkers for psychosis using convergent functional genomics.

There are to date no objective clinical laboratory blood tests for psychotic disease states. We provide proof of principle for a convergent functional genomics (CFG) approach to help identify and prioritize blood biomarkers for two key psychotic symptoms, one sensory (hallucinations) and one cognitive (delusions). We used gene expression profiling in whole blood samples from patients with schizophrenia and related disorders, with phenotypic information collected at the time of blood draw, then cross-matched the data with other human and animal model lines of evidence. Topping our list of candidate blood biomarkers for hallucinations, we have four genes decreased in expression in high hallucinations states (Fn1, Rhobtb3, Aldh1l1, Mpp3), and three genes increased in high hallucinations states (Arhgef9, Phlda1, S100a6). All of these genes have prior evidence of differential expression in schizophrenia patients. At the top of our list of candidate blood biomarkers for delusions, we have 15 genes decreased in expression in high delusions states (such as Drd2, Apoe, Scamp1, Fn1, Idh1, Aldh1l1), and 16 genes increased in high delusions states (such as Nrg1, Egr1, Pvalb, Dctn1, Nmt1, Tob2). Twenty-five of these genes have prior evidence of differential expression in schizophrenia patients. Predictive scores, based on panels of top candidate biomarkers, show good sensitivity and negative predictive value for detecting high psychosis states in the original cohort as well as in three additional cohorts. These results have implications for the development of objective laboratory tests to measure illness severity and response to treatment in devastating disorders such as schizophrenia.

The LEARn model: an epigenetic explanation for idiopathic neurobiological diseases.

Neurobiological disorders have diverse manifestations and symptomology. Neurodegenerative disorders, such as Alzheimer's disease, manifest late in life and are characterized by, among other symptoms, progressive loss of synaptic markers. Developmental disorders, such as autism spectrum, appear in childhood. Neuropsychiatric and affective disorders, such as schizophrenia and major depressive disorder, respectively, have broad ranges of age of onset and symptoms. However, all share uncertain etiologies, with opaque relationships between genes and environment. We propose a 'Latent Early-life Associated Regulation' (LEARn) model, positing latent changes in expression of specific genes initially primed at the developmental stage of life. In this model, environmental agents epigenetically disturb gene regulation in a long-term manner, beginning at early developmental stages, but these perturbations might not have pathological results until significantly later in life. The LEARn model operates through the regulatory region (promoter) of the gene, specifically through changes in methylation and oxidation status within the promoter of specific genes. The LEARn model combines genetic and environmental risk factors in an epigenetic pathway to explain the etiology of the most common, that is, sporadic, forms of neurobiological disorders.

Coming to grips with complex disorders: genetic risk prediction in bipolar disorder using panels of genes identified through convergent functional genomics.

We previously proposed and provided proof of principle for the use of a complementary approach, convergent functional genomics (CFG), combining gene expression and genetic data, from human and animal model studies, as a way of mining the existing GWAS datasets for signals that are there already, but did not reach significance using a genetics-only approach [Le-Niculescu et al., 2009b]. CFG provides a fit-to-disease prioritization of genes that leads to generalizability in independent cohorts, and counterbalances the fit-to-cohort prioritization inherent in classic genetic-only approaches, which have been plagued by poor reproducibility across cohorts. We have now extended our previous work to include more datasets of GWAS, and more recent evidence from other lines of work. In essence our analysis is the most comprehensive integration of genetics and functional genomics to date in the field of bipolar disorder. Biological pathway analyses identified top canonical pathways, and epistatic interaction testing inside these pathways has identified genes that merit future follow-up as direct interactors (intra-pathway epistasis, INPEP). Moreover, we have put together a panel of best P-value single nucleotide polymorphisms (SNPs), based on the top candidate genes we identified. We have developed a genetic risk prediction score (GRPS) based on our panel, and demonstrate how in two independent test cohorts the GRPS differentiates between subjects with bipolar disorder and normal controls, in both European-American and African-American populations. Lastly, we describe a prototype of how such testing could be used to categorize disease risk in individuals and aid personalized medicine approaches, in psychiatry and beyond.

Role of the APP non-amyloidogenic signaling pathway and targeting alpha-secretase as an alternative drug target for treatment of Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent form of dementia, and its effective disease modifying therapies are desperately needed. Promotion of non-amyloidogenic alpha-secretase cleavage of amyloid precursor protein (APP) to release soluble sAPPalpha, based on the most widely accepted "amyloid model" as a plausible mechanism for AD treatment, is the focus of this review. Modulation of alpha-secretase or "a disintegrin and metalloprotease (ADAM)"s activity via protein kinase C (PKC), calcium ion (Ca(2+)), tyrosine kinase (TK), MAP kinase (MAPK), and hormonal signaling, which regulate catabolic processing of APP, are discussed. The inhibition of amyloidogenic processing of APP by the beta- and gamma-secretase has been considered till now a promising strategy to treat AD. But beta- and gamma-secretase inhibitors, along with the available therapeutic tools for AD, have side effects. These challenges can be circumvented to certain extent; but activation of sAPPalpha release appears to be a potential alternative strategy to reduce cerebral amyloidosis. Drug screens have been performed to identify therapeutics for AD, but an effective screening strategy to isolate activators of alpha-secretase has been rarely reported. Novel reporter-based screens targeted toward APP mRNA 5' untranslated region (UTR), followed by counter-screens to detect alpha-secretase stimulators, could be important in detecting compounds to promote sAPPalpha release and reduce amyloid beta (Abeta) buildup. The primary inflammatory cytokine interleukin-1, which stimulates APP 5'UTR-directed translation of cell-associated APP, enhances processing to sAPPalpha in astrocytes and co-activates ADAM-10/ADAM-17 through MAPK signaling; thus illustrating a novel pathway that could serve as therapeutic model for AD.

Different isoforms of the Wilms' tumour protein WT1 have distinct patterns of distribution and trafficking within the nucleus.

The Wilms' tumour suppressor gene WT1 encodes multiple isoforms of a transcription factor essential for correct mammalian urogenital development. Maintenance of the correct isoform ratio is critical. In humans, perturbation of this ratio causes Frasier syndrome, which is characterized by developmental defects of the kidney and urogenital tract. Different WT1 isoforms are thought to regulate transcription and participate in mRNA processing, functions reflected by a complex sub-nuclear distribution. However, the role of individual WT1 isoforms remains unclear and pathways leading to WT1 sub-nuclear localization are completely unknown. Here we use cells expressing green fluorescent protein-tagged WT1 to demonstrate that the two major WT1 isoforms occupy separate and dynamic intranuclear locations in which one isoform, WT1+KTS, preferentially associates with the nucleolus. The alternatively spliced zinc finger region is found to be critical for the initial sub-nuclear separation of WT1 isoforms, but interactions between different isoforms influence the sub-nuclear distribution of WT1. We illustrate how disruption of WT1 nuclear distribution might result in disease. This study contributes to the emerging picture of intranuclear protein trafficking.

Thyrotropin-releasing hormone (protirelin) inhibits potassium-stimulated glutamate and aspartate release from hippocampal slices in vitro.

Excess excitatory amino acid release is involved in pathways associated with seizures and neurodegeneration. Thyrotropin-releasing hormone (TRH; protirelin), a brain-derived tripeptide, has shown efficacy in the treatment of such disorders, yet its mechanism of neuroprotection is poorly understood. Using superfused hippocampal slices, we tested the hypothesis that TRH could inhibit evoked glutamate/aspartate release in vitro. Rat hippocampal slices were first equilibrated in oxygenated Krebs buffer (KRB) (120 min) then superfused for 10 min with KRB (control), or KRB containing 0.1, 1, or 10 microM TRH respectively, prior to and during 5 min depolarization with high potassium KRB (50 mM [K(+)] +/- TRH). Fractions (1 min) were collected during the 5 min stimulation and for an additional 10 min thereafter and analyzed for glutamate and aspartate by HPLC. TRH had no effect on baseline glutamate/aspartate release, while all three TRH doses significantly (P < 0.05) inhibited peak 50 mM [K(+)]-stimulated glutamate/aspartate release, and glutamate remained below control (P < 0.05) at 15 min post stimulation. A 5 min pulse of TRH (10 microM) had no affect on basal glutamate/aspartate release, whereas the TRH pre-pulsed slices failed to release glutamate/aspartate by [K(+)]-stimulation given 15 min later. These results are the first to show a potent and prolonged inhibitory effect of TRH on evoked glutamate/aspartate release in vitro. These initial studies suggest that exogenous and/or endogenous TRH may function, in part, to modulate excess glutamate release in specific CNS loci. Additional studies are in progress to fully understand the mechanism of this potent effect of TRH and its implication in various CNS disorders.

Melatonin suppression by light in euthymic bipolar and unipolar patients.

BACKGROUND: Previous studies have suggested that bipolar patients are supersensitive to light suppression of melatonin and that this may be a trait marker for genetic vulnerability. The present study was an attempt to replicate and extend this observation. Propranolol hydrochloride effects were compared with light effects because of the documented influence of beta-adrenergic receptors on melatonin production. Nighttime levels of corticotropin and cortisol were also examined as potential trait vulnerability markers. METHODS: Melatonin levels in euthymic bipolar patients (n= 29) were tested before and after 500-lux light was administered between 2 and 4 AM and on a separate night in the dark. Results were compared with those of a group of patients with unipolar depression (n= 24) and with those of a group of non-psychiatrically ill control subjects (n= 50). Lithium effects and propranolol effects were tested in subgroups. RESULTS: No group differences were seen in light suppression among bipolar patients, unipolar patients, and controls; an analysis of the whole group did not reveal differences in propranolol effect, differences in corticotropin or cortisol levels, or evidence for a lithium effect. However, patients with bipolar I affective disorder showed the following: (1) significantly lower melatonin levels on the light night, at baseline and following light exposure; and (2) a later peak time for melatonin on the dark night. CONCLUSIONS: The general hypothesis of increased light sensitivity in bipolar patients was not supported. However, melatonin secretion abnormalities were confirmed in the subgroup with bipolar I disorder. Further assessments of circadian rhythm disruption as a vulnerability marker in bipolar illness are indicated.

Cholinesterase inhibitors, beta-amyloid precursor protein and amyloid beta-peptides in Alzheimer's disease.

The extracellular deposition of amyloid beta-peptide (Abeta) in the form of cerebrovascular amyloid and extracellular plaques is one of the major neuropathological manifestations of Alzheimer's disease (AD). Abeta is generated proteolytically from the large beta-amyloid precursor protein (APP). APP is cleaved by a group of proteases called "secretase" to generate soluble derivatives of APP (sAPP), which are secreted in human plasma, CSF and cultured cells. Neurochemically, there is a severe loss of cholinergic neurons and a decreased synthesis of acetylcholine in neocortex in AD. Current approved AD drugs, such as aricept and tacrine, are based on the use of cholinesterase inhibitors (ChEIs) and have been reported to improve memory deficits and cognitive decline in some patients with AD. To compare the effects of ChEIs on APP processing, we have tested a series of ChEIs such as tacrine, physostigmine, metrifonate, phenserine and cymserine in cultured human neuroblastoma cells. We analyzed levels of sAPP by immunochemical techniques with APP-specific antibodies and assayed levels of Abeta by a sensitive sandwich ELISA. Based on these results, ChEIs can be divided into three groups: the first group of ChEIs had no effect on sAPP secretion, the second decreased the sAPP secretion only, and third group affected the secretion of sAPP and Abeta. The difference in the action of metrifonate, physostigmine, phenserine and tacrine on APP processing is independent of their selectivity for the cholinesterase enzymes. This possibly is due to the different targets that are used by ChEIs. Studying the effects of ChEIs on different targets is useful to maximize the benefit of ChEIs for the treatment of AD subjects.

The experimental Alzheimer drug phenserine: preclinical pharmacokinetics and pharmacodynamics.

Phenserine, a phenylcarbamate of physostigmine, is a new potent and highly selective acetylcholinesterase (AChE) inhibitor, with a > 50-fold activity versus butyrylcholinesterase (BChE), in clinical trials for the treatment of Alzheimer's disease (AD). Compared to physostigmine and tacrine, it is less toxic and robustly enhances cognition in animal models. To determine the time-dependent effects of phenserine on cholinergic function, AChE activity, brain and plasma drug levels and brain extracellular acetylcholine (ACh) concentrations were measured in rats before and after phenserine administration. Additionally, its maximum tolerated dose, compared to physostigmine and tacrine, was determined. Following i.v. dosing, brain drug levels were 10-fold higher than those achieved in plasma, peaked within 5 min and rapidly declined with half-lives of 8.5 and 12.6 min, respectively. In contrast, a high (> 70%) and long-lasting inhibition of AChE was achieved (half-life > 8.25 h). A comparison between the time-dependent plasma AChE inhibition achieved after similar oral and i.v. doses provided an estimate of oral bioavailability of 100%. Striatal, in vivo microdialysis in conscious, freely-moving phenserine-treated rats demonstrated > 3-fold rise in brain ACh levels. Phenserine thus is rapidly absorbed and cleared from the body, but produces a long-lasting stimulation of brain cholinergic function at well tolerated doses and hence has superior properties as a drug candidate for AD. It selectively inhibits AChE, minimizing potential BChE side effects. Its long duration of action, coupled with its short pharmacokinetic half-life, reduces dosing frequency, decreases body drug exposure and minimizes the dependence of drug action on the individual variations of drug metabolism commonly found in the elderly.

Functional identification of the promoter of the gene encoding the Rhesus monkey beta-amyloid precursor protein.

Misregulation of the transcription of the beta-amyloid precursor protein (betaAPP) gene is implicated in the pathogenesis of Alzheimer's disease (AD). Here we characterize the 5'-flanking region, the first exon and intron of the betaAPP gene of the Rhesus monkey (rhbetaAPP). For functional analysis, transient transfection in PC12 cells was performed with a series of 5'-deletion constructs (fused with a reporter gene), that extended as far upstream as -7900 down to -1bp. Chloramphenicol acetyltransferase/promoter fusion assays showed that both -7900/+104 and -75/+104-bp regions possessed strong promoter activity. However, -2542/+104bp had the strongest promoter activity, whereas -204/+104bp showed a major reduction in activity and -47/+104bp showed almost a complete loss of activity. A region from -75 to +104bp was essential for minimal basic promoter activity because mutation at the activating site of an upstream stimulator factor (USF) within this region abolished the promoter activity. The very upstream region (-5529/-3416bp) displayed a negative effect on promoter activity. Two blocks of the sequence, 641bp (-1131 /-490) and 105bp (-309/-204), acted as positive regulators for promoter activity. Another 61-bp block (-204/-143) acted as a negative regulator. Gel shift assay indicated that the -249-242-bp region contains a binding domain for the AP-2 transcription factor. No second promoter or bidirectional promoter was observed. A region spanning the first exon and part of the first intron (+99 to +6800bp) acted as a negative regulator. These results suggest that a region of -75 to +104bp, which contains the pyrimidine-rich initiator element, the 5'-untranslated region and the binding site for USF, constitute the minimal promoter element and that interactions between multiple positive and negative elements, the USF and initiator element are crucial for transcription of the TATA-less betaAPP promoter.

Molecular cloning of the promoter of the gene encoding the Rhesus monkey beta-amyloid precursor protein: structural characterization and a comparative study with other species.

Abnormal regulation of transcription of the beta-amyloid precursor protein (betaAPP) gene is implicated in the pathogenesis of Alzheimer's disease (AD). We have examined 17- kb genomic region which contains the 5'-flanking region (promoter), first exon and on of the betaAPP gene of the Rhesus monkey (rhbetaAPP). A predominant scription start site was tified 146 bp upstream of the translation initiation codon. Sequencing 5848 bp of 5'-flanking revealed the presence of multiple near consensus sequences for binding potential transcriptional regulatory factors, such as activator proteins (AP-1, AP-2), an apolipoprotein E-B1 element, estrogen-responsive element, heat shock element and NF-kappaB. The sequence of the rhbetaAPP promoter also contains several sites for the binding of proteins that serve as signal transducers and activators of transcription (STAT1) (GAS). The rhbetaAPP promoter is highly homologous to the human promoter, but less homologous to the rodents. The homology between human and Rhesus monkey of the further upstream region gradually decreased over its length. A region of 270 bp of the human betaAPP promoter is missing from the Rhesus monkey promoter. Structural analysis of the promoter suggests that it contains characteristics of inducible genes and sites for regulated activity by various transcription factors.

Treatment outcome of tacrine therapy depends on apolipoprotein genotype and gender of the subjects with Alzheimer's disease.

We studied the effects of apolipoprotein E (APOE) genotype and gender on clinical response to tacrine in patients with mild to moderate Alzheimer's disease (AD). We analyzed data from a previously reported 30-week, double-blind, placebo-controlled trial of tacrine, in which APOE genotypes were determined from previously collected plasma samples. Patients were assigned to placebo or tacrine with daily dosages of 80, 120, or 160 mg/day. The outcome measures were Alzheimer's Disease Assessment Scale-Cognitive Component, Clinician Interview Based Impression, Mini-Mental State Examination, and the Caregiver-rated Clinical Global Impression of Change. An intent-to-treat (ITT) analysis of patients with available genotypes (n = 528) did not reveal response differences by genotype, although the effect size was twice as large in the epsilon2-3 as the epsilon4 group (-2.62 versus -1.25). The association of treatment effect with APOE genotype varied significantly according to gender (p < 0.002 for ITT; p < 0.05 for evaluables). The treatment effect was larger in the epsilon2-3 compared with epsilon4 women (ITT, 4.24 points, p = 0.03; evaluable, 7.20 points, p = 0.01). In contrast, treatment effect size was not different between epsilon2-3 and epsilon4 of men with AD. APOE genotype and gender may predict response to tacrine in patients with AD.

Metrifonate treatment of AD: influence of APOE genotype.

OBJECTIVE: To investigate whether an interaction exists between APOE genotype and the response of AD patients to metrifonate treatment and whether APOE genotype independently affects the rate of AD progression. BACKGROUND: Metrifonate is a new acetylcholinesterase inhibitor for the treatment of AD symptoms. METHODS: Data were pooled from four prospective, randomized, double-blind, placebo-controlled clinical trials and analyzed retrospectively. A total of 959 patients who received once-daily placebo (n = 374) or metrifonate (30 to 60 mg based on weight or a 50-mg fixed dose, n = 585) for up to 26 weeks agreed to APOE genotyping. RESULTS: Metrifonate clearly improved the cognitive performance of the AD patients when compared with placebo (Alzheimer's Disease Assessment Scale-Cognitive Subscale [ADAS-Cog], p = 0.0001). The interaction of APOE genotype and the metrifonate effect on cognitive performance were not significant (p = 0.25). Metrifonate also clearly improved the global function of the AD patients when compared with placebo (Clinician's Interview-Based Impression of Change with Caregiver Input [CIBIC-Plus], p = 0.0001). The interaction of APOE genotype with the metrifonate effect on global function also was not significant (p = 0.70). No significant three-way interactions were observed among APOE genotype, gender, and response to metrifonate treatment (ADAS-Cog, p = 0.68; CIBIC-Plus, p = 0.26). APOE genotype did not influence disease progression as evaluated by either cognitive performance (ADAS-Cog, p = 0.93) or global function (CIBIC-Plus, p = 0.64). CONCLUSIONS: The findings from these studies of up to 26 weeks' duration do not clearly support an interaction between APOE genotype and metrifonate treatment effects. They suggest that APOE genotypes do not necessarily predict an AD patient's response to metrifonate treatment and that APOE genotype may not influence the rate of disease progression for patients with mild to moderate AD.

Rationale for the development of cholinesterase inhibitors as anti-Alzheimer agents.

Alzheimer's disease (AD) is characterized by progressive dementia caused by the loss of the presynaptic markers of the cholinergic system in the brain areas related to memory and learning and brain deposits of amyloid beta peptide (A beta) and neurofibrillary tangles (NFT). A small fraction of early onset familial AD (FAD) is caused by mutations in genes, such as the beta-amyloid precursor protein (APP) and presenilins that increase the load of A beta in the brain. These studies together with findings that A beta is neurotoxic in vitro, provide evidence that some aggregates of this peptide are the key to the pathogenesis of AD. The yield of A beta and the processing and turnover of APP are regulated by a number of pathways including apolipoprotein E, cholesterol and cholinergic agonists. Early studies showed that muscarinic agonists increased APP processing within the A beta sequence (sAPP alpha). More recently, we have presented evidence showing that some, but not all, anticholinesterases reduce secretion of sAPP alpha as well as A beta into the media suggesting that cholinergic agonists modulate A beta levels by multiple mechanisms. Herein we review the recent advances in understanding the function of cholinesterase (ChE) in the brain and the use of ChE-inhibitors in AD. We propose and support the position that the influence of cholinergic stimulation on amyloid formation is critical in light of the early targeting of the cholinergic basal forebrain in AD and the possibility that maintenance of this cholinergic tone might slow amyloid deposition. In this context, the dual action of certain cholinesterase inhibitors on their ability to increase acetylcholine levels and decrease amyloid burden assumes significance as it may identify a single drug to both arrest the progression of the disease as well as treat its symptoms. A new generation of acetyl- and butyryl cholinesterase inhibitors is being studied and tested in human clinical trials for AD. We critically discuss recent trends in AD research, from molecular and genetic to clinical areas, as it relates to the effects of cholinergic agents and their secondary effects on A beta. Finally, we examine different neurobiological mechanisms that provide the basis of new targets for AD drug development.