Volume 14, Number 2, June 2008

Pages 127-131
Short Communication
Alexander Navarrete Santos, Andreas Simm, Vjera Holthoff, Gerald Boehm (Communicated by A. Cristina Rego)
A method for the detection of amyloid-β1-40, amyloid-β1-42 and amyloid-β oligomers in blood using magnetic beads in combination with flow cytometry and its application in the diagnostics of Alzheimer’s disease
Abstract: A method for simultaneous quantification of amyloid-β1-40, amyloid-β1-42 and amyloid-β oligomers in human plasma is described. The method consists of a combination of immunoprecipitation using specific antibodies against the different forms of amyloid-β, and immobilization of the immunocomplexes to magnetic beads. Addition of fluorescence-labelled antibodies which recognize the specific antibodies to the amyloid-β subsets allows the peptide/associates detection in the sample by flow cytometry. The clinical assay performance was tested using blood samples from Alzheimer disease’s patients and control donors. A sensitivity of 70% and a specificity of 81% were achieved.

Pages 133-145
Ann-Charlotte Granholm, Heather A. Bimonte-Nelson, Alfred B. Moore, Matthew E. Nelson, Linnea R. Freeman, Kumar Sambamurti
Effects of a saturated fat and high cholesterol diet on memory and hippocampal morphology in the middle-aged rat
Abstract: Diets rich in cholesterol and/or saturated fats (Sat-Fat) have been shown to be detrimental to cognitive performance. Therefore, we fed a high cholesterol (2%) and Sat-Fat (hydrogenated coconut oil, 10%) diet to 16-month old rats for 8 weeks to explore the effects on the working memory performance of middle-aged rats. Lipid profiles revealed elevated plasma triglycerides, total cholesterol, HDL, and LDL for the Sat-Fat group as compared to an iso-caloric control diet (12% soybean oil). Weight increase and food consumption were similar in both groups. Sat-Fat treated rats committed more working memory errors in the water radial arm maze, especially at higher memory loads. Cholesterol, amyloid-β peptide of 40 (Aβ40) or 42 (Aβ42) residues, or nerve growth factor in cortical regions was unaffected, but hippocampal Map-2 staining was reduced in rats fed a Sat-Fat diet, indicating a loss of dendritic integrity. Map-2 reduction correlated with memory errors. Microglial activation, indicating inflammation and/or gliosis, was also observed in the hippocampus of Sat-Fat fed rats. These data suggest that saturated fat, hydrogenated fat and cholesterol can profoundly impair memory and hippocampal morphology.

Pages 147-159
Marcella Reale, Carla Iarlori, Claudio Feliciani, Domenico Gambi (Communicated by Alexandre de Mendonca)
Peripheral Chemokine Receptors, Their Ligands, Cytokines and Alzheimer’s Disease
Abstract: Cerebral inflammation as well as systemic immunological alterations has been reported in Alzheimer’s disease (AD). We aimed to determine whether spontaneous and mitogen stimulated production of peripheral blood mononuclear cell (PBMC) cytokines, chemokines and chemokine receptors in clinically diagnosed patients with AD were unregulated. PBMC were purified from AD patients and from healthy controls. Supernatants were analyzed for cytokine levels by ELISA methods. mRNA expression was determined by RT-PCR. Expression of chemokine receptors CCR2 and CCR5 was determined by cytofluorimetric analysis. Both CCR5 and CCR2 expression were increased in AD patients respect to control subjects and the expression of CCR2 and CCR5 was more frequent on CD4+ and less frequent on CD8+ cells. Levels of Th1-type cytokine IFNg and chemokine RANTES were increased and levels of Th2-type cytokine IL-4 and chemokine MCP-1 were reduced in AD patients compared with those of control subjects. Acetylcholinesterase inhibitor pyridostigmine bromide (AChEI)-therapy reduced CCR2, CCR5, RANTES and IFNg expression and production in AD patients. CCR5, CCL5/RANTES, CCL2/MCP-1 and IFNg expression and production were increased in PBMC treated with amyloid-β1-42. Addition of AChEI to PBMC suppresses CCL5/RANTES and IFNg. The observed patterns of cyto-chemokine involvement strengthen the questions regarding the inflammatory theory in AD, and raise a pathophysiologic role for selective alteration of cyto-chemokine network.

Pages 161-173
Jun Wang, Hideo Hara, Takao Makifuchi, Takeshi Tabira (Communicated by Akihiko Takashima)
Development and characterization of a TAPIR-like mouse monoclonal antibody to amyloid-β
Abstract: Tissue amyloid plaque immuno-reactive (TAPIR) antibody was better related to the effect of immunotherapy in Alzheimer’s disease (AD) than ELISA antibody. Here we used a hybridoma technique to develop a TAPIR-like anti-human amyloid-β (Aβ) mouse monoclonal antibody. The obtained monoclonal antibody, 3.4A10, was an IgG2b isotype and recognized N-terminal portion of Aβ1-42 without binding denatured or native amyloid-β protein precursor. It had higher affinity to Aβ1-42 than to Aβ1-40 by Biacore affinity analysis and stained preferably the peripheral part of senile plaques and recognized the plaque core less than 4G8. It inhibited the Aβ1-42 fibril formation as well as degraded pre-aggregated Aβ1-42 peptide in a thioflavin T fluorescence spectrophotometry assay. The in vivo studies showed that 3.4A10 treatment decreased amyloid burden compared to the control group and significantly reduced Aβ42 levels rather than Aβ40 levels in brain lysates as well as the Aβ*56 oligomer (12mer) in TBS fraction of the brain lysates. 3.4A10 entered brain and decorated some plaques, which is surrounded by more Iba1-positive microglia. 3.4A10 therapy did not induce lymphocytic infiltration and obvious increase in microhemorrhage. We conclude that 3.4A10 is a TAPIR-like anti-human amyloid monoclonal antibody, and has a potential of therapeutic application for AD.

Pages 175-177
   Kumar Sambamurti, Miguel A. Pappolla, K.S. Jagannatha Rao
   Value in Development of a TAPIR-like mouse monoclonal antibody to Aβ

Pages 179-191
Israel Ampuero, Raquel Ros, Ana Royuela, Victor Abraira, Teodoro del Ser, Guillermo García Rivas, Justo García de Yébenes
Risk factors for dementia of Alzheimer type and aging-associated cognitive decline in a Spanish population based sample, and in brains with pathology confirmed Alzheimer’s disease
Abstract: We investigated the environmental and genetic factors for Alzheimer’s disease (AD) in Spain and performed a door to door study of a cohort of more than 500 subjects, over 70 years old, from Leganés, a suburban area near Madrid. The cohort was followed for 6 years by neurologists and other health workers and was divided in three groups: normal controls, subjects with aging-associated cognitive decline (AACD) and probable AD or dementia of Alzheimer’s type (DAT). Biological variables and polymorphisms of different genes, important in neurodegeneration or reported to be associated with AD, were investigated as putative risk modifiers. These polymorphisms have also been analyzed in 94 brains, 39 from patients with pathologically confirmed AD and 55 controls. The statistical investigation included the evaluation of different individual risks and a multinomial logistic regression analysis to detect predictive factors. The risk of AACD and AD increased with age, feminine gender and history of stroke and decreased with education. The allele ApoE4 increased the risk of AD but not of AACD. When the impact of ApoE4 was added to the model, the effect of education and stroke disappeared as risk modifiers.

Pages 193-199
Murat Emre, Patrizia Mecocci, Karina Stender
Pooled analyses on cognitive effects of memantine in patients with moderate to severe Alzheimer’s disease
Abstract: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, constituting the most common cause of dementia. Memantine, an N-methyl-D-aspartate receptor antagonist indicated for the treatment of moderate to severe AD, has been shown to provide benefits in cognitive, functional, and behavioral domains in large, randomized clinical trials. The current analysis combined data from six previously published studies and assessed the effect of memantine on various cognitive functions in 1826 patients (867 on placebo and 959 on memantine) with moderate to severe AD (MMSE < 20). The Alzheimer’s Disease Assessment Scale cognitive subscale (ADAS-cog) and the Severe Impairment Battery (SIB) scores from all six studies were pooled and combined into three clusters representing discrete cognitive domains: language, memory, and praxis. At baseline, there were no clinically significant differences between the memantine- and placebo-treated groups. After 24 weeks, responder analyses revealed that memantine treatment resulted in statistically significantly more patients improving on each of the three clusters, language, memory, and praxis, compared with placebo, and a lower percentage of patients treated with memantine showed any worsening on any of the three clusters compared with patients receiving placebo. It is concluded that treatment with memantine provides benefits in all three cognitive functions.

Pages 201-208
Giuseppe Verdile, Bu B.Yeap, Roger M. Clarnette, Satvinder Dhaliwal, Melanie S. Burkhardt, S.A Paul Chubb, Karl De Ruyck, Mark Rodrigues, Pankaj D. Mehta, Jonathan K. Foster, David G. Bruce, Ralph N. Martins
Luteinizing hormone levels are positively correlated with plasma amyloid-β protein levels in elderly men
Abstract: Dysregulation of the hypothalamic pituitary gonadal (HPG) axis during aging has been associated with increased risk of cognitive decline and developing dementia. Compared to controls, men with Alzheimer’s disease (AD) have been shown to have lower serum testosterone levels and higher serum luteinizing hormone (LH) levels. As serum free testosterone concentration is negatively correlated with LH in older men, the independent contributions of these hormones to the pathogenesis of AD warrants further clarification. To explore this notion, we measured plasma amyloid-β (Aβ, serum testosterone, serum LH and other biochemical parameters in 40 cognitively normal elderly men. Multiple linear regression analysis revealed that serum LH concentration is the only parameter that significantly correlates with plasma Aβ levels in these men (r=0.5, p=0.041). These results suggest that increased serum LH concentration, rather than lower serum free testosterone, is associated with the accumulation of Ab in plasma. Larger, longitudinal human studies are needed to determine the significance of LH in the pathogenesis of AD.

Pages 209-223
Sui Cheung Man, Siva Sundara Kumar Durairajan, Wan Fung Kum, Jia Hong Lu, Jian Dong Huang, Ching Fung Cheng, Vincent Chung, Min Xu, Min Li
Systematic Review on the Efficacy and Safety of Herbal Medicines for Alzheimer’s Disease
Abstract: A systematic review was conducted to assess the efficacy and safety of herbal medications (HM), as either monotherapy or adjunct to orthodox medications (cholinesterase inhibitors and nootropic agents, OM) for Alzheimer’s disease (AD). Sixteen studies testing different HM were included. Out of the 15 HM monotherapy studies, 13 reported HM to be significantly better than OM or placebo; one reported similar efficacy between HM and OM. Only the HM adjuvant study reported significant efficacy. No major adverse events for HM were reported and HMs were found to reduce the adverse effects arising from OM. Imbalance in ethnicity among participants was observed; gender distribution was unclear. Heterogeneity in diagnostic criteria, interventions and outcome measures hindered comprehensive data analysis. Studies comparing HM with OM suggest that HM can be a safe, effective treatment for AD, either alone or in conjunction with OM. Methodological flaws in the design of the studies, however, limited the extent to which the results could be interpreted. Among various HMs, the safety and tolerability of EGb761 was best established. Further multi-center trials with large sample size, high methodological qualities and standardized HM ingredients are necessary for clinical recommendations on the use of HM in treating AD.

Pages 225-234
Teri L. Wadsworth, James A. Bishop, Anuradha S. Pappu, Randall L. Woltjer, Joseph F. Quinn (Communicated by Paula I. Moreira)
Evaluation of Coenzyme Q as an Antioxidant Strategy for Alzheimer’s Disease
Abstract: Increasing evidence suggests that Alzheimer’s disease (AD) is associated with oxidative damage that is caused in part by mitochondrial dysfunction. Here we investigated the feasibility of modifying Alzheimer pathology with the mitochondrial antioxidant coenzyme Q (CoQ). Exogenous CoQ protected MC65 neuroblastoma cells from amyloid-β protein precursor C-terminal fragment (APP CTF)-induced neurotoxicity in a concentration dependent manner, with concentrations of 6.25 μM and higher providing near complete protection. Dietary supplementation with CoQ at a dose of 10 g/kg diet to C65/Bl6 mice for one month significantly suppressed brain protein carbonyl levels, which are markers of oxidative damage. Treatment for one month with 2 g lovastatin/kg diet, which interferes with CoQ synthesis, resulted in a significant lowering of brain CoQ10 levels. Mitochondrial energetics (brain ATP levels and mitochondrial membrane potential) were unaffected by either CoQ or lovastatin treatment. Our results suggest that oral CoQ may be a viable antioxidant strategy for neurodegenerative disease. Our data supports a trial of CoQ in an animal model of AD in order to determine whether a clinical trial is warranted.

Pages 235-245
Joanna F. Collingwood, Ryan K.K. Chong, Takeshi Kasama, Lionel Cervera-Gontard, Rafal E. Dunin-Borkowski, George Perry, Mihaly Pósfai, Sandra L. Siedlak, Edward T. Simpson, Mark A. Smith, Jon Dobson
Three-Dimensional Tomographic Imaging and Characterization of Iron Compounds Within Alzheimer’s Plaque Core Material
Abstract: Although it has been known for over 50 years that abnormal concentrations of iron are associated with virtually all neurodegenerative diseases, including Alzheimer’s disease, its origin, nature and role have remained a mystery. Here, we use high-resolution transmission electron microscopy (HR-TEM), energy dispersive X-ray (EDX) spectroscopy and electron energy-loss spectroscopy (EELS), electron tomography, and electron diffraction to image and characterize iron-rich plaque core material – a hallmark of Alzheimer’s disease pathology – in three dimensions. In these cores, we unequivocally identify biogenic magnetite and/or maghemite as the dominant iron compound. Our results provide an indication that abnormal iron biomineralization processes are likely occurring within the plaque or the surrounding diseased tissue and may play a role in aberrant peptide aggregation. The size distribution of the magnetite cores implies formation from a ferritin precursor, implicating a malfunction of the primary iron storage protein in the brain.

Pages 247-255
Andrew P. Lieberman, Diane M. Robins (Communicated by Rudy Castellani)
The androgen receptor’s CAG/glutamine tract in mouse models of neurological disease and cancer
Abstract: The androgen receptor (AR) is a ligand-activated transcription factor that is central to androgen dependent development and diseases. Activity of the receptor is influenced by the length of a CAG/glutamine tract in its N-terminal transactivating domain. Expansions of this tract cause Kennedy disease, a protein aggregation degenerative disorder of motor neurons that occurs only in men, and shorter length tracts have been linked to increased risk of prostate cancer. Here we review recent data from mouse models in which gene targeting was used to humanize the mouse Ar gene and introduce CAG/glutamine tracts of varying lengths. Insertion of an expanded tract encoded by 113 CAG repeats modeled Kennedy disease and revealed an important myopathic contribution to the disease phenotype. Variations in CAG tract length within the range of normal human alleles influenced the onset and progression of prostate cancer when targeted Ar mice were crossed to a transgenic prostate cancer model. This series of mice with different Ar alleles has provided insights into the mechanisms by which variations in the CAG/glutamine tract length influence the occurrence of human disease.