Volume 20, Number 4, July 2010

Pages 953-966
Review Article
Fabio Coppedè and Lucia Migliore (Handling Associate Editor: Alessandro Serretti)
Evidence Linking Genetics, Environment, and Epigenetics to Impaired DNA Repair in Alzheimer’s Disease
Abstract: Increasing evidence suggests that the repair of DNA lesions, particularly oxidative DNA lesions, might be compromised in Alzheimer’s disease (AD). Studies performed in brains and peripheral tissues of both AD patients and individuals affected by mild cognitive impairment (MCI) revealed that oxidative DNA damage is one of the earliest detectable events during the progression from healthy aging to dementia. Moreover, the increase in DNA damage is paralleled by a decrease in DNA repair activities. Several hypotheses are currently tested in order to explain the decreased DNA repair activity observed in MCI and AD subjects. Some authors have suggested that mutations or polymorphisms in DNA repair genes might impair DNA repair. However, this hypothesis does not seem to be confirmed by recent genetic association studies. Others suggest that DNA repair proteins might be inactivated by oxidative induced post-translational modifications or degradation. There is also indication that different isoforms of the same repair protein might be involved in the progression from early to late stages AD. Moreover, a widespread activation of DNA repair pathways might generate death signals ending with neuronal apoptosis. A link between environmental induced epigenetic modification, oxidation, and repair of AD related genes has been also proposed. Most of these studies have been performed during the last few years, and we are still at the beginning of the understanding of the complex interplay between oxidative DNA damage, DNA repair, and neuronal death in the brain leading to Alzheimer’s dementia, making this topic an exciting and promising field for future investigation.

Pages 967-980
Review Article
Raluca Mihaescu, Symone B. Detmar, Martina C. Cornel, Wiesje M. van der Flier, Peter Heutink, Elly M. Hol, Marcel G.M. Olde Rikkert, Cornelia M. van Duijn, A. Cecile J.W. Janssens (Handling Associate Editor: Inga Zerr)
Translational Research in Genomics of Alzheimer’s Disease: A Review of Current Practice and Future Perspectives
Abstract: Alzheimer’s disease (AD) is the most prevalent form of dementia and the number of cases is expected to increase exponentially worldwide. Three highly penetrant genes (AβPP, PSEN1, and PSEN2) explain only a small number of AD cases with a Mendelian transmission pattern. Many genes are analyzed for the association with non-Mendelian AD, but the only consistently replicated finding is APOE. At present, possibilities for prevention, early detection, and treatment of the disease are limited. Predictive and diagnostic genetic testing is available only in Mendelian forms of AD. Currently, APOE genotyping is not considered clinically useful for screening, presymptomatic testing, or clinical diagnosis of non-Mendelian AD. However, clinical management of the disease is expected to benefit from the rapid pace of discoveries in the genomics of AD. Following a recently developed framework for the continuum of translation research that is needed to move genetic discoveries to health applications, this paper reviews recent genetic discoveries as well as translational research on genomic applications in the prevention, early detection, and treatment of AD. The four phases of translation research include: 1) translation of basic genomics research into a potential health care application; 2) evaluation of the application for the development of evidence-based guidelines; 3) evaluation of the implementation and use of the application in health care practice; and 4) evaluation of the achieved population health impact. Most research on genome-based applications in AD is still in the first phase of the translational research framework, which means that massive research is still needed before their implementation can be considered.

Pages 981-990
Review Article
Weiming Xia (Handling Associate Editor: Xiongwei Zhu)
Exploring Alzheimer’s Disease in Zebrafish
Abstract: The amyloid β protein (Aβ)-containing neuritic plaques and hyperphosphorylated tau-containing neurofibrillary tangles are two invariable characteristics of Alzheimer’s disease (AD). Three genes encoding amyloid-β protein precursor (AβPP), presenilin (PS) 1 and 2 are linked to early onset familial AD, and the apolipoprotein E (ApoE) ε4 allele is a major risk factor for sporadic AD. The zebrafish AβPP, PS, and ApoE genes have been identified, and the essential components of the γ-secretase complex that mediates cleavage of AβPP to generate Aβ have been examined in zebrafish. The transgenic zebrafish expressing mutant tau has been created, and the transparent animals exhibit the phenotype of neurodegeneration that could be readily characterized by capturing microscopic images. The application of zebrafish as a model system for AD research has expanded our knowledge of Aβ and tau, two components of AD pathological inclusions.

Pages 991-996
Short Communication
Beatrice Arosio, Chiara Viazzoli, Luigina Mastronardi, Claudio Bilotta, Carlo Vergani and Luigi Bergamaschini
Adenosine A2A Receptor Expression in Peripheral Blood Mononuclear Cells of Patients with Mild Cognitive Impairment
Abstract: Adenosine suppresses immune responses through the adenosine A2A receptors (A2AR). We evaluated the expression of A2AR in blood mononuclear cells (PBMCs) of patients with mild cognitive impairment (MCI), Alzheimer’s disease (AD), and controls in order to verify if it may help distinguish different forms of cognitive decline. There was a significant linear increase in both mRNA levels and receptor density from multiple cognitive domain MCI (mcd-MCI) to amnestic MCI (a-MCI), spanning through AD and controls, without any significant difference between the latter two groups of subjects. These data, which need to be confirmed in a larger number of patients, suggest that expression of A2AR in PBMCs may be a valuable means of differentiating a-MCI and mcd-MCI.

Pages 997-1002
Short Communication
Rosaria A. Cavallaro, Andrea Fuso, Vincenzina Nicolia, Sigfrido Scarpa (Handling Associate Editor: Othman Gribi)
S-Adenosylmethionine Prevents Oxidative Stress and Modulates Glutathione Metabolism in TgCRND8 Mice Fed with B Vitamin Deficient Diet
Abstract: Oxidative stress, altered glutathione levels, and hyperhomocysteinemia play critical roles in Alzheimer’s disease. We studied the relationships between hyperhomocysteinemia, glutathione, and oxidative stress in TgCRND8 mice maintained in conditions of folate, B12, and B6 deficiency and the effect of S-adenosylmethionine supplementation. We found that hyperhomocysteinemia was correlated with increased reduced/oxidized brain glutathione ratio, with decreased glutathione S-transferase activity and increased lipid peroxidation. S-adenosylmethionine potentiated superoxide dismutase and glutathione S-transferase activity and restored altered brain glutathione and erythrocytes lipid peroxidation. These results underline the importance of S-adenosylmethionine as neuroprotective compound, acting both on methylation and oxidation metabolism.

Pages 1003-1008
Short Communication
Brian A. Couch, George J. DeMarco, Shannon L. Gourley, Anthony J. Koleske (Handling Associate Editor: Shu G. Chen)
Increased Dendrite Branching in AβPP/PS1 Mice and Elongation of Dendrite Arbors by Fasudil Administration
Abstract: Amyloid-β (Aβ) overproduction and dendrite arbor atrophy are hallmarks of Alzheimer’s disease. The RhoA GTPase (Rho) signals through Rho kinase (ROCK) to control cytoskeletal dynamics and regulate neuron structure. Hyperactive Rho signaling destabilizes neurons leading to dendritic regression that can be rescued by genetic or pharmacological reduction of ROCK signaling. To understand what effect reduced ROCK signaling has on the dendrite arbors of mice that overproduce Aβ, we administered the ROCK inhibitor fasudil to AβPP/PS1 transgenic mice. We report that increased dendrite branching occurs in AβPP/PS1 mice and that fasudil promotes lengthening of the dendrite arbors of CA1 pyramidal neurons.

Pages 1009-1013
Short Communication
Cara J. Westmark, Pamela R. Westmark and James S. Malter (Handling Associate Editor: Gary Arendash)
Alzheimer’s Disease and Down Syndrome Rodent Models Exhibit Audiogenic Seizures
Abstract: Amyloid-β protein precursor (AβPP) is overexpressed in Alzheimer’s disease (AD), Down syndrome (DS), autism, and fragile X syndrome. Seizures are a common phenotype in all of these neurological disorders, yet the underlying molecular mechanism(s) of seizure induction and propagation remain largely unknown. We demonstrate that AD (Tg2576) and DS (Ts65Dn) mice exhibit audiogenic seizures, which can be attenuated with antagonists to metabotropic glutamate receptor 5 (mGluR5) or by passive immunization with anti-amyloid-β antibody. Our data strongly implicates AβPP or a catabolite in seizure susceptibility and suggests that mGluR5 mediates this response.

Pages 1015-1028
Leena Aho, Maria Pikkarainen, Mikko Hiltunen, Ville Leinonen, Irina Alafuzoff
Immunohistochemical Visualization of Amyloid-β Protein Precursor and Amyloid-β in Extra- and Intracellular Compartments in the Human Brain
Abstract: Amyloid-β (Aβ) peptide, a cleavage product of the amyloid-β protein precursor (AβPP), has been reported to be detected in the intracellular compartment. Most studies reporting the presence of intracellular Aβ are based on the use of immunohistochemistry. In this study, the presence of AβPP and Aβ was assessed by applying immunohistochemistry in postmortem human brain tissue samples obtained from 10 neurologically intact subjects, the youngest being 2 years of age, one aged with mild cognitive impairment, 14 neurologically diseased, and in one brain biopsy sample obtained from a subject with normal pressure hydrocephalus. Intracellular immunoreactivity was detected in all ages independent of the disease state or existence of extracellular Aβ aggregates with all antibodies directed to AβPP, with three Aβ antibodies (4G8, 6E10, and 82E1), clones that are unable to distinguish Aβ from AβPP. These results suggest that it is AβPP rather than Aβ that is detected intracellularly when using the antibodies listed above. Furthermore, the staining results varied when different pretreatment strategies were applied. Interestingly intracellular Aβ was detected with antibodies directed to the C-terminus of Aβ (neoepitope) in subjects with Alzheimer’s disease. The lack of intracellular immunoreactivity in unimpaired subjects, when using antibodies against neoepitopes, may be due to a lack or a low level of the protein that is thus undetectable at light microscopic level by immunohistochemistry method. The staining results and conclusions depended strongly on the chosen antibody and the pretreatment strategy and thus multiple antibodies must be used when assessing the intracellular accumulation of Aβ.

Pages 1029-1037
Francesca Mangialasche, Miia Kivipelto, Patrizia Mecocci, Debora Rizzuto, Katie Palmer, Bengt Winblad, Laura Fratiglioni (Handling Associate Editor: D. Allan Butterfield)
High Plasma Levels of Vitamin E Forms and Reduced Alzheimer’s Disease Risk in Advanced Age
Abstract: In this study we investigated the association between plasma levels of eight forms of vitamin E and incidence of Alzheimer’s disease (AD) among oldest-old individuals in a population-based setting. A dementia-free sample of 232 subjects aged 80+ years, derived from the Kungsholmen Project, was followed-up to 6 years to detect incident AD. Plasma levels of vitamin E (α-, β-, γ, and δ-tocopherol; α-, β-, γ-, and δ-tocotrienol) were measured at baseline. Vitamin E forms-AD association was analyzed with Cox proportional hazard model after adjustment for several potential confounders. Subjects with plasma levels of total tocopherols, total tocotrienols, or total vitamin E in the highest tertile had a reduced risk of developing AD in comparison to persons in the lowest tertile. Multi-adjusted hazard ratios (HRs) and 95% confidence interval (CI) were 0.55 (0.32-0.94) for total tocopherols, 0.46 (0.23-0.92) for total tocotrienols, and 0.55 (0.32-0.94) for total vitamin E. When considering each vitamin E form, the risk of developing AD was reduced only in association with high plasma levels of β-tocopherol (HR: 0.62, 95% CI 0.39-0.99), whereas α-tocopherol, α- tocotrienol, and β-tocotrienol showed only a marginally significant effect in the multiadjusted model [HR (95% CI): α-tocopherol: 0.72 (0.48-1.09); α-tocotrienol: 0.70 (0.44-1.11); β-tocotrienol: 0.69 (0.45-1.06)]. In conclusion, high plasma levels of vitamin E are associated with a reduced risk of AD in advanced age. The neuroprotective effect of vitamin E seems to be related to the combination of different forms, rather than to α-tocopherol alone, whose efficacy in interventions against AD is currently debated.

Pages 1039-1049
Niklas Mattsson, Per Johansson, Oskar Hansson, Anders Wallin, Jan-Ove Johansson, Ulf Andreasson, Oluf Andersen, Sara Haghighi, Maria Olsson, Mats Stridsberg, Johan Svensson, Kaj Blennow, Henrik Zetterberg
Converging Pathways of Chromogranin and Amyloid Metabolism in the Brain
Abstract: Much is unknown regarding the regulation of Alzheimer-related amyloid-β protein precursor (AβPP)-processing in the human central nervous system. It has been hypothesized that amyloidogenic AβPP-processing preferentially occurs in the regulated secretory pathway of neurons. To test this hypothesis we looked for correlations of AβPP-derived molecules in cerebrospinal fluid (CSF) with chromogranin (Cg) derived peptides, representing the regulated secretion. Patients with Alzheimer’s disease (AD, N=32), multiple sclerosis (MS, N=50), and healthy controls (N=70) were enrolled. CSF was analyzed for the amyloid peptides Aβ1-42, Aβx-42, Aβx-40, Aβx-38, α-cleaved soluble AβPP (sAβPPα), β-cleaved soluble AβPP (sAβPPβ), and peptides derived from CgB and SgII (Secretogranin-II, CgC). We investigated CSF levels of the protease BACE1, which processes AβPP into Aβ, in relation to Cg-levels. Finally, we measured Cg levels in cell media from untreated and BACE1-inhibited SH-SY5Y human neuroblastoma cells. CSF Cg levels correlated to sAβPP and Aβ peptides in AD, MS, and controls, and to CSF BACE1. Cell medium from BACE1-inhibited cells had decreased CgB levels. These results suggest that a large part of AβPP in the human central nervous system is processed in the regulated secretory pathway of neurons.

Pages 1049-1068
Ivan Rattray, Alain Pitiot, James Lowe, Dorothee P. Auer, Sarah-Jane Lima, Mirjam I. Schubert, Malcolm J.W. Prior, Charles A. Marsden, Fernando Pérez Diaz, David A. Kendall, Marie-Christine Pardon (Handling Associate Editor: Gemma Casadesus)
Novel Cage Stress Alters Remote Contextual Fear Extinction and Regional T2 Magnetic Resonance Relaxation Times in TASTPM Mice Overexpressing Amyloid
Abstract: We have previously shown that repeated exposure to mild novel cage stress prevents the onset of recent contextual fear memory deficits and attenuated amyloid deposition in the TASTPM mouse model of Alzheimer’s disease. Here, we extended this investigation to remote contextual fear memory and extinction. TASTPM and wild-type mice acquired contextual fear at 4 months of age. Retention and extinction of contextual fear were assessed at 5.5 months prior to in vivo MRI assessment of regional T2 relaxation times and brain volumes followed by immunostaining of amyloid plaque load. Remote contextual fear memory was preserved in TASTPM mice regardless of the stress condition. Stress impaired extinction in wild-type mice but facilitated this process in TASTPM mice. Genotype-dependent effects of stress were observed on regional T2 times which were prolonged in the subiculum and thalamus of stressed TASTPM, possibly reflecting improved amyloid pathology. Amyloid plaque load was particularly decreased in the retrosplenial cortex of stressed TASTPM mice, which also showed an overall reduction in the number of diffuse plaques. These findings support the hypothesis that repeated mild levels of stress induced by novel activities can delay the progression of pathological changes relevant to Alzheimer’s disease.

Pages 1069-1082
Lotta Agholme, Tobias Lindström, Katarina Kågedal, Jan Marcusson, Martin Hallbeck (Handling Associate Editor: Thomas Shea)
An In Vitro Model for Neuroscience: Differentiation of SH-SY5Y Cells into Cells with Morphological and Biochemical Characteristics of Mature Neurons
Abstract: Neuroscience, including research on Alzheimer’s disease, is hampered by the lack of suitable in vitro models to study the human nervous system. To counteract this, many attempts to differentiate cell lines into more neuron-like cells have been performed, resulting in partial expression of neuronal features. Furthermore, it has been reported that neuroblastoma cell lines lack mature isoforms of tau. Our aim was to develop an improved in vitro model, generating sustainable cells with morphology and biochemistry of human, mature neurons. To obtain cells with neuronal differentiation and function, we investigated the effect of combining three-dimensional culturing of SH-SY5Y cells in extracellular matrix (ECM) gel with several factors reported to have neuro-differentiating effects. This resulted in cells with apparent neuronal morphology with long, extensively branched neurites. Further investigation revealed expression of several neurospecific markers including synapse protein Sv2 and nuclear marker NeuN, as well as the presence of synapses and axonal vesicle transport. In addition, these cells expressed mature tau isoforms, and tau protein expression was significantly increased compared to undifferentiated cells, reaching levels found in adult human brain. In conclusion, we found that pre-treatment with retinoic acid followed by ECM gel culturing in combination with brain derived neurotrophic factor, neuregulin β1, nerve growth factor, and vitamin D3 treatment generated sustainable cells with unambiguous resemblance to adult neurons. These cells also expresses adult splicing forms of tau with neuronal localization, making this cellular in vitro model useful in many areas of neuroscience research, particularly the Alzheimer’s disease field.

Pages 1083-1090
Maartje I. Kester, Wiesje M. van der Flier, Gorana Mandic, Marinus A. Blankenstein, Philip Scheltens, Majon Muller (Handling Associate Editor: Jose Luchsinger)
Joint Effect of Hypertension and APOE Genotype on CSF Biomarkers for Alzheimer’s Disease
Abstract: We examined the impact of hypertension on cerebrospinal fluid (CSF) biomarkers amyloid-β1-42 (Aβ42), total tau (tau), and phosphorylated tau at threonine 181 (ptau-181), and assessed the modifying role of APOE genotype in this relation in 546 patients (mean age 65 ± 10, 47% female) from our memory-clinic. Of these patients 150 had subjective complaints, 140 were diagnosed with mild cognitive impairment, and 256 with Alzheimer’s disease. Linear regression analyses adjusted for age, gender, and diagnosis showed that the association of hypertension with tau and ptau-181 was modified by APOE genotype (p-values for interaction <0.05). In APOE ε4 homozygotes (n=74), and to a lesser extent in APOE ε4 heterozygotes, hypertension was associated with higher tau and ptau-181 levels; β (95%CI) were 188 (11; 364) pg/mL and 22 (3; 42) pg/mL for the APOE ε4 homozygotes. Hypertension was not associated with Aβ42 levels, and APOE genotype did not modify this relation. Our findings suggest that hypertension is directly related to tau pathology in APOE ε4 homozygous carriers.

Pages 1091-1106
Jia Yu, Miao Sun, Zheng Chen, Jiangyang Lu, Yi Liu, Liang Zhou, Xuemin Xu, Dongsheng Fan, Dehua Chui (Handling Associate Editor: Chengxin Gong)
Magnesium Modulates Amyloid-β Protein Precursor Trafficking and Processing
Abstract: Alzheimer’s disease (AD), the most common form of dementia, is characterized by the presence of excessive deposits of aggregated amyloid-β (Aβ), which is derived from the amyloid-β protein precursor (AβPP) following processing by β- and γ-secretase. Metal elements are implicated in the pathophysiology of AD. Magnesium affects many biochemical mechanisms vital for neuronal properties and synaptic plasticity, and magnesium levels were reported to be decreased in various tissues including brain of AD patients. However, the exact role of magnesium in the neurodegenerative process of AD remains elusive. In this study, we investigated the effects of physiological (0.8 mM, as normal control), low (0-0.4 mM), and high (1.2-4.0 mM) concentrations of extracellular magnesium ([Mg2+]o) on AβPP processing and Aβ secretion. Here we show the effects of varying [Mg2+]o on AβPP processing is time- and dose-dependent. After 24 h treatment, high [Mg2+]o increased C-terminal fragment-α (CTFα) levels and soluble α-secretase cleaved AβPP (sAβPPα) release via enhancing retention of AβPP on plasma membrane. In contrast, low [Mg2+]o enhanced CTFβ accumulation and Aβ secretion, and reduced cell surface AβPP level. Varying [Mg2+]o did not alter protein contents of full length AβPP. However, decreased total intracellular magnesium level by magnesium deprivation over 24 hr impaired cell viability. Normal AβPP processing could be restored when magnesium was adjusted back to physiological concentration. These data demonstrate that AβPP processing can be modulated by magnesium and at high [Mg2+]o, AβPP processing favors the α-secretase cleavage pathway. Our findings suggest that supplementation of magnesium has a therapeutic potential for preventing AD.

Pages 1107-1118
Jean-Charles Lambert, Benjamin Grenier-Boley, Vincent Chouraki, Simon Heath, Diana Zelenika, Nathalie Fievet, Didier Hannequin, Florence Pasquier, Olivier Hanon, Alexis Brice, Jacques Epelbaum, Claudine Berr, Jean-Francois Dartigues, Christophe Tzourio, Dominique Campion, Mark Lathrop, Philippe Amouyel (Handling Associate Editor: Daniela Galimberti)
Implication of the Immune System in Alzheimer’s Disease: Evidence from Genome-Wide Pathway Analysis
Abstract: The results of several genome-wide association studies (GWASs) in the field of Alzheimer’s disease (AD) have recently been published. Although these studies reported in detail on single-nucleotide polymorphisms (SNPs) and the neighboring genes with the strongest evidence of association with AD, little attention was paid to the rest of the genome. However, complementary statistical and bio-informatics approaches now enable the extraction of pertinent information from other SNPs and/or genes which are only nominally associated with the disease risk. Two different tools (the ALIGATOR and GenGen/KEGG software packages) were used to analyze a large GWAS dataset containing 2,032 AD cases and 5,328 controls. Convergent outputs from the two gene set enrichment approaches suggested an immune system dysfunction in AD. Furthermore, although these statistical approaches did not adopt a priori hypotheses concerning a biological function’s putative role in the disease process, genes associated with AD risk were overrepresented in the “Alzheimer’s disease” KEGG pathway. In conclusion, a systematic search for biological pathways using GWAS data set seems to comfort the primary causes already suspected but may specifically highlight the importance of the immune system in AD.

Pages 1119-1132
Ira Espuny Camacho, Diana Dominguez, Pascal Merchiers, Luc Van Rompaey, Dennis Selkoe, Bart De Strooper
Peroxisome Proliferator-Activated Receptor Gamma Enhances the Activity of an Insulin Degrading Enzyme-Like Metalloprotease for Amyloid-β Clearance
Abstract: Peroxisome proliferator-activated receptor gamma (PPARγ) activation results in an increased rate of amyloid-β (Aβ) clearance from the media of diverse cells in culture, including primary neurons and glial cells. Here, we further investigate the mechanism for Aβ clearance and found that PPARγ activation modulates a cell surface metalloprotease that can be inhibited by metalloprotease inhibitors like EDTA and phenanthroline, but also by the hormone peptides insulin and glucagon. The metalloprotease profile of the Aβ-degrading mechanism is surprisingly similar to insulin-degrading enzyme (IDE). This mechanism is maintained in hippocampal and glia primary cultures from IDE loss-of-function mice. We conclude that PPARγ activates an IDE-like Aβ degrading activity. Our work suggests a drugable pathway that can clear Aβ peptide from the brain.

Supplementary Data for Espuny Camacho et al. article (PDF)

Pages 1133-1141
Silvia C. Lenzken, Serena Stanga, Cristina Lanni, Fabio De Leonardis, Stefano Govoni, Marco Racchi
Recruitment of Casein Kinase 2 is Involved in AβPP Processing Following Cholinergic Stimulation
Abstract: The amyloid-β protein precursor (AβPP) is an integral membrane protein subjected to constitutive and regulated proteolytic processing. We have previously demonstrated that protein kinase C ε (PKCε) plays a key role in the regulation of AβPP metabolism via cholinergic receptors. The purpose of the present work is to clarify whether other putative signaling systems are involved in the same pharmacological pathway. We focused particularly on casein kinase 2 (CK2), demonstrating a direct interaction between PKCε and CK2 following cholinergic stimulation. Treatment of human neuroblastoma SH-SY5Y cells with a selective inhibitor of CK2 reduced the effect of carbachol on the release of sAβPPα. This treatment did not influence the activation and translocation of PKCε suggesting that the latter is located upstream of CK2. On the basis of our results, we add another player to the complex cellular mechanisms regulating non-amyloidogenic processing of AβPP.

Pages 1143-1157
Yong Cheng and Long-Chuan Yu (Handling Associate Editor: Shengdi Chen)
Galanin Protects Amyloid-β-Induced Neurotoxicity on Primary Cultured Hippocampal Neurons of Rats
Abstract: The neuropeptide galanin and its receptors are found to be upregulated in brain associated with Alzheimer’s disease (AD), while the role of galanin in AD is still unclear. The present study was performed to explore the neuroprotective role of galanin both in vitro and in vivo. Our results demonstrated that galanin inhibited the neurotoxicity induced by amyloid-β25-35 (Aβ25-35) or Aβ1-42 in primary cultured hippocampal neurons of rats. Moreover, Gal2-11 (an agonist of GalR2/3) also inhibited the neurotoxicity induced by Aβ25-35 in the cultured neurons. We further found that galanin inhibited the activation of p53, Bax, and caspase-3 induced by Aβ25-35 in the cultured hippocampal neurons. Moreover, galanin reversed the down regulation of Bcl-2 induced by Aβ25-35 in the neurons. Interestingly, in the Morris water maze task we found that intra-CA1 injection of Aβ25-35-induced spatial learning deficits in rats were blocked by galanin. In addition, galanin inhibited Aβ25-35-induced dysregulation of p53, Bax, and MAP2 in rat hippocampus. Our results strongly demonstrate that galanin plays neuroprotective roles in nerve cells and in AD-induced learning and memory deficits.

Pages 1159-1165
Christopher Exley, Emily House, Joanna F. Collingwood, Mark R. Davidson, Danielle Cannon, Athene M. Donald
Spherulites of Amyloid-β42 In Vitro and in Alzheimer’s Disease
Abstract: Several amyloidogenic proteins including insulin, β-lactoglobulin, and albumin form spherulites in vitro under non-physiological conditions. These micrometer-sized, roughly spherical structures are composed of ordered arrays of amyloid fibrils in radial arrangements which, characteristically, show a typical Maltese cross pattern of light extinction under the polarizing microscope. The physiological significance of amyloid spherulites is unknown though in Alzheimer’s disease, senile plaques composed primarily of β sheets of amyloid-β (Aβ)42 have, very occasionally, been shown to give a Maltese cross pattern of light extinction under crossed polarizers. Herein we describe the first observation of the formation in vitro of spherulites of Aβ42. They were formed under near-physiological conditions in which the β sheet conformation of pre-formed aggregates of Aβ42 had been abolished following the addition of an excess of copper. Incubation of these preparations at 37°C for up to 9 months resulted in the formation of globular structures, 5–20 μm in diameter, which exhibited a Maltese cross pattern of light extinction typical of spherulites. Near-identical spherulitic structures were also observed in abundance in 30 μm thick sections of Alzheimer’s disease brain tissue. Synchrotron x-ray fluorescence showed that the location of these spherulites in AD tissue coincided with locally elevated concentrations of tissue copper. The formation in vitro of spherulites of Aβ42 which morphologically appeared analogous to spherulitic structures observed in vivo strongly supports the hypothesis that spherulites and senile plaques in AD tissue are one and the same structures and that their ultimate formation may involve copper.

Pages 1167-1180
Michael Willis, Walter A. Kaufmann, Georg Wietzorrek, Birgit Hutter-Paier, Sven Moosmang, Christian Humpel, Franz Hofmann, Manfred Windisch, Hans-Günther Knaus, Josef Marksteiner
L-Type Calcium Channel CaV1.2 in Transgenic Mice Overexpressing Human AβPP751 with the London (V717I) and Swedish (K670M/N671L) Mutations
Abstract: Cumulative evidence indicates that amyloid-β peptides exert some of their neurodegenerative effects through modulation of L-type voltage gated calcium channels, which play key roles in a diverse range of central nervous system functions. In this study we examined the expression of CaV1.2 L-type voltage gated calcium channels in transgenic mice overexpressing human AβPP751 with the London (V717I) and Swedish (K670M/N671L) mutations by immunohistochemistry in light and electron microscopy. In hippocampal layers of wild type and transgenic mice, CaV1.2 channels were predominantly localized to somato-dendritic domains of neurons and to astrocytic profiles with an age-dependent increase in labeling density. In transgenic animals, CaV1.2-like immunoreactive clusters were found in neuronal profiles in association with amyloid-β plaques. Both the number and density of these clusters depended upon age of animals and number of plaques. The most striking difference between wild type and transgenic mice was the age-dependent expression of CaV1.2 channels in reactive astrocytes. At the age of 6 month, CaV1.2 channels were rarely detected in reactive astrocytes of transgenic mice, but an incremental number of CaV1.2 expressing reactive astrocytes was found with increasing age of animals and number of amyloid-β plaques. This study demonstrates that CaV1.2 channels are highly expressed in reactive astrocytes of 12-month old transgenic mice, which might be a consequence of the increasing amyloid burden. Further studies should clarify which functional implications are associated with the higher availability of CaV1.2 channels in late stage Alzheimer's disease.

Pages 1181-1188
Geoffroy Laumet, Vincent Chouraki, Benjamin Grenier Boley, Vanessa Legry, Simon Heath, Diana Zelenika, Nathalie Fievet, Didier Hannequin, Marc Delepine, Florence Pasquier, Olivier Hanon, Alexis Brice, Jacques Epelbaum, Claudine Berri, Jean-Francois Dartigues, Christophe Tzourio, Dominique Campion, Mark Lathrop, Lars Bertram, Philippe Amouyel, Jean-Charles Lambert (Handling Associate Editor: Daniela Galimberti)
Systematic Analysis of Candidate Genes for Alzheimer’s Disease in a French, Genome-Wide Association Study
Abstract: We selected twenty genes from the "Top Results" list on the AlzGene database website and assessed their association with risk of developing Alzheimer’s disease (AD) in a large, genome-wide association study (using 526 SNPs from 2,032 AD cases and 5,328 controls) performed in France. The APOE, CLU, PICALM, and CR1 loci were excluded, since they had already been extensively analyzed. Ten genes/loci (TFAM, SORL1, CHRNB2, SORCS1, DAPK1, MTHFR, GWA 14q32.13, BDNF, NEDD9, and CH25H) showed weak nominal association with AD risk, in line with previous studies. In the remaining ten genes/loci (TNK1, ACE, CST3, IL1B, hCG2039140, PRNP, GAB2, LOC651924, IL1A, and TF), no single nucleotide polymorphisms were associated in our dataset. Of the genes showing nominal association in our cohorts, TFAM and CHRNB2 appear particularly interesting and warrant further genetic and functional follow-up analyses.

Supplementary Data for Laumet et al. article (PDF)

Pages 1189-1199
Hong-Mei Wang , Yan-Xin Zhao , Shi Zhang, Gui-Dong Liu , Wen-Yan Kang, Hui-Dong Tang, Sheng-Di Chen, Jian-Qing Ding (Handling Associate Editor: Xiongwei Zhu)
PPARγ Agonist Curcumin Reduces the Amyloid-β-Stimulated Inflammatory Responses in Primary Astrocytes
Abstract: Alzheimer’s disease (AD) is the most common age-related neurodegenerative disorder. Accumulating data indicate that astrocytes play an important role in the neuroinflammation related to the pathogenesis of AD. It has been shown that microglia and astrocytes are activated in AD brain and amyloid-β (Aβ) can increase the expression of cyclooxygenase 2 (COX-2), interleukin-1, and interleukin-6. Suppressing the inflammatory response caused by activated astrocytes may help to inhibit the development of AD. Curcumin is a major constituent of the yellow curry spice turmeric and proved to be a potential anti-inflammatory drug in arthritis and colitis. There is a low age-adjusted prevalence of AD in India, a country where turmeric powder is commonly used as a culinary compound. Curcumin has been shown to suppress activated astroglia in amyloid-β protein precursor transgenic mice. The real mechanism by which curcumin inhibits activated astroglia is poorly understood. Here we report that the expression of COX-2 and glial fibrillary acidic protein were enhanced and that of peroxisome proliferator-activated receptor γ (PPARγ) was decreased in Aβ25–35-treated astrocytes. In line with these results, nuclear factor-κB translocation was increased in the presence of Aβ. All these can be reversed by the pretreatment of curcumin. Furthermore, GW9662, a PPARγ antagonist, can abolish the anti-inflammatory effect of curcumin. These results show that curcumin might act as a PPARγ agonist to inhibit the inflammation in Aβ-treated astrocytes.

Pages 1201-1213
Peter Holm, Anders Ettrup, Anders B. Klein, Martin A. Santini, Mona El-Sayed, Anders B. Elvang, Tine B. Stensbøl, Jens D. Mikkelsen, Gitte M. Knudsen, Susana Aznar
Plaque Deposition Dependent Decrease in 5-HT2A Serotonin Receptor in AβPPswe/PS1dE9 Amyloid Overexpressing Mice
Abstract: Intrahippocampal injections of aggregated amyloid-β (Aβ)1-42 in rats result in memory impairment and in reduction of hippocampal 5-HT2A receptor levels. In order to investigate how changes in 5-HT2A levels and functionality relate to the progressive accumulation of Aβ protein, we studied 5-HT2A receptor regulation in double transgenic AβPPswe/PS1dE9 mice which display excess production of Aβ and age-dependent increase in amyloid plaques. Three different age-groups, 4-month-old, 8- month-old, and 11-month-old were included in the study. [3H]-MDL100907, [3H]-escitalopram, and [11C]-PIB autoradiography was performed for measuring 5-HT2A receptor, serotonin transporter (SERT), and Aβ plaque levels in medial prefrontal cortex (mPFC), prefrontal cortex (PFC), frontoparietal cortex (FPC), dorsal and ventral hippocampus, and somatosensory cortex. To investigate 5-HT2A receptor functionality, animals were treated with the 5-HT2A receptor agonist DOI and head-twitch response (HTR) subsequently recorded. Expression level of the immediate early gene c-fos was measured by in situ hybridization. We found that the age-related increase in Aβ plaque burden was accompanied by a significant decrease in 5-HT2A receptor binding in mPFC in the 11-month-old group. The changes in 5-HT2A receptor binding correlated negatively with [11C]-PIB binding and were not accompanied by decreases in SERT binding. Correspondingly, 11-month-old transgenic mice showed diminished DOI-induced HTR and reduced increase in expression of c-fos mRNA in mPFC and FPC. These observations point towards a direct association between Aβ accumulation and changes in 5-HT2A receptor expression that is independent of upstream changes in the serotonergic system.

Pages 1215-1231
Elzbieta Kojro, Petra Füger, Claudia Prinzen, Anna Maria Kanarek, Dorothea Rat, Kristina Endres, Falk Fahrenholz, Rolf Postina (Handling Associate Editor: Angelika Bierhaus)
Statins and the Squalene Synthase Inhibitor Zaragozic Acid Stimulate the Non-Amyloidogenic Pathway of Amyloid-β Protein Precursor Processing by Suppression of Cholesterol Synthesis
Abstract: Cholesterol-lowering drugs such as statins influence the proteolytic processing of the amyloid-β protein precursor (AβPP) and are reported to stimulate the activity of α-secretase, the major preventive secretase of Alzheimer’s disease. Statins can increase the α-secretase activity by their cholesterol-lowering properties as well as by impairment of isoprenoids synthesis. In the present study, we elucidate the contribution of these pathways in α-secretase activation. We demonstrate that zaragozic acid, a potent inhibitor of squalene synthase which blocks cholesterol synthesis but allows synthesis of isoprenoids, also stimulates α-secretase activity. Treatment of human neuroblastoma cells with 50 µM zaragozic acid resulted in a ~3 fold increase of α-secretase activity and reduced cellular cholesterol by ~30%. These effects were comparable to results obtained from cells treated with a low lovastatin concentration (2 µM). Zaragozic acid-stimulated secretion of α-secretase-cleaved soluble AβPP was dose dependent and saturable with an EC50 value of 27 µM. Lovastatin- or zaragozic acid-stimulated increase of α-secretase activity was completely abolished by a selective ADAM10 inhibitor. By targeting the α-secretase ADAM10 to lipid raft domains via a glycosylphosphatidylinositol anchor, we demonstrate that ADAM10 is unable to cleave AβPP in a cholesterol-rich environment. Our results indicate that inhibition of cholesterol biosynthesis by a low lovastatin concentration is sufficient for α-secretase activation.

Pages 1233-1242
James K. Lui*, Simon M. Laws*, Qiao-Xin Li, Victor L. Villemagne, David Ames, Belinda Brown, Ashley I. Bush, Karl De Ruyck, Jasmin Dromey, Kathryn A. Ellis, Noel G. Faux, Jonathan Foster, Christopher Fowler, Veer Gupta, Peter Hudson, Katrina Laughton, Colin L. Masters, Kelly Pertile, Alan Rembach, Mira Rimajova, Mark Rodrigues, Christopher C. Rowe, Rebecca Rumble, Cassandra Szoeke, Kevin Taddei, Tania Taddei, Brett Trounson, Vanessa Ward, Ralph N. Martins, for the AIBL Research Group
*These authors contributed equally to this manuscript.
Plasma Amyloid-β as a Biomarker in Alzheimer’s Disease: The AIBL Study of Aging
Abstract: Amyloid-β (Aβ) plays a central role in the pathogenesis of Alzheimer’s disease (AD) and has been postulated as a potential biomarker for AD. However, there is a lack of consensus as to its suitability as an AD biomarker. The objective of this study was to determine the significance of plasma Aβ as an AD biomarker and its relationship with Aβ load and to determine the effect of different assay methods on the interpretation of Aβ levels. Plasma Aβ1-40, Aβ1-42, and n-terminal cleaved fragments were measured using both a commercial multiplex assay and a well-documented ELISA in 1032 individuals drawn from the well-characterized Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging. Further, Aβ levels were compared to Aβ load derived from positron-emission tomography (PET) with the Pittsburgh compound B (PiB). Lower Aβ1-42 and Aβ1-42/1-40 ratio were observed in patients with AD and inversely correlated with PiB-PET derived Aβ load. However, assay methodology significantly impacted the interpretation of data. The cross-sectional analysis of plasma Aβ isoforms suggests that they may not be sufficient per se to diagnose AD. The value of their measurement in prognosis and monitoring of AD interventions needs further study, in addition to future longitudinal comparisons together with other predictors, which will determine whether plasma Aβ has diagnostic value in a panel of biomarkers.

Supplementary Data for Lui et al. article (PDF)

Pages 1243-1253
Annamalai Prakasam, Anusuya Muthuswamy, Zsolt Ablonczy, Nigel H. Greig, Abdul Fauq, Kosagisharaf Jagannatha Rao, Miguel A. Pappolla, Kumar Sambamurti
Differential Accumulation of Secreted AβPP Metabolites in Ocular Fluids
Abstract: Amyloid-β (Aβ) accumulates in several types of retinal degeneration and in Alzheimer’s disease (AD), but its source has been unclear. We detected the neuronal 695 amino acid form of amyloid-β protein precursor (AβPP) in the normal retina and AβPP751 in the retinal pigment epithelium (RPE) and anterior eye tissues. Similar to the brain, α- and β-secretases cleaved AβPP to soluble derivatives (sAβPP) α or β and membrane-bound C-terminal fragments α or β in the retina and RPE. Levels of sAβPP were particularly high in the vitreous and low in aqueous humor revealing a molecular barrier for AβPP. In contrast, Aβ40 and Aβ42 levels were only 50% lower in the aqueous than the vitreous humor, indicating relatively barrier-free movement of Aβ. These studies demonstrated a relatively high yield of AβPP and Aβ in the ocular fluids, which may serve as a trackable marker for AD. In addition, failure of free clearance from the eye may trigger retina degeneration in a manner similar to Aβ-related neurodegeneration in AD.

Pages 1255-1260
Meeting Report from the Alzheimer Research Forum
The 7th Leonard Berg Symposium, Part 2

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