Volume 13, Number 2, March 2008

Pages 111-122
Amalia M. Dolga, Ingrid M. Nijholt, Anghelus Ostroveanu, Quirine ten Bosch, Paul G.M. Luiten, Ulrich L.M. Eisel (Communicated by Angelika Bierhaus)
Lovastatin induces neuroprotection through tumor necrosis factor receptor 2 signaling pathways
Abstract: Statins are widely used as medication to lower cholesterol levels in human patients. In addition, it was recently reported that they also reduce the incidence of stroke and progression of Alzheimer's disease when prophylactically administered. To date there is only limited information available on how statins exert this beneficial effect. In this study we investigated the neuroprotective effect of lovastatin in primary cortical neurons. We found that lovastatin protects cortical neurons in a concentration-dependent manner against glutamate-mediated excitotoxicity. Interestingly, lovastatin with or without glutamate and/or tumor necrosis factor-alpha (TNF-α) increased TNF receptor 2 (TNF-R2) expression in cortical neurons. It was previously shown that activation of TNF-R2 signaling, which includes phosphorylation of protein kinase B (PKB)/Akt and activation of nuclear factor-kappa B (NF-κB), protects neurons against ischemic or excitotoxic insults. To investigate if the lovastatin-induced neuroprotection is mediated by TNF-R2 signaling, primary cortical neurons were isolated from TNF-R1-/- or TNF-R2-/- mice. We could show that lovastatin is neuroprotective in TNF-R1-/- neurons, while protection is completely absent in TNF-R2-/- neurons. Furthermore, lovastatin-mediated neuroprotection led to an increase in PKB/Akt and NF-κB phosphorylation, whereas inhibition of PKB/Akt activation entirely abolished lovastatin-induced neuroprotection. Thus, lovastatin induced neuroprotection against glutamate-excitotoxicity via activation of TNF-R2-signaling pathways.

Pages 123-135
Michael Willis, Manuela Prokesch, Birgit Hutter-Paier, Manfred Windisch, Mats Stridsberg, Sushil K. Mahata, Rudolf Kirchmair, Georg Wietzorrek, Hans-Günther Knaus, Kurt Jellinger, Christian Humpel and Josef Marksteiner (Communicated by Thomas Bayer)
Chromogranin B and secretogranin II in transgenic mice overexpressing human APP751 with the London (V717I) and Swedish (K670M/N671L) mutations and in Alzheimer patients
Abstract: Chromogranin B and secretogranin II are major soluble constituents of large dense core vesicles of presynaptic structures and have been found in neuritic plaques of Alzheimer patients. We examined the distribution and expression of these peptides in both transgenic mice over-expressing human amyloid-β protein precursor APP751 with the London (V717I) and Swedish (K670M/N671L) mutations and in human post-mortem brain. In transgenic mice, the number of amyloid-β plaques and chromogranin immunopositive plaques increased from 6 to 12 months. About 60% of amyloid-β plaques were associated with chromogranin B, and about 40% with secretogranin II. Chromogranin immunoreactivity appeared mainly as swollen dystrophic neurites. Neither synaptophysin- nor glial fibrillary acidic protein- immunoreactivity was expressed in chromogranin immunoreactive structures at any timepoint. Density of chromogranin peptides in hippocampal structures did not change in transgenic animals at any timepoint, even though animals had a poorer performance in the Morris water maze task. In conclusion, transgenic animals partly resembled findings in Alzheimer patients. Chromogranin peptides were associated with amyloid-β plaques, but were not reduced in specific brain areas as previously reported by our group. Therefore specific changes of chromogranin peptides observed Alzheimer patients can be related to amyloid-β pathology only.

Pages 137-146
Mira Rimajova, Nat P. Lenzo, Jing-Shan Wu, Kristyn A. Bates, Andrew Campbell, Satvinder S. Dhaliwal, Michael McCarthy, Mark Rodrigues, Athena Paton, Christopher Rowe, Jonathan K. Foster, Ralph N. Martins
Fluoro-2-deoxy-D-glucose (FDG)-PET in APOEε4 carriers in the Australian population
Abstract: Apolipoprotein E-e4 (APOEε4) has been associated with increased risk of developing Alzheimer's disease (AD) and regional cerebral glucose hypometabolism, as measured by fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET). We report here preliminary data from studies that aim to determine whether cerebral glucose hypometabolism is observed in APOEe4 positive, cognitively intact individuals between the ages of 50 and 80, and whether there is an additional impact of subjective memory complainer (SMC) status on glucose metabolism determined by NeuroStat analysis. FDG-PET was conducted in 30 community dwelling, APOE-e4 carriers without clinical evidence of dementia and objective cognitive impairment as assessed using a neuropsychological battery. Neurological soft-signs (NSS) were also assessed. Glucose hypometabolism was demonstrated in the anterior and posterior cingulate cortex and in the temporal association cortices in APOEe4 carriers compared to the normative NeuroStat database. This pattern was particularly evident in APOEe4 heterozygous individuals. SMC showed hypometabolism in the aforementioned brain regions, whereas non-SMC showed no significant pattern of glucose hypometabolism. FDG-PET with NeuroStat analysis showed that APOEe4 carriers have mild glucose hypometabolism in areas associated with AD. SMC may be associated with AD-related differences in regional cerebral glucose metabolism. These findings are currently being investigated in a larger group of APOEe4 carriers.

Pages 147-150
Short Communication
Prashant Bharadwaj, Lynne Waddington, Jose Varghese, Ian G. Macreadie
A new method to measure cellular toxicity of non-fibrillar and fibrillar Alzheimer’s Aβ using yeast
Abstract: The 42 amino acid amyloid-β (Aβ) can exist in multiple physical states including oligomers and fibrils. This study shows that fibril formation is hastened by the biological buffers required to support the growth of mammalian cells, but is prevented if Aβ is maintained in water. Here we describe a method to produce Aβ in oligomeric form and the comparison of stable fibrillar and non-fibrillar forms in cell toxicity studies in water, achieved through the use of yeast. We show that extracellular, non-fibrillar Aβ causes a dose dependent loss of cell viability while fibrillar Aβ has low toxicity.

Pages 151-160
Antonietta Gentile, Giuseppina Amadoro, Veronica Corsetti, Maria T. Ciotti, AnnaLucia Serafino, Pietro Calissano (Communicated by Antonino Cattaneo)
Spontaneous aggregation and altered intracellular distribution of endogenous α-synuclein during neuronal apoptosis
Abstract: The precursor of the non-amyloid-β component of Alzheimer's disease amyloid, also known as α-synuclein, is a presynaptic terminal molecule that accumulates in the senile plaques of Alzheimer's disease. Aberrant accumulation of this protein into insoluble aggregates has also been implicated in the pathogenesis of many other neurodegenerative diseases, collectively referred to as synucleinopathies. However, the precise pathogenetic mechanism that leads to aggregates formation and the consequent cellular damage remains elusive. Analyzing differentiated primary cultures of cerebellar granule neurons undergoing apoptosis due to K+ reduction from 25 mM to 5.0 mM, a neuronal model widely used to study event linking apoptosis and neurodegeneration, we assessed that endogenous monomeric α-synuclein decreases and spontaneously aggregates into detergent-insoluble high molecular species. Apoptosis is also correlated with a marked redistribution/accumulation of this protein from terminal neurites to perikaria, with formation of compact inclusion bodies in juxta-nuclear area. In addition, secretion of monomeric α-synuclein decreases in response to apoptotic stimulus, while part of it aggregates into fibrillar structures and becomes detectable by immunogold-electron microscope analysis. The data presented in this study demonstrate that an apoptotic event caused by a “physiological” trigger, such as neuronal membrane repolarization of cultured cerebellar granule neurons, induces α-synuclein intracellular redistribution and aggregation, two molecular events reminiscent of those occurring in different human neurodegenerative diseases all characterized by α-synuclein-positive inclusions. Our study prospects this in vitro neuronal system as an excellent model to dissect the pathogenic mechanism(s).

Pages 161-172
Rosa Guerrero, Paloma Navarro, Eva Gallego, Jesus Avila, Justo G. de Yebenes, Marina P. Sanchez (Communicated by Pilar Gomez Ramos)
Park2-null/tau transgenic mice reveal a functional relationship between parkin and tau
Abstract: Mutations, haplotypes, and polymorphisms of tau and Park-2 genes constitute risk factors for developing tauopathies. In order to analyze the possible relationship between parkin and tau we generated a double-mutant mouse deficient for Park-2 expression and overexpressing a mutant tau protein (hTauVLW). Mice develop normally, although the median survival rate is considerably reduced with respect to wild type (45%). Aggregates of phosphorylated tau in neurons and reactive gliosis are quite abundant in cortex and hippocampus of these mice. Moreover, while in young transgenic mice the hTauVLW immunostained transgene product is observed in both cell bodies and dendrites, the hTauVLW mutant protein is only detected in the neuronal cell bodies when Park-2 gene is additionally deleted. Moreover, DNA fragmentation was detected by the TUNEL method, and cerebral atrophy is also present in these regions. The levels of phosphorylated tau and Hsp70 are increased in the double-mutant mice, while CHIP expression in hippocampus is lower when the Park-2 gene is deleted. Thus, the combination of Park-2 gene deletion with hTauVLW transgene overexpression in mice produces serious neuropathological effects, which reflect the existence of some relationship between both proteins.

Pages 173-176
Short Communication
Patrícia Natália Oliveira Silva, Carolina Oliveira Gigek, Mariana Ferreira Leal, Paulo Henrique Ferreira Bertolucci, Roger W de Labio, Spencer Luiz Marques Payão, Marília de Arruda Cardoso Smith (Communicated by Jesus Avila)
Promoter Methylation Analysis of SIRT3, SMARCA5, HTERT and CDH1 Genes in Aging and Alzheimer’s Disease
Abstract: Longevity related genes were investigated concerning promoter methylation. SIRT3, SMARCA5, HTERT and CDH1 promoters were analyzed in peripheral blood in relation to gender, age and Alzheimer’s disease (AD). Methylation Specifc PCR assay (MSP) was used. There were no significant differences in methylation frequencies of SIRT3, SMARCA5 and CDH1 among young, elderly and AD groups (p>0.05), showing no association with aging or AD. On the other hand, HTERT methylation frequency was associated with the aging process, in that AD patients differed from elderly controls (p=0.0086), probably due to telomere and immune dysfunctions involved in AD pathogenesis.

Pages 177-185
Ismael Santa-Maria, Raquel Cuadros, Francisco J. Moreno, Victor Muńoz, Jesús Ávila, Félix Hernández
Binding of Tau protein to the Ends of ex vivo Paired Helical Filaments
Abstract: Human recombinant tau can bind to the end, of isolated human paired helical filaments (PHF). The sequential binding of tau protein to PHF could result in an elongation of the previously polymerized PHF. However, we have observed that the elongation takes place in a different way on different types of PHF. At least, there are three populations of PHF. For one population tau protein is able to bind to the ends of the filament and to elongate that filament. In other PHF population, tau protein binds but not elongates the filament. In the third PHF no tau binding, no elongation was observed.

Pages 187-197
Cynthia M. Carlsson, Carey E. Gleason, Timothy M. Hess, Kimberly A. Moreland, Hanna M. Blazel, Rebecca L. Koscik, Nathan T.N. Schreiber, Sterling C. Johnson, Craig S. Atwood, Luigi Puglielli, Bruce P. Hermann, Patrick E. McBride, James H. Stein, Mark A. Sager, Sanjay Asthana
Effects of Simvastatin on Cerebrospinal Fluid Biomarkers and Cognition in Middle-Aged Adults at Risk for Alzheimer’s Disease
Abstract: Background: Statins reduce amyloid-β (Aβ) levels in the brain and cerebrospinal fluid (CSF) in animals and may thereby favorably alter the pathobiology of AD. It is unclear if statins modify Aβ metabolism or improve cognition in asymptomatic middle-aged adults at increased risk for AD. Methods: In a 4-month randomized, double-blind, controlled study, we evaluated the effects of simvastatin 40 mg daily vs. placebo on CSF Aβ42 levels and cognition in 57 asymptomatic middle-aged adult children of persons with AD. Results: Compared to placebo, individuals randomized to simvastatin for 4 months had similar changes in CSF Aβ42 (p=0.344) and total tau levels (p=0.226), yet greater improvements in some measures of verbal fluency (p=0.024) and working memory (p=0.015). APOE4 genotype, gender, and vascular risk factors were associated with CSF biomarker levels, but did not modify treatment effects. Conclusions: In asymptomatic middle-aged adults at increased risk for AD, simvastatin use improved selected measures of cognitive function without significantly changing CSF Aβ42 or total tau levels. Further studies are needed to clarify the impact of higher dose and/or longer duration statin therapy on not only Aβ metabolism, but also other preclinical processes related to the development of AD.

Pages 199-223
Amir Nazem, G. Ali Mansoori
Nanotechnology Solutions for Alzheimer's Disease: Advances in research tools, diagnostic methods and therapeutic agents.
Abstract: A century of research has passed since the discovery and definition of Alzheimer’s disease (AD), the primary common dementing disorder worldwide. However, AD lacks definite diagnostic approaches and effective cure at the present. Moreover, the currently available diagnostic tools are not sufficient for an early screening of AD in order to start preventive approaches. Recently the emerging field of nanotechnology has promised new techniques to solve some of the AD challenges. Nanotechnology refers to the techniques of designing and manufacturing nanosize (1-100 nm) structures through controlled positional and / or self-assembly of atoms and molecules. In this report, we present the promises that nanotechnology brings in research on the AD diagnosis and therapy. They include its potential for the better understanding of the AD root cause molecular mechanisms, AD’s early diagnoses, and effective treatment. The advances in AD research offered by the atomic force microscopy, single molecule fluorescence microscopy and NanoSIMS microscopy are examined here. In addition, the recently proposed applications of nanotechnology for the early diagnosis of AD including bio-barcode assay, localized surface plasmon resonance nanosensor, quantum dot and nanomechanical cantilever arrays are analyzed. Applications of nanotechnology in AD therapy including neuroprotections against oxidative stress and anti-amyloid therapeutics, neuroregeneration and drug delivery beyond the blood brain barrier (BBB) are discussed and analyzed. All of these applications could improve the treatment approach of AD and other neurodegenerative diseases. The complete cure of AD may become feasible by a combination of nanotechnology and some other novel approaches, like stem cell technology.

Pages 225-232
Manuel Lavados, Marta Guillón, María Cristina Mujica, Leonel E. Rojo, Patricio Fuentes, Ricardo B. Maccioni
Mild Cognitive Impairment and Alzheimer patients display different levels of Redox-active CSF Iron
Abstract: Oxidative stress constitutes a hallmark of Alzheimer’s disease (AD). Recent studies also point to redox active metal such as iron, copper and zinc in mediating oxidative stress in AD pathogenesis. However, the reactivity of cerebrospinal fluid (CSF) iron and its possible correlation with the severity of cognitive decline in both, Alzheimer’s patients and subjects with mild cognitive impairment (MCI) is still unknown. Here we show that different stages of cognitive and functional impairment are associated with changes in CSF reactive iron. In this work, we compared CSF samples from 56 elders, classified into 4 groups according to their scores in Clinical Dementia Rating scale (CDR). Total CSF iron was analyzed by atomic absorption spectrometry. Redox-active iron was analyzed by a novel fluorimetric assay. One-way ANOVA was used to test differences in mean values, and Newman-Keuls Multiple Comparison Test was used for multi group comparisons. No difference in total CSF iron was found between different groups. Significant amounts of redox-active iron were found in CSF and their levels correlated with the extent of cognitive impairment. Redox-active CSF iron levels increased with the degree of cognitive impairment from normal to MCI subjects, while AD patients showed an abrupt decrease to levels close to zero. Given the relevance of oxidative damage in neurodegeneration, it might be possible to associate the development of cognitive and functional decline with the presence of redox-active iron in the CSF. The decrease in redox-active iron found in AD patients may represent a terminal situation, whereby the central nervous system attempts to minimize iron-associated toxicity.

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