Volume 33, Number 1, January 2013

Pages 1-2
In Memoriam
Alejandra D. Alonso, Cheng-Xin Gong, George Perry, Jesus Avila
Inge Grundke-Iqbal: A Legacy of Tau in the Etiology of Alzheimer Disease (1937-2012)

Pages 3-16
Review
Siddhita D. Mhatre, Brie E. Paddock, Aleister J. Saunders, Daniel R. Marenda
Invertebrate Models of Alzheimer’s Disease
Abstract: A majority of the genes linked to human disease belong to evolutionarily conserved pathways found in simpler organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The genes and pathways of these simple organisms can be genetically and pharmacologically manipulated to better understand the function of their orthologs in vivo, and how these genes are involved in the pathogenesis of different diseases. Often these manipulations can be performed much more rapidly in flies and worms than in mammals, and can generate high quality in vivo data that is translatable to mammalian systems. Other qualities also make these organisms particularly well suited to the study of human disease. For example, developing in vivo disease models can help illuminate the basic mechanisms underlying disease, as in vitro studies do not always provide the natural physiological complexity associated with many diseases. Invertebrate models are relatively inexpensive, easy to work with, have short lifespans, and often have very well characterized and stereotypical development and behavior. This is particularly true for the two invertebrate model organisms that this review will focus on: Caenorhabditis elegans and Drosophila melanogaster. In this review, we will first describe an overview of modeling Alzheimer’s disease in flies and worms, and will then highlight some of the more recent advances that these “simple” animals have contributed to our understanding of Alzheimer’s disease in recent years.

Pages 17-21
Short Communication
Jogender Mehla, Monika Pahuja, Yogendra Kumar Gupta
Streptozotocin-Induced Sporadic Alzheimer’s Disease: Selection of Appropriate Dose
Abstract: The long term effect of single (day 1) and twice (day 1 & 3) injections of intracerebroventricular (ICV) streptozotocin (STZ) at the doses of 1 and 3 mg/kg on the cognitive functions of male Wistar rats was evaluated. Elevated plus maze, passive avoidance, and Morris water maze tests were used to assess the cognitive functions. A significant cognitive deficit was found at the 2nd week onwards, which persisted up to the 14th week with single and twice ICV-STZ (3 mg/kg) injections, whereas no cognitive impairment was found in ICV-STZ (1 mg/kg) treated groups after 8-10 weeks.

Pages 23-27
Short Communication
Anna Kamalainen*, Jayashree Viswanathan*, Teemu Natunen, Seppo Helisalmi, Tarja Kauppinen, Maria Pikkarainen, Juha-Pekka Pursiheimo, Irina Alafuzoff, Miia Kivipelto, Annakaisa Haapasalo, Hilkka Soininen, Sanna-Kaisa Herukka, Mikko Hiltunen (Handling Associate Editor: Henrik Zetterberg) *These authors contributed equally
GRN Variant rs5848 Reduces Plasma and Brain Levels of Granulin in Alzheimer’s Disease Patients
Abstract: Genetic variants in the granulin (GRN) gene have been shown to increase the risk of Alzheimer’s disease (AD). Here, we report that the A allele of rs5848 in GRN reduces plasma granulin levels in a dose-dependent manner in a clinically-defined AD sample cohort. Similarly, the mRNA levels of granulin were decreased with respect to A allele of rs5848 in the inferior temporal cortex of neuropathologically confirmed AD patients. Our findings suggest that the A allele of rs5848 is functionally relevant by reducing the expression of granulin.

Supplementary Data for Kamalainen et al. article (PDF)

Pages 29-33
Short Communication
Susan A. Austin, Livius V. d’Uscio, Zvonimir S. Katusic
Supplementation of Nitric Oxide Attenuates AβPP and BACE1 Protein in Cerebral Microcirculation of eNOS-Deficient Mice
Abstract: Recently, we demonstrated in endothelial nitric oxide synthase deficient (eNOS-/-) mice that loss of endothelial NO led to increased protein levels of amyloid-β protein precursor (AβPP), β-site AβPP cleaving enzyme 1 (BACE1), and amyloid-β (Aβ) peptide. Therefore, our aim was to determine if NO supplementation in vivo would attenuate AβPP and BACE1 protein levels. cGMP levels were significantly increased while AβPP and BACE1 protein levels were statistically lower in cerebral microvessels from nitroglycerin-treated eNOS-/- mice as compared to vehicle-treated mice. Our findings support the concept that preservation of NO/cGMP signaling is an important modulator of expression and processing of AβPP.

Supplementary Data for Austin et al. article (PDF)

Pages 35-44
Roberto Carlos Agis-Balboa, Zsuzsa Pavelka, Cemil Kerimoglu, Andre Fischer (Handling Associate Editor: Israel Ampuero)
Loss of HDAC5 Impairs Memory Function: Implications for Alzheimer’s Disease
Abstract: Epigenetic mechanisms such as histone-acetylation have been implicated with learning and memory and are believed to contribute to the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease (AD). Histone-deacetylase (HDAC) inhibitors were shown to exhibit neuroprotective and neurodegenerative properties in AD animal models, and targeting HDACs appears to be a promising therapeutic strategy for brain diseases. The role of the distinct HDAC proteins in the adult brain is, however, not well understood and so far only pan-HDAC inhibitors have been tested in preclinical settings. Understanding the role of individual HDACs in cognition and AD pathogenesis is therefore vital to develop more selective HDAC inhibitors for the treatment of AD. In this study we investigated the role of HDAC5 in memory function and AD pathogenesis. We show that loss of HDAC5 impairs memory function but has little impact on pathogenesis in a mouse model for amyloid pathology. Our data reveals a novel role of HDAC5 in memory consolidation and shows that future approaches to develop more selective HDAC inhibitors for the treatment of AD should avoid targeting HDAC5.

Pages 45-53
Bob Olsson, Joakim Hertze, Ronald Lautner, Henrik Zetterberg, Katarina Nagga, Kina Hoglund, Hans Basun, Peter Annas, Lars Lannfelt, Niels Andreasen, Lennart Minthon, Kaj Blennow, Oskar Hansson (Handling Associate Editor: Sanna-Kaisa Herukka)
Microglial Markers are Elevated in the Prodromal Phase of Alzheimer’s Disease and Vascular Dementia
Abstract: Microglia manage immunosurveillance and mediate inflammation, both suggested to be important in Alzheimer’s disease (AD). The aim of this study was to investigate if microglial markers could differentiate, firstly between AD and controls, and secondly between stable mild cognitive impairment (MCI) and those progressing to AD and vascular dementia (VaD). Furthermore, we investigated if these markers were sufficiently stable to be used in clinical trials. We quantified YKL-40 and sCD14 in cerebrospinal fluid (CSF) from 96 AD patients, 65 healthy controls, and 170 patients with MCI from baseline and over 5.7 years. For the stability analysis, two CSF samples were collected from 52 AD patients with a six-month interval in between. YKL-40, but not sCD14, was significantly elevated in AD compared with healthy controls (p=0.003). Furthermore, YKL-40 and sCD14 were increased in MCI patients who converted to VaD (p=0.029 and p=0.008), but not to AD according to NINCDS-ADRDA. However, when stratified according to CSF levels of tau and Aβ42, YKL-40 was elevated in those with an AD-indicative profile compared with stable MCI with a normal profile (p=0.037). In addition, YKL-40 and sCD14 were very stable in AD patients with good correlation between time-points (r=0.94, p=3.4 x 10-25; r=0.77, p=2.0 x 10-11) and the cortical damage marker T-tau. Thus, microglial markers are stable and may be used as safety markers for monitoring CNS inflammation and microglia activation in clinical trials. Moreover, YKL-40 differentiates between AD and controls and between stable MCI to AD and those that convert to AD and VaD.

Pages 55-68
Robert M. Chapman, Anton P. Porsteinsson, Margaret N. Gardner, Mark Mapstone, John W. McCrary, Tiffany C. Sandoval, Maria D. Guillily, Elizabeth DeGrush, Lindsey A. Reilly
C145 as a Short-Latency Electrophysiological Index of Cognitive Compensation in Alzheimer’s Disease
Abstract: Brain plasticity and cognitive compensation in the elderly are of increasing interest, and Alzheimer’s disease (AD) offers an opportunity to elucidate how the brain may overcome damage. We provide neurophysiological evidence of a short-latency event-related potential (ERP) component (C145) linked to stimulus relevancy that may reflect cognitive compensation in early-stage AD. Thirty-six subjects with early-stage, mild AD and 36 like-aged normal elderly (controls) had their EEG recorded while performing our Number-Letter task, a cognitive/perceptual paradigm that manipulates stimulus relevancies. ERP components, including C145, were extracted from ERPs using principal components analysis. C145 amplitudes and spatial distributions were compared among controls, AD subjects with high performance on the Number-Letter task, and AD subjects with low performance. Compared to AD subjects, control subjects showed enhanced C145 processing of visual stimuli in the occipital region where differential processing of relevant stimuli occurred. AD high performers recruited central brain areas in processing task relevancy. Controls and AD low performers did not show a significant task relevancy effect in these areas. We conclude that short-latency ERP components can detect electrophysiological differences in early-stage AD that reflect altered cognition. Differences in C145 amplitudes between AD and normal elderly groups regarding brain locations and types of task effects suggest compensatory mechanisms can occur in the AD brain to overcome loss of normal functionality, and this early compensation may have a profound effect on the cognitive efficiency of AD individuals.

Pages 69-76
Luisa Benussi, Rosa Rademakers, Nicola J. Rutherford, Aleksandra Wojtas, Michela Glionna, Anna Paterlini, Valentina Albertini, Thomas Bettecken, Giuliano Binetti, Roberta Ghidoni (Handling Associate Editor: Rosanna Squitti)
Estimating the Age of the Most Common Italian GRN Mutation: Walking Back to Canossa Times
Abstract: Mutations in the progranulin gene (GRN) were first implicated in frontotemporal lobar degeneration in 2006. The GRN p.Leu271LeufsX10 mutation is one of the most common GRN mutations worldwide. To gain further insight into the origin of this mutation in Italy, we performed a haplotype sharing analysis (32 families, residents of Lombardy) and refined the GRN p.Leu271LeufsX10 mutation dating. We showed that almost all families (30/32) can be traced to a single founder. We further estimated the age of this mutation using different methods and population growth rates both for Italy and Lombardy. Using DMLE, we dated the origin of this mutation to the Middle Ages, at the turn of the first millennium (phased families only, Italy: 39 and Lombardy: 32 generations ago; all families Italy: 45 and Lombardy 38 generations ago). Mutation dating was slightly postdated using Estiage (phased families only: 15 generations ago; all families: 20 generation ago). From a translational perspective, targeting mutation carriers offers a unique model to test disease-modifying drugs in clinical trials.

Pages 77-83
Mari-Carmen Badia, Ana Lloret, Esther Giraldo, Francisco Dasi, Gloria Olaso, Maria-Dolores Alonso, Jose Vina
Lymphocytes from Young Healthy Persons Carrying the ApoE4 Allele Overexpress Stress-Related Proteins Involved in the Pathophysiology of Alzheimer’s Disease
Abstract: Apolipoprotein E4 (ApoE4) is a major genetic risk factor for the development of Alzheimer’s disease (AD). The aim of this work was to find if carrying ApoE4 alleles correlates with molecular changes associated with specific processes involved in AD pathophysiology and whether they are useful as early biomarkers of AD. Fifty four young healthy adults (aged 20-55) were recruited. Of these, 33 carried at least one ApoE4 allele and 21 did not (ApoE 3/3). We also recruited eleven patients with clinical diagnoses of probable AD and nine persons of similar age without dementia who served as controls of the AD patients. Using peripheral lymphocytes, we measured RNA expression of glycogen synthase kinase 3β (GSK3β), the regulator of calcineurin 1 (RCAN1), calcineurin, and RNA-dependent protein kinase (PKR) by PCR and protein levels of RCAN1, calcineurin, GSK3β, and phospho-tau by western blotting. Young healthy persons carrying the ApoE 4/4 genotype express more RNA for RCAN1, calcineurin, and PKR and higher protein levels of calcineurin, RCAN1, GSK3β, and phospho-tau than controls (ApoE 3/3). Moreover, we found that carrying one or two alleles for ApoE4 is associated with subjective cognitive impairment. We conclude that lymphocytes from young, non-demented persons carrying the ApoE 4/4 genotype show molecular changes that are involved in specific processes associated with the pathophysiology of AD such as increased phosphorylation of tau or increased expression of stress-related proteins like calcineurin, GSK3β, or RCAN1. These changes may help to understand the development of AD and in the early diagnosis of the disease.

Pages 85-93
Erik Portelius*, Maria Olsson*, Gunnar Brinkmalm, Ulla Ruetschi, Niklas Mattsson, Ulf Andreasson, Johan Gobom, Ann Brinkmalm, Mikko Holtta, Kaj Blennow, Henrik Zetterberg *These authors contributed equally to this work
Mass Spectrometric Characterization of Amyloid-β Species in the 7PA2 Cell Model of Alzheimer’s Disease
Abstract: The Chinese hamster ovary cell line 7PA2, stably transfected with the 751 amino acid isoform of amyloid-β protein precursor (AβPP) containing the Val ? Phe mutation at residue 717, is one of the most used models to study the biochemistry and toxicity of secreted amyloid-β (Aβ) peptides, particularly Aβ oligomers, which are considered to be of relevance to the pathogenesis of Alzheimer´s disease. Here, we present a detailed immunochemical and mass spectrometric characterization of primary structures of Aβ peptides secreted by 7PA2 cells. Immunoprecipitation and western blot of 7PA2 cell culture media revealed abundant anti-Aβ immunoreactive bands in the molecular weight range of 4–20 kDa. Mass spectrometric analysis showed that these bands contain several AβPP/Aβ peptides, starting at the N-terminal of the Aβ sequence and extending across the BACE1 cleavage site. Treatment of cells with a BACE1 inhibitor decreased the abundance of the Aβ monomer band by western blot and resulted in lower levels of Aβ1-40, Aβ1-42, and sAβPPβ as measured by ELISA. However, western blot bands thought to represent oligomers of Aβ increased in response to BACE1 inhibition. This increase was paralleled by the emergence of N-terminally truncated Aβ species (Aβ5-40 in particular) and Aβ species that spanned the β-secretase site in AβPP according to mass spectrometric analyses. The formation of these AβPP/Aβ peptides may have implications for the use of the 7PA2 cell line as a model for Aβ pathology. The enzyme(s) responsible for this particular BACE1-independent AβPP-processing remains to be identified.

Supplementary Data for Portelius et al. article (PDF)

Pages 95-110
Wan Adriyani W. Ruzali, Patrick G. Kehoe, Seth Love
Influence of LRP-1 and Apolipoprotein E on Amyloid-β Uptake and Toxicity to Cerebrovascular Smooth Muscle Cells
Abstract: Vascular deposition of amyloid-β (Aβ) leads to the death of cerebrovascular smooth muscle cells (CVSMCs) in cerebral amyloid angiopathy (CAA). Apolipoprotein E (APOE) genotype influences the severity of CAA and development of vasculopathic complications. We have studied the relationship between uptake of Aβ into human CVSMCs and cell death, and the influence of ApoE isoforms on this process. We found that both Aβ42 and Aβ40 were taken up by human CVSMCs, and that this uptake-particularly that of Aβ42-caused disruption to smooth muscle actin and cell death. Uptake of Aβ42 was partially blocked by the addition of receptor-associated protein (RAP), implicating low-density lipoprotein receptor-related protein-1 (LRP-1) as the cell surface receptor. RAP significantly reduced the death of CVSMCs exposed to Aβ42. In further experiments, CVSMCs were exposed to Aβ42 in the presence of the different isoforms of exogenous ApoE and high-density lipoprotein (HDL). All three isoforms of ApoE in the presence of HDL (HDL-ApoE) reduced the uptake of fluorescein-tagged Aβ42 but only HDL-ApoE3 significantly decreased cell death. We conclude that HDL-ApoE3, acting as an Aβ chaperone molecule, significantly reduces the death of CVSMCs that result from LRP-1-mediated uptake of Aβ. This may contribute to the differential effects of APOE genotype on severity of CAA and the risk of rupture of Aβ-laden blood vessels.

Pages 111-116
Marwan Sabbagh, Michael Malek-Ahmadi, Ian Levenson, D. Larry Sparks
KIF6 719Arg Allele is Associated with Statin Effects on Cholesterol Levels in Amnestic Mild Cognitive Impairment and Alzheimer’s Disease Patients
Abstract: KIF6 719Arg allele carriers are thought to have a greater lipid lowering response from statin therapy than non-carriers. Given the continued interest in the relationship between cholesterol, statin use, amnestic mild cognitive impairment (aMCI), and Alzheimer’s disease (AD), investigating the role of KIF6 719Arg carrier status in these relationships may be of importance. Data from 86 patients (36 aMCI, 50 AD) with an average age of 76.87±8.22 years were used for this study. Total cholesterol (TC), low density lipoprotein (LDL), high density lipoprotein (HDL), and triglycerides were the outcome variables. 719Arg carriers taking statins had significantly lower TC (p < 0.001) and LDL (p < 0.001) levels than 719Arg carriers not taking statins. In addition, 719Arg carriers not taking statins had significantly higher TC (p = 0.004) and LDL (p <0.001) than 719Arg non-carriers taking statins. Additional analyses indicated that ApoE ε4 carrier status and statin use interaction is also associated with lower TC (p = 0.04), but not LDL (p = 0.06). The interaction between 719Arg and ApoE ε4 carrier status on TC and LDL was not significant. This study is the first to demonstrate an association between lower cholesterol levels and statin use among KIF6 719Arg allele carriers with aMCI and AD. Accounting for 719Arg carrier status may be important in future studies investigating the link between cholesterol and AD and also for AD and aMCI clinical trials using statins as a treatment.

Pages 117-131
Nathalie Le Bastard, Els Coart, Hugo Vanderstichele, Eugeen Vanmechelen, Jean-Jacques Martin, Sebastiaan Engelborghs (Handling Associate Editor: Piotr Lewczuk)
Comparison of Two Analytical Platforms for the Clinical Qualification of Alzheimer’s Disease Biomarkers in Pathologically-Confirmed Dementia
Abstract: Combined analysis of the Alzheimer’s disease (AD) biomarkers amyloid-β1-42 (Aβ1-42), total tau (T-tau), and hyperphosphorylated tau (P-tau181P) in cerebrospinal fluid (CSF) reduces the uncertainty associated with clinical dementia diagnosis. The present study evaluated the diagnostic accuracy of the CSF biomarker concentrations obtained with a multi-analyte Luminex assay (INNO-BIA AlzBio3) in comparison to single-analyte ELISA tests (INNOTEST). Data from 66 pathologically-confirmed dementia patients (51 AD and 15 non-AD) and 95 controls were included. Cut-off values were determined for each individual biomarker determined using both methods for different diagnostic challenges (dementia-controls; AD-controls; AD-non-AD). Comparing the diagnostic accuracy of individual cut-off values between INNO-BIA and INNOTEST, no relevant differences could be identified. Logistic regression was used in addition to identify the best combination of predictor variables (biomarkers). Discrimination of dementia patients from controls using Aβ1-42 and T-tau yielded a diagnostic accuracy of 0.87 and 0.90 for INNO-BIA and INNOTEST, respectively. Discriminating AD patients from controls, the diagnostic accuracy was 0.90 and 0.93 for INNO-BIA and INNOTEST, respectively. Optimal discrimination of AD and non-AD patients was achieved by combining Aβ1-42 and P-tau181P (diagnostic accuracy=0.86). In conclusion, which AD biomarkers or combination thereof are most informative is dependent on the differential diagnosis, but the clinical value of these markers in each of the differential diagnoses is independent of the method by which concentrations are determined. Since the clinical value of the ELISA (INNOTEST) and Luminex (INNO-BIA) tests is comparable, further research to select the most suitable analytical platform for routine CSF biomarker measurements is needed.

Supplementary Data for Le Bastard et al. article (PDF)

Pages 133-144
Simone Pinton, Tuane Bazanella Sampaio, Rita M. Ramalho, Cecília M.P. Rodrigues, Cristina Wayne Nogueira
p,p’-Methoxyl-Diphenyl Diselenide Prevents Neurodegeneration and Glial Cell Activation Induced by Streptozotocin in Rats
Abstract: The purpose of this study was to investigate possible molecular targets involved in the neuroprotective effect of p,p’-methoxyl-diphenyl diselenide [(MeOPhSe)2], using a streptozotocin (STZ)-induced sporadic dementia of Alzheimer’s type rat model. Male Wistar rats were injected with STZ (1.0 mg/8 µl; 4 µl/ventricle). After 21 days of STZ injection, regular diet-fed rats were supplemented with 10 ppm of (MeOPhSe)2 during 30 days. At the end of this period, rats performed object recognition and step-down passive avoidance tasks. Apoptosis was assessed by TUNEL staining and active caspase-3. Glial fibrillary acidic protein, ionized calcium binding adaptor molecule 1, and microtubule associated protein 2 were determined by immunofluorescence in rat hippocampus. The results demonstrate that the (MeOPhSe)2 dietary supplementation reversed STZ-induced memory impairment by enhancing memory in sham rats. (MeOPhSe)2 was also effective in reducing STZ-induced apoptosis and preserving dendrites and synapses. Moreover, (MeOPhSe)2 inhibited activation of microglia and astrogliosis induced by STZ in the rat hippocampus. We conclude that the (MeOPhSe)2 neuroprotective action is related to inhibition of apoptosis and suppression of inflammation.

Pages 145-155
Tobias E. Karlsson, Alexandra Karlén, Lars Olson, Anna Josephson (Handling Associate Editor: James Bennett)
Neuronal Overexpression of Nogo Receptor 1 in APPswe/PSEN1(ΔE9) Mice Impairs Spatial Cognition Tasks without Influencing Plaque Formation
Abstract: In the search for molecules that may alter the formation of amyloid-β (Aβ) protofibrils, it has been shown that the Nogo-system can interact and bind to amyloid-β protein precursor and thus affect the amount of Aβ that is formed and deposited in the brain. To further address this issue in vivo, we crossed mice that overexpress Nogo receptor 1 (NgR1), “MemoFlex”, in forebrain neurons, with plaque forming APPswe/PSEN1(ΔE9) mice, to investigate if increased levels of NgR1 would influence plaque load or cognitive function in the resulting MemoFlex/APPswe/PSEN1(ΔE9) transgenic mice. We used a radial arm water maze and the Morris water maze to measure cognitive function. We did not find any significant effect of NgR1 overexpression on the performance of APPswe/PSEN1(ΔE9) mice in the radial arm water maze test. However, MemoFlex/APPswe/PSEN1(ΔE9) mice were found to be significantly impaired in the Morris water maze. We also analyzed the amount of plaques in the two mouse models without finding any significant difference in plaque load in the cerebral cortex or the hippocampal formation. It therefore appears that overexpression of NgR1 in APPswe/PSEN1(ΔE9) mice does not have any marked effects on Aβ levels, yet appears to impair spatial cognitive abilities. We conclude that strong overexpression of NgR1 in forebrain neurons impairs aspects of cognitive function but does not markedly alter plaque load in plaque-forming APPswe/PSEN1(ΔE9) mice. Thus high levels of membrane-bound NgR1 present since early postnatal life does not influence the development of plaques in mice carrying the two human plaque-causing mutations APPswe and PSEN1(ΔE9).

Pages 157-164
Eija Lönnroos, Pentti Kyyrönen, J Simon Bell, Tischa JM van der Cammen, Sirpa Hartikainen
Risk of Death among Persons with Alzheimer’s Disease: A National Register-Based Nested Case-Control Study
Abstract: Few studies have reported the risk of death related to Alzheimer’s disease (AD) in large population-based cohorts. The objective of this study was to analyze the impact of AD on all-cause mortality in a nationwide sample of persons with AD. Community-dwelling persons with AD and an equal number of individually matched (age, gender, and region of residence) control persons without AD were identified from the registers of Social Insurance Institution of Finland at the end of 2005. Deaths in this sample (n=56,041, mean age 79.7 years, 67.8% women) during a 57-month follow-up period were recorded. Using a nested case-control design, unadjusted and adjusted (cardiovascular disease, cancer, diabetes, and asthma and/or COPD) hazard ratios (HR) with 95% confidence intervals (CI) were computed using proportional hazards regression. The results were categorized according to age at death (< 80, 80 to 89, ≥ 90 years) and duration of AD (≤ 3, 4 to 6, &ge; 7 years). The unadjusted HR for death associated with AD was 2.03 (95% CI: 1.97 to 2.09). The HR was highest in the youngest age category [HR=3.46 (95% CI: 3.18 to 3.77)], and still significantly elevated in the oldest age category [HR=1.50 (95% CI: 1.41 to 1.60)]. Comorbidity adjustments did not change the HRs, and even a short duration of AD (≤ 3 years) was associated with a significantly increased risk of death. In conclusion, AD was associated with an increased risk of death that was more pronounced at younger ages and existed even after a recent diagnosis of AD.

Pages 165-176
Florin V. Chirila, Tapan K. Khan, Daniel L. Alkon
Spatiotemporal Complexity of Fibroblast Networks Screens for Alzheimer's Disease
Abstract: Drugs to treat Alzheimer’s disease (AD) have been unsuccessful in preventing its devastating cognitive deficits and progressive neurodegeneration. The lack of a definitive diagnostic for AD has been a major obstacle to AD drug discovery. Here, we describe a novel, highly accurate peripheral diagnostic for AD patients based on quantitatively measured complexity of skin-sampled fibroblast networks. A significant number of samples were studied under double-blind conditions and had autopsy and/or genetic validation. An additional sample confirmed the diagnostic discrimination on freshly obtained skin samples. A sub-sample of these diagnostic differences were induced by oligomerized amyloid-β1-42. Based on the accuracy of these measures that utilize physical principles such as fractal dimension and lacunarity as well as the significant correlation with disease duration, this biomarker profile appears to identify accurately AD patients for therapeutic intervention.

Supplementary Data for Chirila et al. article (PDF)

Pages 177-190
Laus M. Broersen, Almar A.M. Kuipers, Martin Balvers, Nick van Wijk, Paul J.M. Savelkoul, Martijn C. de Wilde, Eline M. van der Beek, John W.C. Sijben, Robert J.J. Hageman, Patrick J.G.H. Kamphuis, Amanda J. Kiliaan (Handling Associate Editor: Danny Michaelson)
A Specific Multi-Nutrient Diet Reduces Alzheimer-Like Pathology in Young Adult AβPPswe/PS1dE9 Mice
Abstract: Diet is an important lifestyle factor implicated in the etiology of Alzheimer’s disease (AD), but so far it is not fully elucidated to which nutrients the suggested protective effect of diet can be attributed. Recent evidence obtained in the amyloid-β 1-42 (Aβ42) infusion model in rats has shown that a multi-nutrient intervention known as Fortasyn™ Connect (FC) may protect the central cholinergic system against Aβ42-induced toxicity. FC comprises the nutritional precursors and cofactors for membrane synthesis, viz. docosahexaenoic acid (DHA), eicosapentaenoic acid, uridine-mono-phosphate (UMP), choline, phospholipids, folic acid, vitamins B6, B12, C, E, and selenium. In order to investigate whether the combined administration of these nutrients may also affect AD-like pathology, we now evaluated the effects of the FC diet intervention in the transgenic AβPPswe/PS1dE9 mouse model with endogenous Aβ production. In addition we evaluated the effects of diets containing the individual nutrients DHA and UMP and their combination in this model. Between the age of 3 and 6 months, FC diet decreased brain Aβ levels and amyloid plaque burden in the hippocampus of AβPP/PS1 mice. The FC diet also reduced ongoing disintegrative degeneration in the neocortex, as indicated by Amino Cupric Silver staining. Although all three DHA-containing diets were equally effective in changing brain fatty acid profiles, diets differentially affected amyloid-related measures, indicating that effects of DHA may depend on its dietary context. The current data, showing that dietary enrichment with FC reduces AD-like pathology in AβPP/PS1 mice, confirm and extend our previous findings in the Aβ42 infusion model and favor the combined administration of relevant nutrients.

Pages 191-203
Sandra Rubial-Álvare, Susana de Sola, María-Clara Machado, Elena Sintas, Peter Böhm, Gonzalo Sánchez-Benavides, Klaus Langohr, Rubén Muñiz, Jordi Peña-Casanova *Both authors contributed equally to this work.
The Comparison of Cognitive and Functional Performance in Children and Alzheimer’s Disease Supports the Retrogenesis Model
Abstract: The retrogenesis model states that the progression of brain aging and Alzheimer’s disease (AD) deterioration proceeds inversely to human ontogenic acquisition patterns. Our aim was to assess if the progressive decline of cognitive abilities and functional capacity in AD follows an inverse sequence of acquisition compared to normal developmental patterns. One hundred eighty one children ranging in age from 4 to 12 years and 148 adults (cognitively normal, subjects with mild cognitive impairment, and mild-moderately severe AD) were assessed with the same cognitive and functional tools. The statistical analyses showed a progressive and inverse distribution on cognitive, functional, and mental age scores when comparing results of children classified by chronological age and patients by dementia staging. The pattern of cognitive acquisition in children showed a progressive development of overall cognitive function along all age ranges, in addition to a simultaneous acquisition of instrumental and basic daily living activities in the functional domain. AD patients showed a progressive decline in cognitive and functional domains, which concurs with the sequence of impairment reported in this dementia. Our findings provide support to the inverse and progressive pattern of functional and cognitive decline observed in AD patients compared to the developmental acquisition of these capacities in children, as stated by the retrogenesis model. Nonetheless, certain differences should be considered when comparing the sequence of acquisition during ontogenic development with that of progressive loss during the course of AD. Retrogenesis may account for the progressive loss of neocortical-related functions in AD.

Supplementary Data for Rubial-Alvare et al. article (PDF)

Pages 205-215
Teodoro del Ser, Klaus C. Steinwachs, Hermann J. Gertz, María V. Andrés, Belén Gómez-Carrillo, Miguel Medina, Joan A. Vericat, Pilar Redondo, David Fleet, Teresa León
Treatment of Alzheimer’s Disease with the GSK-3 Inhibitor Tideglusib: A Pilot Study
Abstract: This pilot, double-blind, placebo-controlled, randomized,escalating dose trial explored the safety and efficacy of tideglusib, an inhibitor of glycogen synthase kinase-3, in Alzheimer's disease (AD) patients. Thirty mild-moderate AD patients on cholinesterase inhibitor treatment were administered escalating doses (400, 600, 800, 1,000 mg) of tideglusib or placebo (ratio 2:1) for 4, 4, 6, and 6 weeks, respectively. The primary objective was to evaluate the safety and tolerability of tideglusib with strict criteria for drug escalation or withdrawal. Mini-Mental Status Examination (MMSE), Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog+), word fluency, Geriatric Depression Scale (GDS), and a final Global Clinical Assessment (GCA) were assessed as secondary objectives. Treatment was well tolerated. Adverse events were as frequent in active and placebo groups, except for some moderate, asymptomatic, and fully reversible increases (>2.5xULN) of serum transaminases in 6 active cases (p=0.001). Tideglusib produced positive trends in MMSE, ADAS-cog, GDS, and GCA without statistical significance in this small sample. Responders in MMSE were significantly higher in the active group (p=0.05). Patients escalated up to 1000 mg/day had a benefit of increases of 1.68 points in the MMSE and 4.72 points in the ADAS-cog+ when compared to placebo. This small pilot study provides valuable safety and efficacy estimates for the treatment of AD patients with tideglusib, currently being confirmed in a larger clinical trial. Due to escalating doses and the small sample size, this trial provides insufficient evidence to support or reject a benefit of tideglusib in AD.

Pages 217-229
Ju Wang, Zi-Qi Shi, Xiaojun Xu, Gui-Zhong Xin, Jun Chen, Lian-Wen Qi, Ping Li (Handling Associate Editor: Yi Zhun Zhu)
Triptolide Inhibits Amyloid-β Production and Protects Neural Cells by Inhibiting CXCR2 Activity
Abstract: Triptolide, a biologically active natural product from Tripterygium wilfordii, protects neurons from inflammation-mediated damage. Our results showed for the first time that triptolide inhibited the expression of CXCR2 and presenilin in a neuroblastoma cell line SHSY5Ysw. Moreover, triptolide potently inhibited amyloid-β1-42 production with IC50 value of 30 pM in HEK293sw cells or 2 nM in SHSY5Ysw cells, respectively. We also demonstrated that triptolide prevented primary cortical neurons from chemokine CXCL1-induced cytotoxicity. Therefore, our study indicates that the neural protective effect of triptolide is largely mediated by inhibiting CXCR2 activity.

Supplementary Data for Wang et al. article (PDF)

Pages 231-247
Irina Lonskaya, Ashot R. Shekoyan, Michaeline L. Hebron, Nicole Desforges, Norah K. Algarzae, Charbel E-H Moussa
Diminished Parkin Solubility and Co-Localization with Intraneuronal Amyloid-β are Associated with Autophagic Defects in Alzheimer’s Disease
Abstract: Alzheimer’s disease (AD) is an aging disorder characterized by amyloid-β (Aβ) accumulation in extracellular plaques and formation of intracellular tangles containing hyperphosphorylated tau (p-Tau). Autophagic defects, leading to accumulation of autophagosomes, are recognized in AD. Parkin is an E3 ubiquitin ligase involved in degradation of proteins via autophagy and the proteasome. We investigated the role of parkin in postmortem brain tissues from 21 AD patients and 15 control subjects. We detected decreased parkin solubility in AD cortex and parkin co-localization with intraneuronal Aβ1-42 in the hippocampus and cortex of AD patients. Parkin accumulation with intraneuronal Aβ and p-Tau was detected in autophagosomes in AD brains. To determine the role of parkin in Aβ clearance, we generated gene transfer animals expressing lentiviral Aβ1-42 with and without parkin and examined autophagic mechanisms. Lentiviral expression of Aβ1-42 led to p-Tau accumulation and induced autophagic defects, leading to accumulation of autophagic vacuoles. However, co-expression of wild type parkin facilitated autophagic clearance and promoted deposition of Aβ1-42 and p-Tau into the lysosome. Taken together, these data suggest that Aβ1-42 alters normal autophagy and parkin enhances autophagic clearance. In conclusion, decreased parkin solubility may lead to co-localization with intraneuronal Aβ1-42 and compromise the cell autophagic clearance ability. Parkin may clear autophagic defects via autophagosome degradation.

Pages 249-263
Naruhiko Sahara, Michael DeTure, Yan Ren, Abdul-Shukkur Ebrahim, Dongcheul Kang, Joshua Knight, Christiane Volbracht, Jan Torleif Pedersen, Dennis W. Dickson, Shu-Hui Yen, Jada Lewis
Characteristics of TBS-Extractable Hyperphosphorylated Tau Species: Aggregation Intermediates in rTg4510 Mouse Brain
Abstract: Conditional overexpression of four-repeat human tau containing the P301L missense mutation in the rTg4510 mouse model of tauopathy leads to progressive accumulation of neurofibrillary tangles and hyperphosphorylated, sarkosyl-insoluble tau species, which are biochemically comparable to abnormal tau characteristic of hereditary tauopathies termed FTDP-17. To fully understand the impact of tau species at different stages of self-assembly on neurodegeneration, we fractionated rTg4510 brain representing several stages of tauopathy to obtain TBS-extractable (S1), high salt/sarkosyl-extractable (S3), and sarkosyl-insoluble (P3) fractions. Under reducing condition, the S1 fraction was demonstrated by western blotting to contain both 50-60 kDa normally-sized and 64 kDa tau. Both are thermo-stable, but the 64 kDa tau showed a higher degree of phosphorylation. Under non-reducing condition, nearly all TBS-extractable 64 kDa tau were detected as ~130 kDa species consistent with the size of dimer. Quantitative analysis showed ~80 times more 64 kDa tau in S1 than P3 fraction. Immunoelectron microscopy revealed tau-positive granules/short filaments in S1 fraction. These structures displayed MC1 immunoreactivities indicative of conformational/pathological change of tau. MC1 immunoreactivity was detected by dot blotting in samples from 2.5 month-old mice, whereas Ab39 immunoreactivity indicative of late stages of tau assembly was detected only in P3 fraction. Quantitative analysis also demonstrated a significant inverse correlation between brain weight and 64 kDa tau, but the level of TBS-extractable 64 kDa tau reflects neurodegeneration better than that of sarkosyl-insoluble 64 kDa tau. Together, the findings suggest that TBS-extractable 64kDa tau production is a potential target for therapeutic intervention of tauopathies.

Supplementary Data for Sahara et al. article (PDF)

Pages 265-272
Luca Rosario La Rosa, Carmela Matrone, Caterina Ferraina, Maria Beatrice Panico, Silvia Piccirilli, Maria Grazia Di Certo, Georgios Strimpakos, Nicola Biagio Mercuri, Pietro Calissano, Marcello D’Amelio#, Robert Nisticò# (Handling Associate Editor: Bruno Imbimbo) #Senior authors
Age-Related Changes of Hippocampal Synaptic Plasticity in AβPP-Null Mice are Restored by NGF through p75NTR
Abstract: Amyloid-β protein precursor (AβPP) is a ubiquitous protein found in all cell types, suggesting basic and yet important roles, which still remain to be fully elucidated. Loss of function of AβPP has been linked to abnormal neuronal morphology and synaptic function within the hippocampus and alterations in spatial learning, suggesting a neurotrophic role for this protein. Besides AβPP, nerve growth factor (NGF) and other neurotrophins have also been shown to finely modulate neuronal excitability, synaptic plasticity, and cognitive functions. In addition, recent data support the hypothesis of a functional interconnection between AβPP and NGF pathway. Here, we demonstrated that loss of AβPP function, leading to progressive decrease of choline acetyltransferase expression in the septum, correlates with age-related impairment of long-term potentiation (LTP) in the dentate gyrus. We next addressed whether impaired hippocampal plasticity in AβPP-null mice can be restored upon NGF treatment. Notably, NGF, as well as Pro-NGF, can fully revert LTP deficits in AβPP-null mice through p75NTR and JNK pathway activation. Overall the present study may unveil a new mechanism by which, in the absence of AβPP, NGF treatment may preferentially direct p75-neurotrophin-dependent JNK activation toward regeneration and plasticity in functionally relevant brain circuits.

Pages 273-280
Alexandra Varjassyová, Daniel Horínek, Ross Andel, Jana Amlerova, Jan Laczó, Katerina Sheardová, Hana Magerová, Iva Holmerová, Martin Vyhnálek, Ondrej Bradác, Yonas E. Geda , Jakub Hort (Handling Associate Editor: Marwan Sabbagh)
Recognition of Facial Emotional Expression in Amnestic Mild Cognitive Impairment
Abstract: We examined whether recognition of facial emotional expression would be affected in amnestic mild cognitive impairment (aMCI). A total of 50 elderly persons met the initial inclusion criteria; 10 were subsequently excluded (Geriatric Depression Score >5). 22 subjects were classified with aMCI based on published criteria (single domain aMCI [SD-aMCI], n = 10; multiple domain aMCI [MD-aMCI], n = 12); 18 subjects were cognitively normal. All underwent standard neurological and neuropsychological evaluations as well as tests of facial emotion recognition (FER) and famous faces identification (FFI). Among normal controls, FFI was negatively correlated with Mini-Mental Status Examination scores and positively correlated with executive function. Among patients with aMCI, FER was correlated with attention/speed of processing. No other correlations were significant. In a multinomial logistic regression model adjusted for age, gender, and education, a poorer score on FER, but not on FFI, was associated with greater odds of being classified as MD-aMCI (odds ratio [OR], 3.82; 95% confidence interval [CI], 1.05–13.91; p = 0.042). This association was not explained by memory or global cognitive score. There was no association between FER or FFI and SD-aMCI (OR, 1.13; 95% CI, 0.36–3.57; p = 0.836). Therefore, FER, but not FFI, may be impaired in MD-aMCI. This implies that in MD-aMCI, the tasks of FER and FFI may involve segregated neurocognitive networks.

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