Bernard Michel

Lithostathine and pancreatitis-associated protein are involved in the very early stages of Alzheimer's disease

According to one of the theories formulated to explain the etiology of Alzheimers disease (AD), amylosis may reflect a specific inflammatory response. Two inflammatory proteins, lithostathine and PAP, were evidenced by immunohistochemistry in senile plaques and neurofibrillary tangles of patients with AD. In addition, lithostathine and PAP were significantly increased in the cerebrospinal fluid of patients with AD when compared to patients with multiple sclerosis, another inflammatory disease, and to normal control subjects. However, no correlation was observed with age of occurrence. Furthermore, lithostathine and PAP were increased even at the very early stages of AD, and their level remained elevated during the course of the AD unlike TNFalpha whose level, very high at very early stages, regularly decreased. Finally, if part of lithostathine and PAP are synthesized in the brain, a large part comes from serum by passage over the blood-brain barrier. These results indicate (i) the existence of an acute phase response followed by a chronic inflammation in AD, and (ii) that lithostathine and PAP are involved even at the first pre-clinical biochemical events of AD. In addition, because lithostathine undergoes an autolytic cleavage leading to its precipitation and the formation of fibrils, we believe that it may be involved in amyloidosis and tangles by allowing heterogeneous precipitation of other proteins.

Lithostathine quadruple-helical filaments form proteinase K-resistant deposits in Creutzfeldt-Jakob disease.

Autocatalytic cleavage of lithostathine leads to the formation of quadruple-helical fibrils (QHF-litho) that are present in Alzheimers disease. Here we show that such fibrils also occur in Creutzfeldt-Jakob and Gerstmann-Sträussler-Scheinker diseases, where they form protease-K-resistant deposits and co-localize with amyloid plaques formed from prion protein. Lithostathine does not appear to change its native-like, globular structure during fibril formation. However, we obtained evidence that a cluster of six conserved tryptophans, positioned around a surface loop, could act as a mobile structural element that can be swapped between adjacent protein molecules, thereby enabling the formation of higher order fibril bundles. Despite their association with these clinical amyloid deposits, QHF-litho differ from typical amyloid fibrils in several ways, for example they produce a different infrared spectrum and cannot bind Congo Red, suggesting that they may not represent amyloid structures themselves. Instead, we suggest that lithostathine constitutes a novel component decorating disease-associated amyloid fibrils. Interestingly, [6,6′]bibenzothiazolyl-2,2′-diamine, an agent found previously to disrupt aggregates of huntingtin associated with Huntingtons disease, can dissociate lithostathine bundles into individual protofilaments. Disrupting QHF-litho fibrils could therefore represent a novel therapeutic strategy to combat clinical amyloidoses.

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre‐clinical stages of Alzheimers disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimers disease. After self‐proteolysis, the N‐terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three‐dimensional X‐ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple‐helical filament (QHF‐litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.

Regenerating Islet-derived 1α (Reg-1α) Protein Is New Neuronal Secreted Factor That Stimulates Neurite Outgrowth via Exostosin Tumor-like 3 (EXTL3) Receptor

Background: Reg-1α is a small secretory protein overexpressed during the early stages of Alzheimer disease. Results: Secreted Reg-1α stimulates axon outgrowth, and this paracrine effect is mediated by its receptor EXTL3. Conclusion: Reg-1α emerges as an important actor in brain plasticity and the regenerative process. Significance: Learning how Reg-1α regulates the nerve cells is important for understanding its implications in early stages of Alzheimer disease. Regenerating islet-derived 1α (Reg-1α)/lithostathine, a member of a family of secreted proteins containing a C-type lectin domain, is expressed in various organs and plays a role in proliferation, differentiation, inflammation, and carcinogenesis of cells of the digestive system. We previously reported that Reg-1α is overexpressed during the very early stages of Alzheimer disease, and Reg-1α deposits were detected in the brain of patients with Alzheimer disease. However, the physiological function of Reg-1α in neural cells remains unknown. Here, we show that Reg-1α is expressed in neuronal cell lines (PC12 and Neuro-2a) and in rat primary hippocampal neurons (E17.5). Reg-1α is mainly localized around the nucleus and at the membrane of cell bodies and neurites. Transient overexpression of Reg-1α or addition of recombinant Reg-1α significantly increases the number of cells with longer neurites by stimulating neurite outgrowth. These effects are abolished upon down-regulation of Reg-1α by siRNA and following inhibition of secreted Reg-1α by antibodies. Moreover, Reg-1α colocalizes with exostosin tumor-like 3 (EXTL3), its putative receptor, at the membrane of these cells. Overexpression of EXTL3 increases the effect of recombinant Reg-1α on neurite outgrowth, and Reg-1α is not effective when EXTL3 overexpression is down-regulated by shRNA. Our findings indicate that Reg-1α regulates neurite outgrowth and suggest that this effect is mediated by its receptor EXTL3.

Association of APOE promoter but not A2M polymorphisms with risk of developing Alzheimer's disease.

The APOE4 allele is widely accepted as a major risk factor for late-onset Alzheimers disease (AD). Recently, it has been reported that polymorphisms in the APOE promoter and in the α2-macroglobulin gene (A2M) are associated with AD. We have analyzed the distribution of APOE alleles, −219T/G APOE promoter polymorphism, and A2M/A2Mdel polymorphism in a large case-control study. Our results showed that APOE genotype was the only informative marker of AD risk contrary to −219T/G and A2M/A2Mdel polymorphism. In AD patients however, a strong linkage disequilibrium was observed between the T allele of −219T/G polymorphism and APOE4 allele. This result indicates that −219T/G APOE promoter polymorphism is a risk factor for AD by increasing the APOE4-associated risk.

Distinct Transcriptome Expression of the Temporal Cortex of the Primate Microcebus murinus during Brain Aging versus Alzheimer's Disease-Like Pathology

Aging is the primary risk factor of neurodegenerative disorders such as Alzheimers disease (AD). However, the molecular events occurring during brain aging are extremely complex and still largely unknown. For a better understanding of these age-associated modifications, animal models as close as possible to humans are needed. We thus analyzed the transcriptome of the temporal cortex of the primate Microcebus murinus using human oligonucleotide microarrays (Affymetrix). Gene expression profiles were assessed in the temporal cortex of 6 young adults, 10 healthy old animals and 2 old, “AD-like” animals that presented ß-amyloid plaques and cortical atrophy, which are pathognomonic signs of AD in humans. Gene expression data of the 14,911 genes that were detected in at least 3 samples were analyzed. By SAM (significance analysis of microarrays), we identified 47 genes that discriminated young from healthy old and “AD-like” animals. These findings were confirmed by principal component analysis (PCA). ANOVA of the expression data from the three groups identified 695 genes (including the 47 genes previously identified by SAM and PCA) with significant changes of expression in old and “AD-like” in comparison to young animals. About one third of these genes showed similar changes of expression in healthy aging and in “AD-like” animals, whereas more than two thirds showed opposite changes in these two groups in comparison to young animals. Hierarchical clustering analysis of the 695 markers indicated that each group had distinct expression profiles which characterized each group, especially the “AD-like” group. Functional categorization showed that most of the genes that were up-regulated in healthy old animals and down-regulated in “AD-like” animals belonged to metabolic pathways, particularly protein synthesis. These data suggest the existence of compensatory mechanisms during physiological brain aging that disappear in “AD-like” animals. These results open the way to new exploration of physiological and “AD-like” aging in primates.

APOE ε4 Genotype and the Risk for Subjective Cognitive Impairment in Elderly Persons

The authors compared the risk for subjective cognitive impairment (SCI) between carriers of the apolipoprotein E ε4 (APOE ε4) allele (cases) and APOE ε4 noncarriers (controls). SCI was assessed by a validated self-reported questionnaire. The authors used multivariable logistic regression analyses to compute odds ratios and 95% confidence intervals adjusted for age, sex, education, and marital status. Data were available on 114 participants (83 women; 47 APOE ε4 carriers; mean age, 69 years). The risk for SCI was significantly higher among cases than controls, particularly for those 70 years of age and older. These findings should be considered preliminary until confirmed by a prospective cohort study.

Aging Stereotypes Must be Taken Into Account for the Diagnosis of Prodromal and Early Alzheimer Disease.

Because of a dramatic increase of older people worldwide, screening for prodromal state of Alzheimer disease (AD) is a major societal challenge. Many individuals diagnosed with prodromal AD, do not convert to AD, some remaining stable and others reversing back to normal. We argue that an important source of this overdiagnosis comes from negative aging stereotypes (eg, the culturally shared beliefs that aging inescapably causes severe cognitive decline and diseases). Many laboratory studies show that such stereotypes impair memory performance in healthy older adults, producing inflated age differences. Research is needed to examine how aging stereotypes implicitly permeate neuropsychological testing and contribute to false positives.

ADFLAG ® , A DIAGNOSTIC BLOOD TEST FOR PRE-DEMENTIA STAGES OF ALZHEIMER’S DISEASE

P3-214 ADFLAG , A DIAGNOSTIC BLOOD TEST FOR PRE-DEMENTIA STAGES OF ALZHEIMER’S DISEASE Beatrice Blanc, Nicolas Pelletier, Clotilde Biscarrat, Pauline Picamal, Nathalie Compagnone, Samantha Galluzzi, Moira Marizzoni, Jorge Jovicich, Giovanni B. Frisoni, Gianluigi Forloni, Diego Albani, Jill Richardson, Lucilla Parnetti, Magda Tsolaki, Flavio Nobili, David Bartrez-faz, Mira Didic, Peter Schoenknecht, Pierre Payoux, Andrea Soricelli, Paolo Rossini, Pieter Jelle Visser, Regis Bordet, Ute Fiedler, Olivier Blin, Julien Dupouey, Joelle Micallef, Laura Lanteaume, Bernard Michel, ICDD, Gemenos, France; IRCCS Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; University of Trento, Trento, Italy; Memory Clinic and LANVIE Laboratory of Neuroimaging of Aging, University Hospitals and University of Geneva, Geneva, Switzerland; Department of Neuroscience, Mario Negri Institute for Pharmacological Research, Milan, Italy; IRCCS, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy; GSK R&D, China-United Kingdom, Middlesex, United Kingdom; Lab of Clinical Neurochemistry, University of Perugia, Perugia, Italy; Aristotle University of Thessaloniki, Thessaloniki, Greece; University of Genoa, Italy, Genoa, Italy; Department of Psychiatry and Clinical Psychobiology, Faculty of Medicine, University of Barcelona and Institut d’Investigacions Biom ediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; Service de Neurologie et Neuropsychologie, Marseille, France; Department of Psychiatry and Psychotherapy, University of Leipzig, Leipzig, Germany; INSERM, Imagerie C er ebrale et Handicaps Neurologiques, Toulouse, France; Fondazione SDN per la Ricerca e l’Alta Formazione in Diagnostica Nucleare, Naples, Italy; Department of Gerontology, Neurosciences & Orthopedics, Catholic University, Rome, Italy; VU University Medical Center, Amsterdam, Netherlands; Service de Pharmacologie-Hôpital Huriez-CHRU, Lille, France; Institutes and Clinics of the University Duisburg-Essen, Essen, Germany; Mediterranean Institute of Cognitive Neurosciences, Marseille, France; Service de Neurologie et Neuropsychologie, CHU la Timone, Marseille, France; Assistance Publique Hôpitaux de Marseille, Marseille, France; Service de Neurologie Comportementale, Hôpital Sainte Marguerite, Marseille, France, CNRS LNIAUMR 7260 FR3C FR 3512, Aix-Marseille Universit e, Marseille, France. Contact e-mail: bblanc@icdd-sas.com

SUBJECTIVE COGNITIVE IMPAIRMENT, APOE ɛ4 STATUS, AND COGNITIVE AGING: THE ARIZONA APOE COHORT

clinically significant depression, defined by GDS> 10, were excluded from participation at screening. General linear regression models with backward elimination were used to evaluate the cross-sectional relations of GDS measures to HVor to DMN connectivity, in separate analyses. Predictors subjected to backward elimination (using a p<0.1 threshold) included GDS, age, sex, premorbid intelligence (AMNART IQ), a binary amyloid variable (determined by a PiB distribution volume ratio cut-off of 1.20) and a interaction term for GDS and amyloid to consider a differential effect in participants with low compared to high amyloid burden. Results: Higher GDS score (p1⁄40.02), greater age (p<0.0001) and lower AMNART IQ (p1⁄40.04) were significantly associated with lower HV (p<0.0001; R 2 1⁄40.22 for the model). Greater age (p<0.03) but no other predictors were related to lower DMN connectivity. Neither amyloid status nor the interaction terms were significantly related to DMN or to HV in regression models. Conclusions: In cognitively normal elderly,SSD was associated with low HV independent of amyloid status, but was not related to DMN connectivity disruption. SSD may accompany neurodegeneration characteristic of early AD but this association may be independent of amyloid pathology.