Endoplasmic reticulum plays important roles in initiating autophagy in cultured hippocampal neurons after exposure to beta-amyloid peptide

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Oral Sessions: O2-09: Cellular and Molecular Mechanisms: ER and Expression Studies

a protocol was developed that (a) used prescription and non-prescription products with the best evidence of effectiveness in prevention and treatment of gingivitis, dental plaque, and dental caries, and (b) trained care providers in techniques to reduce resistive and agitated behaviors through the application of person-centered behavioral principles of dementia care. In three US nursing homes, subjects who needed mouth care assistance (85% of whom had dementia) were recruited and nursing assistants who cared for them trained in the protocol. Pre- and post-intervention measures of dental plaque and gingivitis were obtained by a geriatric dentist not involved in the intervention using established instruments; additional data were collected by record review. Results: 97 nursing home residents entered the trial; 8-week outcome data were obtained on 88 (91%). For persons who had teeth, significant reductions were observed in the amount of plaque present (P <0.001) and the extent to which gingivitis was present (P
0.001). Among persons with dentures, plaque accumulation was similarly reduced (P-value ranged from 0.02 to <0.001 depending on the quadrant evaluated). Success varied among the three facilities and was influenced by the selection process and degree of administrative support for the study nursing assistants. Conclusions: Oral health of nursing home residents with dementia can be significantly improved by a mouth care program that combines innovative dental care products and person-centered behavioral techniques. However, administrative support and motivated, reliable staff are needed to make it maximally effective. O2-08-06

THE EARTHQUAKE- AND TSUNAMIEXACERBATED DEMENTIA IN JAPAN

Katsutoshi Furukawa, Mari Ootsuki, Hiroyuki Arai, Tohoku University, Sendai, Japan. Background: A magnitude 9.0 earthquake hit Japan on March 11, followed by enormous tsunamis which caused numerous casualties and serious damage in the coastal cities. After the disaster many elderly people including patients with dementia evacuated to asylums such as gymnasiums and halls because their houses were destroyed. Many of the patients visited our clinic after evacuation, and most of them got significantly worse with their dementia symptoms including behavioral and psychological symptoms of dementia (BPSD). We here examined changes in cognitive functions and BPSD with minimental state examination (MMSE) and neuropsychiatric inventoryquestionnaire (NPI-Q), respectively, in patients with Alzheimer’s disease after the earthquake. Methods: We analyzed three groups including subjects who did not suffer from the disaster (group 1, M/F ¼ 9/11, Age 74.3 +/8.3), subjects who had experienced the disaster but did not stay at an evacuation site (group 2, M/F ¼ 9/9, Age 75.6 +/- 7.8), and subjects who were forced to stay at an evacuation site (group 3, M/F ¼ 8/9, Age 77.8 +/- 8.5). Results: We compared the altered values (after - before) of each test in the three groups. Both groups 2 and 3 showed a decline in MMSE compared to group 1 although a significant difference was observed only between “group 1 and 2” and “group 1 and 3”. Concerning the NPI-Q both group 2 and 3 exhibited exacerbation of BPSD and the significant difference was observed between “group 1 and 2”, “group 1 and 3” and “group 2 and 3”. Conclusions: Disasters such as earthquakes, tsunamis, hurricanes, and tornados must be extremely stressful to elderly people. On the other hand, to change their circumstances to inferior conditions such as evacuation sites should affect their mental and cognitive condition more severely. Nobody likes to live together with other many people in a big hall with poor hygiene. Not only physical but also mental care to the elderly who suffered from the disaster is now needed. ORAL SESSIONS: O2-09 CELLULAR AND MOLECULAR MECHANISMS: ER AND EXPRESSION STUDIES O2-09-01

ENDOPLASMIC RETICULUM (ER) STRESS AND ABNORMAL TAU CONVERGE IN A UBIQUITINRELATED VICIOUS CYCLE: EARLY EVENTS IN NEURONAL DAMAGE

Jose Abisambra, Sarah Brady, Lily Wang, Karthik Arulselvam, Mathew Cockman, Laura Blair, Chantal Guidi, Chad Dickey, University of South Florida, Tampa, Florida, United States.

Background: A recent genome-wide association study linked a single nucleotide polymorphism (SNP) with increased risk for progressive supranuclear palsy (PSP). As in Alzheimer’s disease, PSP is pathologically characterized by the abnormal intraneuronal aggregation of tau. The SNP is on the EIF2AK3 gene, which codes for the ER-stress sensing protein PERK. Previous reports indicate that pre-tangle neurons in PSP and Alzheimer’s patients show elevated levels of activated PERK. However, the impact of pPERK in disease and its relationship with tau has not been studied. We investigated the possible connection between these two proteins. Methods: Tau transgenic mice and tau inducible-HEK cells were utilized. Assessment of protein levels and localization were performed using immunoblots and immuno-fluorescent confocal microscopy, respectively. An ex vivo functional assay method was employed: brains of mice were extracted and maintained alive in artificial CSF for incubation with chemical manipulators of the proteasome and apoptosis. Results: We assessed whether tau induced ER stress in tau transgenic brains and tau-overexpressing cell cultures. Active PERK and ER-resident chaperones were significantly increased; in fact, these ER stress markers were directly proportional to tau levels. ER-associated degradation (ERAD) is an ubiquitin-assisted clearance mechanism by which the ER removes proteins that cannot be folded/refolded. Alterations to ERAD elicit ER stress, so we assessed whether changes in tau affected ubiquitin: levels were increased in vivo and in vitro. Furthermore, while excess ubiquitin co-localized with the ER, tau did not. We performed ex vivo functional assays and established that ubiquitinylated proteins accumulate in tau mice. However, inhibitors of proteasomal function caused potent upregulation of ubiquitinylated species in control brains, while further ubiquitinylation was impaired in tau brains. Since chronic ERAD initiates apoptosis, we explored for apoptosis markers and found that tau brains have elevated cleaved-PARP. Similarly, further chemical induction of apoptosis did not manifest in tau brains. Conclusions: Our data suggest that tau impairs protein ubiquitinylation eliciting ER stress. Consequent initiation of apoptosis is also evident in tau-bearing brains. This work adds critical information to the mechanism by which tau induces cell damage. Further elucidation of this process may reveal potential therapeutic targets for tauopathies. O2-09-02

ENDOPLASMIC RETICULUM PLAYS IMPORTANT ROLES IN INITIATING AUTOPHAGY IN CULTURED HIPPOCAMPAL NEURONS AFTER EXPOSURE TO BETA-AMYLOID PEPTIDE

Raymond Chuen-Chung Chang, Natalie Qishan Zhang, Clara Hung, Suthicha Wuwongse, Yuen-shan Ho, The University of Hong Kong, Hong Kong, China. Background: Autophagy has long been considered to be one of the mechanisms for cells to clear damaged intracellular organelles. It has not been clear of how autophagy or formation of autophagic vacuoles is initiated in Alzheimer’s disease (AD) or b-amyloid (Ab) peptide neurotoxicity in neurons. We have reported that cultured neurons exposed to oligomeric Ab peptide display aggregation and collapse of the endoplasmic reticulum (ER). This is due to the instability of microtubules leading to detachment of the ER from microtubules. Concomitantly, increase of autophagic vacuoles can be observed after aggregation of the ER. The aim of this study is to investigate molecular mechanisms of how aggregation of the ER initiates formation of autophagosomes. Methods: Cultured hippocampal neurons were employed in this study. Oligomeric Ab1-42 peptide was used as toxin model. Different fluorescent-conjugated DNA constructs were transfected into neurons for live-cell imaging by multiphoton confocal imaging LSM510meta, Carl Zeiss). Together with immunofluorescent staining, immunoprecipitation, we were able to examine colocalization of different signaling molecules clustering on the aggregated ER. Results: Our results show that oligomeric Ab-induced autophagy was induced by initiation complex (Atg14L-Beclin1-Vps34) on the ER. The initiation complex triggered formation of omegasomes, the phosphatidylinositol-3 phosphate enriched-ER subcompartments which were found to be accumulated at the ER aggregation site. Aggregation of the ER facilitated the clustering of

Oral Sessions: O2-09: Cellular and Molecular Mechanisms: ER and Expression Studies Atg14L that is normally localized on the ER; and in turn to facilitate translocation of Beclin1-Vps34 to form a constitutional complex with enhanced lipid kinase activity for generating omegasomes. Also, sequestration of p62, the ubiquitin- and LC3-binding protein, at the ER aggregation site may facilitate aggregated ER membrane or proteins to autophagic degradation. Conclusions: This is among the first study to demonstrate that aggregation of the ER promotes the formation of Atg14L-Beclin1-Vps34 complex to initiate autophagy. In addition p62 on the ER further promote clearance of damaged ER for autophagic degradation. O2-09-03

ENDOPLASMIC RETICULUM STRESS LINKS IMPAIRED INSULIN SIGNALING AND ALZHEIMER’S DISEASE

Mychael Lourenc¸o1, Cesar Silva1, Luciana Sathler1, Leticia FornyGermano1, Theresa Bomfim1, Sheila Ara
ujo2, Paula Campello-Costa3, William Klein4, Douglas Mu~noz5, Sergio Ferreira1, Fernanda De Felice1, 1 Federal University of Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil; 2Fluminense Federal University, Niter
oi, Rio de Janeiro, Brazil; 3 Fluminense Federal University, Niter
oi, Rio de Janeiro, Brazil; 4 Northwestern University, Evanston, Illinois, United States; 5Queen’s University, Kingston, Ontario, Canada. Background: Alzheimer’s disease (AD) has been linked to impaired brain insulin signaling. In type 2 diabetes, endoplasmic reticulum (ER) stress plays a key role in triggering peripheral insulin resistance. AD brains also present elevated levels of ER stress markers. Therefore, it is possible that common mechanisms underlie impaired brain insulin signaling in AD and peripheral insulin resistance in diabetes. Methods: We investigated whether Ab oligomers (AbOs), synaptotoxins recently implicated in neuronal insulin resistance, induce ER stress response in hippocampal neurons in vitro and in vivo. Pharmacological approaches were employed to further address the mechanisms by which AbOs exert this toxic effect. Furthermore, next-generation GLP-1 analogues were used to bolster neuroprotective signaling and to possibly promote beneficial effects in different AD models. Results: Here, we found that Ab oligomers markedly increase neuronal ER stress. Levels of phosphorylated eukaryotic initiation factor 2^I (peIF2^I) and ER membrane-associated inositol-requiring enzyme 1 (pIRE1) were elevated in mature cultured hippocampal neurons exposed to oligomers. Elevated ER stress markers were also verified in hippocampi of APPSwe,PS1deltaE9 AD transgenic mice. Importantly, intracerebroventricular injection of Ab oligomers triggered ER stress in the hippocampi of adult cynomolgus monkeys. We further found that oligomer-induced phosphorylation of eIF2a is mediated by TNF-a/JNK proinflammatory signaling. Double-stranded RNA-dependent protein kinase (PKR), recently described as a critical component that responds to ER stress in peripheral tissue, was here found to be activated by oligomers in neuronal cultures, as well as in transgenic mice and in monkey brains. Stimulation of insulin signaling by GLP-1 receptor agonists or insulin prevented oligomer-induced neuronal pathologies in vitro. GLP-1 agonists further decreased hippocampal ER stress and improved cognition in transgenic mice. Conclusions: We demonstrate that ER stress in AD is mediated by inflammatory mechanisms and reverted by insulinotropic signaling. Results establish that pathological ER stress in AD is linked to impaired brain insulin signaling and provide insight into the protective mechanisms of anti-diabetic agents in the brain. O2-09-04

DIFFERENTIAL MICROGLIAL EXPRESSION OF NEW ALZHEIMER’S DISEASE ASSOCIATED GENES MS4A4A AND MS4A6A

Julia Kofler, Stephanie Bissel, Clayton Wiley, Mark Stauffer, Geoffrey Murdoch, University of Pittsburgh, Pittsburgh, Pennsylvania, United States. Background: Genome-wide association studies (GWAS) have identified nine novel loci associated with late-onset Alzheimer’s disease (AD). These genes have been linked to b-amyloid metabolism, cholesterol metabolism, synaptic function and immune system responses. However, detailed infor-

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mation about cellular expression, function and transcriptional regulation is lacking for many of these genes. The aim of our study was to assess expression of these GWAS genes in primary human microglial cells under baseline conditions and following activation. Methods: Microglial cultures isolated from postmortem adult human brain tissue were either classically stimulated by IFN-g and LPS or alternatively stimulated by IL-4. RNA was collected at 48 hours and gene expression profile determined by Affymetrix U133 Plus 2.0microarrays. Expression of select genes was confirmed by quantitative RT-PCR following treatment with various pathway inhibitors. Results: Primary human adult microglial cells express CLU, PICALM, BIN1, ABCA7, CD2AP and CD33, but not CR1 and EPHA1. Of the MS4A gene cluster, only members located close to the GWAS SNP loci were expressed in microglia (MS4A4A, MS4A6A, MS4A7, MS4A14), whereas other members were not expressed (MS4A1, MS4A2, MS4A3, MS4A5, MS4A6E, MS4A12). Following classical stimulation, there was significant downregulation of MS4A4A and MS4A6A and slight upregulation of CLU. After alternative activation, MS4A6A showed slight upregulation, but none of the other genes was differentially regulated. Treatment with NF-kB and STAT6 inhibitors did not affect microglial expression of MS4A4A or MS4A6A. Conclusions: Microglial cells show expression of several AD-associated genes identified in GWAS studies. MS4A gene family members MS4A4A and MS4A6A, which are located close to the GWAS SNP loci, show differential expression following microglial activation. These findings provide support for an important role of neuroinflammatory pathways in the pathogenesis of AD. We speculate that MS4A4A and MS4A6A genotype may possibly affect AD pathology by modulating microglial functions. O2-09-05

EPIGENETIC MODIFICATION IN METABOLIC SYNDROME-INDUCED BRAIN ENERGY IMPAIRMENT

Merina Varghese, Jun Wang, Alice Cheng, Lap Ho, Cheuk Tang, Giulio Pasinetti, Mount Sinai School of Medicine, New York, New York, United States. Background: Metabolic syndrome, the combination of obesity, insulin resistance, high blood pressure and hypercholesterolemia, is on the rise in the developed world. Diabetes is a known risk factor for Alzheimer’s disease and reduced brain glucose utilization occurs in those at increased risk for Alzheimer’s. We investigated the expression of genes linked to energy metabolism in the brain of mice with diet-induced metabolic syndrome (DIM) and found that epigenetic regulators may be responsible for the observed functional deficits. Methods: Metabolic syndrome was induced in mice by administration of a high-fat diet and control mice were given normal diet. The mice were subjected to micro functional magnetic resonance imaging (fMRI) to assess differences in the brain activity. Differential gene expression in the brain of the DIM mice as compared to control mice was performed by microarray analysis. Mitochondrial respiration was measured using the Seahorse XF24 extracellular flux analyzer, in isolated brain mitochondria or in primary neurons. Results: In the diet-induced animal model of metabolic syndrome (DIM) that develops impaired glucose tolerance, hypercholesterolemia, hypertension and obesity with increased mesentery fat, the four essential elements of metabolic syndrome when fed a high fat diet, we found that brain energy metabolism was severely impaired. Pilot micro MRI imaging studies showed that resting state fMRI abnormalities were evident in DIM-mice. Furthermore, these impairments were associated with altered gene expression in multiple pathways including changes in the expression of mitochondrial glucose metabolism genes and of chromatin modification enzymes, specifically histone deacetylase 5 (HDAC5). Our findings indicate that DIM might induce epigenetic modification of DNA that controls energy metabolism. To test the hypothesis that modulation of HDAC activity may improve mitochondrial function in neurons, we analyzed mitochondrial respiration in primary neurons treated with the HDAC inhibitor trichostatin A and found an increase in oxygen consumption rate. Conclusions: Epigenetic modulation may play a role in the energy metabolism deficits seen in the brain in metabolic syndrome and presents a new target for treatment of neurodegeneration induced by mitochondrial deficits.