Possible involvements of beta-amyloid, insulin signaling and tau ...

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Background: Fused in sarcoma (FUS) has been identified as the causative gene for autosomal-dominant familial amyotrophic lateral sclerosis (ALS) type 6.
Sunday, July 14, 2013: Poster Presentations: P1 been reported in degenerative diseases of the retina. For instance, altered levels of tau protein have been detected in the retina and optic nerve of patients with glaucoma, suggesting that retina and brain degeneration share similar pathogenic mechanisms. We have recently demonstrated that P301S mutant human tau mice (tau P301S) develop tau filamentous inclusions and axonopathy in retinal ganglion neurons (RGCs), in the absence of neuronal loss and that transgenic retinal explants do not respond to neurotrophic stimuli in vitro. Methods: We investigated the impact of tau pathology on BDNF signaling pathway in RGC of adult wild type (WT) and tau P301S mice. Protein and mRNA expression of neurotrophins and their receptors was investigated by western blot and quantitative RT-PCR analyses in retina extracts of WT and tau P301S mice. The activation of the BDNF signaling pathway in vivo was evaluated under basal conditions and upon stimulation with the ligand. Results: Protein levels of BDNF and TrkB receptor were significantly altered in the retina of 5-month old tau P301S mice. The change was not due to altered expression and protein synthesis as the levels of mRNA and pro-BDNF were similar in WT and transgenic retinas. The activation of TrkB signaling was evaluated using antibodies directed towards specific phospho-tyrosine residues. Altered activation state was detected both in basal and BDNF-stimulated retinas of tau P301S mice. Conclusions: These data indicate that the presence of tau pathology is associated with alteration of neurotrophin signaling in the retina of tau P301S mice.

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ALTERATIONS OF NEUROTROPHIN SIGNALING IN THE RETINA OF THE P301S MUTANT HUMAN TAU TRANSGENIC MOUSE

Erica Barini1, Nadia Mazzaro1, Maria Grazia Spillantini2, Michel Goedert3, Paolo Medini1, Laura Gasparini1, 1Istituto Italiano di Tecnologia, Genova, Italy; 2University of Cambridge, Cambridge, United Kingdom; 3MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. Contact e-mail: [email protected] Background: Intracellular inclusions made of microtubule-associated tau protein are a defining pathological hallmark of tauopathies, which include Alzheimer disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17. Altered levels of tau protein have been detected in the retina and optic nerve of patients with glaucoma, suggesting that retina degeneration and tauopathies share similar pathogenic mechanisms. We have recently demonstrated that P301S mutant human tau mice (tau P301S) develop tau filamentous inclusions and axonopathy in retinal ganglion neurons (RGCs), in the absence of neuronal loss and that transgenic retinal explants do not respond to neurotrophic stimuli in vitro. Neurotrophins, and especially BDNF, are important modulators of neuronal survival and function in the visual system. Altered levels of TrkB receptors result in neuronal degeneration and TrkB proteins are associated with tau neuronal inclusions and amyloid plaques in AD brain. Methods: We investigated the BDNF signaling pathway as a function of tau pathology in retinas of wild type (WT) and tau P301S mice at 1-5 months of age. Protein expression of neurotrophins and their receptors was investigated by western blot using specific antibodies. The cellular and subcellular localization of TrkB receptor and phospho-tau was investigated by immunohistochemistry. Results: Protein levels of BDNF and TrkB receptor were significantly altered in the retina of 5-month old tau P301S mice, in the absence of changes of TrkA and p75NTR receptors. Changes of BDNF levels were detected as early as 1 month of age in tau P301S retinas, while the expression of TrkB was significantly altered only at 5 months of age. Immunohistochemistry analyses revealed that the localization of TrkB was altered in transgenic retinas and co-localized with phospho-tau immunoreactivity in RGCs. Conclusions: These data indicate that alterations of neurotrophin signaling occur early during tau pathology development in the retina of tau P301S mice. Funded by a grant of Compagnia di San Paolo awarded to LG.

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ELUCIDATING THE RELATIONSHIP BETWEEN b2 ADRENERGIC RECEPTOR, TAU AND MTOR

Elena Wisely, Salvatore Oddo, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States. Contact e-mail: wiselye@ livemail.uthscsa.edu Background: Accumulation of tau is a critical event in several neurodegenerative disorders, collectively known as tauopathies, which include Alzheimer’s disease (AD) and frontotemporal dementia. Pathological tau is hyperphosphorylated and accumulates to form neurofibrillary tangles. However, the molecular mechanisms leading to tau aggregation remain unclear and need to be elucidated. mTOR signaling, which is linked to tau pathology, has been shown to couple to b2 adrenergic receptors (b2ARs). Genetic studies indicate that polymorphisms in the gene encoding the b2AR are linked to a higher risk of late onset AD. Furthermore, epidemiological studies demonstrate that the use of b2AR antagonists correlates with a lower incidence of AD. Age is another major risk factor for all tauopathies, suggesting that molecular changes contributing to the aging process may facilitate tau accumulation and represent common mechanisms across different tauopathies. Methods: We are using complementary genetic and pharmacological approaches to dissect the relation among b2AR, mTOR and tau. Results: We found that pharmacologically reducing mTOR signaling with rapamycin ameliorates tau pathology and the associated behavioral deficits in a mouse model overexpressing mutant human tau. Complementary to it, we show that increasing mTOR signaling leads to an increase in tau levels and phosphorylation. Conclusions: In summary, we show that increasing mTOR signaling facilitates tau pathology while reducing mTOR signaling ameliorates tau pathology. Given the overwhelming evidence showing that reducing mTOR signaling increases lifespan and health span, our data have profound clinical implications for aging and tauopathies and provide the molecular basis for how aging may contribute to tau pathology. Additionally, these results provide pre-clinical data indicating that reducing mTOR signaling may be a valid therapeutic approach for tauopathies.

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POSSIBLE INVOLVEMENTS OF BETA-AMYLOID, INSULIN SIGNALING AND TAU PHOSPHORYLATION IN THE PATHOLOGICAL INTERACTION BETWEEN ALZHEIMER’S DISEASE AND DIABETES

Naoyuki Sato1, Mari Mori-Ueda1, Toshihisa Tanaka2, Shuko Takeda3, Kozue Uchio-Yamada4, Hironori Ueda1, Mitsuru Shinohara1, Shigeo Murayama5, Masatoshi Takeda1, Hiromi Rakugi1, Ryuichi Morishita1, 1Osaka University, Suita, Japan; 2Osaka University Graduate School of Medicine, Suita, Japan; 3Osaka University, Osaka, Japan; 4National Institute of Biomedical Innovation, Ibaraki, Japan; 5Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan. Contact e-mail: [email protected] Background: Emerging evidence suggests that diabetes mellitus increases the risk of onset of Alzheimer disease (AD). However, the mechanisms by which diabetes increases the risk of AD have not been fully elucidated. Retrospective studies using AD brain indicate that diabetes does not increase amyloid plaques. In contrast, a Japanese cohort, the Hisayama study suggested that insulin resistance in midlife increased the risk of development of amyloid plaques. Therefore, our aim is to understand the mechanisms whereby diabetes increase the risk of AD by dividing them into two phases; before and after the development of amyloid plaques. Moreover, we aimed to understand the mechanisms of the mutual pathological interaction between AD and diabetes. Methods: To test whether insulin resistance increases Abeta accumulation in the brain and the plasma, we fed wild-type mice with a high-fat diet and measured the levels of Abeta in the brain and the plasma. To investigate the effects of diabetes on AD, we further analyzed the phenotypes of APP+ ob/ob mice, which showed the increased cerebral amyloid angiopathy and impaired insulin signaling (Takeda, Sato, et al. Proc Natl Acad Sci U S A, 107, 7036-41, 2010), especially focusing on

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Sunday, July 14, 2013: Poster Presentations: P1

tau phosphorylation. Results: Six months high fat diet increased Abeta40 in the brains ofB6C wild type mice. On the other hand, high fat diet increased plasma Abeta levels in APP/PS1 mice, but not in wild type mice. Moreover, tau phosphorylation was highly increased in the brains of 18 months old APP+ ob/ob mice. Conclusions: Tau phosphorylation is increased by diabetes in APP mice, suggesting that Abeta i s prerequisite, but insufficient to cause tau phosphorylation in vivo. A beta accumulation, insulin signaling and tau phosphorylation might play essential roles in the pathological interaction between AD and diabetes. Of note, a vicious cycle likely underlie the interaction between AD and diabetes. High fat diet-induced elevation of plasma Abeta level might be involved in this interaction between AD and diabetes.

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TWO DISTINCT PATHWAYS LEADING TO RETINAL DEGENERATION IN TRANSGENIC DROSOPHILA MELANOGASTER OVEREXPRESSING HUMAN FUS

Tadafumi Hashimoto, Hayato Kunugi, Koji Matsukawa, Hirokazu Uchigami, Ryoko Ihara, Takahiro Chihara, Masayuki Miura, Tomoko Wakabayashi, Takeshi Iwatsubo, University of Tokyo, Tokyo, Japan. Contact e-mail: [email protected] Background: Fused in sarcoma (FUS) has been identified as the causative gene for autosomal-dominant familial amyotrophic lateral sclerosis (ALS) type 6. FUS is an RNA-binding protein that plays multiple roles in RNA metabolism. Wild-type FUS is predominantly localized in the nuclei of neurons, whereas cytoplasmic FUS-immunoreactive inclusions are observed in degenerating neurons of sporadic or FUS-linked familial ALS (fALS), or frontotemporal lobar degeneration (FTLD). However, the molecular mechanism whereby FUS leads to neurodegeneration, as well as its relationship with that of the subcellular localization of FUS is still unclear. Methods: We generated transgenic Drosophila melanogaster (tg fly) overexpressing human FUS in retinal photoreceptor neurons using GAL4/ UAS system under the control of the GMR driver. Results: We found that overexpressed wild-type FUS is predominantly localized in the nuclei and that wild-type FUS tg flies exhibited ommatidial disorganization, vacuolar degeneration and thinning of the retina. Strikingly, these degenerative phenotypes were completely normalized by the deletion of RNA recognition motif (RRM) of FUS that abolishes the RNA binding ability, suggesting that the RNA binding ability of FUS is required for the neurodegeneration caused by overexpression of FUS in the nuclei. To examine whether cytoplasmic localization of FUS causes neurodegeneration through RNA-binding as well, we crossed tg flies that overexpress wild-type or RRM-deleted FUS with a transgenic RNAi line for transportin, which mediates the nuclear import of FUS. Knockdown of transportin led to the relocation of wild-type or RRM-deleted FUS to the cytoplasmic granular structures. Strikingly, cytoplasmic localization of wild-type FUS aggravated the degenerative phenotypes of the retina, which was not completely rescued by the deletion of RRM. Moreover, we found that fALS mutants of FUS (R514G and R521C) also were predominantly localized to the cytoplasmic granular structures and that the degenerative phenotypes of the retina of tg flies that overexpress the fALS mutant FUS were more severe compared to those expressing wild-type FUS. Conclusions: These findings led us to speculate that FUS in the nuclei causes neuronal degeneration through its RNA-binding ability, whereas those localized to cytoplasm leads to neurodegeneration independently of RNA-binding. Our study raises the possibility of two distinct pathogenic mechanisms causing neurodegenetaion in FUSopathies.

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DETECTING PROGRANULIN MUTATIONS IN PEOPLE WITH ATYPICAL ALZHEIMER’S DISEASE

Welmoed Krudop1, Elise Dopper2, Charlotte Teunissen3, Sisi Durieux-Lu4, Wiesje Van Der Flier5, Philip Scheltens5, John Van Swieten6, Yolande Pijnenburg5, 1Alzheimer Center, VU Medical Center, Amsterdam, Netherlands; 2Erasmus Medical Center, Rotterdam, Netherlands; 3Alzheimer Center, VU Medical Center, Amsterdam, Netherlands; 4VU Medical Centre, Amsterdam, Netherlands; 5 VU University Medical Center, Amsterdam, Netherlands; 6Erasmus University Medical Center, Rotterdam, Netherlands. Contact e-mail: w. [email protected] Background: Progranulin mutations cause frontotemporal dementia (FTD) in 2-8% of FTD cases. The clinical manifestations of these mutations are heterogeneous and about 25% of progranulin mutation carriers develop a clinical picture similar to Alzheimer’s disease (AD). Measuring serum progranulin levels has recently been shown to be a highly sensitive and specific screening method for identifying progranulin loss of function mutations. We set out to identify progranulin mutation carriers among patients with clinically probable AD, not supported by their atypical CSF amyloid-beta (1-42) level. Methods: Serum progranulin levels were measured using a thoroughly validated test in 48 patients from the memory clinic based Amsterdam Dementia Cohort and 20 patients from the Rotterdam, Erasmus medical centre memory clinic with a clinical diagnosis of probable AD and an atypical CSF amyloid-beta(1-42) level (amyloid beta(1-42) 600 ng/L). Two patients known to have a pathogenic progranulin mutation served as controls. Results: Serum progranulin levels corresponding with a progranulin loss of function mutation were found in the 2 known progranulin mutation carriers and in one atypical AD case (figure 1). Apart from an atypical CSF profile, this case had an atypical clinical presentation as well, consisting of word finding problems and decreased semantic knowledge, memory impairment, apraxia, visuocontructional impairment and compulsive behaviour. On MRI predominantly left sided temporal and parietal atrophy was seen in this case. Conclusions: We found evidence for a progranulin mutation in 1.5% of atypical AD cases (serum progranulin 25ng/ml). The remaining 68 AD patients (abeta1-42 600 ng/L) showed serum progranulin levels ranging from 62-199 ng/ml with a mean of 110 ng/ml (figure 1).The prevalence of progranulin mutations among atypical AD cases therefore appears to be lower than their prevalence among FTD patients. Nevertheless, with the availability of a simple and reliable test, screening for progranulin mutations among atypical AD cases remains something to consider.