TREM2-MEDIATED EARLY RESPONSE BY RESIDENT MICROGLIA LIMITS DIFFUSION AND TOXICITY OF AMYLOID PLAQUES

Podium Presentations: Monday, July 25, 2016

hydroxymethylation patterns in AD as well as separating these patterns from the true DNA methylation signal at single base pair resolution. We have identified a number of novel loci that display epigenetic dysregulation in AD brain. This study was funded by an Alzheimer’s Association New Investigator Grant.

MONDAY, JULY 25, 2016 ORAL SESSIONS O2-07 MOLECULAR AND CELL BIOLOGY: THE IMMUNE CELL ROLE IN ALZHEIMER’S DISEASE PATHOLOGY O2-07-01

O2-06-06

HIGHER PERIPHERAL TREM2 MRNA EXPRESSION LEVELS ARE RELATED TO COGNITIVE DEFICITS AND HIPPOCAMPAL ATROPHY IN ALZHEIMER’S DISEASE AND AMNESTIC MCI

Yi Jayne Tan1, Adeline Su Lyn Ng2, Joseph K. W. Lim1, Russell J. Chander3, Ji Fang1, Yingwei Qiu1, Simon Ting4, Shahul Hameed4, Nagaendran Kandiah2, Juan Zhou1,5, 1Duke-NUS Graduate Medical School, Singapore, Singapore; 2National Neuroscience Institute, Tan Tock Seng Hospital, Singapore, Singapore; 3National Neuroscience Institute, Singapore, Singapore; 4National Neuroscience Institute (SGH campus), Singapore, Singapore; 5Agency for Science, Technology and Research, Singapore, Singapore. Contact e-mail: [email protected] Background: Studies have reported strong association of the triggering receptor expressed on myeloid cells 2 (TREM2) gene with Alzheimer’s disease (AD), with the rs75932628 (R47H) variant reaching genome-wide significance. In AD, TREM2 is hypothesized to attenuate inflammation-induced neuronal damage and activate phagocytosis of damaged cells. Preliminary studies in noncarriers of the R47H allele have found higher peripheral TREM2 mRNA and protein expression levels in AD and schizophrenics compared to healthy controls, with higher levels correlating with lower cognitive scores. Whether higher levels reflect pathological or compensatory mechanisms remains unclear. We aimed to determine if peripheral TREM2 expression levels differ between healthy controls and early-stage AD, and if they correlate with cognitive and brain structural changes. Methods: We measured peripheral TREM2 mRNA levels in 65 AD subjects, 22 amnestic mild cognitive impairment (aMCI) subjects, and 68 healthy controls using real-time quantitative polymerase chain reaction. All participants underwent detailed neuropsychological testing and structural MRI imaging. Association of TREM2 expression with cognitive scores and brain imaging measures were assessed using linear regression models, with further accounting for APOE4 status. Results: TREM2 mRNA levels differed across all three groups (mean levels 6 SEM in AD, aMCI, and controls were 1.0360.07, 0.8060.09, and 0.8160.05 respectively, p¼0.006), controlling for age, gender and ethnicity. TREM2 mRNA levels were higher in AD compared with controls (p¼0.002) and aMCI (p¼0.020). Levels did not differ between aMCI and controls. Group differences remained significant after controlling for APOE4 status. In aMCI and AD, higher TREM2 levels correlated with lower MMSE (b¼ -0.269, p¼0.009) and Montreal cognitive assessment (MoCA) scores (b¼ -0.261, p¼0.010), and poorer episodic memory (b¼ -0.340, p¼0.0017) and visuospatial function (b¼ -0.396, p¼0.0192) on further neuropsychological testing. In AD and aMCI, TREM2 levels correlated significantly with left hippocampal volume (b¼ -0.249, p¼0.038), controlling for age, gender, ethnicity and total intracranial volume. Conclusions: Peripheral TREM2 mRNA levels are higher in early-stage AD compared with aMCI and controls and is associated with AD-specific cognitive deficits and hippocampal volume loss. Peripheral TREM2 expression levels are a potential biomarker for Alzheimer’s-related neurodegeneration, particularly when CSF and amyloid-based imaging are not readily available.

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PERTURBED PURINERGIC SIGNALING CAUSES DYSFUNCTION OF PLAQUE-ASSOCIATED MICROGLIA IN A MOUSE MODEL OF ALZHEIMER’S DISEASE

Matthias Brueckner1, Jan N. Hansen1, Ligang Gu1, Sangyong Lee2, Nicole Reichenbach3, Gabor C. Petzold3, Christa E. Mueller2, Annett Halle1, 1Center of Advanced European Studies and Research, Bonn, Germany; 2University of Bonn, Bonn, Germany; 3German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany. Contact e-mail: [email protected] Background: Recent studies have highlighted the importance of micro-

glia in Alzheimer’s disease (AD), implicating microglia as a promising target for AD treatment. Microglial cells in mouse models of AD show signs of functional impairment. Whether alterations in purinergic signaling, which under physiological conditions is critically involved in shaping microglial functions, contribute to this phenomenon and whether microglial dysfunction is reversable by targeting purinergic signaling has remained unclear. Methods: Dynamics, morphology and phagocytic capacity of microglial cells from APP/PS1-CX3CR1EGFP mice were assessed using intravital 2-photon microscopy and acute cerebral slice phagocytosis assays. Microglial cells were subgrouped into beta-amyloid plaque-associated and -distant cells depending on their spatial relation to plaques. Cellular dynamics, morphology and phagocytic capacity of microglial cells were quantified upon perfusion with specific pharmacological agonists and antagonists of purinergic signaling pathways. Furthermore, microglial subgroups were harvested from APP/PS1-CX3CR1-EGFP mice by laser-capture microdissection and expression of purinergic signaling pathway genes were investigated by qPCR array. Results: Microglia from APP/PS1 mice showed impaired phagocytic capacity and cell motility compared to wildtype animals. We found a striking difference in microglial functional parameters between plaque-associated and plaque-distant microglia with severe functional impairment in plaque-associated and only minor impairment in plaque-distant cells. Transcriptional analysis of microglial cells harvested by laser-microdissection revealed increased expression of several purine degrading enzymes and purinoreceptors in plaque-associated cells. Importantly, purinoreceptor agonists or pharmacological blockage of nucleotide-degrading enzymes rapidly normalized phagocytic and dynamic function of plaque-associated microglia in slices from APP/PS1 mice. Conclusions: We have identified perturbed purinergic signaling as an important cause for the impairment of cellular dynamics, morphology and phagocytic capacity of plaque-associated microglia in the APP/PS1 model. Therefore, targeting microglial purinergic signaling may represent a promising novel approach for the treatment of AD. O2-07-02

TREM2-MEDIATED EARLY RESPONSE BY RESIDENT MICROGLIA LIMITS DIFFUSION AND TOXICITY OF AMYLOID PLAQUES

Yaming Wang1, Tyler Ulland2, Jason D. Ulrich2, Wilbur Song2, Peng Yuan3, Thomas E. Mahan2, Yang Shi2, Susan Gilfillan2, Marina Cella2, Ronald B. DeMattos1, Jaime Grutzendler3, John R. Cirrito2, David M. Holtzman2, Marco Colonna2, 1Eli Lilly and Company, Indianapolis, IN, USA; 2Washington University in St. Louis, St. Louis, MO, USA; 3Yale University School of Medicine, New Heaven, CT, USA. Contact e-mail: [email protected]

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Podium Presentations: Monday, July 25, 2016

Background: TREM2 is a microglial receptor that recognizes changes in the lipid microenvironment, which may result from Ab accumulation and neuronal degeneration in Alzheimer’s disease (AD). Rare TREM2 variants that impair lipid/lipoprotein recognition lead to an increased risk of developing AD. In murine models of AD, TREM2 deficiency prevents Iba-1+ myeloid cells clustering around Ab deposits. However, the origin of these cells and their impact on Ab accumulation and Ab-associated neurotoxicity are a matter of debate. Methods: Using parabiosis, we determined the contribution of peripheral blood monocytes to the pool of myeloid cells that are associated with Ab deposits in 5XFAD and APPPS1-21 mice. To understand the role of microglia in Ab accumulation and Ab-associated neurotoxicity, we examined the impact of TREM2 deficiency at the onset of Abrelated pathology in 5XFAD mice. Results: We found that amyloidassociated myeloid cells were derived from expansion of brain-resident microglia rather than from recruitment of peripheral monocytes. TREM2 deficiency impaired the clustering of microglia around amyloid plaques even at the onset of Ab-related pathology. Remarkably, morphological analysis revealed that plaques from TREM2-deficient 5XFAD mice were drastically distinct, appearing more diffuse and less dense than those from 5XFAD mice. Moreover, Ab plaques in TREM2-deficient mice were associated with significantly greater neuritic dystrophy. Conclusions: We concluded that peripheral blood monocytes have a minimum contribution to the expansion of microglia under normal physiological condition in two AD transgenic models. One mechanism via which TREM2 protects from AD is by enabling the proliferation of amyloid-associated microglia to prevent the physical diffusion of Ab deposits, thereby limiting their toxicity. O2-07-03

REGULATORY T CELLS DELAY DISEASE PROGRESSION IN ALZHEIMER’S-LIKE PATHOLOGY

Cira Dansokho1, Dylla Ait Ahmed1, Saba Aid1, Cecile Toly-Ndour1, Thomas Chaigneau1, Vanessa Calle1, Nicolas Cagnard2, Martin Holzenberger1, Eliane Piaggio3, Pierre Aucouturier1, Guillaume Dorothee1, 1INSERM-UPMC UMRS 938, Centre de Recherche Saint-Antoine, Paris, France; 2Faculte de Medecine Paris Descartes, Hopital Necker, Paris, France; 3INSERM U932, Institut Curie, Paris, France. Contact e-mail: [email protected] Background: Recent studies highlight the implication of innate and

adaptive immunity in the pathophysiology of Alzheimer’s disease (AD), and foster immunotherapy as a promising strategy for its treatment. Vaccines targeting amyloid-beta (Abeta) peptide provided encouraging results in mouse models, but severe side effects attributed to T cell responses in the first clinical trial AN1792 underlined the need for better understanding adaptive immunity in AD. We previously showed that regulatory T cells (Tregs) critically control Abetaspecific CD4+T cell responses in both physiological and pathological settings. Here, we analyzed the impact of Tregs on spontaneous disease progression in a murine model of AD. Methods:Preclinical studies were carried out in the APPPS1 mouse model. Treg cells were either depleted using anti-CD25 antibodies, or selectively amplified through low-dose IL-2 treatment. Impact of early Treg modulation on cognitive functions, neuropathology, and disease-related gene expression was assessed. Results: Early transient depletion of Tregs accelerated the onset of cognitive deficits in APPPS1 mice, without altering Abeta deposition. Earlier cognitive impairment correlated with reduced recruitment of microglia towards amyloid deposits and altered disease-related gene expression profile. Conversely, amplification of Tregs through peripheral low-dose IL-2 treatment increased numbers of plaque-associated microglia, and restored cognitive functions in

APPPS1 mice. Conclusions: These data suggest that Tregs play a beneficial role in the pathophysiology of AD, by slowing disease progression and modulating microglial response to Abeta deposition. Our study highlights the therapeutic potential of repurposed IL-2 for innovative immunotherapy based on Tregs modulation in AD. O2-07-04

SPECIFIC INHIBITION OF ASTROCYTE REACTIVITY IMPROVES SOME DISEASE OUTCOMES IN ALZHEIMER’S DISEASE MICE

Kelly Ceyzeriat1,2, Lucile Ben Haim1,2, Thomas Saint-Georges1,2, Fanny Petit3,4, Diane Houitte1,2, Martine Guillermier1,2, Aurelie Berniard1,2, Julien Mitja1,2, Charlene Josephine1,2, Philippe Hantraye1,2, Emmanuel Brouillet1,2, Karine Cambon1,2, Marc Dhenain3,4, Gilles Bonvento1,2, Carole Escartin1,2, 1Commissariat a l’Energie Atomique et aux Energies Alternatives (CEA), MIRCen, Fontenay-aux-Roses, France; 2Centre National de la Recherche Scientifique (CNRS), Fontenay-aux-Roses, France; 3Commissariat a l’Energie Atomique et aux Energies Alternatives (CEA), Fontenay-aux-Roses, France; 4Centre National de la Recherche Scientifique (CNRS), Fontenay-aux-Roses, France. Contact e-mail: [email protected] Background: Astrocytes play crucial supportive roles in the brain. In

Alzheimer’s disease (AD), astrocytes become reactive. Astrocyte reactivity is essentially characterized by morphological changes and overexpression of intermediate filament proteins. But how the normal supportive functions of astrocytes are changed by their reactive state is unclear. Any alteration in these functions could have important consequences on neuronal activity and viability. In this study, we aim to understand the contribution of reactive astrocytes to neuronal dysfunction in the context of AD. We reported recently that the JAK2/STAT3 pathway (Janus Kinase 2/ Signal Transducer and Activator of Transcription 3) is responsible for astrocyte reactivity in models of AD (Ben Haim et al., 2015). Here, we used adeno-associated viral vectors (AAV) to modulate this pathway specifically in astrocytes in two mouse models of AD (APP/PS1dE9 and 3xTg-AD mice). Methods: 3 month-old APP/PS1dE9 mice and 6 month-old 3xTg-AD mice were injected bilaterally in the hippocampus with AAV, with a GFA-ABC1D promoter encoding SOCS3, the endogenous inhibitor of the JAK2/STAT3 pathway, to de-activate astrocytes. 3xTg-AD mice were tested at 15 months on elevated plus maze test and on the Y maze test. Postmortem analysis were performed at 7 months for APP/PS1dE9 and 16 months for 3xTg-AD mice and included immunohistology, MSD ELISA tests and western blot. Results:Hippocampal injection of an AAV encoding SOCS3 efficiently de-activated astrocytes in both models. We showed that the number of amyloid plaques and the concentration of soluble amyloid-beta were decreased in APP/ PS1dE9 mice injected with AAV-SOCS3. De-activation of astrocytes also impacted microglia response. Finally, we evaluated the effects of astrocyte reactivity inhibition on anxiety and memory in 3xTg-AD mice. We found that anxiety tended to be reduced by AAV-SOCS3, but memory was not restored. Conclusions: Overall, blocking astrocyte reactivity improved several alterations linked to AD pathology. By which mechanisms reactive astrocytes influence disease progression? This is what we are currently studying. Our potent approach to modulate astrocyte reactivity in the mouse brain makes it possible to directly assess the contribution of reactive astrocytes to AD. O2-07-05

KV1.3 INHIBITION REDUCES AB-INDUCED MICROGLIAL NEUROTOXICITY

Lee-Way Jin1, Izumi Maezawa2, Hai Minh Nguyen3, Jacopo Di Lucente4, Srikant Rangaraju5, Allan I. Levey5, Heike Wulff3, 1UC Davis, Davis, CA, USA; 2UC Davis, Sacramento, CA, USA; 3University of California Davis,