CN autoinhibitory domain located near the C terminus of the CN catalytic subunit. Commercially available antibodies that target the N terminus of the CN catalytic subunit reveal the presence of a highly-active 48 kDa pro- teolytic fragment in human brain tissue during early stages of cognitive decline and also in a variety of experimental models of neurodegeneration. While useful for determining the extent of CN proteolysis in Western blot applications, N terminus antibodies do not reveal the cellular location of the proteolysis. Knowing where CN is proteolyzed in nervous tissue seems critical to understanding the mechanistic basis of its many deleterious ac- tions, particularly because CN is found in both neurons and glial cells where it is involved in different cellular functions. Methods: To address this gap in our understanding of CN regulation, we generated custom rabbit polyclonal antibodies to CN A based on previously identified calpain-dependent cleav- age sites. One of these antibodies (referred to as “DCN48”) detects a 48 kDa fragment in Western blot assays, but does not detect full-length (60 kDa) CN. The DCN48 antibody was then used for DAB immunohistochemical la- beling of human brain sections characterized by AD and mixed AD/vascular pathologies. Results: The anatomical features labeled by the DCN48 anti- body included astrocyte clusters and vascular-associated elements and/or processes. We also observed numerous DCN48-positive astrocytes associ- ated with microinfarcts. Surprisingly, we have seen very little neuronal la- beling with this antibody. Conclusions: The results suggest that astrocytes and, perhaps astrocyte end-feet, are a primary locus for CP- dependent CN proteolysis in injured or diseased nervous tissue. CN prote- olysis appears to be especially pronounced in astrocytes associated with vascular pathology. This work may provide new mechanistic insights into the impact of Ca 2+ dysregulation on neurodegenerative diseases. P4-246 ACTIVATION OF THE SIGMA-1 RECEPTOR BY SPECIFIC LIGANDS INHIBITS HUMAN INFLAMMATORY DENDRITIC CELL FUNCTIONS AND EFFECTOR T-LYMPHOCYTE RESPONSES Attila Szabo, Attila Kovacs, Ede Frecska, Eva Rajnavolgyi, Faculty of Medicine, University of Debrecen, Debrecen, Hungary. Contact e-mail: szattila@med.unideb.hu Background: Neuropsychiatric diseases have recently been attributed to chronic inflammation in the central nervous system, and correlation between gene polymorphisms of innate immune receptors and the frequency of late onset of Alzheimer’s disease (AD) has also been shown. Ligation of murine maternal Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs) by LPS or PolyI:C have been shown to cause decreased neurogenesis, cognitive deficits, and increased deposition of Ab aggregates in the brain of the off- springs. These data in line with the accumulation of monocyte-derived den- dritic cells (moDCs) and macrophages during chronic inflammation suggest an activation-induced disease promoting mechanism. In contrast, the orphan receptor sigma-1 has been shown to mediate anti-inflammatory responses in rodent in vivo models, but the molecular background has not been eluci- dated. Methods: Western blot was used to monitor protein level expression of SIGMAR1 in human primary monocytes, macrophages and moDCs. Gene expression of sigma-1 receptor (SIGMAR1), and IL-1b, IL-6, TNFa, IL-8, IL-10 cytokines was assessed by Q-PCR. Concentration of secreted cytokines was measured by ELISA. ELISPOT was used to assess the numbers of moDC-primed autologous na € ıve Th1 and Th17 cells. Gene-specific RNA-interference was performed to silence sigmar-1 gene. N,N-dimethyltryptamine, 5-methoxy-N,N-dimethyltryptamine, and PRE- 084 hydrochloride were used to trigger SIGMAR1 in moDCs. To mimic different inflammatory conditions, we used TLR/RLR ligands (LPS, PolyI:C) and inactivated pathogens (E. coli and influenza virus). Results: In this study we used endogenous ligands (NN-DMT, 5-MeO-DMT) and high affinity synthetic PRE-084-hydrochloride to trigger sigma-1 in human moDCs and monitored their effects on LPS- and polyI:C-induced inflamma- tory responses. Co-administration of sigma-1 ligands with these activators inhibited the production of pro-inflammatory cytokines and chemokines (IL-1b, IL-6, TNFa, IL-8), while increased the secretion of anti-inflamma- tory IL-10. The antigen-presenting capacity of moDCs was also inhibited and co-administration of sigma-1 ligands with E. coli or influenza virus decreased the differentiation of moDC-induced Th1 and Th17 inflammatory effector cells in a sigma-1 receptor specific manner confirmed by gene silencing. Conclusions: These results demonstrate the inhibitory potential of stimulated sigma-1 receptor in brain-resident moDCs that could be har- nessed for the pharmacological treatment of AD and other chronic inflam- matory conditions in the CNS. P4-247 IMMUNE-CHALLENGED TNF-DEFICIENT MICE SHOWA DECREASE IN HIPPOCAMPAL LEARNING Christa Løth Myhre 1 , Alicia A. Babcock 2 , Kate Lambertsen 2 , Laura Ilkjær 2 , Morten Skovgaard Jensen 3 , Zengqiang Yuan 4 , Bente Finsen 5 , 1 Institute of Molecular Medicine, Odense, Denmark,; 2 Institute of Molecular Medicine, Odense, Denmark; 3 Department of Biomedicine, Aarhus, Denmark; 4 Institute of Biophysics, CAS, Beijing, China; 5 Institute of Molecular Medicine, Odense, Denmark. Contact e-mail: cmyhre@ health.sdu.dk Background: A dominant feature of Alzheimer ^ A’s disease (AD) is activa- tion of microglial cells, which can produce the pro-inflammatory cytokine TNF. TNF is deregulated in AD, and can be induced upon immune-chal- lenge with endotoxin. TNF may also be involved in hippocampal neurogen- esis, which is the basis for spatial learning. Spatial learning can be induced in mice by repeated exposure to the Barnes maze. Aim: The aim was to eval- uate the involvement of TNF in hippocampal learning in mice during aging and after immune-challenge with endotoxin. Methods: To validate the Barnes maze paradigm, 3-month-old C57BL/6 mice and C57BL/6 mice in- jected with Scopolamine, a drug that interferes with learning, were exposed to the Barnes Maze with 4 trials/day for 6 days and killed on day 7. Three- and 9-month-old TNF-KO and C57BL/6 mice were then exposed to the Barnes Maze. To challenge the immune system of the mice, 9-month-old TNF-KO and C57BL/6 mice were twice injected with endotoxin, a strong inducer of TNF, and exposed to the Barnes maze 4 days later, after sickness behavior had ceased. Locomotor activity was assessed using Open Field. Results: The C57BL/6 mice injected with Scopolamine showed a signifi- cantly impaired learning ability compared with vehicle-injected C57BL/6 mice, as expected. Surprisingly, TNF deficiency did not affect the ability to learn in either 3- or 9-month-old mice. Interestingly, 9-month-old TNF- KO mice injected with endotoxin showed a significantly impaired learning ability, whereas endotoxin treatment had no effect on learning in C57BL/6 mice. There was no difference in activity level between the two groups. Conclusions: We conclude that the TNF pathway is important for hippo- campal learning when the immune system is activated, as takes place during systemic infection and pathological aging. Studies in progress focus on un- derstanding the homeostatic role of TNF in hippocampal-dependent learning during normal and pathological aging. P4-248 THE ROLE OF TREM2 EXPRESSION ON MYELOID CELLS IN ALZHEIMER’S DISEASE Bruce Lamb 1 , Crystal Miller 1 , Taylor Jay 1 , Leah Graham 2 , Shane Bemiller 1 , Guixiang Xu 1 , Daniel Margevicius 1 , Colleen Karlo 3 , Gregory Sousa 2 , Bunny Cotleur 1 , Lynn Bekris 4 , Susan Staugaitis 1 , James Leverenz 5 , Sanjay W. Pimplikar 6 , Gary Landreth 3 , Gareth R. Howell 2 , Richard Ransohoff 1 , 1 The Cleveland Clinic, Cleveland, Ohio, United States; 2 The Jackson Laboratory, Bar Harbor, Maine, United States; 3 Case Western Reserve University, Cleveland, Ohio, United States; 4 The Cleveland Clinic, Seattle, Washington, United States; 5 The Cleveland Clnic, Cleveland, Ohio, United States; 6 Cleveland Clinic, Cleveland, Ohio, United States. Contact e-mail: lambb@ccf.org Background: Recent genetic studies implicate inflammatory genes and pathways in the etiology of Alzheimer’s disease (AD).Notably, mutations in TREM2, a gene expressed in myeloid cells, were recently demonstrated to confer high risk for developing AD. Mutations in TREM2 have also been identified as the genetic basis of Nasu-Hakola disease, and confer risk for frontotemporal dementia (FTD), Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS). Since microglia are the primary myeloid cell type within brain, it has been proposed that TREM2 likely functions in AD Poster Presentations: P4 P876