INTRODUCTION Mutations in Presenilin (PS) 1 and 2 cause familial Alzheimer’s Disease (AD) through undetermined mechanisms. PS’s are intramembranous aspartyl proteases capable of cleaving amyloid precursor protein (APP), however their full function in normal and injured cell physiology is not yet known. PS’s catalyze a growing number of identified substrates by regulated intramembranous proteolysis such as the developmental and cell fate regulator, Notch and the tyrosine kinase receptor ErbB4. PS’s are cleaved into C-terminus (CTF) and N-terminus (NTF) fragments to form an active heterodimer enzyme. Inflammation contributes to the neurodegenerative process in a number of human diseases including AD, Parkinson’s Disease, ALS and HIV Associated Dementia (HAD) PS2 knockout mice have normal APP metabolism yet develop age-related mild pulmonary fibrosis a process associated with chronic inflammation in humans. We hypothesize that PS2 participates in the CNS inflammatory process in human neurodegenerative disease. CONCLUSIONS Microglial PS2 is increased in the human neurodegenerative diseases AD and HIV Associated Dementia. HIV glycoprotein gp120, a known activator of microglia, leads to enhanced expression of PS2 protein. Hypotheses PS2 mediated  -secretase activity or the PS2 holoprotein participates in intracellular signaling processes of microglia activation. Inhibition of PS2  -secretase activity or non-canonical PS2 function can interfere with microglial activation. Presenilin 2 is Upregulated During Microglia Activation S. Jayadev, H. Nguyen and G. Garden Neurology, University of Washington, Seattle Microglia from AD brain express enhanced PS2 AD Control AD Ctrl % PS2 Positive Microglia * B) A) PS2 (brown) strongly labels microglia (purple) in AD brain (arrows) whereas microglia in control brain (arrowheads) do not demonstrate similar PS2 immunopositivity The proportion of microglia demonstrating PS2 immunoreactivity is significantly higher ( * = p<.001) in AD brain compared to control. 5 fields each were counted from 4 AD brains and 4 control brains. 45kDa 4. Cultured microglia and microglia cell lines stimulated with the HIV coat protein gp120 show increased PS2 Microglia treated with gp120 demonstrate a dose dependent increase in cell line PS2 expression on western blot (A). Boiled gp120 (control) does not result in increased PS2 expression (B) HIV/gp120 PS2 actin 50kDa 400pM0 boiled Primary Microglia RAW cells BV-2 cells 200pM00 400pM800pM1000pM 200pM +LPS 50kDa 45kDa PS2 antibody : Calbiochem Loop (1:1000) 50 kDa 25 kDa 37 kDa PS 2 20 kDa PS 2 NTF A) actin gp120 Control gp120 B) actin PS 2 CTF gp120 Control gp PS2 holoprotein, NTF and CTF are all increased in response to gp120 stimulation in primary microglia Western blot analysis using antibody to the PS2 N-terminus demonstrates increased PS2 holoprotein and N-terminus after gp120 stimulation of primary microglia (A). Antibody to C-terminus reveals similar enhancement of protein after gp120 treatment (B) When normalized to actin and quantified using Image J densitometry software, PS2 holoprotein is increased by 41%, CTF by 92% PS2 N-terminus : APS 21 AbCam (12  g/ml), PS2 CTF : 2192 Cell Signaling (1:1000) WT mouse PS2 KO Specificity of PS2 antibody using PS2 KO mouse PS2 antibody employed in (A) labels glial cells in wildtype mice but not PS2 KO mouse (tissue gift from Dr. Reuven Stein) 1. PS2 is markedly increased in glial cells in AD brain A) PS2 (brown) immunolabeling is increased in the white matter (co-labeled with microglia marker CD68) of AD brain compared to age- matched control. B) Similar increase in immunopositivity is not seen for PS1 (brown) in AD brain compared to control (D). Both antibodies labeled neuron cell bodies in cortex. Control-PS2Control-PS2 AD-PS2AD-PS2 Control-PS1Control-PS1AD-PS1AD-PS1 Samples are autopsy tissue paraffin embedded slides obtained from Harborview Medical Center Pathology Department. Slides underwent antigen retrieval by microwave heating. PS2 antibody : 2192 Cell Signaling (1:50), PS1 antibody : APS 11 ABR (75  g/ml), CD68 antibody: DAKO (1:750) A) B) PS2 antibody : 2192 Cell Signaling (1:50), CD68 antibody: DAKO (1:750) HAD Control HAD Control 3. Microglia PS2 is increased in HIV Associated Dementia A) Paraffin embedded autopsy cortical tissue from control and HAD colabeled for PS2 (brown) and microglia (purple) demonstrate enhanced PS2 immunoreactivity in HAD brain, particularly in activated microglia (arrows). Ramified microglia with little PS2 immunoreactivity are seen in control (arrowhead). B) PS1 protein (brown, empty arrows) is not markedly increased in HAD compared to control. Samples are autopsy tissue paraffin embedded slides obtained from National NeuroAids Tissue Consortium. Slides underwent antigen retrieval by microwave heating. PS2 antibody : 2192 Cell Signaling (1:50), PS1 antibody : APS 11 ABR (75  g/ml), CD68 antibody: DAKO (1:750) A) B) A)B)