F1‐02‐02: Pyroglutamate formation influences the solubility and amyloidogeneity of amyloid peptides

not available. F1-02-03 PYROGLUTAMATE 3 ABETA IMMUNOREACTIVITY IN BRAINS OF MICE, MONKEYS AND HUMANS Cynthia A. Lemere, Eva M. Luo, Jeffrey L. Frost, Roberta M. Palmour, Frank R. Ervin, Keith G. Mansfield, Susan V. Westmoreland, Takaomi C. Saido, Hans-Ulrich Demuth, Brigham & Women’s Hospital; Harvard Medical School, Boston, MA, USA; Behavioral Science Foundation, Eastern Caribbean, St. Kitts & Nevis; New England Regional Primate Research Center, Southboro, MA, USA; RIKEN Brain Science Institute, Wako, Japan; Probiodrug AG, Halle Saale, Germany. Contact e-mail: clemere@rics.bwh.harvard.edu Background: Amyloid beta protein (Aß) starting with pyroglutamate (pE) at the third position (AßN3pE) is a major form of Aß deposition in plaques and vascular amyloid in Alzheimer’s disease (AD). Glutaminyl cyclase (QC) is an enzyme responsible for the cyclization leading to pyroglutamate formation of the glutamate at position 3. Methods: Paraffin brain sections from 12 AD patients (ave. 84 yr), 10 Aged Controls (AC) (ave. 71 yr), 3 Down syndrome (DS) individuals (ave. 49 yr), 22 vervet brains (13-32 yr), 22 tamarin brains (12-21 yr), 17 J20 APP tg mice (3-27 mo), and 13 3xTg-AD mice (7-26 mo) were immunostained with a general Aß polyclonal antibody (R1282) and an AßN3pE monoclonal antibody following formic acid pretreatment and visualized using an ABC Elite kit (Vector Labs) and diaminobenzidine. A polyclonal AßN3pE antibody was used to confirm the mAB immunoreactivity. In addition, human and vervet brain sections were immunostained with a polyclonal antibody against human QC. Results: Intense AßN3pE immunoreactivity (IR) was observed in plaques and blood vessels containing vascular amyloid in cortex and hippocampus in all 12 AD and 3 DS brains, whereas only 7/10 AC brains had any AßN3pE IR, often in lesser quantities. AßN3pE IR was detected in cortical plaques in 7/22 vervets (>15 yr) and 7/22 tamarins (>17 yr) and, in blood vessels in 8/22 vervets (> 13 yr) and 16/22 tamarins (>12 yr). In general, AßN3pE IR colocalized with most R1282-positive plaques and vessels in humans and monkeys. However, in tg mice, AßN3pE IR occurred in a smaller subset of R1282-positive plaques. While plaques were first detected with R1282 by 6 mo in J20 APP tg mice, AßN3pE-IR plaques were first detected at 16 mo and increased with age. R1282and anti-AßN3pE-IR plaques were detected starting at 11 mo in 3xTg-AD mice. QC IR was detected focally in a subset of neurons, often in Layer III of cortex in humans and vervets, but no differences were observed between AD and AC brains. Conclusions: AßN3pE is a major species of plaques and vascular amyloid in humans and non-human primates but less so in AD-like tg mouse models. F1-02-04 INHIBITION OF GLUTAMINYL CYCLASE REDUCES AMYLOID PATHOLOGY IN TRANSGENIC ANIMALS Steffen Rossner, Stephan Schilling, Ulrike Zeitschel, Torsten Hoffmann, Manfred Windisch, Gunter Reuter, Dirk Montag, Hans-Ulrich Demuth, Paul Flechsig Institute for Brain Research, Leipzig, Germany; Probiodrug AG, Halle/S., Germany; JSW CNS Research, Graz, Austria; Institute of Genetics, Halle/S., Germany; Institute for Neurobiology, Magdeburg, Germany. Contact e-mail: rossn@medizin. uni-leipzig.de Background: Extensive data support a causative role of amyloid b (Ab) peptides in the pathophysiology of Alzheimer’s disease (AD). Due to their abundance, stability against proteolytical degradation and rapid aggregation, N-terminally truncated and in particular pyroglutamate (pE)-modified Ab peptides have been suggested to play a pivotal role in the initiation of pathological cascades resulting in the development of AD. The pE modification of Ab is catalyzed by glutaminyl cyclase (QC) in vitro. However, an in vivo proof of concept for the role of QC in amyloidogenesis is still lacking. Methods: We used two different transgenic mouse models of AD, Tg2576 and TASD-41 mice, and chronically inhibited QC activity by oral application of a QC inhibitor via food pellets. After treatment periods for 6 to 10 months, Ab3(pE)-42 and total Ab peptides were quantified by ELISA and amyloid plaque formation and gliosis were monitored histochemically. The performance of mice was tested in context memory and in spatial learning. Additionally, a novel Drosophila model was generated and investigated. Results: Inhibition of QC resulted in reduced Ab3(pE)-42 and total Ab burden, diminished amyloid plaque formation, gliosis and in improved performance in context memory and in spatial learning. Moreover, QC inhibition alleviated Ab3(pE)-42 generation in a new Drosophila model. Conclusions: These results demonstrate that QC is the enzyme catalyzing Ab3(pE)-42 formation in vivo. Furthermore, the observations are consistent with the hypothesis that Ab3(pE)-42 acts as a seed for Ab aggregation by self-aggregation and by co-aggregation of Ab1-40/42. Therefore, Ab3(pE)42 peptides appear to represent Ab forms with exceptional potency to disturb neuronal function. The reduction of brain Ab3(pE)-42 by inhibition of QC offers a novel therapeutic option for the causative treatment of AD. F1-02-05 INTRANEURONAL ABETA TRIGGERS NEURODEGENERATION: NEW EVIDENCE FROM MOUSE MODELS Thomas A. Bayer, University of Göettingen, Department of Psychiatry, Göttingen, Germany. Contact e-mail: tbayer@gwdg.de Background: The concept of the ß-amyloid cascade in Alzheimer’s disease (AD) provides the basis for current therapeutic strategies in AD. It is well established that only a fraction of Aß peptides in the brain of AD patients contain N-terminal aspartate (Aß1D) which is generated by proteolytic processing of amyloid precursor protein (APP). N-terminally truncated and pyroglutamate modified Aß3(pE)-42 have been previously shown to be a major peptide in AD brains. While it is unknown which peptidases remove the first two N-terminal amino acids, the cyclization of Aß with N-terminal glutamate and glutamine can be catalyzed by glutaminyl cyclase (QC) in vitro. Methods: Transgenic mouse models; behavioral tests; neuropathology, neuron count (stereology); electronmicroscopy; ELISA, Western blotting. Results: Here we show that Aß3(pE)-42 induces neurodegeneration and concomitant neurological deficits in transgenic mice. The mouse models are based on neuron specific expression of Aß3E-42 and Aß3Q-42, which were fused to the pre-pro-sequence of murine thyrotropin-releasing hormone. Eight weeks after birth, massive neurological impairments of the mice become apparent, which correlate with Purkinje cell degeneration. The results suggest that intraneuronal Aß3(pE)-42 due to its high stability and aggregation propensity triggers Aß accumulation providing strong evidence that this modified Ab peptide is neurotoxic in vivo. In addition, APP/PS1KI mice develop a robust neuron loss, axonopathy, synaptic deficits and learning deficits correlating with intraneuronal aggregation of AßX-42 peptides. APP/PS1KI mice express human mutant APP751SL, and murine PS1 with two FAD-linked mutations (PS1M233T and PS1L235P). Conclusions: In summary, these observations support the idea for a pivotal role of intraneuronal Aß as a trigger in AD-typical neurodegeneration. F1-02-06 AMYLOIDOSIS AND INFLAMMATION: INDICATIONS FOR A DUAL FUNCTION OF GLUTAMINYL CYLCASE IN ALZHEIMER’S DISEASE Holger Cynis, Stephan Schilling, Hans-Ulrich Demuth, Probiodrug AG, Halle (Saale), Germany. Contact e-mail: holger.cynis@probiodrug.de Background: Pyroglutamate (pGlu)-modified b-amyloid is abundant in Alzheimer’s disease brains and, recently, its appearance was correlated to Oral O1-01: Neuroimaging Progression and Correlation P76 neurological deficits in patients and transgenic animals. N-terminal pGlumodifications are a characteristic feature of several neuropeptides or of chemokines, such as ‘‘monocyte-chemoattractant protein 1’’ (MCP-1, CCL2). In this regard, we have shown, that the zinc-dependent metalloenzyme Glutaminyl Cyclase (QC) is capable of catalyzing pyroglutamyl formation in vivo, with a high preference for the glutaminyl substrates. However, due to the exceptionally high amyloidogenicity and neurotoxicity, we have suggested, that the reduction of pGlu-Ab peptides might be a novel treatment strategy against Alzheimer’s disease. Further, we focused on studying the role of QC in generation of pGlu-CCL2. Methods: The degradation of CCL2 by aminopeptidases and its effect on chemotactic potency and CCR2 receptor activation was studied in vitro using Maldi-TOF MS, chemotaxis assays and FACS analysis. Ab-stimulation of neuronal cells was analyzed using ELISA and qPCR. An application of specific inhibitors was used to study the importance of QC activity on monocyte-recruitment in a model of thioglycollate-induced peritonitis in mice. In addition, gliosis was studied in Tg2576 and TASD41 AD mouse models treated with a QC-specific inhibitor. Results: Here, we present a novel, highly specific approach to inhibit CCL2 maturation and, thus, its inactivation by proteases through the application of the same QC-inhibitors preventing pGlu-Ab formation. QC plays a pivotal role in stabilization of the NTerminus of CCL2. QC-inhibition prevents monocyte-recruitment to inflamed tissues and approaches targeting pGlu-Ab formation show also a reduction in brain inflammation. Conclusions: Based on these experiments, we propose a dual function of QC-inhibitors in Alzheimer’s disease, which might not only reduce the amount of highly toxic pGlu-Ab peptides, but also ameliorate the exaggerated neuroinflammation by reduction of active CCL2. SUNDAY, JULY 12, 2009 ORAL O1-01 NEUROIMAGING PROGRESSION AND CORRELATION O1-01-01 RELATION BETWEEN AMYLOID BURDEN, BRAIN ATROPHY AND MEMORY IN ALZHEIMER’S DISEASE Pierrick Bourgeat, Victor L. Villemagne, Jurgen Fripp, Kerryn E. Pike, Parnesh Raniga, Oscar Acosta, Cassandra Szoeke, Sebastien Ourselin, David Ames, Kathryn A. Ellis, Colin L. Masters, Chris C. Rowe, Olivier Salvado, Australian eHealth Research Centre, CSIRO ICT Centre