- Email: [email protected]
DEVELOPMENT OF A FLUORESCENT PROBE FOR IMAGING PATHOLOGICAL TAU AGGREGATES IN LIVE CELLS
Poster Presentations: Wednesday, July 19, 2017 P4-051
DEVELOPMENT OF A FLUORESCENT PROBE FOR IMAGING PATHOLOGICAL TAU AGGREGATES IN LIVE CELLS
Sungsu Lim, KIST, Seoul, Republic of South Korea. Contact e-mail: [email protected] Background: Neuronal accumulation of tau aggregates is a patho-
logical hallmark in multiple neurodegenerative disorders including Alzheimer’s disease, collectively called tauopathies. Due to the pathological implication, effort has been made in understanding tau aggregation processes. However, progress has been slow due to the lack of a reliable tool to investigate tau aggregation in live cells. A tau aggregation sensor that can monitor abnormal tau aggregation in cells would facilitate the study of tau aggregation processes and the discovery of tau aggregation inhibitors. Methods: Tau aggregation mixture (125 mg/mL in 25 mL PBS) was transferred to a black 384-well plate with 25 mL of PBS containing 2 mM of BODIPY probes. After 10 min, fluorescence intensity was measured at lex ¼ 525 nm and lem ¼ 560-690 nm in a Spectrophotometer. For treatment of the probes to tau aggregation induced cells, HT22 cells or HEK293 tau-BiFC cells were plated in a black transparent 96well plate with or without 40 mM forskolin. After tau aggregation activation, HT22 cells or HEK293 tau-BiFC cells were treated with 300 nM of BODIPY probes for 30 min. The fluorescence responses of the probes were automatically imaged by using OperettaÒ and the fluorescence intensities were analyzed using Harmony 3.1 software. Results: As a fluorescence turn-on sensor, the quantum yield of BD-tau increased 3.8-fold upon binding to tau aggregates in vitro. Compared with other conventional tau probes such as PBB3, ThS and Curcumin, BD-tau has superior selectivity to tau aggregates over beta-sheet protein aggregates. As a cell-permeable imaging probe, BD-tau selectively labeled pathological tau aggregates in live neurons. In comparison, the conventional tau selective probes, ThS, curcumin and PBB3, were not cell-permeable. The live-cell imaging property of BD-tau might be aided by the high cell-permeability property of the BODIPY scaffold. Conclusions: We develope a BODIPY-fluorescence sensor that selectively responds to pathological tau aggregates in live cells.
P4-052
PARTIAL VOLUME EFFECTS AND MEDIAL TEMPORAL LOBE TAU QUANTITATION WITH PET
Suzanne L. Baker1, Samuel N. Lockhart2, Anne Maass2, William J. Jagust1,2, 1Lawrence Berkeley National Laboratory, Berkeley, CA, USA; 2University of California Berkeley, Berkeley, CA, USA. Contact e-mail: [email protected] Background: Because tau accumulation is thought to begin in the en-
torhinal cortex, it is of considerable interest in aging and dementia. However, medial temporal lobe tau measurements may be contaminated by tracer retention in the choroid plexus. We explored partial volume effects (PVE) in the medial temporal lobe to evaluate this issue. Methods: 80 healthy controls (44F/36M, age range ¼ 2094, mean age¼73) received a [18F]AV-1451 (SUVR 80-100), which were coregistered to T1-weighted MRI. The MRIs were segmented using Freesurfer5.3. The inferior portion of the cerebellar gray was used as a reference region. The choroid plexus was subdivided into anterior and posterior portions. Mean SUVR values were calculated for Freesurfer ROIs. Binary ROIs were smoothed by the resolution of the PET data to understand their intermingled PVEs. Rousset partial volume correction (PVC) was applied to the data. To eval-
P1277
uate PVE effects on a biological variable of interest, Spearman correlation was calculated between age and amount of ROI bleed-in from a neighboring ROI, and between age and mean ROI values before and after PVC; we specifically examined entorhinal cortex, hippocampus and the posterior portion of choroid plexus, and ROIs with signal that bled into the entorhinal cortex. Results: 3564% of the entorhinal cortex PET signal comes from itself; other ROIs contributing to the entorhinal cortex PET signal are white matter (1562%), CSF (2764%), fusiform (661%), hippocampus (461%), amygdala (361%), temporal pole (361%), and parahippocampus (261%). Choroid plexus contributes 361% to the signal in the hippocampus. Before PVC, SUVRs and age were correlated in hippocampus (p<0.05), amygdala (p<0.001), and choroid plexus (p<0.01). After PVC age correlations disappeared in right hippocampus and were unchanged in other regions. Also after PVC, additional correlations appeared between age and SUVR in parahippocampus (p<0.05), and entorhinal cortex (p<0.05). Conclusions: PVC slightly reduced age correlations with hippocampus, perhaps related to removal of choroid plexus contributions to the ROI. Partial volume effects also have complex effects on correlations with important biological variables such as age that should be taken into account when interpreting tau data.
P4-053
TAU PATHOLOGY PROPAGATES THROUGH NEURAL NETWORKS IN TAU22 MICE AFTER INJECTION OF SYNTHETIC TRUNCATED TAU AGGREGATES IN DORSAL HIPPOCAMPUS
Veronique Mary1, Marie-Carmen Obinu2, Jean-Marie Miquet2, Stephanie Eyquem2, Irene Mafhouz2, Mati Lopez-Grancha1, Elisabeth Genet1, Veronique Blanchard2, Pascal Chaillou2, Agnes Hubert2, Nadine Michot3, Mireille Meunier3, Valerie Fauchey2, 1Sanofi R&D, Chilly-Mazarin, France; 2 Sanofi R&D, Chilly-Mazarin, France; 3 Sanofi R&D, Vitry-sur-Seine, France. Contact e-mail: veronique. [email protected] Background: Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of tau protein aggregates, for which there is a large unmet medical need for disease-modifying therapies. The concept of transmission and propagation of tau pathology has emerged in the clinic and has been reinforced by studies in cellular and animal models showing that exogenous tau aggregates may trigger the misfolding and aggregation of endogenous tau (seeding) and propagate to interconnected neurons (spreading). Here we characterized such an in vivomodel of tau propagation in TAU22 mice with cognitive, biochemical and histological markers. Methods: Two month old transgenic Tau22 heterozygous male mice (Thy1.2-promotor, 2N4R tau, G272V/P301S) were injected unilaterally in the dorsal hippocampus with mutated-microtubule binding repeat (mut-MTBR) domain tau aggregates (6.75mg/2.5ml) or vehicle. Tau pathology was evaluated 2, 4, 9 and 12 weeks after injection by quantification of AT8 and Gallyas staining in several brain regions by immunohistochemistry. The impact on cognition was evaluated 4 weeks after injection using the object recognition task and tissue tau aggregates were measured in several brain structures using HTRFÒtechnology. Results: Injection of mut-MTBR aggregates in the dorsal hippocampus induced a progressive accumulation of Gallyas and AT8 positive cells in the ipsi- and contralateral CA3/ hippocampus, the ipsilateral entorhinal cortex and the ipsi- and contralateral lateral septum. Four weeks after injection, mice exhibited a significant cognitive deficit compared to
DEVELOPMENT OF A FLUORESCENT PROBE FOR IMAGING PATHOLOGICAL TAU AGGREGATES IN LIVE CELLS
Sungsu Lim, KIST, Seoul, Republic of South Korea. Contact e-mail: [email protected] Background: Neuronal accumulation of tau aggregates is a patho-
logical hallmark in multiple neurodegenerative disorders including Alzheimer’s disease, collectively called tauopathies. Due to the pathological implication, effort has been made in understanding tau aggregation processes. However, progress has been slow due to the lack of a reliable tool to investigate tau aggregation in live cells. A tau aggregation sensor that can monitor abnormal tau aggregation in cells would facilitate the study of tau aggregation processes and the discovery of tau aggregation inhibitors. Methods: Tau aggregation mixture (125 mg/mL in 25 mL PBS) was transferred to a black 384-well plate with 25 mL of PBS containing 2 mM of BODIPY probes. After 10 min, fluorescence intensity was measured at lex ¼ 525 nm and lem ¼ 560-690 nm in a Spectrophotometer. For treatment of the probes to tau aggregation induced cells, HT22 cells or HEK293 tau-BiFC cells were plated in a black transparent 96well plate with or without 40 mM forskolin. After tau aggregation activation, HT22 cells or HEK293 tau-BiFC cells were treated with 300 nM of BODIPY probes for 30 min. The fluorescence responses of the probes were automatically imaged by using OperettaÒ and the fluorescence intensities were analyzed using Harmony 3.1 software. Results: As a fluorescence turn-on sensor, the quantum yield of BD-tau increased 3.8-fold upon binding to tau aggregates in vitro. Compared with other conventional tau probes such as PBB3, ThS and Curcumin, BD-tau has superior selectivity to tau aggregates over beta-sheet protein aggregates. As a cell-permeable imaging probe, BD-tau selectively labeled pathological tau aggregates in live neurons. In comparison, the conventional tau selective probes, ThS, curcumin and PBB3, were not cell-permeable. The live-cell imaging property of BD-tau might be aided by the high cell-permeability property of the BODIPY scaffold. Conclusions: We develope a BODIPY-fluorescence sensor that selectively responds to pathological tau aggregates in live cells.
P4-052
PARTIAL VOLUME EFFECTS AND MEDIAL TEMPORAL LOBE TAU QUANTITATION WITH PET
Suzanne L. Baker1, Samuel N. Lockhart2, Anne Maass2, William J. Jagust1,2, 1Lawrence Berkeley National Laboratory, Berkeley, CA, USA; 2University of California Berkeley, Berkeley, CA, USA. Contact e-mail: [email protected] Background: Because tau accumulation is thought to begin in the en-
torhinal cortex, it is of considerable interest in aging and dementia. However, medial temporal lobe tau measurements may be contaminated by tracer retention in the choroid plexus. We explored partial volume effects (PVE) in the medial temporal lobe to evaluate this issue. Methods: 80 healthy controls (44F/36M, age range ¼ 2094, mean age¼73) received a [18F]AV-1451 (SUVR 80-100), which were coregistered to T1-weighted MRI. The MRIs were segmented using Freesurfer5.3. The inferior portion of the cerebellar gray was used as a reference region. The choroid plexus was subdivided into anterior and posterior portions. Mean SUVR values were calculated for Freesurfer ROIs. Binary ROIs were smoothed by the resolution of the PET data to understand their intermingled PVEs. Rousset partial volume correction (PVC) was applied to the data. To eval-
P1277
uate PVE effects on a biological variable of interest, Spearman correlation was calculated between age and amount of ROI bleed-in from a neighboring ROI, and between age and mean ROI values before and after PVC; we specifically examined entorhinal cortex, hippocampus and the posterior portion of choroid plexus, and ROIs with signal that bled into the entorhinal cortex. Results: 3564% of the entorhinal cortex PET signal comes from itself; other ROIs contributing to the entorhinal cortex PET signal are white matter (1562%), CSF (2764%), fusiform (661%), hippocampus (461%), amygdala (361%), temporal pole (361%), and parahippocampus (261%). Choroid plexus contributes 361% to the signal in the hippocampus. Before PVC, SUVRs and age were correlated in hippocampus (p<0.05), amygdala (p<0.001), and choroid plexus (p<0.01). After PVC age correlations disappeared in right hippocampus and were unchanged in other regions. Also after PVC, additional correlations appeared between age and SUVR in parahippocampus (p<0.05), and entorhinal cortex (p<0.05). Conclusions: PVC slightly reduced age correlations with hippocampus, perhaps related to removal of choroid plexus contributions to the ROI. Partial volume effects also have complex effects on correlations with important biological variables such as age that should be taken into account when interpreting tau data.
P4-053
TAU PATHOLOGY PROPAGATES THROUGH NEURAL NETWORKS IN TAU22 MICE AFTER INJECTION OF SYNTHETIC TRUNCATED TAU AGGREGATES IN DORSAL HIPPOCAMPUS
Veronique Mary1, Marie-Carmen Obinu2, Jean-Marie Miquet2, Stephanie Eyquem2, Irene Mafhouz2, Mati Lopez-Grancha1, Elisabeth Genet1, Veronique Blanchard2, Pascal Chaillou2, Agnes Hubert2, Nadine Michot3, Mireille Meunier3, Valerie Fauchey2, 1Sanofi R&D, Chilly-Mazarin, France; 2 Sanofi R&D, Chilly-Mazarin, France; 3 Sanofi R&D, Vitry-sur-Seine, France. Contact e-mail: veronique. [email protected] Background: Tauopathies are neurodegenerative diseases characterized by the pathological accumulation of tau protein aggregates, for which there is a large unmet medical need for disease-modifying therapies. The concept of transmission and propagation of tau pathology has emerged in the clinic and has been reinforced by studies in cellular and animal models showing that exogenous tau aggregates may trigger the misfolding and aggregation of endogenous tau (seeding) and propagate to interconnected neurons (spreading). Here we characterized such an in vivomodel of tau propagation in TAU22 mice with cognitive, biochemical and histological markers. Methods: Two month old transgenic Tau22 heterozygous male mice (Thy1.2-promotor, 2N4R tau, G272V/P301S) were injected unilaterally in the dorsal hippocampus with mutated-microtubule binding repeat (mut-MTBR) domain tau aggregates (6.75mg/2.5ml) or vehicle. Tau pathology was evaluated 2, 4, 9 and 12 weeks after injection by quantification of AT8 and Gallyas staining in several brain regions by immunohistochemistry. The impact on cognition was evaluated 4 weeks after injection using the object recognition task and tissue tau aggregates were measured in several brain structures using HTRFÒtechnology. Results: Injection of mut-MTBR aggregates in the dorsal hippocampus induced a progressive accumulation of Gallyas and AT8 positive cells in the ipsi- and contralateral CA3/ hippocampus, the ipsilateral entorhinal cortex and the ipsi- and contralateral lateral septum. Four weeks after injection, mice exhibited a significant cognitive deficit compared to