In vivo studies confirmed that PARP 1 gene depletion reduces Ab induced microglial activation, and studies in mice expressing human amyloid precursor protein with familial AD mutations showed ameliorated neuronal and behavioral deficits when crossed Tivantinib to PARP 1 mice. These results suggest that PARP 1 inhibition reduces deleterious effects of Ab induced microglial activation. Methods Materials Cell culture reagents were obtained from Cellgro Media tech, unless otherwise stated. Culture plates and 75 cm2 polystyrene culture flasks were from Falcon Becton Dickinson. N N, N dimethylacetamide was obtained from Sigma. 3 2 propenenenitrile Inhibitors,Modulators,Libraries was obtained from Alexis Biochemicals. Amy loid beta 1 42, reverse amyloid beta 42 1, and carboxyfluorescein labeled amyloid beta 1 42, were obtained from Biopeptide Co.
Inc. Primary antibodies used were, rabbit poly clonal anti poly, rabbit polyclonal anti mouse ionized calcium binding adapter molecule 1, rabbit polyclonal anti glial fibrillic acid protein, rabbit polyclonal anti microtubule Inhibitors,Modulators,Libraries associated protein 2, mouse monoclonal anti amyloid b 3D6 and rabbit polyclonal anti Calbindin D 28k. Secondary antibodies used were, anti rabbit IgG conjugated with Alexa Fluor 488 or 594. Mice All animal studies were approved by the San Francisco Veterans Affairs Medical Center animal Inhibitors,Modulators,Libraries studies commit tee and follow NIH guidelines. PARP Inhibitors,Modulators,Libraries 1 mice were derived from the 29S Adprt1tm1Zqw strain, originally developed by Z. Q. Wang, and obtained from Jack son Laboratory. PARP 1 mice used for cell culture studies were backcrossed for over 10 generations with wt CD 1 mice, and wt CD 1 mice were used as their controls.
PARP 1 mice used for in vivo studies and for generating the hAPPJ20 PARP 1 mice were backcrossed to the C57BL 6 strain for over 10 gen erations. The hAPPJ20 mice on the C57BL 6 background were obtained from Dr. Lennart Mucke. These mice express a hAPP minigene with the familial AD linked Swedish and Indiana mutations, under Inhibitors,Modulators,Libraries control of the plate let derived growth factor b chain promoter. The hAPPJ20 mice were crossed with the PARP 1 mice to obtain the breeder genotypes, PARP and hAPPJ20 PARP 1. These were in turn crossed to generate sub sequent generation breeder genotype mice along with the four genotypes of interest, wt, PARP 1, hAPPJ20 and hAPPJ20 PARP 1. Male mice 5 6 months of age were used for in vivo studies.
Genotype was re con firmed on each mouse using tissue obtained at euthanasia. Neuron cultures Neuron cultures were prepared as described Fluoro-Sorafenib previously. In brief, cortices were removed from embryonic day 16 wt mice, dissociated into Eagles minimal essential medium containing 10 mM glucose and supple mented with 10% fetal bovine serum and 2 mM glutamine, and plated on poly D lysine coated 24 well plates at a density of 7 �� 105 cells per well.