Journal: The Journal of Neuroscience

Article Title: Aβ Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease

doi: 10.1523/JNEUROSCI.3501-06.2007

Figure Lengend Snippet: Characterization of ADDL-bound neurons. ADDLs (red) bind to neurons expressing NMDA-R subunits, such as NR1 (A, green) and NR2B (B, green) but not to GABAergic neurons identified as GAD-positive cells (C, green). Cultured hippocampal cells exposed to 500 nm ADDLs for 15 min were fixed and immunolabeled using an oligomer-raised antibody and NNDA-R subunits 1 and 2B or glutamic acid decarboxylase (GAD65/67) antibodies. ADDLs are highly concentrated along dendritic processes rather than on the cell soma, suggesting preferential binding to excitatory synapses. Images are representative of several replicate experiments. Scale bar, 30 μm.

Article Snippet: Aβ 1–40 prepared in DMSO, filtrates, and retentates obtained by 10 and 50 kDa cutoff filters were added to the cell culture at a final concentration of 500 n m . The uncompetitive NMDA receptor (NMDA-R) antagonist memantine (3,5-dimethyl-amantadine hydrochloride) (Sigma, St. Louis, MO) was added to cell culture media, at the final concentration of 5 μ m , 30 min before ADDL or vehicle treatment.

Techniques: Expressing, Cell Culture, Immunolabeling, Binding Assay

Journal: The Journal of Neuroscience

Article Title: Aβ Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease

doi: 10.1523/JNEUROSCI.3501-06.2007

Figure Lengend Snippet: NMDA-R and EphB2 surface expressions are decreased after ADDL exposure. Surface expression of NR1 and NR2B subunits and EphB2 were measured using antibodies against an extracellular epitope at the N-terminal portion of the receptor for immunolabeling under nonpermeabilizing conditions and analyzed using MetaMorph. A, Quantification represents the number of NR1-labeled puncta per length of dendrite (number of NR1 puncta/10 μm dendrite). Patterned bars represent ADDL treatment at the indicated incubation time, and black bar represents vehicle treatment at 3 h. Density of NR1 tends to decrease after AD 1 h (10.1 ± 2.2; n = 11; NS) and highly decrease after AD 3 h (3.3 ± 0.7; n = 11; p = 0.002) compared with controls represented by AD 2 min (14.3 ± 4.8; n = 9) and VEH 3 h (13.1 ± 1.4; n = 9). The follwoing dendritic lengths were measured over the different groups of treatment: AD 2 min, 383 ± 36 μm; AD 1 h, 325 ± 25 μm; AD 3 h, 348 ± 32 μm; VEH 3 h, 252 ± 29 μm. B, A significant decrease in the number of NR2B puncta per field was observed after AD 3 h (p < 0.001; n = 10). The average number of puncta for treated neurons was normalized against the mean value of vehicle-treated ones. A similar decrease was observed after 1 h biotinylated ADDL (bADDLs) treatment. Black bar represents vehicle, and white bar represents ADDL or bADDL. C, A significant decrease in the number of EphB2-labeled puncta per length of dendrite was observed after AD 6 h (p = 0.0005; n = 15), although the difference at 3 h was not significant (p = 0.55). The average number of puncta per length of dendrite for ADDL treated was normalized against the mean value of vehicle treated. Black bar represents vehicle, and white bar represents ADDL. Right panel shows representative images of dendritic branches labeled with NR1 (A), NR2B (B), and EphB2 (C) antibodies at indicated time points of treatment. Scale bar, 8 μm. **p = 0.0005.

Article Snippet: Aβ 1–40 prepared in DMSO, filtrates, and retentates obtained by 10 and 50 kDa cutoff filters were added to the cell culture at a final concentration of 500 n m . The uncompetitive NMDA receptor (NMDA-R) antagonist memantine (3,5-dimethyl-amantadine hydrochloride) (Sigma, St. Louis, MO) was added to cell culture media, at the final concentration of 5 μ m , 30 min before ADDL or vehicle treatment.

Techniques: Expressing, Immunolabeling, Labeling, Incubation

Journal: The Journal of Neuroscience

Article Title: Aβ Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease

doi: 10.1523/JNEUROSCI.3501-06.2007

Figure Lengend Snippet: Memantine prevents ADDL-induced dendritic drebrin loss. A, Confocal microscopy images of drebrin immunofluorescent labeling in 21 DIV cultured rat hippocampal neurons. Grayscale intensities ranging from black to white were converted in a pseudocolor lookup table with increasing values of gray intensity. Black (0) is representing the darkest intensity, and white (255) is the brightest. Intermediary values are as follows: 20, violet; 65, cyan; 80, turquoise; 115, green; 155, yellow; 220, red. As indicated by the arrows, dendrites of neurons treated with ADDLs for 24 h (AD) exhibit decreased drebrin immunofluorescence when compared with the abundance of drebrin hot spots (yellow–red) present in the neurons treated with F-12 vehicle for 24 h (VEH). Whereas neurons treated with memantine 30 min before 24 h ADDL treatment (MEM-AD) exhibit dendritic drebrin immunofluorescence similar to that of vehicle-treated neurons and neurons treated with memantine before 24 h F-12 vehicle treatment (MEM-VH). B, Cells treated for 24 h with 500 nm ADDL (AD) or memantine before ADDLs (MEM-AD) were double labeled for ADDLs using M94 (red) and drebrin (green). Similar distribution of M94-immunoreactive hot spots is observed in both groups of treatment, demonstrating that memantine prevents ADDL-induced drebrin loss but does not alter ADDLs binding to synapses. C, Bar graph illustrating quantification of drebrin immunofluorescence integrated density from confocal image sets as shown in A. Values are normalized to vehicle-treated cells (VEH) for 24 h. Difference between VEH (n = 25) versus AD (n = 31) and AD versus MEM-AD (n = 31) are highly significant (**p < 0.001), whereas VEH versus MEM-AD are comparable (p > 0.05). Memantine by itself did not have any effect on drebrin (MEM-VEH, n = 10). D, Western blotting of hippocampal neuron extracts treated in the same manner as neurons imaged in A. Blots were probed with anti-drebrin, and antibody against cyclophilin was used as control of protein loaded in each lane.

Article Snippet: Aβ 1–40 prepared in DMSO, filtrates, and retentates obtained by 10 and 50 kDa cutoff filters were added to the cell culture at a final concentration of 500 n m . The uncompetitive NMDA receptor (NMDA-R) antagonist memantine (3,5-dimethyl-amantadine hydrochloride) (Sigma, St. Louis, MO) was added to cell culture media, at the final concentration of 5 μ m , 30 min before ADDL or vehicle treatment.

Techniques: Confocal Microscopy, Labeling, Cell Culture, Immunofluorescence, Binding Assay, Western Blot