Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 2. Knockdown of HAP1 causes a loss of synaptic AMPAR responses. A and B, cumulative distribution plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, HAP1 siRNA, or a scrambled control siRNA. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 3. Dominant negative HAP1 (DN-HAP1) occludes the reducing effect of polyQ-htt on synaptic AMPAR responses. A and B, cumulative distribu- tion plot of the mEPSC amplitude and inter-event interval in cortical neurons transfected with GFP, polyQ-htt, DN-HAP1, or DN-HAP1 plus polyQ-htt. C, representative mEPSC traces. Scale bar: 25 pA, 2 s. D, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Dominant Negative Mutation, Transfection

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 4. The polyQ-htt-induced impairment of synaptic AMPAR responses is occluded by knockdown of KIF5, but not KIF-17. A, representative Western blots in HEK293 cells transfected with FLAG-tagged rat KLC1 in the absence or presence of a control siRNA or a KLC1 siRNA. B–D, representative mEPSC traces (B) and cumulative distribution plot of the mEPSC amplitude (C) and inter-event interval (D) from cortical neurons transfected with control siRNA, polyQ-htt, KLC1 siRNA, or KLC1 siRNA plus polyQ-htt. Scale bar: 25 pA, 1 s. E and F, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA. NS, no significance.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 5. The WT-htt-induced enhancement of synaptic AMPAR responses is blocked by knockdown of KIF5. A–C, representative mEPSC traces (A) and cumulative distribution plot of the mEPSC amplitude (B) and inter-event interval (C) from cortical neurons transfected with control siRNA, WT-htt, KLC1 siRNA, or KLC1 siRNA plus WT-htt. Scale bar: 25 pA, 1 s. D and E, summary data (mean S.E.) of mEPSC amplitude and frequency in cortical neurons with different transfections. *, p 0.05, ANOVA. NS, no significance.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Knockdown, Transfection, Control

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 7. HAP1/GluR2/KIF5/microtubule complex is disrupted in the mouse model of HD. A–C, co-immunoprecipitation assays showing the asso- ciation between tubulin and KIF5 (A), GluR2 and KIF5 (B), tubulin and GluR2 (B), GluR2 and HAP1 (C) from striatal slices of WT versus HD mice (4-month- old). Each experiment was repeated in 3–5 pairs of mice. D, immunoblots and quantification analysis showing the surface and total GluR2 and GluR1 sub- units in lysates of striatal slices taken from WT versus HD mice (4-month-old). *, p 0.01, ANOVA. E, co-immunoprecipitation assays showing the associa- tion between KIF5 and GluR1 or GluR2 in brain lysates.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Impaired α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Trafficking and Function by Mutant Huntingtin

doi: 10.1074/jbc.m111.236521

Figure Lengend Snippet: FIGURE 8. A schematic model demonstrating the potential mechanism for huntingtin regulation of AMPAR trafficking. A, in normal conditions, WT-htt binds to HAP1 and enables the binding of HAP1 to KIF5 motor protein. KIF5 heavy chain interacts with GRIP1/GluR2-containing vesicles, and the wholecomplexisanterogradelytransportedalongthemicrotubule(MT)rails. B, in HD conditions, polyQ-htt interacts abnormally with HAP1 (Step 1), thereby interrupting the binding of GluR2/KIF5 (Step 2), and KIF5/MT (Step 3). Consequently, KIF5 is unable to transport the GluR2 cargo along MTs (Step 4), leading to insufficient synaptic delivery of AMPAR channels.

Article Snippet: Cultured neurons (12–14 DIV) were transfected with various constructs or siRNA using Lipofectamine 2000 method, which included EGFP-tagged N-terminal fragment of huntingtin containing the first 480 amino acids with 17Q (WT-htt) or 68Q (polyQhtt), HAP1 siRNA (15), and KLC1 (Kinesin Light Chain 1) siRNA (Santa Cruz Biotechnology).

Techniques: Binding Assay