Htt is present in the PSD fraction. (A) Schematic diagram of the procedures used for subcellular fractionation of adult rat forebrains. (B) Representative immunoblot (n = 4) of subcellular fractions (40 µg of total protein) prepared from adult rat forebrains by differential and density-gradient centrifugation. PSD-95 (a postsynaptic density marker), clathrin heavy chain (CHC, a clathrin-coated vesicle marker), Huntingtin (Htt) and Huntingtin-associated protein 1 (HAP1) were detected as described in Materials and Methods. (C) Representative immunoblot (n = 2) of sequential fractions (40 µl), collected beginning from the bottom of the tube, after ultracentrifugation of a PSD fraction through an OptiPrep (Axis-Shield) density gradient (lanes 1–8, from high to low density). PSD-95, Htt and HAP1 were detected as described in Materials and Methods. H, forebrain homogenate; S1, post-nuclear supernatant; P1, nuclear pellet; S2, medium speed supernatant of S1; P2, crude synaptosomal pellet; SS, purified synaptosomal fraction derived from P2; SUP, high-speed supernatant of SS extracted with Triton X-100; PSD, postsynaptic density fraction; S3, cytosol; P3, microsomal pellet; CCV, clathrin-coated vesicles derived from P3.

NF-κB is present in the PSD fraction. (A) Representative immunoblot (n = 4) of subcellular fractions (20 µg of total protein) prepared from a single adult mouse forebrain by differential and density gradient centrifugation. PSD-95 (a postsynaptic marker) and Synaptophysin (a presynaptic marker) were detected as described in Materials and Methods. M, molecular weight marker; H, forebrain homogenate; SS, purified synaptosomal fraction derived from H; PSD, postsynaptic density fraction derived from SS extracted with Triton X-100. (B) Representative immunoblot (n = 4) of subcellular fractions (40 µg of total protein) prepared from a single adult mouse forebrain by differential and density gradient centrifugation. The p65 and p50 subunits of NF-κB, and I-κBα phosphorylated on serine 32 (I-κBα-pS32) were detected as described in Materials and Methods. H, forebrain homogenate; SS, purified synaptosomal fraction derived from H; PSD, postsynaptic density fraction derived from SS extracted with Triton X-100.

Htt interacts preferentially with active NF-κB. (A) Representative immunoblot of proteins immunoprecipitated (IP) from HeLa cell lysates (INPUT, 10% of total input; 40 µg of total protein) by incubation with protein G magnetic beads pre-coated with antibody against Htt (+, lanes 1–2 and 4–5) or with an isotype control antibody (−, lanes 3 and 6). The p65 subunit of NF-κB, total I-κBα, Importin-α2 (Imp-α2) and mouse IgG (IgG H) were detected as described in Materials and Methods. Lanes 2–3 and 5–6 contain precipitates from beads covalently bound to Htt and control antibodies by a cross-linking reagent to enable elution of the immunoprecipitated proteins without contamination by heavy and light chains of IgG. Lanes 1–3 (−TNF-α) contain precipitates from lysates of cells that were not treated TNF-α. Lanes 4–6 (+TNF-α) contain precipitates from lysates of cells that had been treated with TNF-α to activate NF-κB, as described in Materials and Methods. The experiment was performed twice under the conditions shown and once under slightly different detergent conditions with the same result (n = 3). (B) Immunoblot of subcellular fractions (40 µg of total protein) prepared from a single adult mouse forebrain by differential and density gradient centrifugation. Importin-α2 (Imp-α2) was detected in the PSD as described in Materials and Methods and as previously shown by Thompson et al. (51). H, forebrain homogenate; SS, purified synaptosomal fraction derived from H; PSD, postsynaptic density fraction derived from SS extracted with Triton X-100; MW, molecular weight marker. (C) Representative immunoblot of proteins immunoprecipitated (IP) from lysates of HeLa cells that had been treated with TNF-α as described in (B; INPUT, 10% of total input; 40 µg of total protein) by incubation with protein G magnetic beads pre-coated with antibody against Imp-α2 (+) or isotype control antibody (−). The p65 subunit of NF-κB was detected as described in Materials and Methods. The immunoblot was performed twice under the conditions shown and once under slightly different detergent conditions with the same result (n = 3). MW, molecular weight markers.

Both loss of Htt function and the HD mutation reduce the concentration of active NF-κB in neuronal nuclei. Representative immunoblots of active NF-κB purified from neuronal nuclear extracts (100 µg of total protein) by incubation with streptavidin magnetic beads pre-coated with biotinylated double-stranded DNA oligonucleotides containing the κB site (+) or a negative control sequence (−). The p65 subunit of NF-κB and Lamin A/C (nuclear markers) were detected as described in Materials and Methods. Extracts (INPUT, 10% of total input; 10 µg of total protein) were obtained from neuronal nuclear fractions prepared from forebrains of (A) 4-month-old Htt knock-out (KO) and control (CTRL) mice (n = 2 non-sex matched, littermate pairs) and (B) 6-month-old HD knock-in (HD) and wild-type control (WT) mice (n = 2 non-sex matched, littermate pairs). See text for quantification of the immunoblots.

Htt interacts with PSD-95 in the PSD fraction. (A) Representative immunoblot of proteins immunoprecipitated (IP) from a solubilized PSD fraction prepared from adult rat forebrains (INPUT, 5% of total input; 20 µg of total protein) by incubation with protein G magnetic beads pre-coated with an antibody against Htt (+) or with an isotype control antibody (−). Huntingtin (Htt), synGAP and PSD-95 were detected as described in Materials and Methods. The experiment was performed twice under the conditions shown and twice under slightly different detergent conditions with the same result (n = 4). (B) Binding of full-length PSD-95 to the N-terminal portion of Htt corresponding to exon 1, immobilized in a microplate well. Data (n = 3) were obtained by a modified enzyme-linked sorbent assay (ELSA) in which the integrated intensity of the fluorescence emitted by a fluorophore-labeled antibody against PSD-95 is measured with a fluorescence microplate reader.

The HD mutation impairs localization of Htt in the PSD fraction. (A) Mean amount of indicated proteins in PSD fractions (40 µg of total protein) prepared from forebrains of 6-month-old (6 mo) HD knock-in (HD) mice, plotted as percentage relative to wild-type control (WT) mice (n = 4 non-sex matched, littermate pairs). Protein quantitation was performed by immunoblotting as described in Materials and Methods. Representative blots are shown below the graph. Error bars represent SEM. *P < 0.05, **P < 0.01; two-tailed paired t-test. (B) Same as (A) but using 2-year-old (2 yr) mice (n = 4 non-sex matched, littermate pairs).

Both loss of Htt function and the HD mutation slow the rate of movement of NF-κB out of activated dendritic spines. (A) Dendritic segment of a cultured cortical pyramidal neuron (15 days in vitro) that was transiently transfected with mCherry (red channel, left) and with photoactivatable green fluorescent protein fused to the p65 subunit of NF-κB (paGFP–p65; green channel, right), as described in Materials and Methods. Circles indicate dendritic spines in which paGFP–p65 was photoactivated by targeted two-photon laser radiation. The green channel on the right shows the fluorescence of paGFP–p65 before (top) and after (bottom) photoactivation. (B) Time course of paGFP–p65 fluorescence after photoactivation in single dendritic spines of live cortical pyramidal neurons derived from Htt knock-out (red dots, KO) and control (yellow dots, CTRL) mice, in a representative time-lapse experiment. Decay of paGFP–p65 fluorescence due to photobleaching (black dots, BL) was measured as the time course of paGFP–p65 fluorescence after photoactivation in single dendritic spines of the same neurons after chemical fixation. paGFP–p65 fluorescence is plotted over time (t) as percent of fluorescence at the time of photoactivation (F/F₀). (C) Mean half-life (t_½) of paGFP–p65 fluorescence decay (fitted with a single exponential and corrected for photobleaching) after photoactivation in single dendritic spines (n = 3 non-sex matched, littermate pairs [21/59 neurons/spines]). (D and E) Same as (B) and (C), except that neurons were prepared from HD knock-in (HD) and wild-type control (WT) mice (n = 5 non-sex matched, littermate pairs [32/93 neurons/spines]). Error bars represent SEM. *P < 0.05, **P < 0.01; two-tailed paired t-test.