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

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Analysis of mRNA that encode store-operated calcium entry (SOCE) proteins in day in vitro 21 (DIV21) medium spiny neuron (MSN) cultures from YAC128 and wildtype mice. (A) Gene expression analysis of mRNA that encode SOCE proteins and proteins that are involved in Ca 2+ homeostasis in MSNs from wildtype (control) and YAC128 mice using real-time polymerase chain reaction (PCR). The gene expression results for all genes were normalized to Gapdh . Data from wildtype MSNs were normalized to 1. (B) Relative mRNA levels of genes that encode SOCE proteins, measured as 2 ∧ -dCt. (C) Immunoblots of HTT-associated protein-1 (HAP1) normalized to GAPDH and CacyBP/SIP (both monomers and dimers) normalized to Vinculin in YAC128 and wildtype MSN cultures. On the bottom of each panel, the expression levels of HAP1 and CacyBP/SIP proteins are plotted. The results are expressed as mean ± SEM and represent data from three independent mRNA and protein preparations of three different MSN cultures. ns, not significant.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: In Vitro, Expressing, Real-time Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Parameters of Ca 2+ homeostasis in YAC128 MSNs that overexpress HAP1A. (A) Immunoblots of HAP1 and green fluorescent protein (GFP) in YAC128 MSN cultures that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP and pLenti-GFP. (B) GFP fluorescence that indicates the overexpression of HAP1A-pLenti-GFP and HAP1B-pLenti-GFP and pLenti-GFP in YAC128 MSNs. (C) Protocol to induce SOCE in YAC128 MSNs that overexpressed HAP1 isoforms using the eight-well system. (D) Protocol to induce SOCE in wildtype MSNs that overexpressed HAP1 isoforms. Cultures of MSNs on DIV14 from YAC128 and wildtype mice (control) that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP, or pLenti-GFP using lentiviruses were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the endoplasmic reticulum (ER) was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (E) Ca 2+ release from the ER in YAC128 MSNs that overexpressed HAP1 isoforms. (F) Ca 2+ release from the ER in wildtype MSNs that overexpressed HAP1 isoforms. (G) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed HAP1 isoforms. (H) Ca 2+ influx via DHPG-induced SOCE in wildtype MSNs that overexpressed HAP1 isoforms. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of at least three independent MSN culture preparations are shown. * p < 0.05, *** p < 0.001. ns, not significant.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Western Blot, Fluorescence, Over Expression, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Parameters of Ca 2+ homeostasis in YAC128 MSNs with silencing of HAP1A. (A) Immunoblots of HAP1 and beta-actin in YAC128 MSN cultures that overexpressed short-hairpin RNA (shRNA) against HAP1. shRNAs against HAP1 are named a, b, c, d, control scrambled shRNA, and control MSN cultures as indicated on the blot. (B) Protocol to induce SOCE in YAC128 MSNs with silencing of HAP1 using the eight-well system. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed different shRNAs against HAP1 in pLenti-GFP were loaded with the Ca 2+ indicator Fura-2AM and incubated in Ca 2+ -free medium (0.1 mM EGTA). Ca 2+ release from the ER was induced by 20 μM DHPG. SOCE was activated by the addition of 2 mM Ca 2+ to the medium. KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (C) Ca 2+ release from the ER in YAC128 MSNs that overexpressed different shRNAs against HAP1. (D) Ca 2+ influx via DHPG-induced SOCE in YAC128 MSNs that overexpressed different shRNAs against HAP1. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. The results of three independent MSN culture preparations are shown. *** p < 0.001.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Western Blot, shRNA, Incubation

Journal: Frontiers in Cellular Neuroscience

Article Title: Huntingtin-Associated Protein 1A Regulates Store-Operated Calcium Entry in Medium Spiny Neurons From Transgenic YAC128 Mice, a Model of Huntington’s Disease

doi: 10.3389/fncel.2018.00381

Figure Lengend Snippet: Effect of HAP1A on ER Ca 2+ content in YAC128 MSNs. Cultures of MSNs on DIV14 from YAC128 mice that overexpressed HAP1A-pLenti-GFP, HAP1B-pLenti-GFP, or pLenti-GFP using lentiviruses that were loaded with the Ca 2+ indicator Fura-2AM. (A) The figure shows the protocol to induce ionomycin-induced Ca 2+ release from the ER in YAC128 MSNs that overexpressed HAP1 isoforms using the eight-well system. YAC128 MSNs that overexpressed HAP1A isoforms were incubated in Ca 2 -free medium (0.1 mM EGTA), followed by 15 μM ionomycin application to induce the release of Ca 2+ . KCl (56 nM) in 2 mM Ca 2+ was applied to distinguish neurons from glial cells. (B) Ionomycin-induced ER Ca 2+ release in YAC128 MSNs that overexpressed HAP1 isoforms. The results are expressed as mean ± SEM. The number of cells is shown on the top of the bars. * p < 0.05. ns, not significant. The results were obtained from three independent MSN culture preparations.

Article Snippet: HAP1A or HAP1B mouse cDNA clones in pCMV6-ENTRY that originated from Origene (catalog no. NM010404 or NM177981) were cloned between the Sgf I and Mlu I sites into pLenti-C-mGFP (catalog no. PS100071, Origene) for HAP1 overexpression.

Techniques: Incubation