Huntingtin and huntingtin-associated protein 1 influence neuronal calcium signaling mediated by inositol-(1,4,5) triphosphate receptor type 1

Abstract

Huntington's disease (HD) is caused by polyglutamine expansion (exp) in huntingtin (Htt). The type 1 inositol (1,4,5)-triphosphate receptor (InsP3R1) is an intracellular calcium (Ca2+) release channel that plays an important role in neuronal function. In a yeast two-hybrid screen with the InsP3R1 carboxy terminus, we isolated Htt-associated protein-1A (HAP1A). We show that an InsP3R1-HAP1A-Htt ternary complex is formed in vitro and in vivo. In planar lipid bilayer reconstitution experiments, InsP3R1 activation by InsP3 is sensitized by Httexp, but not by normal Htt. Transfection of full-length Httexp or caspase-resistant Httexp, but not normal Htt, into medium spiny striatal neurons faciliates Ca2+ release in response to threshold concentrations of the selective mGluR1/5 agonist 3,5-DHPG. Our findings identify a novel molecular link between Htt and InsP3R1-mediated neuronal Ca2+ signaling and provide an explanation for the derangement of cytosolic Ca2+ signaling in HD patients and mouse models.

Figure 1. The InsP₃R1 Binds HAP1A and Htt in the Yeast Two-Hybrid System and In Vitro

(A) The strength of interaction between each InsP₃R1 IC bait and clone 11 (Q273–L599 of rat HAP1A) was determined by a liquid Y2H assay. The β-galactosiadase activity is plotted as a percentage relative to the IC/clone 11 interaction (mean ± SE, n = 3). On the top, predicted secondary structure elements within the IC sequence are shown. The predicted minimal fragment of the rat InsP₃R1 carboxy terminus (F2627–G2736) required for association with HAP1A is shaded. The minimal 4.1N binding domain (F2627–R2676) (Maximov et al., 2003) and minimal PP1α binding region (R2731–A2749) (Tang et al., 2003) are also shown. (B) The strength of interaction between full-length rat HAP1A, rat HAP1B, and clone 11 with IC10 InsP₃R1 bait was determined by liquid Y2H assay. Data are presented in percentages relative to the observed interaction between clone 11 and IC10 (mean ± SE, n = 3). The carboxy-terminal regions unique to HAP1A and HAP1B isoforms are depicted by striped boxes. Also shown are the Htt binding region of HAP1 (L277–K370) (Li et al., 1995, 1998b) and motifs identified in HAP1 by computer analysis. (C) GST-IC8/IC10 pull-down experiments of HA-HAP1A or HA-HAP1B from COS7 cells extracts. (D) Lysates from RT1-infected Sf9 cells and COS7 cells overexpressing either HA-HAP1A or HA-HAP1B were mixed for 2 hr and analyzed by immunoprecipitation with anti-InsP₃R1 polyclonal antibodies or corresponding preimmune sera (IP/S). (E) GST-IC8/IC10 pull-down experiments of Htt-23Q or Htt-82Q from HEK293 cells extracts. COS7 lysate containing overexpressed HA-HAP1A was added to GST-IC10 pull-down reactions as indicated. The input lanes on (C)–(E) contain 1/50 of the COS7 and HEK293 cell lysates used in GST pull-down or immunoprecipitation experiments. The precipitated fractions were analyzed by Western blotting with anti-HA monoclonal antibodies (C and D), or anti-Htt monoclonal antibodies (E).

Figure 2. InsP₃R1 Is Associated with Htt In Vivo

(A) Rat cerebellar (left) or cortical (right) lysates were used in GST-IC8/IC10 pull-down experiments. (B) Rat cerebellar (left) or cortical (right) lysates were used in coimmunoprecipitation experiments with anti-InsP₃R1 polyclonal antibodies or corresponding preimmune sera (IP/S). GST-IC8 and GST-IC10 proteins (200 µg/ml final concentration) were included in the immunoprecipitation reactions as indicated. The precipitated fractions on (A) and (B) were analyzed by Western blotting with anti-Htt monoclonal antibodies. (C and D) Cortical lysates from wild-type (left panel) or HAP1^−/− (right panel) mice (at postnatal day 3) were used in immunoprecipitation experiments with the anti-InsP₃R1 polyclonal antibodies or with the corresponding preimmune sera (IP/S). The precipitated proteins were analyzed by Western blotting with the anti-Htt monoclonal antibodies (C) or with the anti-HAP1 monoclonal antibodies (D). An additional 0.5 M KCl wash step was included in the immunoprecipitation protocol as indicated. The input lanes on (A)–(D) contain 1/50 of lysates used in immunoprecipitation and GST pull-down experiments.

Figure 3. The Htt^exp Amino Terminus Sensitizes the InsP₃R1 to InsP₃

(A) Lysates from full-length InsP₃R1 (RT1)-infected Sf9 cells were used in pull-down experiments with GST, Htt-N-15Q, and Htt-N-138Q GST-fusion proteins as indicated. The precipitated fractions were analyzed by Western blotting with anti-InsP₃R1 polyclonal antibodies (B) Effects of GST, Htt-N-15Q, and Htt-N-138Q on activity of recombinant InsP₃R1 in planar lipid bilayers at 100 nM InsP₃. Each current trace corresponds to 10 s (2 s for expanded traces) of current recording from the same experiment. (C) The average InsP₃R1 open probability (P_o) in the presence of 100 nM InsP₃ is calculated for a 5 s window of time and plotted for the duration of an experiment. Data from the same experiment are shown on (B) and (C). Similar results were obtained in four independent experiments. (D) The average InsP₃R1 P_o plot for the experiment performed in the presence of 2 µM InsP₃. Similar results were obtained in two independent experiments. The times of InsP₃, GST, Htt-N-15Q, and Htt-N-138Q additions on (C) and (D) are shown above the P_o plot.

Figure 4. The Htt Amino Terminus Facilitates InsP₃R1 Activity in the Presence of HAP1A

(A) Effects of HAP1A, Htt-N-15Q, and Htt-N-138Q on activity of recombinant InsP₃R1 in planar lipid bilayers in the presence of 100 nM InsP₃. Each current trace corresponds to 10 s (2 s for expanded traces) of current recording from the same experiment. (B) The average InsP₃R1 open probability (P_o) in the presence of 100 nM InsP₃ is calculated for a 5 s window of time and plotted for the duration of an experiment. The times of InsP₃, HAP1A, Htt-N-15Q, and Htt-N-138Q additions to the bilayer are shown above the P_o plot. Data from the same experiment are shown on (A) and (B). Similar results were obtained in two independent experiments or in experiments when HAP1A and Htt-N-15Q (n = 4) or HAP1A and Htt-N-138Q (n = 4) were premixed.

Figure 5. Full-Length Htt^exp Sensitizes InsP₃R1 to Activation by InsP₃

(A) Microsomes from Sf9 cells coinfected with RT1 and Htt-23Q or Htt-82Q baculoviruses were solubilzed in 1% CHAPS, precipitated with anti-InsP₃R1 polyclonal antibodies, and blotted with the anti-Htt monoclonal antibodies. The input lanes contain 1/10 of lysates used in the immunoprecipitation experiments. (B) Activity of InsP₃R1 coexpressed in Sf9 cells with Htt-23Q and reconstituted into planar lipid bilayers. Responses to application of 100 nM InsP₃ and 2 µM InsP₃ are shown. Each current trace corresponds to 10 s (2 s for expanded traces) of current recording from the same experiment. (C) The average InsP₃R1 open probability (P_o) was calculated for a 5 s window of time and plotted for the duration of an experiment. The times of 100 nM and 2 µM InsP₃ additions to the bilayer are shown above the P_o plot. Data from the same experiment are shown on (B) and (C). Similar results were obtained in four independent experiments. (D and E) Same as (B) and (C) for InsP₃R1 coexpressed in Sf9 cells with Htt-82Q. Similar results were obtained in six independent experiments.

Figure 6. Effects of Htt^exp on DHPG-Induced Ca²⁺ Release in Transfected Medium Spiny Neurons

Representative images showing Fura-2 340/380 ratios in transfected rat medium spiny neurons (MSNs). The pseudocolor calibration scale for 340/380 ratios is shown on the right. Ratio recordings are shown for 10 µM DHPG-induced Ca²⁺ transients in MSN neurons transfected with EGFP (first row), EGFP + Htt-23Q (second row), EGFP + Htt-82Q (third row), and EGFP + Htt-138Q (fourth row). The recordings were performed in Ca²⁺-free ACSF containing 100 µM EGTA. GFP images (1st column) were captured before Ca²⁺ imaging to identify transfected cells (arrowheads). 340/380 ratio images are shown for MSN neurons 1 min before (2^nd column), and 8 s, 30 s, 1 min, 2 min, and 3 min after application of 10 µM DHPG as indicated. The GFP-negative cell that responds to 10 µM DHPG in the third row (triangle arrow) is interpreted to correspond to a neuron transfected with Htt-82Q plasmid alone. We used Htt:EGFP plasmids at a 3:1 ratio during transfections, which probably resulted in the expression of Htt in some GFP-negative MSNs. Only GFP-positive MSNs were considered for quantitative analyses shown on Fig 7.

Figure 7. Htt^exp Facilitates DHPG-Induced Ca²⁺ Release in Medium Spiny Neurons

Basal and peak 340/380 ratios are shown for individual MSN neurons transfected with (A), EGFP, (C), EGFP+Htt-23Q, (E), EGFP+Htt-82Q, (G), EGFP+Htt-138Q. The experiments were performed as described in Figure 6. Only GFP-positive MSNs were considered for quantitative analysis for each group of cells. The basal ratios were determined 1 min prior to DHPG application (−1 min). The peak ratios were measured from maximal signals observed within 30 s after DHPG application. 340/380 ratio traces for representative cells (marked *) are shown in (B), (D), (F), and (H). Time of DHPG application is shown. Similar results were obtained in four independent transfections. (I) Summary of MSN Ca²⁺ imaging experiments with Htt and Htt(R) constructs. Average basal and DHPG-evoked peak 340/380 ratios from four independent transfections are shown as mean + SEM (n = number of cells). These averages represent the data shown in this figure in addition to data from other experiments. The peak ratios in MSNs transfected with EGFP + Htt-82Q, EGFP + Htt-138Q, or EGFP + Htt(R)-138Q are significantly (***p < 0.001, paired t test) higher than the basal ratios in the same cells.

Figure 8. Proposed Mechanism Linking Htt^exp to Increased InsP₃R1- and NMDAR-Mediated Neuronal Ca²⁺ Signaling in Medium Spiny Neurons of HD Patients

Glutamate released from corticostriatal projection neurons stimulates NR1A/NR2B NMDAR and mGluR5 receptors on striatal medium spiny neurons (MSNs). (A) In normal MSNs, NMDAR activation triggers Ca²⁺ influx from extracellular sources. Activation of mGluR5 stimulates phospholipase C (PLC) via a heterotrimeric G protein (G) pathway to generate diacylglycerol (DAG) and inositol triphosphate (InsP₃) second messengers. Activation of mGluR5 cooperates with NMDAR activation largely through the actions of DAG, which activates PKC. The InsP₃ generated downstream of mGluR5 does not trigger robust Ca²⁺ release from the endoplasmic reticulum (ER) in response to low levels of glutamate, as InsP₃R1 sensitivity to InsP₃ is low. The association of Htt with the cytosolic carboxy-terminal tail of the InsP₃R1 is weak and with the PSD95-NR1A/NR2B is strong. In this schematic, only the interaction of Htt with the InsP₃R1 carboxy-terminal tail is represented. This interaction appears to be stabilized by the presence of HAP1A (labeled as HAP1) in normal MSNs. (B) In MSNs of Huntington’s disease (HD) patients, Htt^exp perturbs Ca²⁺ signaling via two synergistic mechanisms. Htt^exp enhances NMDAR function, possibly through decreased interaction with the PSD95-NR1A/NR2B complex. In addition, Htt^exp strongly binds to InsP₃R1 carboxy terminus and sensitizes the InsP₃R1 to activation by InsP₃. As a result, low levels of glutamate released from corticostriatal projection neurons lead to supranormal Ca²⁺ influx via NMDAR and Ca²⁺ release via the InsP₃R1. The net result is elevated intracellular Ca²⁺ levels in MSNs that eventually trigger pathogenic Ca²⁺-dependent downstream pathways such as increased caspase and calpain activity, increased Htt proteolysis, activation of the apoptotic program, and MSN degeneration.