Differential Levels and Phosphorylation of Type 1 Inositol 1,4,5-Trisphosphate Receptor in Four Different Murine Models of Huntington Disease

Abstract

Background:

The intracellular ion channel type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1) releases Ca²⁺ from the endoplasmic reticulum upon stimulation with IP₃. Perturbation of IP₃R1 has been implicated in the development of several neurodegenerative disorders, including Huntington disease (HD).

Objective:

To elucidate the putative role of IP₃R1 phosphorylation in HD, we investigated IP₃R1 levels and protein phosphorylation state in the striatum, hippocampus and cerebellum of four murine HD models.

Methods:

Quantitative immunoblotting with antibodies to IP₃R1 protein and its phosphorylated serines 1589 and 1755 was applied to brain homogenates from R6/1 mice to study early-onset aggressive HD. To determine if IP₃R1 changes precede overt pathology, we immunostained tissues from the regions of interest and several control regions for IP₃R1 in tgHD_CAG51n rats and BACHD and zQ175DNKI mice, all recognized models for late-onset HD.

Results:

R6/1 mice had reduced total IP₃R1 immunoreactivity, variably reduced serine1755-phosphorylation in all regions investigated, and reduced serine1589-phosphorylation in cerebellum. IP₃R1 levels were decreased relative to cell-specific marker proteins. In tgHD_CAG51n rats we found reduced IP₃R1 levels in the cerebellum, but otherwise unchanged IP₃R1 phosphorylation and protein levels. In BACHD and zQ175DNKI mice only age-dependent decline of IP₃R1 was observed.

Conclusion:

The level and phosphorylation of IP₃R1 is reduced to a variable degree in the different HD models relative to control, indicating that earlier findings in more aggressive exon 1-truncated HD models may not be replicated in models with higher construct validity. Further analysis of possible coupling of reduced IP₃R1 levels with development of neuropathological responses and cell-specific degeneration is warranted.

Keywords

Cerebellum Huntington disease neurodegeneration phosphorylation transgenic model

INTRODUCTION

The inositol 1,4,5-trisphosphate (IP₃) receptor is a ubiquitously expressed ligand-gated intracellular Ca²⁺ channel situated in endoplasmic reticulum (ER) membranes (for reviews, see [1 –3]). Three isoforms from different genes have been characterised, and an alternatively spliced long form of the type 1 IP₃ receptor (IP₃R1) is predominantly expressed in neurons, particularly in cerebellar Purkinje cells [2]. Between the N-terminal IP₃-binding domain and the C-terminal Ca²⁺ channel-containing domain, this large transmembrane protein of ∼2750 amino acids has a regulatory part, containing sites for phosphorylation by several kinases, as well as binding and interaction sites for a vast number of regulatory ions, proteins and other molecules [4]. A number of reports suggests a role for the IP₃R1 in a range of neurodegenerative conditions: Disruption of Ca²⁺ signalling and altered levels of IP₃ receptor protein have been reported in several neuropathological conditions, including Alzheimer disease [5 –7], ischaemic/hypoxic damage [8 –10], Parkinson disease [11], Huntington disease (see below) and several cerebellar ataxias [12, 13]. Mechanisms by which the IP₃R1 may mediate neurodegeneration are summarized by Banerjee and Hasan [14].

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder which primarily affects GABAergic medium-sized spiny neurons (MSNs) in the striatum, but also affects connected brain regions including other parts of the basal ganglia, the cerebral cortex, thalamus, the hippocampal formation and cerebellum [15]. A polyglutamine expansion (CAG^exp) in the huntingtin protein (HTT) has been shown to induce death in striatal cells [16], but the molecular link between CAG^exp and selective neuronal death remains elusive. Disturbances of the IP₃R1 in HD pathology were first suggested by [³H]IP₃ binding studies in HD patients, with significantly reduced binding of [³H]IP₃ in striatal tissue [17, 18]. We have previously reported selectively reduced levels of the IP₃R1 protein in a pharmacological rat model for HD, in which striatal neurons were destroyed by the excitotoxic agent quinolinic acid (QA) [19]. Furthermore, IP₃R1 has been shown to interact with mutant huntingtin (mHTT) and the huntingtin-associated protein HAP1 [20], and injection of a viral vector mediating expression of a C-terminal IP₃R1 fragment into brains of HD transgenic mice was shown to protect neurons against cell death and alleviate symptoms [16, 21]. Further work from the same group has recently shown that binding of mHTT to IP₃R1 mediates sensitization to IP₃, increased Ca²⁺ release from the ER and as a result, overstimulated store-operated calcium release leading to neuronal cell degeneration both in corticostriatal co-cultures and in HD model mice [22]. Another proposed mechanism whereby IP₃R1 may be involved in HD pathology, is through irreversible allosteric inhibition of channel properties induced by the enzyme transglutaminase 2 (TTG2; [23]). In addition, dysfunction of the IP₃R1 has been shown to promote cell death during ER stress, and its interaction with the ER chaperone GRP78 was impaired in the brain of HD model mice, pointing towards the IP₃R1 as central in ER stress-induced brain damage [24]. For the calcium-dependent phosphatase 2B (PP2B, calcineurin; EC 3.1.3.16), which also regulates the activity of the IP₃R1, altered levels were recently suggested to participate in excitotoxicity and in HD pathology [25].

Cell types with high IP₃R1 expression include MSNs of the striatum, pyramidal neurons of the hippocampus and some other cortical areas and cerebellar Purkinje cells [26, 27]. These regions are important in several neurodegenerative diseases, including HD. A selective loss of IP₃R1 expression is seen following striatal lesions induced by injection of QA in rats [19], and disturbed IP₃R1-mediated Ca²⁺ signalling has been associated with apoptosis of striatal MSNs in genetic mouse models (HAP1-/-; YAC128) of HD [2 , 29]. IP₃R1 was therefore suggested as a potential therapeutic target in HD[21, 28]. Gene expression profiling in a transgenic rat model of HD revealed a considerable number of Ca²⁺-regulatory proteins to be affected age-dependently [30], and also in HD transgenic mice, the calcium-regulated proteins calmodulin kinase II and IV and nitric oxide synthase showed age-dependent alteration [31]. This suggests that transgenic HD models may provide important insights into the dynamics of the disease processes.

cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of the IP₃R1, which occurs on two main phosphorylation sites (Ser1589 and Ser1755) [26, 32], also increases the Ca²⁺ transport through the channel by increasing its sensitivity to IP₃ [33]. This represents an interesting interaction point between two major second messenger systems. In striatal MSNs, activation of D1 dopamine receptors results in Ca²⁺ oscillations due to interactions of IP₃- and cAMP-mediated signalling pathways, i.e., by PKA-mediated phosphorylation of IP₃R1 [34]. Conversely, dephosphorylation of IP₃R1 may be mediated by protein phosphatase 1α (PP1α) [33] and PP2A [35], both of which are found to interact with IP₃R1 in macromolecular signalling complexes. Calcium-dependent dephosphorylation by calcineurin has also been shown to affect IP₃R1 activity [36, 37]. Differential phosphorylation of IP₃R1 (variations in regional and subcellular distribution) is found in normal rat brain hippocampus and cortex, and in pathological conditions (ischaemia/excitotoxicity), the subcellular distribution of phosphorylated and non-phosphorylated IP₃R1 is changed both in rat and human brain [9]. Also, members of the B-cell lymphoma 2 (BCL-2) gene family that control apoptosis, regulate IP₃R1 phosphorylation on Ser1755 in order to control the rate of Ca²⁺ leak from intracellular stores [38]. Taken together, the present evidence indicates that both regulation of expression levels and phosphorylation of IP₃R1 may affect intracellular calcium ion homeostasis in both physiologyand pathology.

We here investigated possible HD-induced effects on IP₃R1 expression levels and phosphorylation state at cyclic nucleotide-regulated serines in relevant brain regions in transgenic rodent models of HD. To maximise the chance of detecting neuropathological changes, we investigated a model with pronounced early disease manifestations: the R6/1 mouse model, which reflects early onset HD with considerable manifestations of neuropathology [39, 40], also in the cerebellum [31, 41]. However, since our previous study of Alzheimer patients suggested an involvement of IP₃R1 down-regulation in early neurodegeneration [6, 19], we also wanted to identify changes in IP₃R1 levels and phosphorylation occurring before development of fulminant HD. For this purpose we chose a transgenic rat model which exhibits more subtle late-onset HD-like symptoms and neurodegenerative changes in aged animals [42 –44], as well as BACHD and zQ175 mice, which are also recognized as models for late onset HD [45, 46].

We tested three hypotheses: (1) that IP₃R1 levels would be lower in HD animals as a sign of selective degradation in HD pathology, and that IP₃R1 protein levels would be reduced in a late-onset model, where HD pathology is modest; (2) that the cyclic nucleotide-mediated phosphorylation state of the IP₃R1 in HD model animals would be different compared to their wild-type littermates, as an indicator of disturbed intracellular Ca²⁺ handling in HD; and (3) that regional differences in protein expression could be identified in brain areas with high IP₃R1 levels, such as the striatum, hippocampus and cerebellum.

We report significantly reduced IP₃R1 expression levels and variably reduced phosphorylation in the striatum, hippocampus, and cerebellum of R6/1 mice, and reduced IP₃R1 expression in the cerebellum of transgenic HD rats. BACHD mice show only minor differences and zQ175DNKI knock-in mice show no differences in phosphorylation but an age dependent decline in IP₃R1. The study of IP₃R1 and its phosphorylation across several animal models of HD suggests only minor effect of IP₃R1 regulation on pathophysiology and progression of HD.

MATERIALS AND METHODS

Ethics statement, animal husbandry, and sample isolation

All animal work was performed in accordance with guidelines of the Norwegian or German Animal Welfare Act and current regulations. All research and animal care procedures were approved either by the local institutional animal welfare committee at the University of Oslo, Norway or authorized by the district government of Lower Franconia, Würzburg, Bavaria, Germany.

All mice were kept under standard laboratory conditions, with a 12 hours light/12 hours dark cycle and access to food and tap water ad libitum.

Animals

R6/1 mice

B6CBA-Tg(HDexon1)61pb/J mice of the R6/1 line [39] with ∼119 CAG repeats in exon 1 of the HTT gene and their wild type littermates (described in [40]) were a kind gift from Dr. Linda Møllersen (Dep. Microbiology, Oslo University Hospital). At 19 weeks of age, 14 male and female mice (20–25 g body weight) were sacrificed by cervical dislocation, after which the cerebella, striata and hippocampi from both hemispheres were carefully isolated and immersed in liquid nitrogen. Samples were stored at –80°C until homogenisation.

BACHD mice

The BACHD mice derive from a FvB/NJ background and were generated by injection of a bacterial artificial chromosome-based construct into a founder oocyte leading to an overexpression of an additional full-length human mutant huntingtin (mHTT) gene with 97 CAA/CAG repeats in exon1 under endogenous regulatory elements. BACHD mice develop progressive motor and cognitive deficits starting at 2 months and show late-onset neuropathology at around 12 months of age [45]. For the present studies, BACHD mice were backcrossed on C57BL6 N background (International strain information: Tg(HTT*97Q)IXwy/J_C57BL/6N).

zQ175 mice

The zQ175DN knock-in mouse model was bred under a C57B/l6J background and derived from spontaneous expansion of the CAG repeat region in a CAG140 knock-in colony. This model expresses full-length chimeric mouse/human HTT exon1 containing 188 CAG repeats under regulation of the endogenous mouse Htt promoter. zQ175 mice demonstrate early-onset behavioral impairment as well as decreased striatal markers at beginning of 4 months [47]. Those mice were then genetically reengineered/improved [48]. These mice (B6J.129S1-Htttm1.1Mfc/190ChdiJ – also known as zQ175 neo-deleted knock-in, i.e., zQ175DN KI) were then backcrossed to C57BL6N (F7) in our laboratory (International code: B6J.129S1-Htttm1.1Mfc/190ChdiJ_C57BL/6N) in order to reduce the incidence of porto-caval-shunts in this mouse model of HD.

For this study, we used brains of wildtype and heterozygous BACHD and zQ175 mice at the age of 12, 43, (zQ175) and 86 weeks (BACHD). Mice were anaesthetized with isoflurane. As soon as no toe pinch reflex was observable, transcardiac perfusion with ice-cold perfusion buffer was performed for 2 min. The whole brain was removed and further processed. For immunohistochemistry, brains were fixed in 4 % paraformaldehyde in 0.1 M phosphate buffer for 24 h, equilibrated in 30 % sucrose for additional 48–72 h and snap-frozen in –70°C isopentane for 50 sec. Cryosections were cut in 40 μm sections along the coronal axis on a microtome at –21°C (Leica CM3050S Research Cryostat) and stored in anti-freezing solution (0.1 M PBS, supplemented with 13.3 mM NaH₂PO₄, 38.5 mM Na₂HPO₄, 876.4 mM sucrose, 90 mM polyvinylpyrrolidone and 4.8 M ethylene glycol) at –20°C until further usage.

Rats

TgHDCAG51n rats

12 male transgenic HD rats carrying a truncated huntingtin cDNA fragment with 51 CAG repeats under the control of the native rat huntingtin promoter [44, 49], and 12 wild-type littermates were bred and genotyped at the Franz–Penzoldt Centre, Experimental Therapy, Friedrich–Alexander University of Erlangen–Nuremberg, Germany. This transgenic rat line was originally derived from a Sprague-Dawley (SD) founder oocyte (Max Delbrück Center, Berlin-Buch, Germany; MDC). This model is a so-called “fragment model” carrying a transgene spanning exon 1 to exon 15 and expressing 727 amino acids of the HD gene with 51 CAG repeats (cDNA position 324–2321, corresponding to 22% of full length) [49]. At 17 months of age (well beyond the age at which HD rats are known to display HD-like symptoms [43, 50], rats (250–300 g body weight) were anaesthetised by intraperitoneal injection with 120 mg kg^–1 sodium pentobarbital, and transcardially perfused with 0.9% wt vol^–1 saline (37°C), followed by freshly depolymerised 4% wt vol^–1 paraformaldehyde (PFA; 37°C) in 0.1 M sodium phosphate buffer (PB; pH 7.4), and finally, 10% wt vol^–1 sucrose (4°C). After perfusion, brains were isolated and cryoprotected by immersion in 30% wt vol^–1 sucrose in PB at 4°C until no longer buoyant. They were then rapidly frozen with 30% wt vol^–1 sucrose in PB onto the stage of a freezing microtome (Microm HM450), and 50 μm sections were cut in the coronal plane at –25°C. Sections were managed free-floating, collected individually and kept in a solution containing 0.1% wt vol^–1 PFA in 0.1 M PB at 4°C until labelling procedures.

Production and affinity-purification of antibodies against holoprotein and phospho-specific type 1 inositol 1,4,5-trisphosphate receptor

See Table 1 for a list of all primary antibodies used in this study. Antibodies against the invariant 18-amino acid carboxy-terminal of the IP₃R1 were raised in the Neurochemical Laboratory at the University of Oslo [51]. The antiserum was affinity-purified by means of a BSA-coupled Affi-Gel column (Bio-Rad) onto which the immunising C-terminal peptide was immobilised, and shows highly specific immunoreactivity against the IP₃ receptor (Fig. 1A, 5A, D).

Fig.1

Specificity of anti-IP₃R1 antibodies and in vitro detection of IP₃R1 phosphorylation. (A) Western blot of proteins from rat cerebellar membrane preparations incubated with ATP in the presence of protein kinase A (left lane), protein kinase A succeeded by antarctic phosphatase (middle lane), and in the absence of kinase/phosphatase (right lane). The antibody does not discriminate between phosphorylated and unphosphorylated IP₃R1. (B) Autoradiogram of dried polyacrylamide gel from the experiment in (A) with samples applied in the same order. Incorporation of [γ-³²P]ATP in the 260 kDa-area of the left lane is confirmed, as is dephosphorylation in the middle lane. Faint radiolabelling in the right lane may indicate either autophosphorylation by the IP₃R1 or be the result of endogenous protein kinase activity in the sample. (C,D) Western blots showing that affinity-purified antibodies against the phosphorylated Ser1589 (C) in cerebellar membrane preparations specifically recognise the phosphorylated form of the IP₃R1 (left lane) but not the unphosphorylated form (right lane), whereas in duplicated, respective lanes incubated with anti-phospho-Ser1755 (D), the immunoreactivity developed within few seconds was too strong to show phospho-antibody specificity. This effect was not overcome by further dilution (not shown). (E,F) Dotblot analyses showing specificity of the phospho-specific antibodies for detection of synthetic peptides corresponding to phosphorylated and unphosphorylated Ser1589 (E) and Ser1755 (F), dotted onto a nitrocellulose membrane in different concentrations (a) 800 pmol, (b) 80 pmol and (c) 8 pmol, using bovine serum albumin as negative control (d). Visualization by enhanced chemiluminescence shows that the affinity-purified sera have 100–1,000 times greater affinity for the phosphorylated peptides (PP1589 and PP1755) than for the non-phosphorylated peptides (NP1589 and NP1755). (G,H) Dotblot analyses where anti-Ser1589 (G) and anti-Ser1755 (H) were applied to both phosphopeptides in concentrations (a) 1500 pmol, (b) 150 pmol and (c) 15 pmol, using bovine serum albumin as negative control (d), showed no cross reaction between the antisera.

Table 1

Antibodies used

Antibody Name	Immunogen Structure	Manufacturer
Anti-IP₃R1^18C	Synthetic peptide of the 18 C-terminal aa in rat IP₃R1,	Characterised in Magnusson 1993, Haug 1996, 1998, 1999, Dahl 2000.
	²⁷³¹Igllghpphmnvnpqqpc²⁷⁴⁸ coupled to KLH through an added N-terminal cysteine	Rabbit. Polyclonal, affinity purified
Anti-Ser1589P	Synthetic peptide of 12 aa encompassing the rat IP₃R1^Ser1589P, ¹⁵⁸⁴AARRDSpVLAASC¹⁵⁹⁴ coupled to KLH through an added N-terminal cysteine	Synthetic peptide (The Biotechnology Centre, Oslo). Immunisations and bleedings carried out at the Department of Comparative Medicine at the Institute of Basic Medical Sciences, University of Oslo. Further preparative procedures undertaken in our laboratory.
		Rabbit. Polyclonal, affinity purified
Anti-Ser1755P	Synthetic peptide of 12 aa encompassing the rat IP₃R1^Ser1755P, ¹⁷⁵⁰SGRRESpLTSFGC¹⁷⁶⁰ coupled to KLH through an added N-terminal cysteine	Synthetic peptide (The Biotechnology Centre, Oslo). Immunisations and bleedings carried out at the Department of Comparative Medicine at the Institute of Basic Medical Sciences, University of Oslo. Further preparative procedures undertaken in our laboratory.
		Rabbit. Polyclonal, affinity purified, dialysed
Anti-DARPP-32	Synthetic peptide (CQVEMIRRRRPTPAM) coupled to KLH	Millipore, catalog no. AB1656. Rabbit. Polyclonal, affinity-purified.
Anti-calbindin-D28k	Full length purified cow native calbindin	Abcam, catalog no. ab66185. Mouse. Monoclonal
Anti-parvalbumin	Purified native protein from frog muscle	Millipore, catalog no. MAB1572. Mouse. Monoclonal
Anti-synaptophysin	Cat retina synaptosome	Sigma, catalog no. S 5768. Mouse. Monoclonal, clone SVP-38
Anti-mGluR1/5	Fusion protein of rat mGluR5b (aa 824–1203)	NeuroMab. Mouse. Monoclonal, clone N75/33

KLH, keyhole limpet haemocyanin; IP₃R1, type 1 inositol 1,4,5-trisphosphate receptor; Ser1589P, phosphorylated serine 1589 of the IP₃R1; Ser1755P, phosphorylated serine 1755 of the IP₃R1; DARPP-32, dopamine- and cAMP-regulated neuronal phosphoprotein, M_r 32,000.

New Zealand White rabbits were immunised with synthetic 12-amino acid peptides encompassing the phosphorylated PKA- and PKG (cGMP-dependent protein kinase, EC 2.7.11.12)-specific phosphosites Ser1589 and Ser1755 of IP₃R1 (¹⁵⁸⁴AARRDSpVLAASC¹⁵⁹⁴ and ¹⁷⁵⁰SGRREpSLTSFGC¹⁷⁶⁰, respectively, abbreviated PP1589 and PP1755). Peptides were coupled to keyhole limpet haemocyanine (KLH) [52] through an amino-terminal cysteine by m-maleimidobenzoyl-N-hydroxysuccimide ester (MBS) [52, 53]. To achieve highly specific antibodies recognising phosphorylated Ser1589 and Ser1755 (Fig. 1C–H), crude sera were affinity-purified using a SulfoLink column (Thermo Scientific Inc.) coupled with the phosphopeptide in question. Immunodepletion of crossreacting anti–non-phosphoserine IgG was achieved using columns with the corresponding non-phosphorylated peptides (named NP1589 and NP1755). Eluted fractions were pooled, and antibodies were stored at –80°C until use.

Antibody-specificity control experiments

Microsomal membrane fractions were prepared from rat cerebella as previously described, but with the addition of 4% vol vol^–1 Complete Protease Inhibitor Cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride (PMSF) for protease inhibition [6, 51]. Deoxycholate (DOC)-extracted protein [51] was used to evaluate the specificity of the affinity-purified antibodies (Fig. 1). Microsomal membrane proteins (∼200 μg) were phosphorylated with PKA and γ[³²P]-ATP as described [9, 32], and dephosphorylated using alkaline phosphatase (EC 3.1.3.1; Antarctic phosphatase, New England BioLabs) (final concentration, 3.2 U μL^–1) [9].

Samples containing phosphorylated and dephosphorylated microsomal protein were resolved by SDS-PAGE. Radiolabelled protein was cut out of the gel, stained with Coomassie Brilliant Blue, destained, dried and exposed to phosphoimaging using Storage Phosphor Low-Energy Screens in a Typhoon 9210 Variable Mode Imager (Fig. 1B).

Proteins from the rest of the gel (containing non-radioactive proteins) were electrotransferred to nitrocellulose membranes (Bio-Rad), blocked and rinsed before incubation with a 1:5,000 dilution of anti-IP₃R1^18C, anti-Ser1589P, or anti-Ser1755P (the latter diluted 1:10,000) antibodies and subsequent incubation with a 1:50,000 dilution of horseradish peroxidase (HRP)-conjugated goat–anti-rabbit IgG secondary antibody. Blots were incubated with Super Signal West Pico (Thermo Scientific) for enhanced chemiluminescence (ECL) and immunocomplexes were visualised by exposure to X-ray film (Fig. 1A, C, D).

Specificity of the phosphospecific antibodies was tested using synthetic phospho- and nonphospho-peptides dotted onto nitrocellulose membranes (immuno-dotblot analyses). Membranes were fixated with 1% wt vol^–1 glutaraldehyde, blocked with 5 % wt vol^–1 skimmed milk powder in Tris-buffered saline (pH 7.4) with 0.05 % vol vol^–1 Tween-20 (TBS-T) and subsequently incubated in a 1:5,000 dilution of antibody. Following rinsing and incubation with secondary antibody and ECL reagents as described above, antibody binding to phosphopeptide and non-phosphopeptide was visualised by exposure to X-ray film (Fig. 1E-H).

Quantitative immunoblotting of brain proteins from HD and wild-type mice

Isolated mouse cerebella, striata and hippocampi were thawed and homogenised as described above, followed by careful sonication. Nuclei were pelleted by centrifugation at 1,000×g for 10 min and discarded. Supernatants were treated with DOC (final concentration, 1 % wt vol^–1) on ice for 30 min before centrifugation at 27,000×g for 30 min, to yield a fraction containing both cytosolic and DOC-extractable membrane proteins. Protein concentration in the final supernatant was determined by a dye-binding assay (Bio-Rad).

Samples were dissolved in SDS application buffer and proteins were further denatured at 96°C for 2 min. Equal amounts of DOC–extracted protein (cerebellum, 30 μg; striatum, 50 or 100 μg; hippocampus, 100 μg) were then loaded in each well of 4–20 % gradient gels (Bio-Rad) and subjected to SDS-PAGE. Proteins were electrotransferred to nitrocellulose or PVDF membranes. Irrelevant epitopes were blocked with blocking buffer (5 % wt vol^–1 skimmed milk powder or BSA in TBS-T) before incubation in primary antibodies, then rinsed in TBS-T and incubated with peroxidase-conjugated secondary antibodies in the concentration recommended by the supplier. Following final rinsing in TBS-T, proteins were visualised with Super Signal West Pico and exposure to X-ray film, or with Transilluminator followed by analysis with Image Lab Software. Films were scanned using an Epson Perfection V750 Pro scanner, and immunoreactivity was quantitated by X-ray film densitometry from resulting TIFF-images in Adobe Photoshop CS6. A ratio between values of all normalization lanes was calculated and used as factor to exclude inter-blot differences, and the integrated optical density (IOD) is presented as a percentage of wild-type mean IOD. To ensure quantifiable immunobands with immunoreactivity signals within the linear range of the IOD curve, titration curves using microsomal protein from mouse cerebellum, striatum and hippocampus were made to determine the concentrations of total protein and antibody dilutions needed [6]. All immunoblotting experiments were conducted2–4 times.

Single immunoperoxidase labelling of IP₃R1 in HD and wild type rats

For immunohistochemical labelling, we employed a modified version of the ABC method of Hsu [54]. Sections were rinsed in 0.1 M sodium phosphate buffer (PB; pH 7.4) before free aldehyde groups were blocked by incubation in 1 M ethanolamine in PB for 30 min. Following repeated rinsing in PB, endogenous peroxidase activity was halted by incubation in 1 % wt vol^–1 H₂O₂ in phosphate-buffered saline (PBS, pH 7.4). Irrelevant epitopes were blocked by incubation in a buffer containing 0.1 M NaCl, 0.1 M Tris-HCl (pH 7.4), 0.1 % vol vol^–1 Triton X-100, 10 % vol vol^–1 newborn calf serum (NCS) and 0.1 % wt vol^–1 NaN₃. Sections were then incubated with the appropriate primary antibodies, all diluted in blocking solution. Primary antibody was omitted in negative controls. Sections were then rinsed in a buffer containing 0.1 M NaCl, 0.1 M Tris-HCl (pH 7.4), 0.1 % vol vol^–1 Triton X-100 and 10 % vol vol^–1 NCS before they were treated with biotinylated monkey–anti-rabbit secondary antibody (RPN1004 V, GE Healthcare), followed by an incubation in biotinylated streptavidin–horseradish peroxidase (HRP) complex (RPN1051, GE Healthcare), both diluted 0.01 % vol vol^–1 in 0.1 M NaCl, 0.1 M Tris-HCl (pH 7.4), 0.1 % vol vol^–1 Triton X-100 and 1 % vol vol^–1 NCS. After repeated rinsing in PBS with 0.02 % vol vol^–1 Triton X-100, sections were incubated in 0.5 mg mL^–1 3,3’-diaminobenzidine (DAB) in PB substrate solution before the addition of H₂O₂ to a final concentration of 0.01 % wt vol^–1. Peroxidase reactions were terminated by immersing sections in PBS. Duration of peroxidase reactions was individually optimised for the different antibodies. Sections were mounted onto microscopic slides and coverslipped with glycerol–gelatine (Millipore).

Blocking experiment

Brain slices were treated as described above. Prior to incubation with anti-IP₃R-antibody, the antibody was blocked with a 100-fold concentration of extracts of rat cerebellum for 3 hours at room temperature under constant movement.

Quantification of immunostaining in brain slices from HD rats

High-resolution images of histological sections were acquired using an automated slide scanner system (Mirax Scan, Carl Zeiss Microscopy). Images were inspected using the Panoramic Viewer software (3DHISTECH). MRXS-files were exported as TIFFs with 1:4 JPEG lossy compression before saving as 50 ppi RGB images with lossless data compression onto a standard-sized canvas in Adobe Photoshop CS6. All further image processing was performed in Adobe Photoshop CS6. Quantitative analysis was only performed on images of similarly processed sections. Images were first sorted according to anterioposterior stereotactic coordinates (i.e., distance from bregma levels defined in the atlas of [55]), ensuring that comparable brain areas were sampled in each group. Next, images from different brain areas were superimposed onto respective template sections, then stacked and meticulously aligned and adjusted relative to anatomical landmarks in the template, ensuring overlap of regions of interest (ROIs). Rectangular ROIs of fixed size were then systematically isolated from each image stack on the basis of anatomical landmarks (cerebellum, 1450×180 μm; dorsal striatum, 1935×1210 μm; CA1 of the hippocampal formation, 1290×340 μm). ROI images were converted to 16-bit black and white images before they were inverted to their black and white negatives, and the integrated optical density representing section immunoreactivity was calculated as the product of image size (i.e., the number of pixels in ROI) and the intensity of signal within this area (i.e., the distribution of graytones on a continous black-to-white scale), analogous to our quantifications of immunoband immunoreactivity described for western blotting above. DAB immunoreactivity in rat histological sections was expressed as percentages of the wild-type mean in order to ensure interexperimental comparability.

Quantification of immunostaining of brain slices from BACHD and zQ175 mice

Images were taken with a Keyence BZ9000 Generation II microscope and processed with Adobe Photoshop CC 2015.1.2. Brain slices stained with anti-IP₃R1-antibody from three wildtype versus three transgenic animals for each mouse model and age stage were additionally quantified with FIJI freeware. Rectangular ROIs of a fixed size were analysed in the subventricular zone, piriform cortex and olfactory tubercle. Measured greyvalues were normalised to greyvalues obtained in the cingulate cortex, since staining was most uniform in this region and therefore suitable as internal control. This guarantees that the obtained results are unaffected by staining effects possibly evoked by varying storage time or alterations in perfusion performances.

Data analyses and statistics

All data analyses were carried out using the GraphPad Prism software. For the R6/1 mouse immunoblotting analyses, a non-parametric Mann–Whitney U test was performed for comparisons between HD- and wild-type mice. For the rat immunostaining analyses, where HD- and wild-type sections were analysed in pairs, we used a two-tailed paired Student’s t-test assuming unequal variances. The significance level was set at p < 0.05. Immunohistochemical quantification of slices from BACHD and zQ175 mice was analysed using two-way-ANOVA (genotype:age) with multiple comparison. Fisher’s least significant difference (LSD) pre-planned test was used to quantify statistically significant differences between experimental groups.

RESULTS

Antibody specificity

Antibodies against the C-terminal part of the IP₃R1 are well characterised [6 , 51]. Western blot analyses of antibody specificity showed that affinity-purified anti-IP₃R1^18C does not discriminate between the phosphorylated and the unphosphorylated forms of the IP₃R1 (Fig. 1A,B), while binding of affinity-purified antiserum against Ser1589P is abolished following dephosphorylation (Fig. 1C). Western blots using anti-Ser1755P showed that the affinity-purified antiserum was extremely potent, since even short development times resulted in a strong signal, accompanied by several weak additional bands, mostly representing IP₃R1 breakdown products (Fig. 1D). Also, dephosphorylation of P-Ser1755 was ineffective in these in vitro conditions.

Further characterisation using dotblot analyses showed that both affinity-purified phospho-specific antisera have a 100–1,000 times greater affinity for their respective phosphopeptides (PP1589, PP1755) compared to their non-phosphorylated peptides (NP1589, NP1755), without cross reaction (Fig. 1E,F). Similar dotblot analyses excluded cross-reaction of anti-Ser1589P against NP/PP1755 peptides and of anti-Ser1755P against NP/PP1589 peptides (Fig. 1G,H and data not shown). We thus conclude that the chosen antibodies (anti-IP₃R1^18C, anti- IP₃R1^Ser1589P and anti- IP₃R1^Ser1755P) provided reasonably specific information about total IP₃R1 levels and Ser1589-/Ser1755-phosphorylated IP₃R1.

Decreased levels of IP₃R1 protein in striatum, cerebellum and hippocampus of R6/1 mice

IP₃ receptor protein levels and relevant marker proteins from the striatum of the HD (R6/1) mice were compared to wild-type siblings using quantitative western blots.

Striatum

Both the level of total IP₃R1 protein and the level of IP₃R1 phosphorylated on Ser1755 were significantly reduced in the R6/1 mice, to 16 % (p < 0.01) and 20 % (p < 0.05) of wild-type mean, respectively (Fig. 2A,B; Table 2). When Ser1755-phosphorylation was related to total IP₃R1 protein levels, there was a non-significant increase in the HD group (Fig. 2A–C; Table 3). Phosphorylation on Ser1589 of IP₃R1 was not reliably detected in the striatal samples.

Fig.2

Regional comparison of protein levels in R6/1 HD and wild-type mice. Western blot quantification of protein immunoreactivity in deoxycholate-extracted microsomal protein samples from mouse striatum (A–F), cerebellum (G–L) and hippocampus (M–O). Immunoreactivity bands were related to interblot actin controls, and values expressed as a percentage of control group means for each antibody (mean±SD). Black bars represent wild-type control mice (n = 6) and red bars represent R6/1 transgenic HD mice (n = 6). Left column shows representative western blots of total and phosphorylated IP₃R1 and region specific reference proteins. Graphs in the middle and right columns show absolute and relative levels of total IP₃R1, phosphorylated IP₃R1 and reference proteins. (F,L) To visualise tendency to IP₃R1 downregulation, IP₃R1 levels are shown relative to reference proteins (F, DARPP-32; L, CALB1), displayed as column bars and scatter dot plot. Asterisks indicate statistically significant differences from wild-type controls: *p < 0.05, **p < 0.01 (Mann–Whitney U-test). CALB1, calbindin-D28k; DARPP-32, dopamine- and cAMP-regulated neuronal phosphoprotein; HD, Huntington disease; IP₃R1, type 1 IP₃ receptor; mGluR1/5, metabotropic glutamate receptor 1; PVALB, parvalbumin; SYP, synaptophysin; WT, wild-type.

Table 2

Protein immunoreactivity in R6/1 HD mouse brains compared to wild-type

Brain Region	Protein/epitope	Immunoreactivity (IOD)
Cerebellum (n = 6/group)	IP₃R1^18C	50.8±17.1 % **
	Ser1589P	55.9±49.2 % (p = 0.0931)
	Ser1755P	22.4±18.1 % **
	mGluR1/5	66.9±25.4 % *
	CALB1	62.2±12.9 % **
	PVALB	18.7±17.2 % **
	SYP	114.4±19.0 %
Striatum (n = 6/group)	IP₃R1^18C	16.3±21.5 % **
	Ser1755P	19.7±31.9 % *
	DARPP-32	5.5±5.4 % *
	mGluR1/5	105.1±104.4 %
	CALB1	109.8±78.9 %
	PVALB	18.8±15.7 % **
	SYP	94.9±59.7 %
Hippocampus (n = 6 WT, 5 HD)	IP₃R1^18C	44.5±22.4 % *
	Ser1755P	39.8±31.2 % (p = 0.0519)
	SYP	99.8±23.1 %

IOD is presented as percentage of wild-type mean (100%)±SD and analysed using Mann-Whitney U-test as described in Materials and Methods. *p < 0.05; **p < 0.01. WT, wild-type; CALB1, calbindin D_28k, PVALB, parvalbumin; SYP, synaptophysin.

Table 3

Relative immunoreactivity of R6/1 HD mouse brains compared to wild-type

Brain Region	Protein/epitope	Immunoreactivity (IOD)
Cerebellum (n = 6/group)	Ser1589P/IP₃R1^18C	138.6±113.8 %
	Ser1755P/IP₃R1^18C	49.6±27.1 % *
	IP₃R1^18C /CALB1	71.4±31.8 %
Striatum (n = 6/group)	Ser1755P/IP₃R1^18C	161.1±216.0 %
	IP₃R1^18C /DARPP-32	41.6±102.0 % (p = 0.0649)
	IP₃R1^18C /PVALB	27.3±34.9 % (p = 0.0931)
	DARPP-32/PVALB	35.5±64.8 %
Hippocampus (n = 6 WT, 5 HD)	Ser1755P/IP₃R1^18C	86.3±23.7 %

OD is presented as percentage of wild-type mean (100%)±SD and analysed using Mann-Whitney U-test as described in Materials and methods. *p < 0.05; **p < 0.01. WT, wild-type.

Analysis of relevant striatal marker proteins in the striatum of HD mice showed very low levels of the MSN marker protein dopamine- and cAMP-regulated neuronal phosphoprotein, M_r 32,000 (DARPP-32) (5.5 % of wild-type mean; p < 0.05) [56] and the interneuron-specific Ca²⁺-buffering protein parvalbumin was reduced to 19 % (p < 0.01;Table 2) [57]. However, the levels of the synaptic membrane marker protein group I metabotropic glutamate receptor (mGluR1/5), the calcium-binding protein calbindin D_28k and the synaptic vesicle marker synaptophysin [58] were unchanged (Fig. 2D, E; Table 2). Hence, the population of DARPP32-containing MSNs was particularly sensitive in the HD mice, compared to other striatal neurons including local interneurons [19, 59]. Analysis of relative differences in levels of IP₃R1 compared to parvalbumin and DARPP-32 indicated a tendency of selective reduction in the IP₃R1 levels, albeit not significant (Fig. 2C, F; Table 3).

Cerebellum

The level of total IP₃R1 protein was reduced to 51 % of wild-type mean (p < 0.01), and phosphorylation state was reduced to a similar extent on both Ser1589 and Ser1755 (Fig. 2G, H; Table 2). In contrast to the Ser1589 phosphorylation site, phosphorylation of Ser1755 was significantly reduced to 50 % also when corrected for the reduced levels of total IP₃R1 protein in the cerebellar samples from HD mice (p < 0.05; Fig. 2I; Table 3). Furthermore, the Purkinje cell marker protein calbindin D_28k was decreased to 62% (p < 0.01) and parvalbumin to 19% (p < 0.01; Fig. 2J, K; Table 2). When the amount of total IP₃ receptor protein was normalised to the level of calbindin D_28k, the IP₃R1 level in R6/1 mice was 71±32 % of wild-type mean (not significant; Fig. 2L; Table 3). Finally, the synaptic membrane marker protein mGluR1/5 was reduced by ∼30 % in the cerebellum samples (p < 0.05), whereas the levels of the synaptic vesicle marker synaptophysin were unchanged (Fig. 2J, K; Table 2). Thus, whereas a small decrease of postsynaptic membrane was possible, the number of presynaptic vesicles appeared not to be significantly disturbed [58].

Hippocampus

The mean level of total IP₃ receptor protein in R6/1 mice was 45 % of wild-type levels (p < 0.05; Fig. 2M, N; Table 2), and Ser1755-phosphorylated IP₃R1 was reduced to a similar extent (to 40 %; p = 0.052; Fig. 2O; Table 2). Phosphorylation of Ser1589 was not detected in the hippocampal samples. When synaptophysin was analysed in these samples, no differences were found (Fig. 2M, N; Table 2). This was similar to the data from the other brain areas, indicating a stable presynaptic density throughout all areas in the R6/1 mouse brains [58].

Levels of IP₃R1 protein in striatum of zQ175 mice

Quantitative western blotting performed in this model at two ages, revealed no statistically significant differences between transgenic and wild-type mice, although there was a trend towards increased immunoreactivity of anti-Ser1755 in young animals (Fig. 3A, B).

Fig.3

Comparison of IP₃R1 protein levels in striatum of zQ175 and wild-type mice. Western blot quantification of protein immunoreactivity in striatal samples from zQ175 and wild-type mice aged 12 and 43 weeks, was performed as described in Materials and Methods. Graphs show normalized volume intensity for each age and genotype. Total IP₃R1 is compared using the IP₃R1^18C antibody (A), phosphorylated IP₃R1 is compared using the IP₃R1^Ser1755P antibody (B). Tg, transgenic; wt, wild-type.

Immunohistochemical staining of sections from zQ175 and BACHD mice

Blocking of primary anti-IP₃R1-antibody with rat cerebellum extract was successful for both models since no specific immunoreactivity could be detected in brain sections stained with blocked antibody (Fig. 4A,B). Rat cerebellum extract was used because IP₃R1 protein is one of the most abundant proteins in rat cerebellar tissue, estimated to 0.4 % of total protein [60]. Blocking of phosphospecific antibodies showed clear blocking effect for the 1755P antibody (data not shown), but was unsuccessful for the 1589P antibody, as this antibody showed dominant high affinity for endothelia (Fig. 5B,E). To specifically exclude cross reaction with other brain proteins, blocking of antibodies with the corresponding synthetic phosphopeptides would have been desirable, however, these were not available in the necessary concentrations for IHC.

Fig.4

IP₃R1 immunoreactivity in striatum of zQ175 and BACHD mice. Selected representative microphotographs from brain slices sampled from corresponding locations and stained with anti-IP₃R1^18C -antibody. Left column (A,C,E): Sections from zQ175 mice; right column (B,D,F): Sections from BACHD mice. Top row (A,B): overview pictures of each hemisphere (scale bars, 500 μm) with inserted enlarged sections of striatum (scale bars, 50 μm). Second row (C,D): Rectangular ROIs of a fixed size were analyzed in the subventricular zone (SVZ), olfactory tubercle (olf Tub) and piriform cortex (pir Cor). Scale bars, 50 μm in the large images and 25 μm in the higher magnification inserts. Lower row (E,F): Measured greyvalues were normalized to greyvalues obtained in the cingulate cortex, since staining was most uniform in this region and therefore suitable as internal control. Results are given as mean + standard error of the mean (SEM) from three wildtype versus three transgenic animals. *p < 0.05.

Fig.5

IP₃R1 immunoreactivity in cerebellum of tgHD and wild-type rats. Selected representative microphotographs from immunolabelled coronal histological sections sampled from corresponding locations in the 4th cerebellar lobe of wild-type (WT) and transgenic HD rats. Left column (A,D): Immunolabeling with anti-IP₃R1^18C; middle column (B,E): IP₃R1 phosphorylated on Ser1589; right column (C,F): IP₃R1 phosphorylated on Ser1755. Lower row (G-I): Bar graphs showing quantification of immunolabelling in a region-of-interest, with results in HD rats presented as percent of wild-type mean integrated optical density (IOD)±SD, compared using Students t-test. Arrowheads indicate zones with little or no IP₃R1 labelling; *p < 0.05. Scale bar, 500 μm.

Both zQ175 and BACHD derived brain sections (n = 3 for each genotype and age stage, respectively) presented an ubiquitous immunoreactivity for anti-IP₃R1-antibody (Fig. 4A,B). Clear differences in staining intensity could be detected between ages in both of these models independent of genotype. 12-week-old zQ175 mice showed higher immunoreactivity compared to 43-week-old littermates (Fig. 4A). Most interestingly, BACHD mice displayed the complete opposite reaction with less staining intensity in the young group (Fig. 4B). Age differences were also present in certain subregions of the brain: The subventricular zone (SVZ), the piriform cortex (pir Cor) and the olfactory tubercle (olf Tub) (Fig. 4C,D). Though the visual impression may be different, in the olf Tub of 12-week-old BACHD mice image analysis revealed higher immunoreactivity compared to 86-week-old animals. These regions were quantified and normalized to the cingulate cortex (Fig. 4E,F). Surprisingly, quantification revealed no significant differences in neither region of zQ175 brain slices. BACHD mice, however, showed a trend towards age related decrease of IP₃R1 in SVZ and olf Tub region with a significant difference in transgenic animals in the olf Tub (p = 0.0235), thereby supporting obtained results for R6/1 animals.

Amount of IP₃R1 in transgenic HD Rats

Immunolabelling of histological sections sampled from corresponding locations in the striatum, hippocampus and cerebellum of transgenic HD rats and wild-type controls showed distinct IP₃R1^18C labelling of striatal MSNs and hippocampal pyramidal neurons (Fig. 6), and cerebellar Purkinje cell somata and dendrites (Fig. 5). In the cerebellum, we further observed narrow parasagittal bands with reduced IP₃R1^18C labelling in both wild-type and HD rats, but with visibly lower amounts of labelling in HD rats (Fig. 5A, D), possibly indicating loss of Purkinje cells in HD rats. Comparison of the mean amounts of total IP₃R1 labelling showed significantly reduced levels in HD rats relative to wild-type controls (79.1±25.4 %, p < 0.05; Fig. 5G; Table 4). No differences in total levels of IP₃R1 were observed in the striatum or hippocampus (Fig. 6; Table 4). The amount of IP₃R1 phosphorylated on Ser1589 or Ser1755 was not statistically significant between HD and wild-type rats in any of the regions investigated (Figs. 5 and 6; Table 4).

Fig.6

IP₃R1 immunoreactivity in striatum and hippocampus of tgHD and wild-type rats. Selected representative microphotographs from immunolabelled coronal histological sections sampled from corresponding locations in the dorsal striatum (top section) and hippocampus (bottom section) of wild-type (WT) and transgenic HD (HD) rats. Bar graphs show quantification of immunolabelling in a region of interest (ROI; defined as explained in Materials and methods), with results in HD rats presented as percent of wild-type mean integrated optical density±SD, compared using Students t-test. Left column (A,D,G,J,M,P): Immunolabeling with anti-IP₃R1^18C; Middle column (B,E,H,K,N,Q): IP₃R1 phosphorylated on Ser1589; Right column (C,F,I,L,O,R): IP₃R1 phosphorylated on Ser1755. Frames indicate positions of enlargements superimposed to the left of each image. No qualitative or quantitative differences in amount of total and phosphorylated IP₃R1was found. Scale bars, 500 μm.

Table 4

DAB staining intensity in wild type and HD rat brains

Brain Region	Protein/epitope	Immunoreactivity (IOD)
Cerebellum	IP₃R1^18C	79.1±25.4 % (n = 10) *
	Ser1589P	98.7±12.9 % (n = 9)
	Ser1755P	99.0±12.2 % (n = 10)
Striatum	IP₃R1^18C	95.2±16.1 % (n = 9)
	Ser1589P	100.8±11.4 % (n = 9)
	Ser1755P	94.7±14.7 % (n = 7)
Hippocampus (CA1)	IP₃R1^18C	104.5±19.6 % (n = 10)
	Ser1589P	100.8±14.1 % (n = 12)
	Ser1755P	102.4±13.2 % (n = 11)

OD is presented as percentage of wild-type mean (100%)±SD and analysed using 2-tailed paired t-test as described in Materials and methods. *p < 0.05. n, number of HD & wild-type rat pairs analysed per antibody.

DISCUSSION

We have studied IP₃R1 levels and phosphorylation state in several murine HD models using quantitative immuoblotting and immunohistochemical staining. We observed significant and selective reductions in type 1 IP₃ receptor protein levels in the R6/1 mouse model compared to wildtypes, in all brain areas examined by western immunoblotting (Fig. 2). Similar reduction was not shown when western immunoblotting was performed in zQ175 mice at two different ages, where IP₃ R levels tended to be increased in transgenic animals (Fig. 3A).

Immunohistochemistry revealed small differences between the investigated models: In the zQ175 mouse model no particular differences in IP₃R1 protein levels were detected, whereas in BACHD mice we found an age-dependent decrease in the olfactory tubercle. In the rat HD model, which displays a less severe disease phenotype, we demonstrate a small decrease in IP₃R1 immunoreactivity in the cerebellum.

IP₃R1 immunoreactivity levels in the striatum of the R6/1 mice (Fig. 2A, B) were significantly reduced, and the levels of the MSN marker protein DARPP-32 [61] (Fig. 2D, E) were reduced correspondingly, in accordance with earlier reports indicating loss of striatal neurons in QA-injected rats [19, 59]. Whereas loss of MSNs is not substantial in R6/1 mice [62], as opposed to in the pharmacological QA model, both DARPP-32 (Fig. 2D–F) and parvalbumin levels (Fig. 2C, E) were significantly decreased in the R6/1 striatum at 19 weeks of age, and both IP₃R1 levels and DARPP-32 levels tended to be decreased upon normalisation to parvalbumin. In a recent study of YAC128 transgenic HD mice, in which DARPP-32 was used as the only marker for MSN in corticostriatal cocultures, IP₃R1 was demonstrated as a key player in neuronal loss: Sensitisation of IP₃R1 induced by binding of mHTT lead to depletion of ER Ca²⁺ followed by store-operated calcium entry and synaptic instability [22]. And even more recently, single cell analysis provided evidence for this mHTT/HAP1/IP₃R1-complexing: HAP1A was identified to build up a protein complex with mHTT to elicit Ca²⁺ release from ER [63]. Binding of mHTT to the C-terminal part of IP₃R1 may compete with the IP₃R1 antibody used in our study, providing an alternative explanation for the observed reduction of IP₃R1 levels in R6/1 rodent brain which we cannot exclude. Inhibition of IP₃R1 function in HD models can also be caused by transglutaminase 2-mediated posttranslational modification of Gln2746, resulting in locking of subunit configuration [23].

A common denominator for our immunoblot analyses is the indications of a stable presynaptic density in all studied brain areas, as expressed by the unchanged levels of synaptophysin in HD (R6/1) mouse brains, combined with a variable reduction in postsynaptic membrane protein markers. The hippocampal samples from R6/1 mice show reduced levels of IP₃R1 immunoreactivity in spite of normal synaptophysin levels (Fig. 2M, N). This data is similar to the findings induced by administration of QA [19], and indicates different responses to pathogenetic stimulations between the IP₃R1-containing ER membranes and the presynaptic vesicles. Also, the responses to IP₃R1 sensitisation, ER-depletion and calcium dysregulation in hippocampal areas differ compared to striatal neurons [22]. The role of mHTT binding to the IP₃R1 was not addressed in our study. While the reduction in Ser1755-phosphorylation in this brain area could be caused by the reduced IP₃R1 protein levels as such (Fig. 2O), the pattern of phosphorylation of the IP₃R1 in the hippocampus is similar to that seen in the striatum and distinct from that seen in the cerebellum.

Altered Ca²⁺ signalling is likely to be an early feature in the mHTT-initiated cascade later leading to full HD-like neurodegeneration in the HD rat model, as suggested by gene expression analyses revealing that at 3 and 12 months of age, expression levels of many genes involved in Ca²⁺ signalling, synaptic long term potentiation pathways and HD signalling were changed [30].

Immunostaining using phospho-specific antibodies must be interpreted with caution since blocking experiments were suboptimal, with some blocking effect for the 1755P-antibody but unsuccessful for the 1589P-antibody, due to high affinity for endothelia (Fig. 5B,E). In zQ175 mice and BACHD mice, subtle variation of Ser1755-phosphorylation was seen, the significance of which we consider uncertain (not shown). In the HD transgenic rat model, with relatively modest neuropathology [42 , 64], immunohistochemical staining suggested a relative increase in phosphorylation of the IP₃R1 protein for both phosphorylation sites (Fig. 5). This raises the possibility of different regulatory mechanisms being active in the phosphorylation systems involved in these different models.

Most brain areas except cerebellum showed phosphorylation only of Ser1755 and not Ser1589, whereas phosphorylation of Ser1589 was seen only in the cerebellum. A possible explanation for the latter is that anti-Ser1589P detects Purkinje cell-enriched IP₃R1 phosphorylated by PKG, which is also enriched in these cells [65, 66]. Both in R6/1 and HD tg rats we found reduction of IP₃R1 protein in the cerebellum. Indeed, cerebellar samples from R6/1 mice exhibited significant loss of both IP₃R1, mGluR1/5 and the Purkinje cell marker proteins calbindin D_28k and parvalbumin (Fig. 2G, H, J, K; Table 2). The changes of IP₃R1 expression in association with cerebellar Purkinje neurons is to some extent surprising and could be attributed to associated changes in the Ca²⁺ binding proteins calbindin and parvalbumin being expressed at higher levels in the cerebellar cortex. A selective loss of Purkinje cells has been demonstrated in R6/2 and HdhQ200 knock-in mice [67, 68], and other mouse models of HD have demonstrated other cerebellar signalling defects [31, 69]. Cerebellar neurodegeneration has been reported in HD patients (see e.g.,), thus these findings suggest that cerebellar Ca²⁺ signalling in HD might be an interesting focus for further studies.

We have previously suggested a role for regulation of IP₃R1 in some neurodegenerative diseases, including reduced levels of IP₃R1 both in human post-mortem Alzheimer brain tissue and in experimental mouse models for ischaemia and HD [6 , 19]. Other conditions involving IP₃R1 irregularities include the cerebellar ataxias (e.g., spinocerebellar ataxia; SCA [75]). ∼40 % of SCAs are CAG repeat disorders similar to HD [76], and in addition to huntingtin, the C-terminal part of the IP₃R1 interacts with both ataxin-2 and –3 [77]. Apart from the CAG repeat-caused SCAs, impact on the IP₃R1 has been reported as a feature of many genes involved in cerebellar ataxias [13]. Many of these conditions exhibit premature death of neurons in the CNS; frequently caused by apoptosis and dysregulation of intracellular calcium ion homeostasis. Converging evidence now points to the IP₃R1 as a key player in these cellular processes [14 , 78]. Evidence that the subcellular distribution of phosphorylated and non-phosphorylated IP₃R1 in the rat and human brain can be altered by ischaemia [9], as well as prolonged excitatory synaptic stimulation, suggests that phosphorylation of IP₃R1 may be a regulatory phenomenon involved in neurological disorders [79].

Our data showing differential expression levels of IP₃R1 and phospho-regulation of the channel protein within and between brain areas of the transitional and oldest animal model (R6/1), are partly supported by the tgHD rat model with differences only in cerebellum. Interestingly, while the more recently developed, full-length BACHD mice show only minor differences in IP₃R1 levels, the more advanced zQ175DNKI knock-in mice show age-dependent decline in IP₃R1 levels, but no differences in immunoreactivity of phospho-specific antibodies. Thus, our study of IP₃R1 and its phosphorylation across several animal models of HD provides little evidence for a significant effect within pathophysiology and progression of HD in this preclinical level, perhaps because the methods employed lack sensitivity, i.e., necessary spatial resolution. Thus, further studies, particularly on the cellular level, are warranted.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

Footnotes

ACKNOWLEDGMENTS

This work was funded by The Medical Student Research Programme at the University of Oslo (to JIP) and by The Research Council of Norway (to TBL; FRIBIO project #178571 and the Norwegian Brain Initiative (NORBRAIN)). Histological images were acquired at the NORBRAIN Slide Scanning Facility at the Institute of Basic Medical Sciences, University of Oslo, a resource funded by the Research Council of Norway. We thank Dr. Kirsten Grundt for the initial purification of antisera, Dr. Linda Møllersen for genotyping of mouse brains, Dr. Yvette C. van Dongen for preparation of HD rat tissue sections, and Mrs. Grażyna Babinska for technical assistance with scanning of histological slides. We are grateful to Professor Farrukh A. Chaudhry for hosting parts of the laboratory work.

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Differential Levels and Phosphorylation of Type 1 Inositol 1,4,5-Trisphosphate Receptor in Four Different Murine Models of Huntington Disease

Abstract

Background:

Objective:

Methods:

Results:

Conclusion:

Keywords

INTRODUCTION

MATERIALS AND METHODS

Ethics statement, animal husbandry, and sample isolation

Animals

R6/1 mice

BACHD mice

zQ175 mice

Rats

TgHDCAG51n rats

Production and affinity-purification of antibodies against holoprotein and phospho-specific type 1 inositol 1,4,5-trisphosphate receptor

Antibody-specificity control experiments

Quantitative immunoblotting of brain proteins from HD and wild-type mice

Single immunoperoxidase labelling of IP3R1 in HD and wild type rats

Blocking experiment

Quantification of immunostaining in brain slices from HD rats

Quantification of immunostaining of brain slices from BACHD and zQ175 mice

Data analyses and statistics

RESULTS

Antibody specificity

Decreased levels of IP3R1 protein in striatum, cerebellum and hippocampus of R6/1 mice

Striatum

Cerebellum

Hippocampus

Levels of IP3R1 protein in striatum of zQ175 mice

Immunohistochemical staining of sections from zQ175 and BACHD mice

Amount of IP3R1 in transgenic HD Rats

DISCUSSION

CONFLICT OF INTEREST

Footnotes

ACKNOWLEDGMENTS

References

Single immunoperoxidase labelling of IP₃R1 in HD and wild type rats

Decreased levels of IP₃R1 protein in striatum, cerebellum and hippocampus of R6/1 mice

Levels of IP₃R1 protein in striatum of zQ175 mice

Amount of IP₃R1 in transgenic HD Rats