inositol-1-4-5-trisphosphate and Huntington-Disease

Huntington's disease (HD) and spinocerebellar ataxias (SCAs) are autosomal-dominant neurodegenerative disorders. HD is caused by polyglutamine (polyQ) expansion in the amino-terminal region of a protein huntingtin (Htt) and primarily affects medium spiny striatal neurons (MSN). Many SCAs are caused by polyQ-expansion in ataxin proteins and primarily affect cerebellar Purkinje cells. The reasons for neuronal dysfunction and death in HD and SCAs remain poorly understood and no cure is available for the patients. Our laboratory discovered that mutant huntingtin, ataxin-2 and ataxin-3 proteins specifically bind to the carboxy-terminal region of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1), an intracellular Ca(2+) release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP(3)R1 causes sensitization of IP(3)R1 to activation by IP(3) in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca(2+) signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca(2+) signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca(2+) signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP(3)R and other Ca(2+) signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.

Topics: Animals; Ataxins; Calcium Signaling; Disease Models, Animal; Humans; Huntingtin Protein; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Receptors, N-Methyl-D-Aspartate; Spinocerebellar Ataxias

The modulation of cytoplasmic Ca2+ concentration by release from internal stores through the inositol trisphosphate receptor (InsP3R) Ca2+ release channel is a ubiquitous signaling system involved in the regulation of numerous processes. Because of its ubiquitous expression and roles in regulating diverse cell physiological processes, it is not surprising that the InsP3R has been implicated in a number of disease states. However, relatively few mutations in InsP3R genes have been identified to date. Here, I will discuss mutations in the type 1 InsP3R that have been discovered by analyses of human patients and mice with neurological disorders. In addition, I will highlight diseases caused by mutations in other genes, including Huntington's and Alzheimer's diseases and some spinocerebellar ataxias, where the mutant proteins have been found to exert strong influences on InsP3R function that may link InsP3R to disease pathogenesis.

Topics: Alzheimer Disease; Animals; Biomarkers, Tumor; Calcium; Calcium Channels; Humans; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Models, Biological; Neurodegenerative Diseases; Spinocerebellar Ataxias

The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington's disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD.. To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results.. Functional analysis of generated Htt-null MEF cells revealed that Htt plays a direct role in Ca2+ signaling by modulating InsP3R sensitivity to InsP3. The genome-wide transcriptional profiling of Htt-null cells yielded novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD.

Topics: Animals; Calcium Signaling; Cell Line; Endoplasmic Reticulum; Fibroblasts; Huntingtin Protein; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mice; Mice, Knockout; Mitochondria; Nerve Tissue Proteins; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; RNA, Messenger; Signal Transduction

Huntington's disease is caused by polyglutamine expansion (exp) in huntingtin (Htt). Htt-associated protein-1 (HAP1) was the first identified Htt-binding partner. The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) is an intracellular Ca2+ release channel that plays an important role in neuronal function. Recently, we identified a InsP3R1-HAP1A-Htt ternary complex in the brain and demonstrated that Httexp, but not normal Htt, activates InsP3R1 in bilayers and facilitates InsP3R1-mediated intracellular Ca2+ release in medium spiny striatal neurons [MSN; T.-S. Tang et al. (2003) Neuron, 39, 227-239]. Here we took advantage of mice with targeted disruption of both HAP1 alleles (HAP1 -/-) to investigate the role of HAP1 in functional interactions between Htt and InsP3R1. We determined that: (i) HAP1 is expressed in the MSN; (ii) HAP1A facilitates functional effects of Htt and Htt(exp) on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Htt(exp) overexpression; (iv) HAP1 facilitates potentiation of InsP3R1-mediated Ca2+ release by Htt(exp) in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.

Topics: Animals; Calcium; Cells, Cultured; Corpus Striatum; Huntington Disease; Inositol 1,4,5-Trisphosphate; Kinetics; Lipid Bilayers; Membrane Glycoproteins; Membrane Transport Proteins; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Nerve Tissue Proteins; Neurons; Serotonin Plasma Membrane Transport Proteins

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.

Topics: Action Potentials; Animals; Blotting, Western; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Cerebellum; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Fura-2; Green Fluorescent Proteins; Humans; Huntingtin Protein; Huntington Disease; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lipid Bilayers; Luminescent Proteins; Methoxyhydroxyphenylglycol; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Patch-Clamp Techniques; Peptide Fragments; Plasmids; Protein Binding; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Time Factors; Two-Hybrid System Techniques

Alterations in protein kinase C (PKC) and myo-inositol 1,4,5-trisphosphate (IP3) receptors were studied in the autopsied human striata from 21 patients with Parkinson's disease (PD) (Yahr III, IV, and V), 8 patients with Huntington's disease (HD), and 23 age-matched and postmortem time-matched nonneurological controls. The concentrations of PKC and IP3 receptors were determined using [3H]4 beta-phorbol 12,13-dibutyrate (PDBu) and [3H]IP3 as respective ligands. Both the specific [3H]-PDBu and [3H]IP3 bindings were significantly reduced in the striata of Yahr V patients with dementia (PDD) and in that of HD patients, as compared to findings in the controls. These bindings were unchanged when all the PD patients without dementia, Yahr (III plus IV) patients, or Yahr V patients without dementia were compared with evidence from the controls. Immunoquantification of four PKC subspecies (alpha, beta I, beta II, and gamma) in the HD putamen revealed a selective reduction in the beta II-PKC immunoreactions. These results are supported by immunohistochemical findings in the rat brain that beta II-PKC is expressed in the striatal gabaergic efferent pathway, while the alpha-PKC is present in the nigrostriatal dopaminergic neurons. The neurochemical pathophysiology of PD differs between patients with and without dementia.

Topics: Aged; Animals; Brain; Brain Mapping; Calcium; Cell Death; Corpus Striatum; Female; Humans; Huntington Disease; Inositol 1,4,5-Trisphosphate; Male; Middle Aged; Nerve Degeneration; Neurotransmitter Agents; Parkinson Disease; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Second Messenger Systems; Substantia Nigra

Specific [3H]inositol 1,4,5-trisphosphate [( 3H]InsP3) binding was studied in regions of postmortem brain from 15 patients with Huntington's disease (HD) and 13 nonneurological controls. Single-point binding analyses, using 5.0 nM InsP3, showed statistically significant reductions in specific [3H]InsP3 binding in the caudate (-71%) and putamen (-75%) of HD patients compared with controls. Frontal and occipital cortical [3H]InsP3 binding was not significantly different between HD and controls, a finding suggesting that the reduced [3H]InsP3 binding parallels the brain regional specificity of the neuropathological changes in HD. Scatchard analyses of data from [3H]InsP3 competition binding assays performed on caudate nucleus revealed that the reductions found using single-point binding assays were due to a decrease in both binding density (-57%) and affinity (-50%) in HD brain compared with controls. The concomitant changes in InsP3 receptor density and affinity in HD brain suggest that these alterations may be produced by processes in addition to cell loss. These results suggest the possibility that disturbances in InsP3 receptor function, possibly resulting in altered intracellular calcium flux and homeostasis, occur in HD and may participate in the pathogenesis of this neurodegenerative disorder.

Topics: Brain; Corpus Striatum; Female; Haloperidol; Humans; Huntington Disease; Inositol 1,4,5-Trisphosphate; Male; Middle Aged; Postmortem Changes; Reference Values; Time Factors; Tritium