Huiping Tu

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

Huntingtons 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.

Deranged Calcium Signaling and Neurodegeneration in Spinocerebellar Ataxia Type 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease (MJD), is an autosomal-dominant neurodegenerative disorder caused by a polyglutamine expansion in ataxin-3 (ATX3; MJD1) protein. In biochemical experiments, we demonstrate that mutant ATX3exp specifically associated with the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1), an intracellular calcium (Ca2+) release channel. In electrophysiological and Ca2+ imaging experiments, we show that InsP3R1 was sensitized to activation by InsP3 in the presence of mutant ATX3exp. We found that feeding SCA3-YAC-84Q transgenic mice with dantrolene, a clinically relevant stabilizer of intracellular Ca2+ signaling, improved their motor performance and prevented neuronal cell loss in pontine nuclei and substantia nigra regions. Our results indicate that deranged Ca2+ signaling may play an important role in SCA3 pathology and that Ca2+ signaling stabilizers such as dantrolene may be considered as potential therapeutic drugs for treatment of SCA3 patients.

Familial Alzheimer disease–linked mutations specifically disrupt Ca2+ leak function of presenilin 1

Mutations in presenilins are responsible for approximately 40% of all early-onset familial Alzheimer disease (FAD) cases in which a genetic cause has been identified. In addition, a number of mutations in presenilin-1 (PS1) have been suggested to be associated with the occurrence of frontal temporal dementia (FTD). Presenilins are highly conserved transmembrane proteins that support cleavage of the amyloid precursor protein by gamma-secretase. Recently, we discovered that presenilins also function as passive ER Ca(2+) leak channels. Here we used planar lipid bilayer reconstitution assays and Ca(2+) imaging experiments with presenilin-null mouse embryonic fibroblasts to analyze ER Ca(2+) leak function of 6 FAD-linked PS1 mutants and 3 known FTD-associated PS1 mutants. We discovered that L166P, A246E, E273A, G384A, and P436Q FAD mutations in PS1 abolished ER Ca(2+) leak function of PS1. In contrast, A79V FAD mutation or FTD-associated mutations (L113P, G183V, and Rins352) did not appear to affect ER Ca(2+) leak function of PS1 in our experiments. We validated our findings in Ca(2+) imaging experiments with primary fibroblasts obtained from an FAD patient possessing mutant PS1-A246E. Our results indicate that many FAD mutations in presenilins are loss-of-function mutations affecting ER Ca(2+) leak activity. In contrast, none of the FTD-associated mutations affected ER Ca(2+) leak function of PS1, indicating that the observed effects are disease specific. Our observations are consistent with the potential role of disturbed Ca(2+) homeostasis in Alzheimer disease pathogenesis.

Functional and Biochemical Analysis of the Type 1 Inositol (1,4,5)-Trisphosphate Receptor Calcium Sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.

HAP1 facilitates effects of mutant huntingtin on inositol 1,4,5-trisphosphate-induced Ca2+ release in primary culture of striatal medium spiny neurons

Huntingtons 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 Httexp on InsP3R1 in planar lipid bilayers; (iii) HAP1 is required for changes in MSN basal Ca2+ levels resulting from Htt or Httexp overexpression; (iv) HAP1 facilitates potentiation of InsP3R1‐mediated Ca2+ release by Httexp in mouse MSN. Our present results indicate that HAP1 plays an important role in functional interactions between Htt and InsP3R1.

Functional characterization of the type 1 inositol 1,4,5-trisphosphate receptor coupling domain SII(+/-) splice variants and the Opisthotonos mutant form.

The type 1 inositol (1,4,5)-trisphosphate receptor (InsP3R1) plays a critical role in Ca2+ signaling in cells. Neuronal and nonneuronal isoforms of the InsP3R1 differ by alternative splicing in the coupling domain of the InsP3R1 (SII site). Deletion of 107 amino acids from the coupling domain of the InsP3R1 results in epileptic-like behaviors in opisthotonos (opt) spontaneous mouse mutant. Using Spodoptera frugiperda cells expression system, we compared single-channel behavior of recombinant InsP3R1-SII(+), InsP3R1-SII(-), and InsP3R1-opt channels in planar lipid bilayers. The main results of our study are: 1) the InsP3R1-SII(-) has a higher conductance (94 pS) and the InsP3R1-opt has a lower conductance (64 pS) than the InsP3R1-SII(+) (81 pS); 2) the bell-shaped Ca2+-dependence peaks at 200-300 nM Ca2+ for all three InsP3R1 isoforms; 3) the bell-shaped Ca2+-dependence is wider for the InsP3R1-SII(+) and narrower for the InsP3R1-SII(-) and InsP3R1-opt; 4) the apparent affinity for ATP is sixfold lower for the InsP3R1-SII(-) (1.4 mM) and 20-fold lower for the InsP3R1-opt (5.3 mM) than for the InsP3R1-SII(+) (0.24 mM); 5) the InsP3R1-SII(-) is approximately twofold more active than the InsP3R1-SII(+) in the absence of ATP. Obtained results provide novel information about the molecular determinants of the InsP3R1 function.