Role of Inositol 1,4,5-Trishosphate Receptors in Pathogenesis of Huntington’s Disease and Spinocerebellar Ataxias

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 (IP₃R1), an intracellular Ca²⁺ release channel. Moreover, we found that association of mutant huntingtin or ataxins with IP₃R1 causes sensitization of IP₃R1 to activation by IP₃ in planar lipid bilayers and in neuronal cells. These results suggested that deranged neuronal Ca²⁺ signaling might play an important role in pathogenesis of HD, SCA2 and SCA3. In support of this idea, we demonstrated a connection between abnormal Ca²⁺ signaling and neuronal cell death in experiments with HD, SCA2 and SCA3 transgenic mouse models. Additional data in the literature indicate that abnormal neuronal Ca²⁺ signaling may also play an important role in pathogenesis of SCAl, SCA5, SCA6, SCA14 and SCA15/16. Based on these results I propose that IP₃R and other Ca²⁺ signaling proteins should be considered as potential therapeutic targets for treatment of HD and SCAs.     

Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17

Spinocerebellar ataxia type 17 (SCA17) is an autosomal dominant inherited disorder characterized by degeneration of spinocerebellar tracts and selected brainstem neurons owing to the expansion of a CAG repeat of the human TATA-binding protein (hTBP) gene. To gain insight into the pathogenesis of this hTBP mutation, we generated transgenic mice with the mutant hTBP gene driven by the Purkinje specific protein (Pcp2/L7) gene promoter. Mice with the expanded hTBP allele developed ataxia within 2-5 months. Behavioral analysis of L7-hTBP transgenic mice showed reduced fall latency in a rotarod assay. Purkinje cell degeneration was identified by immunostaining of calbindin and IP3R1. Reactive gliosis and neuroinflammation occurred in the transgenic cerebellum, accompanied by up-regulation of GFAP and Iba1. The L7-hTBP transgenic mice were thus confirmed to recapitulate the SCA17 phenotype and were used as a disease model to explore the potential of granulocyte-colony stimulating factor in SCA17 treatment. Our results suggest that granulocyte-colony stimulating factor has a neuroprotective effect in these transgenic mice, ameliorating their neurological and behavioral deficits. These data indicate that the expression of the mutant hTBP in Purkinje cells is sufficient to produce cell degeneration and an ataxia phenotype, and constitutes a good model for better analysis of the neurodegeneration in SCA17.     

Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2

Distorted neuronal calcium signaling has been reported in many neurodegenerative disorders, including different types of spinocerebellar ataxias (SCAs). Cerebellar Purkinje cells (PCs) are primarily affected in SCAs and the disturbances in the calcium homeostasis were observed in SCA PCs. Our previous results have revealed that 3,5-dihydroxyphenylglycine (DHPG) induced greater calcium responses in SCA2-58Q PC cultures than in wild type (WT) PC cultures. Here we observed that glutamate-induced calcium release in PCs cells bodies is significantly higher in SCA2-58Q PCs from acute cerebellar slices compared to WT PCs of the same age. Recent studies have demonstrated that the stromal interaction molecule 1 (STIM1) plays an important role in the regulation of the neuronal calcium signaling in cerebellar PCs in mice. The main function of STIM1 is to regulate store-operated calcium entry through the TRPC/Orai channels formation to refill the calcium stores in the ER when it is empty. Here we demonstrated that the chronic viral-mediated expression of the small interfering RNA (siRNA) targeting STIM1 specifically in cerebellar PCs alleviates the deranged calcium signaling in SCA2-58Q PCs, rescues the spine loss in these cerebellar neurons, and also improves the motor decline in SCA2-58Q mice. Thus, our preliminary results support the important role of the altered neuronal calcium signaling in SCA2 pathology and also suggest the STIM1-mediated signaling pathway as a potential therapeutic target for treatment of SCA2 patients.     

Defective calcium homeostasis in the cerebellum in a mouse model of Niemann-Pick A disease

We recently demonstrated that calcium homeostasis is altered in mouse models of two sphingolipid storage diseases, Gaucher and Sandhoff diseases, owing to modulation of the activities of a calcium-release channel (the ryanodine receptor) and of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) respectively, by the accumulating sphingolipids. We now demonstrate that calcium homeostasis is also altered in a mouse model of Niemann-Pick A disease, the acid sphingomyelinase (A-SMase)-deficient mouse (ASM-/-), with reduced rates of calcium uptake via SERCA in the cerebellum of 6-7-month-old mice. However, the mechanism responsible for defective calcium homeostasis is completely different from that observed in the other two disease models. Thus, levels of SERCA expression are significantly reduced in the ASM-/- cerebellum by 6-7 months of age, immediately before death of the mice, as are levels of the inositol 1,4,5-triphosphate receptor (IP3R), the major calcium-release channel in the cerebellum. Systematic analyses of the time course of loss of SERCA and IP3R expression revealed that loss of the IP3R preceeded that of SERCA, with essentially no IP3R remaining by 4 months of age, whereas SERCA was still present even after 6 months. Expression of zebrin II (aldolase C), a protein found in about half of the Purkinje cells in the adult mouse cerebellum, was essentially unchanged during development. We discuss possible pathological mechanisms related to calcium dysfunction that may cause Purkinje cell degeneration, and as a result, the onset of neuropathology in Niemann-Pick A disease.     

Loss of Purkinje cells in the PKCγ H101Y transgenic mouse

Spinocerebellar ataxia type 14 (SCA14) is an autosomal, dominant neurodegenerative disorder caused by mutations in PKCγ. The objective of this study was to determine effects of PKCγ H101Y SCA14 mutation on Purkinje cells in the transgenic mouse. Results demonstrated that wild type PKCγ-like Purkinje cell localization of HA-tagged PKCγ H101Y mutant proteins, altered morphology and loss of Purkinje cells were observed in the PKCγ H101Y SCA14 transgenic mouse at four weeks of age. Failure of stereotypical clasping responses in the hind limbs of transgenic mice was also observed. Further, PKCγ H101Y SCA14 mutation caused lack of total cellular PKCγ enzyme activity, loss of connexin 57 phosphorylation on serines, and activation of caspase-12 in the PKCγ H101Y SCA14 transgenic mouse. Results clearly demonstrate a need for PKCγ control of gap junctions for maintenance of Purkinje cells. This is the first transgenic mouse to our knowledge which models a human SCA14 mutation.     

Spinocerebellar ataxia: localization of an autosomal dominant locus between two markers on human chromosome 6.

Inherited spinocerebellar ataxias (SCA) are progressively degenerative neurological diseases. The primary site of degeneration is the cerebellar cortex--in particular, the Purkinje cells. In the present report, the SCA locus, inherited as an autosomal dominant trait in a large kindred, is localized to a region approximately 15 centimorgans telomeric of HLA-A on the short arm of chromosome 6.     

Deranged neuronal calcium signaling and Huntington disease

Huntington disease (HD) is an autosomal-dominant neurodegenerative disorder that primarily affects medium spiny striatal neurons (MSN). HD is caused by polyglutamine (polyQ) expansion (exp) in the amino-terminal region of a protein huntingtin (Htt). The connection between polyQ expansion in Httexp and MSN neurodegeneration remains elusive. Here we discuss recent data that link polyQ expansion in Httexp and deranged Ca2+ signaling in MSN neurons. Experimental evidence indicates that (1) Ca2+ homeostasis is abnormal in mitochondria isolated from lymphoblasts of HD patients and from brains of the YAC72 HD mouse model; (2) Httexp leads to potentiation of NR1/NR2B NMDA receptor activity in heterologous expression systems and in MSN from YAC72 HD mouse model; and (3) Httexp binds to the type 1 inositol 1,4,5-trisphosphate receptor (InsP3R1) carboxy-terminus and causes sensitization of InsP3R1 to activation by InsP3 in planar lipid bilayers and in MSN. Based on these results we propose that Httexp-induced cytosolic and mitochondrial Ca2+ overload of MSN plays an important role in the pathogenesis of HD and that Ca2+ signaling blockers may play a beneficial role in treatment of HD.     

PC-PLC is involved in osteoclastogenesis induced by TNF-α through upregulating IP3R1 expression

The precise mechanism of how TNF-α promotes osteoclast formation is not clear. Previous reports show TNF-α targets molecules that regulate calcium signaling. Inositol-1,4,5-trisphosphate receptors (IP3Rs) are important calcium channel responsible for evoking intracellular calcium oscillation. We found that TNF-α increased the expression of IP3R1 and promoted osteoclastogenesis in RANKL-induced mouse BMMs. Phosphatidylcholine-specific phospholipase C (PC-PLC) specific inhibitor D609 eliminated the upregulation of IP3R1 by TNF-α, and decreased the autoamplification of nuclear factor of activated T-cells 1 (NFATc1), thus resulted in less osteoclasts formation. However, D609 did not inhibit RANKL-induced osteoclastogenesis. Our data suggest TNF-α promotes RANKL-induced osteoclastogenesis, at least partially, through PC-PLC/IP3R1/NFATc1 pathway.     

Modeling and analysis of calcium signaling events leading to long-term depression in cerebellar Purkinje cells

Modeling and simulation of the calcium signaling events that precede long-term depression of synaptic activity in cerebellar Purkinje cells are performed using the Virtual Cell biological modeling framework. It is found that the unusually high density and low sensitivity of inositol-1,4,5-trisphosphate receptors (IP3R) are critical to the ability of the cell to generate and localize a calcium spike in a single dendritic spine. The results also demonstrate the model's capability to simulate the supralinear calcium spike observed experimentally during coincident activation of the parallel and climbing fibers. The sensitivity of the calcium spikes to certain biological and geometrical effects is investigated as well as the mechanisms that underlie the cell's ability to generate the supralinear spike. The sensitivity of calcium release rates from the IP3R to calcium concentrations, as well as IP3 concentrations, allows the calcium spike to form. The diffusion barrier caused by the small radius of the spine neck is shown to be important, as a threshold radius is observed above which a spike cannot be formed. Additionally, the calcium buffer capacity and diffusion rates from the spine are found to be important parameters in shaping the calcium spike.     

Inositol 1,4,5-trisphosphate receptor type 1 phosphorylation and regulation by extracellular signal-regulated kinase

Type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) is a widely expressed intracellular calcium-release channel found in many cell types. The operation of IP(3)R1 is regulated through phosphorylation by multiple protein kinases. Extracellular signal-regulated kinase (ERK) has been found involved in calcium signaling in distinct cell types, but the underlying mechanisms remain unclear. Here, we present evidence that ERK1/2 and IP(3)R1 bind together through an ERK binding motif in mouse cerebellum in vivo as well as in vitro. ERK-phosphorylating serines (Ser 436) was identified in mouse IP(3)R1 and Ser 436 phosphorylation had a suppressive effect on IP(3) binding to the recombinant N-terminal 604-amino acid residues (N604). Moreover, phosphorylation of Ser 436 in R(224-604) evidently enhance its interaction with the N-terminal "suppressor" region (N223). At last, our data showed that Ser 436 phosphorylation in IP(3)R1 decreased Ca(2+) releasing through IP(3)R1 channels.     

ROCK-phosphorylated vimentin modifies mutant huntingtin aggregation via sequestration of IRBIT

ABSTRACT: BACKGROUND: Huntington's Disease (HD) is a fatal hereditary neurodegenerative disease caused by the accumulation of mutant huntingtin protein (Htt) containing an expanded polyglutamine (polyQ) tract. Activation of the channel responsible for the inositol-induced Ca2+ release from ensoplasmic reticulum (ER), was found to contribute substantially to neurodegeneration in HD. Importantly, chemical and genetic inhibition of inositol 1,4,5-trisphosphate (IP3) receptor type 1 (IP3R1) has been shown to reduce mutant Htt aggregation. RESULTS: In this study, we propose a novel regulatory mechanism of IP3R1 activity by type III intermediate filament vimentin which sequesters the negative regulator of IP3R1, IRBIT, into perinuclear inclusions, and reduces its interaction with IP3R1 resulting in promotion of mutant Htt aggregation. Proteasome inhibitor MG132, which causes polyQ proteins accumulation and aggregation, enhanced the sequestration of IRBIT. Furthermore we found that IRBIT sequestration can be prevented by a rho kinase inhibitor, Y-27632. CONCLUSIONS: Our results suggest that vimentin represents a novel and additional target for the therapy of polyQ diseases.     

The hereditary spinocerebellar ataxias in Japan

In Japan, multiple system atrophy (MSA) accounts for 40% of all spinocerebellar ataxias (SCAs) and hereditary disorders account for 30%. Among the latter, autosomal dominant disorders are common and recessive ataxias are rare. Although the frequency of SCA genotypes differs between geographic regions throughout Japan, SCA6, SCA3/MJD, and DRPLA are the three major disorders, while SCA7, SCA8, SCA10, SCA12, and SCA17 are infrequent or almost undetected. SCA1 predominantly occurs in the northern part of Japan. Overall, 20-40% of dominant SCAs are due to unknown mutations. From this cluster, pure cerebellar ataxias linked with the SCA4, SCA14, and SCA16 locus have been isolated. Among the recessive SCAs, patients with AVED and EAOH have been detected. However, FRDA associated with GAA repeat expansion in the frataxin gene has not been reported so far.     

Genetic ablation and chemical inhibition of IP3R1 reduce mutant huntingtin aggregation

Huntington's disease (HD) is a dominantly inherited neurodegenerative disease caused by an expansion of the polyglutamine (polyQ) stretch in huntingtin (htt). Previously, it has been shown that inhibition of the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) activity reduced aggregation of pathogenic polyQ proteins. Experimentally, this effect was achieved by modification of the intracellular IP3 levels or by application of IP3R1 inhibitors, such as 2-aminoethyl diphenylborinate (2-APB). Unfortunately, there are certain concerns about the 2-APB specificity and cytotoxicity. Moreover, a direct link between IP3R1 and polyQ aggregation has not been shown yet. In this study we show, that down-regulation of the IP3R1 levels by shRNA reduced the aggregation of mutant htt. We tested 2-APB analogs in an attempt to identify less toxic and more IP3R1-specific compounds and found that the effect of these analogs on the reduction of the mutant htt aggregation did weakly correlate with their inhibitory action toward the IP3-induced Ca(2+) release (IICR). Their effect on aggregation was not correlated with the store-operated Ca(2+) entry (SOCE), which is another target of the 2-APB related compounds. Our findings suggest that besides functional contribution of the IP3R inhibition on the mutant htt aggregation there are additional mechanisms for the anti-aggregation effect of the 2-APB related compounds.     

Ankyrin B modulates the function of Na,K-ATPase/inositol 1,4,5-trisphosphate receptor signaling microdomain

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.     

A Highlights from MBoC Selection: Praja1 ubiquitin ligase facilitates degradation of polyglutamine proteins and suppresses polyglutamine-mediated toxicity

A network of chaperones and ubiquitin ligases sustain intracellular proteostasis and is integral in preventing aggregation of misfolded proteins associated with various neurodegenerative diseases. Using cell-based studies of polyglutamine (polyQ) diseases, spinocerebellar ataxia type 3 (SCA3) and Huntington’s disease (HD), we aimed to identify crucial ubiquitin ligases that protect against polyQ aggregation. We report here that Praja1 (PJA1), a Ring-H2 ubiquitin ligase abundantly expressed in the brain, is diminished when polyQ repeat proteins (ataxin-3/huntingtin) are expressed in cells. PJA1 interacts with polyQ proteins and enhances their degradation, resulting in reduced aggregate formation. Down-regulation of PJA1 in neuronal cells increases polyQ protein levels vis-a-vis their aggregates, rendering the cells vulnerable to cytotoxic stress. Finally, PJA1 suppresses polyQ toxicity in yeast and rescues eye degeneration in a transgenic Drosophila model of SCA3. Thus, our findings establish PJA1 as a robust ubiquitin ligase of polyQ proteins and induction of which might serve as an alternative therapeutic strategy in handling cytotoxic polyQ aggregates.