Full length mutant huntingtin is required for altered Ca2+ signaling and apoptosis of striatal neurons in the YAC mouse model of Huntington's disease

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Abstract

Huntington's disease (HD) is caused by a progressive loss of striatal medium spiny neurons (MSN). The molecular trigger of HD is a polyglutamine expansion in the Huntingtin protein (Htt). The mutant Htt protein forms insoluble nuclear aggregates which have been proposed to play a key role in causing neuronal cell death in HD. Other lines of investigation suggest that expression of mutant Htt facilitates activity of the NR2B subtype of NMDA receptors and the type 1 inositol 1,4,5-trisphosphate receptors (InsP3R1), and that disturbed calcium (Ca2+) signaling causes apoptosis of MSNs in HD. The YAC128 transgenic HD mouse model expresses the full-length human Htt protein with 120Q CAG repeat expansion and displays an age-dependent loss of striatal neurons as seen in human HD brain. In contrast, the shortstop mice express an amino-terminal fragment of the mutant Htt protein (exons 1 and 2) and display no behavioral abnormalities or striatal neurodegeneration despite widespread formation of neuronal inclusions. Here we compared Ca2+ signals in primary MSN neuronal cultures derived from YAC128 and shortstop mice to their wild-type non-transgenic littermates. Repetitive application of glutamate results in supranormal Ca2+ responses in YAC128 MSNs, but not in shortstop MSNs. In addition, while currents mediated by the NR2B subtype of NMDA receptors were increased in YAC128 MSNs, currents in SS MSNs were found to be similar to WT. Furthermore, YAC128 MSNs were sensitized to glutamate-induced apoptosis. Consistent with these findings, we found that application of glutamate induced rapid loss of mitochondrial membrane potential in YAC128 MSNs. In contrast, SS MSNs do not show increased cell death postglutamate treatment nor accelerated loss of mitochondrial membrane potential following glutamate stimulation. Glutamate-induced loss of mitochondrial membrane potential in YAC128 MSNs could be prevented by inhibitors of NR2B NMDA receptors and mGluR1/5 receptors. Our results are consistent with the hypothesis that disturbed neuronal Ca2+ signaling plays a significant role in the degeneration of MSN containing full-length mutant Httexp. Furthermore, the results obtained with neurons from shortstop mice provide additional evidence that not all fragments of mutant Httexp are toxic to neurons.

Introduction

Huntington's disease (HD) has onset usually between 35 and 50 years with chorea and psychiatric disturbances and gradual but inexorable intellectual decline to death after 15-20 years (Vonsattel and DiFiglia, 1998). Neuropathological analysis reveals selective and progressive neuronal loss in the striatum (Vonsattel and DiFiglia, 1998), particularly affecting the GABAergic medium spiny striatal neurons (MSN). At the molecular level, the cause of HD is a polyglutamine (polyQ) expansion (exp) in the amino-terminus of huntingtin (Htt), a 350 kDa ubiquitously expressed cytoplasmic protein (The Huntington's Disease Collaborative Research Group, 1993). The cellular mechanisms that link the Httexp mutation with the disease are under intense investigation (Tobin and Signer, 2000). The mutant Httexp protein forms insoluble nuclear aggregates which have been proposed to play a key role in causing neuronal cell death in HD (Cooper et al., 1998, Ross, 2002). A number of laboratories are focused on screening for compounds which are able to inhibit the formation of Htt aggregates (Heiser et al., 2002, Apostol et al., 2003, Zhang et al., 2005, Hockly et al., 2006). A decrease in nuclear inclusion formation has been interpreted as a positive outcome in preclinical therapeutic trials with HD mouse models (Smith et al., 2001, Ferrante et al., 2002, Tanaka et al., 2004). However, more recent evidence suggested that Htt-formed nuclear inclusions may not be toxic and may even protect MSN neurons from cell death (Kuemmerle et al., 1999, Arrasate et al., 2004).

Several lines of evidence indicate that glutamate-mediated excitotoxicity plays a role in neurodegeneration of HD MSNs (DiFiglia, 1990). Striatal injection of kainic acid induced death of MSNs and yielded one of the first animal models of HD (Coyle and Schwarcz, 1976, McGeer and McGeer, 1976). More direct evidence for an involvement of NMDAR was obtained when HD-like lesions were observed following striatal injection of the NMDAR agonist quinolinic acid (Beal et al., 1986, Beal et al., 1991, Hantraye et al., 1990). It has been reported that Httexp facilitates activity of the NR2B subtype of NMDA receptors (Chen et al., 1999, Sun et al., 2001, Zeron et al., 2002, Zeron et al., 2004, Fan et al., 2007) and the type 1 inositol 1,4,5-trisphosphate receptors (InsP3R1) (Tang et al., 2005). Moreover, elevated Ca2+ signals have been directly linked to the cell death of striatal MSNs cultured from HD mouse models (Zeron et al., 2002, Tang et al., 2005, Shehadeh et al., 2006. These studies suggested that excitotoxicity and abnormal neuronal Ca2+ signaling play an important role in HD pathogenesis (Bezprozvanny and Hayden, 2004).

A number of transgenic HD mouse models have been generated which reproduce some features of the disease (Menalled and Chesselet, 2002, Rubinsztein, 2002). However, none of the previously generated HD mouse models reproduced selective MSN degeneration, which is a hallmark of HD. Recently, a yeast artificial chromosome (YAC) mouse model of HD has been generated (YAC128) (Slow et al., 2003). In this model, the full-length human Htt protein with 120Q expansion is expressed under the control of the endogenous promoter and regulatory elements. The early-onset motor deficit, and striatal neuronal loss observed in the YAC128 mouse model accurately recapitulate the progression of HD (Slow et al., 2003). Thus, the YAC128 mouse model is ideal for understanding cellular mechanisms that lead to neurodegeneration in HD, as well as for validating potential therapeutic agents. Our laboratory have been extensively using the YAC128 HD mouse model in studies of HD pathogenesis and in the testing of potential HD therapeutic agents Tang et al., 2005, Tang et al., 2007, Wu et al., 2006).

In the process of generating the YAC128 HD mouse model, a mouse expressing a short fragment (exons 1 and 2 of 67) of human Htt with 120 CAG repeat expansion was serendipitously established (shortstop mouse) (Slow et al., 2005). Analysis of the shortstop mice revealed widespread Htt nuclear inclusions. However, despite widespread inclusion burden, these mice do not manifest a behavioral, HD-related phenotype as assessed by rotarod or decreases in brain weight, striatal volume, and striatal neuronal count at 12 or 18 months of age (Slow et al., 2005). The shortstop mouse therefore illustrated in vivo the inability of Htt inclusions to have a toxic effect over the lifespan of an organism, clearly demonstrating that Htt nuclear inclusions are not toxic.

Here we took an advantage of the YAC128 and shortstop HD mouse models to further evaluate the importance of Ca2+ signaling in HD pathogenesis. The results obtained are consistent with the hypothesis that disturbed neuronal Ca2+ signaling plays a significant role in the degeneration of MSNs containing full-length mutant Httexp. Furthermore, the results derived from our analysis of SS MSNs support the premise that not all fragments of mutant Httexp are toxic to neurons.

Section snippets

Materials

Propidium iodide (PI), Fura-2 acetoxymethyl ester (Fura-2 AM) and tetramethylrhodamine methyl ester (TMRM+) were obtained from Molecular Probes. Glutamate, NMDA, CNQX,TTX, MPEP, CPCCOEt, (+) MK801 maleate, and ifenprodil were purchased from Tocris. Cell culture reagents were all from Life Technologies. All other reagents were from Sigma.

Primary neuronal cultures

Generation and characterization of YAC128 and shortstop mice has been previously described (Slow et al., 2003, Slow et al., 2005). In our experiments

Ca2+ signaling is destabilized in YAC128 MSNs but not in shortstop MSNs

In the first series of experiments we compared Ca2+ responses induced by glutamate application to YAC128 and shortstop (ss) MSNs at 13–14 DIV. The corresponding wild-type littermate cultures (WT and ss-WT) were used as an internal control in these experiments. In our studies we adapted an experimental paradigm that we used previously in studies of Ca2+ signals in YAC128 MSNs (Tang et al., 2005, Tang et al., 2007). To mimic physiological conditions more closely, we applied repetitive pulses of

Discussion

The shortstop mouse, which expresses a short fragment (exons 1 and 2) of human mutant Httexp with 120 CAG repeat expansion was serendipitously established (Slow et al., 2005). The striatal and cortical neurons in the shortstop mice accumulate abundant Htt nuclear aggregates. However, these mice do not manifest a behavioral phenotype or striatal neuronal loss (Slow et al., 2005). These results are in contrast to the full-length YAC128 mouse generated on identical FVB/N background, which displays

Acknowledgments

We thank Xiangmei Kong and Huarui Liu for help with maintaining the YAC128 and shortstop mouse colony and Janet Young and Leah Benson for administrative assistance. IB, a Carla Cocke Francis Professor in Alzheimer's Research, is supported by the McKnight Neuroscience of Brain Disorders Award, the Robert A. Welch Foundation, the HighQ foundation, and NINDS R01NS38082 and R01NS056224. MRH, a Killam University Professor, holds a Canada Research Chair in Human Genetics and is supported by grants

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