Suppression of aggregate formation of mutant huntingtin potentiates CREB-binding protein sequestration and apoptotic cell death
Introduction
Huntington's disease (HD), an autosomal dominant neurodegenerative disease, is caused by an abnormal polyglutamine expansion in the N-terminal region of huntingtin protein (Muchowski et al., 2002, Palmer et al., 1999). Although mutant huntingtin is a fundamental cause of HD, the specific molecular mechanisms responsible for the selective neuronal cell death remain to be elucidated (Aiken et al., 2009). HD is characterized by the aggregation of mutant huntingtin into intracellular deposits called aggregates or inclusion bodies. The protein aggregates have been reported in the brains of human patients and transgenic mouse models, as well as in transfected cell models of HD (Nucifora et al., 2001).
Although aggregates of mutant huntingtin are a pathological hallmark of HD, the role of inclusions in the pathogenesis of the disease is still controversial (Bossy-Wetzel et al., 2008). There have been reports indicating that aggregates of mutant huntingtin are the cause of neuronal death since there is a direct correlation between frequency of inclusions in neurons and severity of the disease (Becher et al., 1998, Sieradzan et al., 1999) and since inclusions precede the disease in transgenic mice (Davies et al., 1997). In addition, it has been reported that the formation of aggregates increases the susceptibility to apoptosis in cell models of HD (Hackam et al., 2000, Wellington et al., 1998). However, it has also been suggested that although a conformational change in mutant huntingtin due to the polyglutamine expansion may cause both aggregation and pathogenicity, they occur separately and independently (Kim et al., 1999, Klement et al., 1998, Saudou et al., 1998, Slow et al., 2005). Consistent with this view, in a YAC transgenic mouse model of HD, neuronal death was observed in the absence of inclusions (Leavitt et al., 2006). Furthermore, it has been suggested that the inclusions may be beneficial (Cummings et al., 1999). Indeed, it is possible that the inclusions represent a means to sequester the toxic N-terminal fragments and oligomers of mutant huntingtin which could cause more rapid and severe damage in the soluble form (Ciechanover, 2003, Gunawardena et al., 2003). The formation of inclusions was reported to result in a decrease in the levels of diffuse mutant huntingtin and an increase in neuronal survival (Arrasate et al., 2004).
Aberrant transcriptional regulation is one possible mechanism of mutant huntingtin toxicity because mutant huntingtin interacts with several transcriptional regulators such as TATA-binding protein (TBP) (Schaffar et al., 2004), CREB-binding protein (CBP) (Steffan et al., 2000), Sp-1 (Li et al., 2002), TBP-associated factor II 130 (TAFII130) (Dunah et al., 2002), and p53 (Steffan et al., 2000). CBP has been reported to be recruited into huntingtin aggregates, leading to inhibition of CBP-mediated transcription (Cong et al., 2005, Jiang et al., 2006). More recently, expression of mutant huntingtin has been shown to down-regulate peroxisome proliferator-activated receptorγ (PPARγ) coactivator-1α (PGC-1α), a key regulator of mitochondrial biogenesis and respiration (McGill and Beal, 2006). The decreased expression of PGC-1α is considered due to the fact that CREB signaling pathway is a predominant regulator of PGC-1α expression (Cui et al., 2006). Overexpression of CBP has been reported to rescue cells from polyglutamine-mediated toxicity in neuronal cell culture (McCampbell et al., 2000). In addition, sequestration of Sp1 and TAFII130 into huntingtin aggregates has been also reported, resulting in the inhibition of Sp1-mediated transcription (Li et al., 2002), suggesting that entrapment of transcription factors into mutant huntingtin aggregates might play an important role in the pathogenesis of the disease.
Although transcriptional dysregulation has been suggested as an important pathogenic mechanism of HD, the nature of pathogenic species of mutant huntingtin has been still unclear. In the present study, to further evaluate the role of aggregates of mutant huntingtin in the pathogenesis of the disease, aggregation propensity of exon 1 huntingtin was sterically hindered by linking a yellow fluorescent protein (YFP) either to amino- or carboxy-terminus of exon 1 huntingtin. N-terminally YFP linked mutant huntingtin (YFP-MT) exhibited significantly attenuated frequency of aggregates compared to C-terminally YFP linked mutant huntingtin (MT-YFP). Strikingly, less aggregate-bearing YFP-MT showed increased apoptotic cell death, potentiated transcriptional dysregulation, and increased CBP sequestration compared to more aggregate-bearing MT-YFP. These data clearly demonstrate that unmasked polyglutamine tract is essential in the pathogenic role of mutant huntingtin.
Section snippets
N-terminally YFP-tagged mutant huntingtin (YFP-MT) forms less aggregates compared to C-terminally YFP- tagged mutant huntingtin (MT-YFP)
To manipulate the propensity of aggregate formation of mutant huntingtin, yellow fluorescent protein (YFP), a variant of green fluorescent protein (GFP), was attached to huntingtin as steric hindrance for the aggregation of mutant huntingtin. YFP was connected to either N-terminus or C-terminus of exon 1 wild type or mutant huntingtin protein (Fig. 1A), given the fact that polyglutamine domain is located closer to the N-terminus than to the C-terminus in exon 1 huntingtin. Wild type exon 1
Discussion
Here, we present evidence that suppression of the aggregate formation of mutant huntingtin potentiates the pathology of HD in a transfected cell model. Linkage of YFP to the N-terminus of exon 1 mutant huntingtin resulted in a significantly attenuated frequency of aggregation of mutant huntingtin presumably due to steric hindrance by YFP, given the fact that the polyglutamine domain is located closer to the N-terminus of exon 1 huntingtin. N-terminally YFP-tagged, less aggregate-bearing, mutant
Construction of expression plasmids
The huntingtin of full length were created by subcloning a fragment of the huntingtin cDNA generated by PCR and included KpnI and XbaI restriction sites. The 263 and 191-bp product of interest was digested with KpnI and XbaI, and then subcloned into TA cloning vector. The KpnI-XbaI huntingtin cDNA fragments were also subcloned into pcDNA 3.1(+) vector. The KpnI and XbaI restriction sites were filled in with T4 DNA polymerase before ligation. All DNA constructs were verified by automated
Acknowledgments
This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2008-E00028).
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