Accumulation of ubiquitin conjugates in a polyglutamine disease model occurs without global ubiquitin/proteasome system impairment

Abstract

Aggregation-prone proteins have been suggested to overwhelm and impair the ubiquitin/proteasome system (UPS) in polyglutamine (polyQ) disorders, such as Huntington's disease (HD). Overexpression of an N-terminal fragment of mutant huntingtin (NmutHtt), an aggregation-prone polyQ protein responsible for HD, obstructs the UPS in cellular models. Furthermore, based on the accumulation of polyubiquitin conjugates in brains of R6/2 mice, which express human N-mutHtt and are one of the most severe polyQ disorder models, it has been proposed that UPS dysfunction is a consistent feature of this pathology, occurring in both in vitro and in vivo models. Here, we have exploited transgenic mice that ubiquitously express a ubiquitin fusion degradation proteasome substrate to directly assess the functionality of the UPS in R6/2 mice or the slower onset R6/1 mice. Although expression of N-mutHtt caused a general inhibition of the UPS in PC12 cells, we did not observe an increase in the levels of proteasome reporter substrate in the brains of R6/2 and R6/1 mice. We show that the increase in ubiquitin conjugates in R6/2 mice can be primarily attributed to an accumulation of large ubiquitin conjugates that are different from the conjugates observed upon UPS inhibition. Together our data show that polyubiquitylated proteins accumulate in R6/2 brain despite a largely operative UPS, and suggest that neurons are able to avoid or compensate for the inhibitory effects of N-mutHtt.Huntington ͉ neurodegeneration ͉ protein degradation T he primary proteolytic machinery responsible for the turnover of proteins in the cytosol and nuclei of cells, including the destruction of misfolded or otherwise abnormal proteins, is the ubiquitin/proteasome system (UPS) (1). The UPS is in essence a two-step process: the targeting of proteins through the covalent linkage of polyubiquitin chains (2), and the destruction of ubiquitylated proteins by the proteasome (3). A number of studies have implicated UPS dysfunction in a range of polyglutamine (polyQ) neurodegenerative diseases (4). The aggregation-prone polyQ proteins are postulated to impair the UPS in these diseases, either by overloading the capacity of the cell's UPS machinery (5), by sequestration of essential components of the UPS into inclusions (6), or by obstruction of the proteasome (7). If polyQ proteins themselves inhibit the system crucial to their own degradation, this could elicit a self-perpetuating pathogenic cascade of events, both accelerating the accumulation of the toxic protein, and impairing essential regulatory functions of the UPS (4). With the help of specifically designed reporter substrates, it has been shown that polyQ proteins can cause UPS impairment in cell lines (5,8,9). However, since these experiments rely on acute overexpression of the polyQ protein in cells with limited physiological relevance, it is difficult to extrapolate these findings to the status of the UPS during the progression of the disease in patients.In contrast to the observation with in vitro models, ...

Acute Polyglutamine Expression in Inducible Mouse Model Unravels Ubiquitin/Proteasome System Impairment and Permanent Recovery Attributable to Aggregate Formation

“…However, N-mutHttinduced UPS impairment in vivo is transient and, in good agreement with previous reports combining PolyQ mouse models with the same (Bowman et al, 2005;Maynard et al, 2009) or similar (Bett et al, 2009) reporter mice, it is not detected with constitutive N-mutHtt expression in adult mice. Finally, that aggregate formation correlates with UPS recovery had also been recently reported in a cell model (Mitra et al, 2009), and here we were able to demonstrate causality with the use of antiaggregation compounds in a cell model and also in vivo in mouse models.…”

“…However, similar studies on mouse models failed to detect UPS impairment either by measuring proteasome activity in brain extracts (Diaz-Hernandez et al, 2003;Bett et al, 2006) or by using UPS reporter mice that express specifically designed proteasome substrates (Bett et al, 2009;Maynard et al, 2009). Moreover, we have recently shown that the increase in polyubiquitin conjugates in the brain of R6/2 mice (Bennett et al, 2007) occurs in the absence of a general UPS impairment .…”

Protein misfolding specifies recruitment to cytoplasmic inclusion bodies

“…Although this model is consistent with data showing a strong correlation between mutant htt aggregation and accumulation of synthetic short-lived substrates of the UPS in cell culture (Bence et al, 2001;Bennett et al, 2005), these UPS substrate reporters do not accumulate (Bett et al, 2009;Maynard et al, 2009) or only accumulate transiently (Ortega et al, 2010) in mouse models of HD. In cell culture, mutant htt aggregation and recruitment of Ub conjugates to nascent IBs is not temporally correlated with accumulation of UPS reporters (Hipp et al, 2012).…”

Role of the ubiquitin–proteasome system in nervous system function and disease: using C. elegans as a dissecting tool

“…A recent study also suggested that toxic htt forms do not impair the proteasome and that, instead, the impairment of the UPS arises from the accumulation of N-terminal fragments [200]. Supporting this model, an HD mouse study showed no global impairment of UPS activities, and the forms of polyubiquitylated proteins accumulated in this model were of a different nature than those found upon proteasome inhibition [201]. In contrast, impairment of the UPS was demonstrated in transfected HEK293 cells upon polyQ aggregation, even before inclusion of body formation [183].…”