Live-cell imaging reveals divergent intracellular dynamics of polyglutamine disease proteins and supports a sequestration model of pathogenesis

Abstract

Protein misfolding and aggregation are central features of the polyglutamine neurodegenerative disorders, but the dynamic properties of expanded polyglutamine proteins are poorly understood. Here, we use fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) with green fluorescent protein fusion proteins to study polyglutamine protein kinetics in living cells. Our results reveal markedly divergent mobility states for an expanded polyglutamine protein, ataxin-3, and establish that nuclear inclusions formed by this protein are aggregates. Additional studies of green fluorescent proteintagged cAMP response element binding protein coexpressed with either of two mutant polyglutamine proteins, ataxin-3 and huntingtin, support a model of disease in which coaggregation of transcriptional components contributes to pathogenesis. Finally, studies of a third polyglutamine disease protein, ataxin-1, reveal unexpected heterogeneity in the dynamics of inclusions formed by different disease proteins, a finding which may help explain disease-specific elements of pathogenesis in these neurodegenerative disorders.A t least nine inherited neurodegenerative diseases are caused by CAG triplet repeat expansions that encode expanded polyglutamine (polyQ) in the disease proteins (1). A unifying feature of polyQ diseases is the formation of neuronal intracellular inclusions by the disease protein, most commonly in the nuclear inclusions (NIs). Growing evidence suggests that polyQ expansion promotes protein misfolding, resulting in neuronal dysfunction and degeneration by still unknown mechanisms.Two leading, yet compatible, theories of polyQ pathogenesis are failures in protein homeostasis and perturbations in transcriptional regulation. Evidence implicating problems in protein homeostasis includes the formation of ubiquitin-positive inclusions in disease tissue, the recruitment of molecular chaperones and proteasome components to NI, and the ability of molecular chaperones to suppress toxicity in disease models (2-11). Evidence supporting transcriptional dysregulation includes expanded polyQ-induced changes in gene expression, the inhibition of histone acetyltransferases by polyQ proteins, and the fact that at least two disease proteins are transcription factors (1, 12-13). In addition, the nucleus is recognized to be a critical site for polyQ toxicity (14-16), suggesting that one or more nuclearspecific functions are perturbed in disease.A compelling link between these two leading theories is the fact that many proteins implicated in transcriptional control become redistributed into NI (17-27). One intriguing example is the transcription coactivator, cAMP response element binding protein (CREB)-binding protein (CBP), which colocalizes to the NI formed by several expanded polyQ proteins (18-20, 25, 27). Moreover, overexpressed CBP suppresses polyQ toxicity in cellular models of polyQ disease. These results suggest a model of pathogenesis (25) in which sequestration of CBP by polyQ proteins inhib...

Mutant Protein Kinase Cγ Found in Spinocerebellar Ataxia Type 14 Is Susceptible to Aggregation and Causes Cell Death

“…5). This result is consistent with previous reports that the GFP fluorescence of polyglutamine protein aggregates is not recovered after photobleaching (36,37). These findings indicate that dot-like accumulations of mutant ␥PKC-GFP are indeed aggregates, but are not the simple result of targeting to particular cell organelles.…”

Nuclear Aggresomes Form by Fusion of PML-associated Aggregates

“…Similar dynamic mobility is exhibited by aggregates of poly-Q expanded ataxin-1 molecules (Stenoien et al, 2002). Both contrast with the behavior of aggregates of poly-Q expanded ataxin-3 that are largely immobile and do not exchange their components (Chai et al, 2002). These results indicate that structures defined morphologically as visible inclusions can be true aggregates (as those formed by ataxin-3) or concentrated depositions of rapidly exchanging components (as those formed by ataxin-1 and GFP170*).…”

“…The t 1/2 of fluorescence recovery to steady state levels is ϳ50 s. The recovery of GFP170* is slower than that of a poly-Q expanded GFP-ataxin-1-84Q (t 1/2 of Ͻ2 s) deposited in nuclear inclusions of similar size (Stenoien et al, 2002). However, GFP170* recovers significantly faster than the almost immobile poly-Q expanded ataxin-3 (Chai et al, 2002) or Q82-GFP (Kim et al, 2002). To provide a direct comparison between dynamics of GFP170* and an immobile aggregate, we analyzed FRAP of Q82-GFP.…”

A network of protein interactions determines polyglutamine toxicity

“…Several studies have convincingly demonstrated that the interaction of polyQ-expanded htt proteins with other Q-rich proteins correlates with toxicity in mammalian cells (14,24,25). Because all of the previously studied Q-rich proteins are essential, their sequestration into polyQ aggregates, and the ensuing loss of their function could well have been the sole mechanism accounting for toxicity (26)(27)(28)(29)(30).…”