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The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro. Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies. [ABSTRACT FROM AUTHOR]

Juxtanuclear aggresomes form in cells when levels of aggregation- prone proteins exceed the capacity of the proteasome to degrade them. It is widely believed that aggresomes have a protective function, sequestering potentially damaging aggregates until these can be removed by autophagy. However, most in-cell studies have been carried out over a few days at most, and there is little information on the long term effects of aggresomes. To examine these long term effects, we created inducible, singlecopy cell lines that expressed aggregation-prone polyglutamine proteins over several months. We present evidence that, as perinuclear aggresomes accumulate, they are associated with abnormal nuclear morphology and DNA double-strand breaks, resulting in cell cycle arrest via the phosphorylated p53 (Ser-15)- dependent pathway. Further analysis reveals that aggresomes can have a detrimental effect on mitosis by steric interference with chromosome alignment, centrosome positioning, and spindle formation. The incidence of apoptosis also increased in aggresome-containing cells. These severe defects developed gradually after juxtanuclear aggresome formation and were not associated with small cytoplasmic aggregates alone. Thus, our findings demonstrate that, in dividing cells, aggresomes are detrimental over the long term, rather than protective. This suggests a novel mechanism for polyglutamine-associated developmental and cell biological abnormalities, particularly those with early onset and non-neuronal pathologies. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and a-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in a-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates a-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect a-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-b 1-42 (Ab42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiq proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies. [ABSTRACT FROM AUTHOR]