Huntingtin and Mutant SOD1 Form Aggregate Structures with Distinct Molecular Properties in Human Cells

Abstract

Expression of many proteins associated with neurodegenerative disease results in the appearance of misfolded species that readily adopt alternate folded states. In vivo, these appear as punctated subcellular structures typically referred to as aggregates or inclusion bodies. Whereas groupings of these distinct proteins into a common morphological class have been useful conceptually, there is some suggestion that aggregates are not homogeneous and can exhibit a range of biological properties. In this study, we use dynamic imaging analysis of living cells to compare the aggregation and growth properties of mutant huntingtin with polyglutamine expansions or mutant SOD1 (G85R/G93A) to examine the formation of aggregate structures and interactions with other cellular proteins. Using a dual conditional expression system for sequential expression of fluorescence-tagged proteins, we show that mutant huntingtin forms multiple intracellular cytoplasmic and nuclear structures composed of a dense core inaccessible to nascent polypeptides surrounded by a surface that stably sequesters certain transcription factors and interacts transiently with molecular chaperones. In contrast, mutant SOD1 (G85R/G93A) forms a distinct aggregate structure that is porous, through which nascent proteins diffuse. These results reveal that protein aggregates do not correspond to a single common class of subcellular structures, and rather that there may be a wide range of aggregate structures, perhaps each corresponding to the specific disease-associated protein with distinct consequences on the biochemical state of the cell.Accumulation of abnormal protein deposits as aggregates or inclusion bodies is a common cytological feature of a number of disease states as represented by clinically related neurodegenerative diseases. The mutant proteins that initiate protein aggregates in many of these diseases have been identified: A␤ in Alzheimer disease, PrP in prion diseases, ␣-synuclein in Parkinson disease, huntingtin in Huntington disease, tau in tauopathy, and SOD1 in familial amyotrophic lateral sclerosis (1-3). Although these proteins do not share distinctive common features in their respective primary sequences, they have all been shown to adopt alternate conformational states and form misfolded protein structures that appear visually as aggregates and inclusion bodies that correlate with disease pathology. There is increasing evidence to support a "toxic gain-of-function" mechanism by which misfolded protein and aggregate structures lead to a dominant pathological phenotype (2, 4).A molecular basis for proteotoxicity is the aberrant interactions between aggregation-prone proteins and other cellular proteins. This is in part supported by in vivo polyglutamine disease models in which aggregates have been shown to contain specific transcription factors, cytoskeletal, autophagy, and degradative proteins as well as molecular chaperones (5-14). The recruitment and sequestration of these cellular proteins has been proposed to lead to functional deplet...

Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state

“…Aggregate size and morphology was likewise unaltered (Fig. 2b) similar to what has been reported previously 20,21 . Consistent with the visual increase in aggregate-containing cell numbers was an increase in htt protein detected by the filter trap assay for 7.5-fold (Fig.…”

Compartmentalization of superoxide dismutase 1 (SOD1G93A) aggregates determines their toxicity

“…Other experiments have suggested that familial ALS (fALS)-associated mutant SOD1 is most toxic when localized to an IB (8). It has also been shown that toxic SOD1 IBs are more dynamic than Htt inclusions, and interact with quality control machinery (19,23). The differences between these IBs may represent differences in function (degradation vs. sequestration).…”

“…Given that yeast studies of JUNQ and IPOD (15) as well as several studies in mammalian cells (19) have observed aggregate inclusions with different properties, we set out to coexpress different types of aggregation-prone proteins to track their localization and effect on cell viability. By imaging live cells expressing fluorescently tagged proteins, we observed that in human cells, as in yeast, normal misfolded proteins (VHL and Ubc9 ts ) localize to JUNQ-like compartments (Fig.…”

Different anti-aggregation and pro-degradative functions of the members of the mammalian sHSP family in neurological disorders

“…HSPB6, which only effectively suppresses shorter polyQs [1,107], also plays a role in modulating autophagy [108]. By contrast, HSPB7 does not change the rate of proteasomal degradation and does not increases the autophagic flux; rather, it appears to prevent early aggregates from nucleating into inclusions with sizes that are too large to be handled by the autophagic machinery, probably by marking these early seeds, which enables their shuttling into the autophagosomes [1,57,109,110]. This HSPB7 action thus does not rely on the speed of seed formation but rather on the rate at which these seeds grow (and thus is less dependent on the size of the expansion).…”

“…Indeed, the effects of HSPB members on different aggregation-causing mutants seem to differ widely (table 1). It has been shown that, while polyQ proteins form aggregates with a core that is inaccessible to nascent proteins, mutated SOD1 (G85R/G93A), associated with ALS, forms a porous aggregate, through which nascent proteins can diffuse [110]. As stated above, we have already shown that overexpression of HSPB8 efficiently prevented the aggregation and facilitated the autophagy-mediated degradation of mutated SOD1 and of various mutated forms of TDP43, which is associated with both ALS and FTD [43] (figure 3a).…”