Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates

Abstract

Sequence-specific nucleated protein aggregation is closely linked to the pathogenesis of most neurodegenerative diseases and constitutes the molecular basis of prion formation 1 . Here we report that fibrillar polyglutamine peptide aggregates can be internalized by mammalian cells in culture where they gain access to the cytosolic compartment and become co-sequestered in aggresomes together with components of the ubiquitin-proteasome system and cytoplasmic chaperones. Remarkably, these internalized fibrillar aggregates are able to selectively recruit soluble cytoplasmic proteins with which they share homologous, but not heterologous amyloidogenic sequences and to confer a heritable phenotype upon cells expressing the homologous amyloidogenic protein from a chromosomal locus.Genetic expansion of CAG triplets in protein-coding regions of otherwise unrelated genes is the underlying cause of a family of dominantly inherited neurodegenerative diseases including Huntington disease (HD) and spinocerebellar ataxias 2 . The tracts of polyglutamine (polyQ) homopolymers (Q ≥ 40) encoded by these expanded CAG triplets cause the normally soluble protein products of these genes, or fragments thereof, to form cytotoxic protein aggregates 2 . CAG expansion diseases therefore, belong to a much larger family of protein "conformational diseases," including systemic and organ-specific amyloidosis, Alzheimer's disease and prion encephalopathy. Pathogenesis in these diseases is tightly linked to the formation of high molecular weight, fibrillar, β-sheet rich, insoluble protein aggregates, termed "amyloid," that accumulate in characteristic sites either inside or outside of the cell 1, 3 .In amyloidosis, insoluble protein fibrils derived from normally soluble secreted proteins are deposited in the extracellular milieu causing damage to surrounding viscera, blood vessel walls and connective tissue 4 . Whether organ damage is a consequence of tissue disruption or obstruction due to the sheer mass of deposited protein, as in the case of systemic amyloidosis 4 , or to an intrinsic cytotoxicity of amyloids or their oligomeric precursors, as in the case of neuropathic amyloidosis 5 , remains a critical but unresolved question. In contrast to amyloidosis, most neurodegenerative diseases are caused by alterations in the conformation and oligomeric state of normally well-behaved intracellular proteins that, in diseased states, accumulate within cytoplasmic or nuclear inclusion bodies 6 . Emerging evidence suggests that oligomeric precursors to these large assemblies are cytotoxic and directly impair crucial Many extracellular amyloids and amyloid precursors, including those associated with systemic amyloidosis, neurodegenerative disease, and even those not associated with disease 7 , can be taken-up by a wide variety of cell types including macrophages, neurons, fibroblasts, and epithelial cells [7][8][9][10] . This uptake is reported to occur via phagocytic or endocytic processes that result in delivery to lysosomes which may suppress...

A deadly spread: cellular mechanisms of α-synuclein transfer

“…They were taken up by cultured cells expressing Htt with a nonpathogenic polyQ tract (25Q), inducing the aggregation of the 25Q-Htt, which normally should not fibrillize. 78 These results support the theory of a seeding process. The nucleation capacity of mutated Htt was recently further documented by a novel technique where the fluorescence signal within cultured cells was correlated to the extent of Htt exon 1 protein aggregation.…”

“…79 Finally, the incapacity of other amyloid aggregates, such as the prion domain of the yeast Sup35 prion protein or Ab, to nucleate wild-type Htt underscores the importance of sequence specificity for the nucleation of polyQ fibrils. 78 Such specificity may likely also apply to intracellular nucleationdependent seeding of a-syn, but it remains to be investigated.…”

Prion-Like Propagation of Protein Aggregation and Related Therapeutic Strategies

“…Expanded polyQ peptide amyloids gain access to the cytoplasm of a variety of mammalian cells upon introduction to the culture medium [80]. The fibrils that are taken into the cell can induce the aggregation of other proteins with polyQ tracts, and this misfolding is maintained for multiple passages, implying the aggregates formed from endogenously expressed protein can also seed further intracellular misfolding and aggregation, similar to yeast prions [81].…”

“…Htt aggregate release into the media has not been clearly documented, but cell lystates promote aggregation in an acceptor cell population [81]. Cells can therefore take up Htt, but cell-to-cell transfer of these aggregates appears relatively inefficient compared to the other aggregationprone proteins discussed above.…”

Proteolytic clearance of extracellular α-synuclein as a new therapeutic approach against Parkinson disease

“…45,46 Prion-like propagation of SOD-1 aggregates have also been reported both in vivo and in vitro. 47,48 Polyglutamine peptide aggregates observed in polyglutamine diseases have also been reported to be internalized by cells and become co-sequestered in aggreasomes with cytosolic proteins, 49 implying the possibility of the intercellular spreading of polyglutamine aggregates.…”