Hunting for the cause: Evidence for prion-like mechanisms in Huntington's disease

Abstract

The hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell-to-cell in the brain in a manner akin to infectious prions has gained substantial momentum due to an explosion of research in the past 10-15 years. Here, we review current evidence supporting the existence of prion-like mechanisms in Huntington's disease (HD), an autosomal dominant neurodegenerative disease caused by expansion of a CAG repeat tract in exon 1 of the huntingtin (HTT) gene. We summarize information gained from human studies and in vivo and in vitro models of HD that strongly support prion-like features of the mutant HTT (mHTT) protein, including potential involvement of molecular features of mHTT seeds, synaptic structures and connectivity, endocytic and exocytic mechanisms, tunneling nanotubes, and nonneuronal cells in mHTT propagation in the brain. We discuss mechanisms by which mHTT aggregate spreading and neurotoxicity could be causally linked and the potential benefits of targeting prion-like mechanisms in the search for new disease-modifying therapies for HD and other fatal neurodegenerative diseases.

Keywords: Huntington’s disease; aggregate seed; aggregate spread; mutant huntingtin; polyglutamine; prion-like transmission; protein aggregate.

FIGURE 1

HTT structure and aggregation mechanism. (A) Primary protein structure of full-length human HTT highlighting the N-terminal polyQ tract encoded by exon 1, calpain (clp) and caspase (casp) 3 and 6 cleavage sites, and 7 regions containing 36 HEAT repeat (HR). PolyQ tract lengths associated with wtHTT (n ≤ 36) or mHTT (n ≥ 37) proteins are indicated by green and red, respectively, in and below the protein structure. (B) mHTT aggregation occurs via nucleated growth polymerization. wtHTT proteins achieve their native, functional fold, whereas expanded polyQ tracts cause mHTT proteins to misfold and a stabilize once a critical nucleus is achieved. This rate-limiting step is followed by rapid addition of mHTT monomers via templated misfolding to form soluble oligomers and, ultimately, insoluble, β-sheet-rich, amyloid fibrils. Prion-like conversion of wtHTT also occurs via templated conformational stabilization of natively-folded wtHTT proteins by mHTT aggregate seeds.

FIGURE 2

Experimental approaches to monitor prion-like behavior of mHTT proteins. (A) Spreading of mHTT can be reported as a time-dependent loss of co-localization between mHTT-FP expressed in “donor” neurons and a co-expressed non-transmissible cytoplasmic protein marker such as synaptophysin- or synaptotagmin-GFP. This approach has been used to monitor the transmissibility of mHTT proteins in mouse and fly brains (see Table 1). A similar approach in vitro monitors internalization of exogenous fluorescently-labeled mHTT or polyQ aggregates by several unlabeled “acceptor” cell types, including neuronal cell lines (e.g., SH-SY5Y, Neuro2A, and PC12), COS-7 fibroblast-like cells, and THP1 macrophages. (B) Transfer of mHTT-FP proteins from donor cells can also be detected by monitoring acquisition of mHTT-GFP signal within the cytoplasm of acceptor cells labeled by a soluble FP such as GFP, BFP, or mCherry. This approach has been used to demonstrate cell-to-cell mHTT spreading between neurons in mouse brain slices and cultured neuronal or primary neuron cells. (C) Entry of extracellular polyQ or mHTT fibrils into the cytoplasm of numerous cell types (e.g., HEK, HeLa, and PC12) causes templated aggregation of wtHTT-FP proteins, detected by a phenotypic change in wtHTT-FP expression pattern (e.g., diffuse → punctate) or decreased solubility measured by biochemical methods. (D) The seeding capacity of mHTT-FP aggregates can be measured by examining the aggregation of cytoplasmic wtHTT-FP proteins in acceptor cells templated by mHTT-FP aggregate seeds from donor cells. This approach has been applied to in vitro (e.g., in co-cultured HEK cells) and in vivo (e.g., adult Drosophila brains) experimental systems. (E,F) Physical interaction between mHTT seeds and monomeric HTT proteins originating in donor and acceptor cells, respectively, has been reported using biomolecular fluorescence complementation (BiFC; E), where each HTT protein is fused to a non-fluorescent GFP fragment, or by fluorescence resonance energy transfer (FRET; F), where HTT is fused to FP FRET pairs (e.g., CFP/YFP or GFP/mCherry).

FIGURE 3

Mechanisms for cell-to-cell transmission of mHTT. Pathways reported to mediate inter-cellular transmission of mHTT aggregates are illustrated here and described in more detail in the text. mHTT aggregate release from donor cells (red cell; left) may be coupled to synaptic activity in presynaptic neurons (A), could occur with in exosomes released from a multivesicular body (MVB) (B), or could be passively released from dying cells (C). Entry of prion-like mHTT aggregates into acceptor cells has been reported to occur via bulk-phase or receptor-mediated endocytosis (D), direct penetration of the plasma membrane (E), or alternatively, aggregates may transfer directly from one cell cytoplasm to another via membrane-enclosed tunneling nanotubes (F). Phagocytic glia may play double-edged roles in HD through receptor-mediated engulfment of aggregates from neurons (G), which can lead to either clearance in the lysosome or aggregate “escape” from the glial phagolysosomal system prior to degradation (H). Disruption of normal endosomal or autophagosomal pathways may also underlie mHTT aggregate transmission to the cytoplasm of non-phagocytic cells (I). mHTT aggregates that evade lysosomal degradation as a result of endo/phagolysosomal defects could generate cytoplasmic reservoirs of prion-like mHTT species in “intermediate acceptor cells” (e.g., glia) and enhance aggregate seed transmission to other cells, such as post-synaptic neurons (J).