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A pathological hallmark of many protein-misfolding diseases is the formation of insoluble aggregates. Quantitative methods are needed to better resolve and define the formation, aggregation, and temporal dynamics of soluble misfolded proteins in native settings. In this book chapter we describe simple and sensitive detection methods to characterize high ordered aggregates (AGERA) and subsets of distinct soluble aggregates (SEC-FRET) of mutant huntingtin protein in biological samples.

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the amplification of a polyglutamine stretch at the N terminus of the huntingtin protein. N-terminal fragments of the mutant huntingtin (mHtt) aggregate and form intracellular inclusions in brain and peripheral tissues. Aggregates are an important hallmark of the disease, translating into a high need to quantify them in vitro and in vivo. We developed a one-step TR-FRET-based immunoassay to quantify soluble and aggregated mHtt in cell and tissue homogenates. Strikingly, quantification revealed a decrease of soluble mHtt correlating with an increase of aggregated protein in primary neuronal cell cultures, transgenic R6/2, and HdhQ150 knock-in HD mice. These results emphasize the assay's efficiency for highly sensitive and quantitative detection of soluble and aggregated mHtt and its application in high-throughput screening and characterization of HD models., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The Huntington's disease (HD) mutation leads to a complex process of Huntingtin (Htt) aggregation into multimeric species that eventually form visible inclusions in cytoplasm, nuclei and neuronal processes. One hypothesis is that smaller, soluble forms of amyloid proteins confer toxic effects and contribute to early cell dysfunction. However, analysis of mutant Htt aggregation intermediates to identify conformers that may represent toxic forms of the protein and represent potential drug targets remains difficult. We performed a detailed analysis of aggregation conformers in multiple in vitro, cell and ex vivo models of HD. Conformation-specific antibodies were used to identify and characterize aggregation species, allowing assessment of multiple conformers present during the aggregation process. Using a series of assays together with these antibodies, several forms could be identified. Fibrillar oligomers, defined as having a β-sheet rich conformation, are observed in vitro using recombinant protein and in protein extracts from cells in culture or mouse brain and shown to be globular, soluble and non-sedimentable structures. Compounds previously described to modulate visible inclusion body formation and reduce toxicity in HD models were also tested and consistently found to alter the formation of fibrillar oligomers. Interestingly, these compounds did not alter the rate of visible inclusion formation, indicating that fibrillar oligomers are not necessarily the rate limiting step of inclusion body formation. Taken together, we provide insights into the structure and formation of mutant Htt fibrillar oligomers that are modulated by small molecules with protective potential in HD models.

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington's Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.

Inclusion bodies of aggregated mutant huntingtin (htt) fragments are a neuropathological hallmark of Huntington disease (HD). The molecular chaperones Hsp70 and Hsp40 colocalize to inclusion bodies and are neuroprotective in HD animal models. How these chaperones suppress mutant htt toxicity is unclear but might involve direct effects on mutant htt misfolding and aggregation. Using size exclusion chromatography and atomic force microscopy, we found that mutant htt fragments assemble into soluble oligomeric species with a broad size distribution, some of which reacted with the conformation-specific antibody A11. Hsp70 associated with A11-reactive oligomers in an Hsp40- and ATP-dependent manner and inhibited their formation coincident with suppression of caspase 3 activity in PC12 cells. Thus, Hsp70 and Hsp40 (DNAJB1) dynamically target specific subsets of soluble oligomers in a classic ATP-dependent reaction cycle, supporting a pathogenic role for these structures in HD.

Huntington disease (HD) is caused by an expansion of more than 35-40 polyglutamine (polyQ) repeats in the huntingtin (htt) protein, resulting in accumulation of inclusion bodies containing fibrillar deposits of mutant htt fragments. Intriguingly, polyQ length is directly proportional to the propensity for htt to form fibrils and the severity of HD and is inversely correlated with age of onset. Although the structural basis for htt toxicity is unclear, the formation, abundance, and/or persistence of toxic conformers mediating neuronal dysfunction and degeneration in HD must also depend on polyQ length. Here we used atomic force microscopy to demonstrate mutant htt fragments and synthetic polyQ peptides form oligomers in a polyQ length-dependent manner. By time-lapse atomic force microscopy, oligomers form before fibrils, are transient in nature, and are occasionally direct precursors to fibrils. However, the vast majority of fibrils appear to form by monomer addition coinciding with the disappearance of oligomers. Thus, oligomers must undergo a major structural transition preceding fibril formation. In an immortalized striatal cell line and in brain homogenates from a mouse model of HD, a mutant htt fragment formed oligomers in a polyQ length-dependent manner that were similar in size to those formed in vitro, although these structures accumulated over time in vivo. Finally, using immunoelectron microscopy, we detected oligomeric-like structures in human HD brains. These results demonstrate that oligomer formation by a mutant htt fragment is strongly polyQ length-dependent in vitro and in vivo, consistent with a causative role for these structures, or subsets of these structures, in HD pathogenesis.

Huntington disease (HD) is a neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) domain in the N-terminal region of huntingtin (htt). PolyQ expansion above 35-40 results in disease associated with htt aggregation into inclusion bodies. It has been hypothesized that expanded polyQ domains adopt multiple potentially toxic conformations that belong to different aggregation pathways. Here, we used atomic force microscopy to analyze the effect of a panel of anti-htt antibodies (MW1-MW5, MW7, MW8, and 3B5H10) on aggregate formation and the stability of a mutant htt-exon1 fragment. Two antibodies, MW7 (polyproline-specific) and 3B5H10 (polyQ-specific), completely inhibited fibril formation and disaggregated preformed fibrils, whereas other polyQ-specific antibodies had widely varying effects on aggregation. These results suggest that expanded polyQ domains adopt multiple conformations in solution that can be readily distinguished by monoclonal antibodies, which has important implications for understanding the structural basis for polyQ toxicity and the development of intrabody-based therapeutics for HD.

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.