Elsevier

Methods in Enzymology

Volume 412, 2006, Pages 106-122
Methods in Enzymology

Imaging Polyglutamine Deposits in Brain Tissue

https://doi.org/10.1016/S0076-6879(06)12008-XGet rights and content

Abstract

The formation of polyglutamine aggregates occupies a central role in the pathophysiology of neurodegenerative diseases caused by expanded trinucleotide repeats encoding the amino acid glutamine. This chapter describes sensitive histological methods for detection of tissue sites that are capable of further recruitment of polyglutamine and for sites rich in polyglutamine defined immunohistochemically. These methods have been found to be applicable in a number of diseases and animal models of disease. Recruitment, which is a property of highly ordered, amyloid‐like aggregates, is most commonly found in punctate sites, termed aggregation foci (AF), in the neuronal perikaryonal cytoplasm. As expected, these AF correspond to sites containing polyglutamine aggregates detected using the antibody 1C2. Interestingly, however, many of the latter sites, including most neuropil aggregates and neuronal intranuclear inclusions, exhibit a limited ability to support polyglutamine recruitment. Thus there is limited correlation between the distribution of polyglutamine aggregates and recruitment activity, suggesting functional heterogeneity among polyglutamine aggregates. These methods should prove useful in explaining the relationship between aggregation reactions, aggregate formation, and the development of symptomatic disease and should be adaptable to the study of other protein aggregation disorders.

Introduction

A number of hereditary degenerative diseases are caused by mutations that involve expanded trinucleotide repeats in the affected gene. The largest number of these entails expansion of the trinucleotide, CAG, in a frame that encodes the amino acid glutamine and includes nine neurodegenerative disorders of which Huntington's disease (HD), dentatorubral and pallidoluysian atrophy (DRPLA), and several spinocerebellar atrophies (SCA1, 2, 3, 6, 7, and 17) are conventional autosomal dominant diseases, whereas spinal bulbar muscular atrophy (SBMA) is, in effect, a sex‐limited recessive disorder (Wells et al., 1998).

A common histopathological feature of each of the CAG‐repeat diseases is the detection at autopsy of inclusion bodies. Initially detected in SCA1 (Cummings et al., 1998), SCA3 (Paulson et al., 1997), and HD (DiFiglia et al., 1997) as ubiquitinylated intraneuronal intranuclear inclusions, these structures have invariably been detected in glutamine‐encoding CAG repeat disorders and in transgenic or knock‐in animal models of these diseases and have been found to include the polyglutamine‐containing segment of the affected gene product, most commonly as a proteolytic fragment of the protein (Bates et al., 2002). Although only intranuclear inclusions are seen in most of these diseases, in HD numerous cytoplasmic inclusions are the dominant inclusion body in cortex being found in neuronal axons and dendrites; these inclusion bodies have been termed neuropil aggregates, because they are only rarely seen in the neuronal perikaryon (Gutekunst et al., 1999).

The physical properties of polyglutamine aggregates have been considered to reflect an amyloid‐like structure (i.e., the appearance of fibrillar or ribbon‐like structures in electron micrographs) (Scherzinger et al., 1999) and a composition rich in beta‐pleated sheets (Perutz et al., 1994). Because the inclusion bodies in CAG‐repeat diseases display the tinctorial properties of amyloid (Huang et al., 1998), that is, the appearance of birefringence when stained with Congo red, these inclusions can be considered to meet the formal histopathological criteria for amyloid.

The invariable presence of polyglutamine‐containing inclusion bodies taken together with the discovery that polyglutamine readily aggregates into essentially insoluble amyloid‐like structures gave rise to the idea that aggregate formation is the common cause of the expanded CAG repeat diseases. Subsequently, various studies have been interpreted to suggest that polyglutamine aggregates can be toxic, inert, or even protective (Scherzinger 1997, Arrasate 2004). The frequent presence of intranuclear inclusions in large numbers of seemingly unaffected neurons in these diseases indicates that these structures are not invariably neurotoxic. Nevertheless, there remain no a priori reasons to reject the hypothesis that the propensity of critical lengths of repetitive polyglutamine sequences to readily form aggregates is, indeed, the underlying cause of these diseases. Considerations of aggregate etiology, coupled with mounting experimental evidence, suggest that polyglutamine proteins can form a variety of aggregates of different sizes, morphologies, and functional characteristics (Wetzel, 2006). Thus, although large inclusions may prove to not have a toxic role, other aggregated states, and/or the aggregation process itself, may be implicated in the disease mechanism. Such arguments draw attention to the critical need for improved methods for detection of aggregates, and in particular requirements for enhanced sensitivity and the ability to discriminate among different aggregate types.

Inclusion bodies were initially detected by immunoreactivity with antibodies to ubiquitin or with antibodies to the respective mutant protein (e.g., ataxin‐3 in SCA3, ataxin‐1 in SCA1, and huntingtin in HD). Subsequently, in HD, antibodies with selective reactivity for the aggregated fragments of the protein were used to demonstrate that anti‐ubiquitin antibodies, although reactive with all intranuclear inclusions, only detected a subset of neuropil aggregates (Gutekunst et al., 1999); furthermore, the use of antibodies specific to various regions of the huntingtin molecule showed that only fragments of the N‐terminal region of the molecule was contained within the inclusion bodies (Lunkes et al., 1999). The presence of the polyglutamine‐containing segment in inclusion bodies was confirmed by the use of polyglutamine‐specific monoclonal antibodies and one of these, 1C2, has become the de facto standard reagent for the immunochemical detection of polyglutamine (Trottier et al., 1995).

Because of solubility problems, initial observations on the formation of amyloid‐like fibrils were based on studies with short lengths of polyglutamine peptides (Perutz et al., 1994). Development in this laboratory of novel peptides together with procedures to solubilize and maintain pathological lengths of polyglutamine in solution (Chen and Wetzel, 2001) permitted the study of both solution‐phase and solid‐phase aggregation reactions of such peptides in vitro, demonstrating for the first time the kinetics of recruitment and elongation reactions of various lengths of polyglutamine (Berthelier 2001, Chen 2002). The logical application of these methods to the detection of polyglutamine recruitment in HD brain tissue and in animal models of HD, demonstrating sites at which active aggregate formation was ongoing, was successfully undertaken. Discrete intraneuronal structures were readily demonstrated, and these have been termed aggregation foci. The selective and sensitive methods that have been developed to detect these sites are the subject of this chapter, and the methods should be applicable to all glutamine‐encoding CAG repeat diseases, although these have only been thoroughly investigated in HD and in a number of rodent models of HD (Menalled 2003, Slow 2003, von Horsten 2003) and recently found in a mouse model of Machado‐Joseph disease (spinocerebellar ataxia‐3) (Goti et al., 2004).

Section snippets

Acknowledgments

This work was supported by grants and contracts from the Hereditary Diseases Foundation to R. W. and A. O. The methods were established and validated using human tissues obtained from the Harvard Brain Tissue Resource Center, funded by R24‐MH068855, from the Human Brain and Spinal Fluid Resource Center, and from the New York Brain Bank, and, using brain tissue from rodent models of polyglutamine diseases, from Michael Hayden and Elizabeth Slow, University of British Columbia, from

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