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Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

ABSTRACT: Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthetic polyglutamine peptides as the substrate for amyloid formation, we found that partially purified misfolded HTT obtained from end-stage brain tissue of two Tg HD mouse models and brain tissue of post-mortem human HD patients was capable of specifically accelerating polyglutamine amyloid formation compared with unseeded reactions and controls. Alzheimer and prion disease brain tissues did not do so, demonstrating the specificity of the ASA. It is unclear whether early intermediates or later conformational species in the protein misfolding process act as seeds in the ASA for HD. However, we were able to detect misfolded protein in the brains of YAC128 mice early in disease pathogenesis (11 weeks of age), whereas large inclusion bodies have not been observed in the brains of these mice by histology until 78 weeks of age, much later in the pathogenic process. The sensitive detection of misfolded HTT protein early in the disease pathogenesis in the YAC128 Tg mouse model strengthens the argument for a causative role of protein misfolding in HD.

SUBMITTER: Gupta S

PROVIDER: S-EPMC3323021 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

Gupta Sharad S Jie Shy'Ann S Colby David W DW

The Journal of biological chemistry 20111220 13

Huntington disease (HD) is one of several fatal neurodegenerative disorders associated with misfolded proteins. Here, we report a novel method for the sensitive detection of misfolded huntingtin (HTT) isolated from the brains of transgenic (Tg) mouse models of HD and humans with HD using an amyloid seeding assay (ASA), which is based on the propensity of misfolded proteins to act as a seed and shorten the nucleation-associated lag phase in the kinetics of amyloid formation in vitro. Using synthe ...[more]

PMID: 22187438

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Project description:Huntington disease (HD) is a fatal neurodegenerative movement disorder caused by an expanded CAG repeat in the huntingtin gene (HTT). The mutant huntingtin protein is ubiquitously expressed, but only certain brain regions are affected. The hypothalamus has emerged as an important area of pathology with selective loss of neurons expressing the neuropeptides orexin (hypocretin), oxytocin and vasopressin in human postmortem HD tissue. Hypothalamic changes in HD may have implications for early disease manifestations affecting the regulation of sleep, emotions and metabolism. The underlying mechanisms of selective vulnerability of certain neurons in HD are not fully understood, but excitotoxicity has been proposed to play a role. Further understanding of mechanisms rendering neurons sensitive to mutant huntingtin may reveal novel targets for therapeutic interventions. In the present study, we wanted to examine whether transgenic HD mice display altered sensitivity to excitotoxicity in the hypothalamus. We first assessed effects of hypothalamic injections of the excitotoxin quinolinic acid (QA) into wild-type (WT) mice. We show that neuronal populations expressing melanin-concentrating hormone (MCH) and cocaine and amphetamine-regulated transcript (CART) display a dose-dependent sensitivity to QA. In contrast, neuronal populations expressing orexin, oxytocin, vasopressin as well as tyrosine hydroxylase in the A13 area are resistant to QA-induced toxicity. We demonstrate that the R6/2 transgenic mouse model expressing a short fragment of mutant HTT displays hypothalamic neuropathology with discrete loss of the neuronal populations expressing orexin, MCH, CART, and orexin at 12 weeks of age. The BACHD mouse model expressing full-length mutant HTT does not display any hypothalamic neuropathology at 2 months of age. There was no effect of hypothalamic injections of QA on the neuronal populations expressing orexin, MCH, CART or oxytocin in neither HD mouse model. In conclusion, we find no support for a role of excitotoxicity in the loss of hypothalamic neuronal populations in HD.

Project description:Prion-like seeding and propagation of Tau-pathology have been demonstrated experimentally and may underlie the stereotyped progression of neurodegenerative Tauopathies. However, the involvement of templated misfolding of Tau in neuronal network dysfunction and behavioral outcomes remains to be explored in detail. Here we analyzed the repercussions of prion-like spreading of Tau-pathology via neuronal connections on neuronal network function in TauP301S transgenic mice. Spontaneous and GABA(A)R-antagonist-induced neuronal network activity were affected following templated Tau-misfolding using synthetic preformed Tau fibrils in cultured primary neurons. Electrophysiological analysis in organotypic hippocampal slices of Tau transgenic mice demonstrated impaired synaptic transmission and impaired long-term potentiation following Tau-seed induced Tau-aggregation. Intracerebral injection of Tau-seeds in TauP301S mice, caused prion-like spreading of Tau-pathology through functionally connected neuroanatomical pathways. Electrophysiological analysis revealed impaired synaptic plasticity in hippocampal CA1 region 6 months after Tau-seeding in entorhinal cortex (EC). Furthermore, templated Tau aggregation impaired cognitive function, measured in the object recognition test 6 months post-seeding. In contrast, Tau-seeding in basal ganglia and subsequent spreading through functionally connected neuronal networks involved in motor control, resulted in motoric deficits reflected in clasping and impaired inverted grid hanging, not significantly affected following Tau-seeding in EC. Immunostaining, biochemical and electron microscopic analysis in the different models suggested early pathological forms of Tau, including Tau-oligomers, rather than fully mature neurofibrillary tangles (NFTs) as culprits of neuronal dysfunction. We here demonstrate for the first time using in vitro, ex vivo and in vivo models, that prion-like spreading of Tau-misfolding by Tau seeds, along unique neuronal connections, causes neuronal network dysfunction and associated behavioral dysfunction. Our data highlight the potential relevance of this mechanism in the symptomatic progression in Tauopathies. We furthermore demonstrate that the initial site of Tau-seeding thereby determines the behavioral outcome, potentially underlying the observed heterogeneity in (familial) Tauopathies, including in TauP301 mutants.

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Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

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Protein misfolding detected early in pathogenesis of transgenic mouse model of Huntington disease using amyloid seeding assay.

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