Early detection of exon 1 huntingtin aggregation in zQ175 brains by molecular and histological approaches

Abstract

Huntingtin-lowering approaches that target HTT expression are a major focus for therapeutic intervention for Huntington’s disease. When the cytosine, adenine, guanine repeat is expanded, the huntingtin pre-mRNA is alternatively processed to generate the full-length HTT and HTT1a transcripts. HTT1a encodes the aggregation-prone and highly pathogenic exon 1 HTT protein. In evaluating HTT-lowering approaches, understanding how the targeting strategy modulates levels of both transcripts and the HTT protein isoforms that they encode will be essential. Given the aggregation-propensity of exon 1 HTT, the impact of a given strategy on the levels and subcellular location of aggregated HTT will need to be determined. We have developed and applied sensitive molecular approaches to monitor the levels of aggregated and soluble HTT isoforms in tissue lysates. We have used these, in combination with immunohistochemistry, to map the appearance and accumulation of aggregated HTT throughout the CNS of zQ175 mice, a model of Huntington’s disease frequently chosen for preclinical studies. Aggregation analyses were performed on tissues from zQ175 and wild-type mice at monthly intervals from 1 to 6 months of age. We developed three homogeneous time-resolved fluorescence assays to track the accumulation of aggregated HTT and showed that two of these were specific for the exon 1 HTT protein. Collectively, the homogeneous time-resolved fluorescence assays detected HTT aggregation in the ten zQ175 CNS regions by 1–2 months of age. Immunohistochemistry with the polyclonal S830 anti-HTT antibody showed that nuclear HTT aggregation, in the form of a diffuse nuclear immunostain, could be visualized in the striatum, hippocampal CA1 region and layer IV of the somatosensory cortex by 2 months. That this diffuse nuclear immunostain represented aggregated HTT was confirmed by immunohistochemistry with a polyglutamine-specific antibody, which required formic acid antigen retrieval to expose its epitope. By 6 months of age, nuclear and cytoplasmic inclusions were widely distributed throughout the brain. Homogeneous time-resolved fluorescence analysis showed that the comparative levels of soluble exon 1 HTT between CNS regions correlated with those for HTT aggregation. We found that soluble exon 1 HTT levels decreased over the 6-month period, whilst those of soluble full-length mutant HTT remained unchanged, data that were confirmed for the cortex by immunoprecipitation and western blotting. These data support the hypothesis that exon 1 HTT initiates the aggregation process in knock-in mouse models and pave the way for a detailed analysis of HTT aggregation in response to HTT-lowering treatments.

Evolution of aberrant brain-wide spatiotemporal dynamics of resting-state networks in a Huntington’s disease mouse model

“…Since the cortico-striato-thalamic network is comprised of sub-segregated regional circuitry 88,89 , to have a more granulated assessment of how these regions are altered in their timings during QPPs, future studies should employ a detailed whole brain parcellation to unravel these changes. In our IF study, we did not observe changes in 2B4 spot coverage in cells at 3 months, despite this being shown in this model, using different markers, such as the antibodies S829 or S830, which observe changes starting even from 1-2 months of age 78,90 . Likewise, the use of S100ß as a marker for astrocytes would be a more appropriate replacement compared to GFAP, which only represents inflammatory states of astrogliosis.…”

A CAG repeat threshold for therapeutics targeting somatic instability in Huntington’s disease

“…In zQ175 mice, it is the soluble HTTexon1 protein that is depleted from brain regions during the aggregation process. 7 The N-terminus of HTT contains a potent nuclear export signal, 61,62 and HTT is retained in neuronal nuclei because it aggregates there, appearing first as a diffuse immunoreactive product that fills the nucleus. 15,61,63 The threshold concentration for the nucleation of aggregation will be cell-type and subcellular location specific.…”

“…Homogeneous time-resolved fluorescence (HTRF) was performed as previously described, 7 using n = 8/ genotype/ age with an equal ratio of males to females. The antibody pairings and concentrations for the soluble HTTexon1 assay were donor: 2B7-Tb, 1 ng and acceptor: MW8-d2, 40 ng.…”

“…5,6 This encodes the highly aggregation prone and pathogenic HTTexon1 protein. 5,7,8 Huntington’s disease pathology is characterised by the deposition of nuclear and cytoplasmic inclusions, 9,10 as well as synaptic and neuronal loss in the striatum and other regions of the brain. 11,12 Nuclear huntingtin aggregation is linked to transcriptional dysregulation, a molecular phenotype observed in mouse models and post-mortem brains.…”

“…However, such a model is likely to have been created with a highly expanded CAG repeat that would cause childhood onset of the disease in humans, and therefore, does not model the CAG repeat expansion process that occurs in the adult-onset disease. The zQ175 knock-in mouse of Huntington’s disease is an attractive candidate as it is well characterised 7,37,38 and has been used for HTT lowering approaches. 39 However, the mutant allele in zQ175 generally has approximately (CAG) 190 .…”