Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntington´s Disease: An In Vivo High-Content Imaging Study

April Characterization of HTT Inclusion Size, Location, and Timing in the zQ175 Mouse Model of Huntingtons Disease: An In Vivo High-Content Imaging Study Data Availability Statement: All relevant data are within the paper. 0 1 0 1 Evotec AG , Manfred Eigen Campus, Hamburg, Germany, 2 CHDI Management/CHDI Foundation, Princeton, New Jersey , United States of America 1 Academic Editor: David R Borchelt, University of Florida, UNITED STATES Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis. - Funding: This work was supported by CHDI Foundation, a privately-funded nonprofit biomedical research organization exclusively dedicated to discovering and developing therapeutics that slow the progression of Huntington's disease. CHDI Foundation conducts research in a number of different ways; for the purposes of this manuscript, research was conducted at the contract research organization Evotec AG under a fee-for-service agreements. The authors listed all contributed to the Huntington's disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat encoding a polyglutamine tract within the amino-terminus of the huntingtin (HTT) protein [1]. The disease is clinically characterized by motor, cognitive and psychiatric manifestations [2]. Current understanding of HD is largely based on studies of mutant huntingtin (mHTT)conception, planning, and direction of the research, including generation, analysis, and interpretation of the data. The specific roles of these authors are articulated in the author contributions section. induced pathogenesis in transgenic mammalian models. A number of HD mouse models that have been extensively characterized [3, 4]. Among these, the knock-in (KI) mouse models bearing an expanded polyglutamine tract within the endogenous murine HTT gene locus on chromosome 5q most closely mimic the genetic context of the disorder and express the mutant allele in the endogenous mRNA and protein context [5, 6]. The zQ175 knock-in (KI) mouse line has recently emerged as a particularly useful model to test potential therapeutics since they exhibit extensive behavioral, histopathological, and molecular phenotypes reminiscent of human disease [4, 7]. Amongst the deficits reported, the heterozygote zQ175 mice display age- and region-dependent molecular alterations in genes associated with disease progression, including expression changes in neurotransmitter receptors, BDNF, and DARPP-32, amongst others [8, 9]. zQ175 mice display behavioral deficits, especially in the dark phase of the diurnal cycle at 4.5 months of age, and overt rotarod deficits by 8 months of age, amongst other alterations [8, 9]. Additionally, decreases in striatal Cnr1 and PDE10a mRNA levels are seen in heterozygotes of both sexes by 10 months of age, accompanied by the progressive hyperexcitability of medium spiny neurons, progressive loss of cortical afferent innervation of the striatum, and decreased striatal volumes in heterozygous mice [8]. Given the inverse relationship between age of motoric onset and HTT CAG-repeat length in HD[10], an increase in the CAG length is expected to accelerate and/or magnify HD phenotypes. Here we extend the characterization of the zQ175 KI mice by monitoring HTT expression and inclusion bodies to enable the evaluation of genetic or pharmacological interventions that might affect disease progression [1113]. High content imaging (HCI) of brain sections is a powerful technique that allows automated quantification of histological endpoints and can be used to detect changes in response to treatments or in the context of genetic perturbations. The Opera platform, formerly used in cellular functional screening assays, enables automated multicolor, multi-parametric fluorescence imaging readouts and, in conjunction with custom adaptation of image analysis algorithms, permits quantitation from thousands of cells in brain sections of large numbers of animals. This allows efficient and thorough analysis of in vivo studies following perturbations such as virallymediated gene delivery, where intra group variability between animals is high with respect to area and cells transduced following intraparenchymal injections. Here we investigate immunohistological changes in zQ175 heterozygous animals by HCI and describe the alterations in several striatal histological markers [14]. Specifically, we report on the spatial and temporal analysis of mHTT containing EM48-immunoreactive (EM48-ir) species consisting of inclusion bodies, diffuse nuclear signals, and small nuclear puncta across various ages. We also describe the expression of endogenous mouse HTT as detected with the monoclonal antibody MAB2174. These analyses will enable future studies to evaluate the effects of HTT-lowering therapeutic strategies in a quantitative and spatiallyinformative manner. The zQ175 C57B/L6J knock-in mice, derived from a spontaneous expansion of the CAG copy number in the CAG 140 knock-in mice, were generated at Psychogenics (Tarrytown, NY, USA) and provided for breeding by Jackson Laboratory (Bar Harbor, Maine, USA). Transgenic mice were backcrossed to C57BL/6J to obtain heterozygous zQ175 mice and wild type littermates. Animals were housed in Eurostandard Type II long cages and given access to food and water ad libitum. Environmental conditions were maintained at a temperature of 211C, humidity of 5510% and a 12:12 light:dark cycle with lights on at 7 am and off at 7 pm. During housing, animals were checked daily for health status. All animal work was carried out in accordance with the regulations of the German animal welfare act and the EU legislation (EU directive 2010/63/EU). The protocol wa (...truncated)