Towards a transgenic model of Huntington's disease in a non-human primate

Abstract

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.

Figure 1. Generation of a transgenic model in monkeys of HD

a, b, The transgenic HD monkeys rHD-1 (left) and rHD-2 (right) are shown. Transmission light image (a) and fluorescent image (b) showing GFP expression in HD monkeys. c, Top panel: lentiviral vector that carries exon 1 of the HTT gene with 84 CAG repeats (pLVU-HTT-84Q). Bottom panel: lentiviral vector that carries the GFP gene (pLVU-G). Arrows indicate the positions of PCR primers; arrowheads denote restriction digest sites. Flap, HIV-flap sequence; GFP, green-fluorescent-protein gene; HTT, huntingtin gene; LTR, long terminal repeat; Ubi, ubiquitin promoter; WPRE, woodchuck post-transcriptional regulatory element. d, e, The presence of transgenes in HD monkeys was confirmed by PCR analysis using primer sets specifically for mutant HTT (top panels) and for the GFP gene (bottom panels). PCR of the cord (C) and placental (P) tissues of all HD monkeys (d), and PCR of different tissues collected from rHD-4 and rHD-5 (e). f, Expression of the transgenic mutant HTT was confirmed by western blot analysis using the placental tissues. Immunostaining was performed using mouse-monoclonal-mEM48 antibody (top panel) and an antibody against γ-tubulin (bottom panel).

Figure 2. Expression of mutant HTT in HD monkey peripheral tissues and brains

a-d, mEM48 immunoblot of peripheral and brain tissues reveals high-molecular-mass oligomeric HTT (arrow) and soluble HTT products. The blot was also probed with an antibody to γ-tubulin as an internal control. Shown are immunoblots of peripheral tissues collected from monkey rHD-4 (a) and rHD-5 (b), and samples from different brain regions of monkey rHD-4 (c) and rHD-5 (d), with antibody to mEM48 (top panel) and γ-tubulin (bottom panel). WT, wild-type non-transgenic monkey.

Figure 3. Histopathology of HD monkey brain

a, b, Brain sections of rHD-4 (a) and rHD-5 (b) were immunostained with mEM48 and 1C2, respectively. Low magnification (upper panels; scale bars, 100 mm) shows the abundant distribution of transgenic mutant HTT in the cortex (Ctx) and striatum (Str). High magnification (bottom panels; scale bars, 10 mm) demonstrates that transgenic mutant HTT is distributed in neuronal nuclei and forms neuropil aggregates (arrowheads). Nuclear inclusions (arrows) are evident in sections stained with mEM48 or 1C2.