Expression of Mutated Huntingtin Fragment in the Putamen Is Sufficient to Produce Abnormal Movement in Non-human Primates
Authors: Stéphane Palfi, Emmanuel Brouillet, Béchir Jarraya, Jocelyne Bloch, Caroline Jan, Masahiro Shin, Françoise Condé, Xiao-Jiang Li, Patrick Aebischer, Philippe Hantraye, Nicole Déglon
Journal: Molecular Therapy (2007)

Huntington's disease (HD) is a neurological disorder characterized by striatal degeneration, motor symptoms and complex neuropsychiatric alterations. There is currently no genetic model of HD in non-human primates (NHPs). In this study we investigated neuropathological and behavioral changes following injections of lentiviral vectors encoding a fragment of mutated huntingtin (Htt171-82Q) into the dorsolateral sensorimotor putamen of macaques. In the first study, we injected Htt171-82Q into one hemisphere and a lentiviral vector encoding Htt171-19Q or saline into the other, and studied the animals for 9 weeks. During this period, when apomorphine was administered into Htt171-19Q/82Q animals, it induced progressive chorea, dystonia and ipsilateral turning behavior, whereas animals infected with Htt171-19Q/19Q showed no abnormal behavior. After 9 weeks, the putamen of animals infected with Htt171-82Q presented neuritic and nuclear Htt aggregates, reactive astrocytes and loss of the neuronal marker NeuN. In a second study, we injected Htt171-82Q bilaterally into the dorsolateral putamen. From week 15 after infection, these animals progressively developed spontaneous dyskinesia of the legs, arms, and trunk and, in one case, tics that persisted for up to 30 weeks. The present study constitutes a proof-of-principle for the development of a genetic model of HD in NHP. Huntington's disease (HD) is a neurological disorder characterized by striatal degeneration, motor symptoms and complex neuropsychiatric alterations. There is currently no genetic model of HD in non-human primates (NHPs). In this study we investigated neuropathological and behavioral changes following injections of lentiviral vectors encoding a fragment of mutated huntingtin (Htt171-82Q) into the dorsolateral sensorimotor putamen of macaques. In the first study, we injected Htt171-82Q into one hemisphere and a lentiviral vector encoding Htt171-19Q or saline into the other, and studied the animals for 9 weeks. During this period, when apomorphine was administered into Htt171-19Q/82Q animals, it induced progressive chorea, dystonia and ipsilateral turning behavior, whereas animals infected with Htt171-19Q/19Q showed no abnormal behavior. After 9 weeks, the putamen of animals infected with Htt171-82Q presented neuritic and nuclear Htt aggregates, reactive astrocytes and loss of the neuronal marker NeuN. In a second study, we injected Htt171-82Q bilaterally into the dorsolateral putamen. From week 15 after infection, these animals progressively developed spontaneous dyskinesia of the legs, arms, and trunk and, in one case, tics that persisted for up to 30 weeks. The present study constitutes a proof-of-principle for the development of a genetic model of HD in NHP. IntroductionHuntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin (Htt), a ubiquitously distributed protein.1The Huntington's Disease Collaborative Research Group A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes.Cell. 1993; 72: 971-983Abstract Full Text PDF PubMed Scopus (6958) Google Scholar Studies of Htt's protein interactors and subcellular localization indicate that Htt may play a functional role in clathrin-mediated endocytosis, apoptosis, vesicle transport, cell signaling, morphogenesis, and transcriptional regulation. One of the hallmarks of HD is the proteolytic production of an N-terminal fragment of Htt, containing the polyglutamine repeat, which forms aggregates in the nucleus and cytoplasm of affected neurons.2Li SH Li XJ Huntingtin-protein interactions and the pathogenesis of Huntington's disease.Trends Genet. 2004; 20: 146-154Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar Clinically, HD is characterized by personality changes and cognitive decline, and is associated with motor disturbances, progressing from chorea and tics to rigidity and dystonia.3Harper PS Huntington's disease. WB Saunders Company Ltd., London1991Google Scholar The neuropathological features of HD include a severe loss of GABAergic projection neurons in the striatum, followed by the selective degeneration of neurons in extrastriatal regions, such as the substantia nigra and several cortical areas.3Harper PS Huntington's disease. WB Saunders Company Ltd., London1991Google ScholarVarious transgenic mouse models, including conditional and knock-in models, have been generated in order to study HD pathogenesis and the temporal relationships between neuronal protein aggregation, motor dysfunction and the occurrence of cell death.4Menalled LB Chesselet MF Mouse models of Huntington's disease.Trends Pharmacol Sci. 2002; 23: 32-39Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar These transgenic mouse models display many of the typical pathological features of early HD, including striatal atrophy, decrease in brain size, and changes in neurotransmitter receptor levels,5Cha JH Frey AS Alsdorf SA Kerner JA Kosinski CM Mangiarini L et al.Altered neurotransmitter receptor expression in transgenic mouse models of Huntington's disease.Philos Trans R Soc Lond B Biol Sci. 1999; 354: 981-989Crossref PubMed Scopus (187) Google Scholar but do not replicate either the typical pattern of neurodegeneration or the motor syndrome including chorea, dyskinesia and dystonia that is observed in HD patients. Consequently, despite the useful information gained from such models, critical questions concerning HD pathogenesis and disease progression, and their relationship to the motor and cognitive symptoms typical of HD remain largely unanswered.Primate models based on the use of excitotoxins or mitochondrial inhibitors have proved valuable in increasing our understanding of neurodegenerative diseases, such as Parkinson's disease6Bloem BR Irwin I Buruma OJ Haan J Roos RA Tetrud JW et al.The MPTP model: versatile contributions to the treatment of idiopathic Parkinson's disease.J Neurol Sci. 1990; 97: 273-293Abstract Full Text PDF PubMed Scopus (145) Google Scholar,7Burns RS Chiueh CC Markey SP Ebert MH Jacobowitz DM Kopin IJ A primate model of parkinsonism: selective destruction of dopaminergic neurons in the pars compacta of the substantia nigra by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Proc Natl Acad Sci USA. 1983; 80: 4546-4550Crossref PubMed Scopus (1999) Google Scholar,8Langston JW Forno LS Rebert CS Irwin I Selective nigral toxicity after systemic administration of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyrine (MPTP) in the squirrel monkey.Brain Res. 1984; 292: 390-394Crossref PubMed Scopus (838) Google Scholar,9Schneider JS Pope-Coleman A Cognitive deficits precede motor deficits in a slowly progressing model of parkinsonism in the monkey.Neurodegeneration. 1995; 4: 245-255Crossref PubMed Scopus (97) Google Scholar and HD,10Brouillet E Hantraye P Ferrante RJ Dolan R Leroy-Willig A Kowall NW et al.Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.Proc Natl Acad Sci USA. 1995; 92: 7105-7109Crossref PubMed Scopus (425) Google Scholar,11Palfi S Ferrante RJ Brouillet E Beal MF Dolan R Guyot MC et al.Chronic 3-nitropropionic acid treatment in baboons replicates the cognitive and motor deficits of Huntington's disease.J Neurosci. 1996; 16: 3019-3025Crossref PubMed Google Scholar and for the assessment of new treatments.12Palfi S Conde F Riche D Brouillet E Dautry C Mittoux V et al.Fetal striatal allografts reverse cognitive deficits in a primate model of Huntington disease.Nat Med. 1998; 4: 963-966Crossref PubMed Scopus (144) Google Scholar,13Kendall AL Rayment FD Torres EM Baker HF Ridley RM Dunnett SB Functional integration of striatal allografts in a primate model of Huntington's disease.Nat Med. 1998; 4: 727-729Crossref PubMed Scopus (139) Google Scholar,14Kordower JH Emborg ME Bloch J Ma SY Chu Y Leventhal L et al.Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease.Science. 2000; 290: 767-773Crossref PubMed Scopus (1139) Google Scholar,15Mittoux V Joseph JM Conde F Palfi S Dautry C Poyot T et al.Restoration of cognitive and motor functions by ciliary neurotrophic factor in a primate model of Huntington's disease.Hum Gene Ther. 2000; 11: 1177-1187Crossref PubMed Scopus (128) Google Scholar Comparative analyses of movements in primate models have shown strong similarities between the motor and cognitive patterns observed in these animal models and the patterns involved in human disease. Significant progress has been made toward identifying primate equivalents of bradykinesia, tremor, postural deficits, dyskinesia and even frontal-type cognitive deficits, in models relevant to various neurodegenerative disorders, including Parkinson's disease, delayed dystonia and HD.10Brouillet E Hantraye P Ferrante RJ Dolan R Leroy-Willig A Kowall NW et al.Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates.Proc Natl Acad Sci USA. 1995; 92: 7105-7109Crossref PubMed Scopus (425) Google Scholar,11Palfi S Ferrante RJ Brouillet E Beal MF Dolan R Guyot MC et al.Chronic 3-nitropropionic acid treatment in baboons replicates the cognitive and motor deficits of Huntington's disease.J Neurosci. 1996; 16: 3019-3025Crossref PubMed Google Scholar In HD models, the intrastriatal injection of various excitotoxins in primates has been shown to lead to the progressive appearance of abnormal choreiform movements under apomorphine stimulation,16Hantraye P Riche D Maziere M Isacson O A primate model of Huntington's disease: behavioral and anatomical studies of unilateral excitotoxic lesions of the caudate-putamen in the baboon.Exp Neurol. 1990; 108: 91-104Crossref PubMed Scopus (139) Google Scholar,17Burns LH Pakzaban P Deacon TW Brownell AL Tatter SB Jenkins BG et al.Selective putaminal excitotoxic lesions in non-human primates model the movement disorder of Huntington disease.Neuroscience. 1995; 64: 1007-1017Crossref PubMed Scopus (76) Google Scholar thereby suggesting that chorea and dyskinesia may be features specific to primates. Although the underlying cell death mechanisms in these models may encompass related, but distinct, molecular pathways of degeneration, the genetic nature of HD has always called into question the relevance of these neurotoxic lesion models as far as the pathogenic mechanisms of the human disorder are concerned.In this study, we investigated the spatial relationship between polyglutamine-Htt and HD neuropathology, and studied the time-course of behavioral deficits resulting from the overproduction of mutated Htt in the primate striatum. We used a lentiviral-mediated delivery of a short N-terminal fragment of Htt, previously described in rodents,18de Almeida LP Ross CA Zala D Aebischer P Déglon N Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length.J Neurosci. 2002; 22: 3473-3483Crossref PubMed Google Scholar,19Régulier E Trottier Y Perrin V Aebischer P Déglon N Early and reversible neuropathology induced by tetracycline-regulated lentiviral overexpression of mutant huntingtin in rat striatum.Hum Mol Genet. 2003; 12: 2827-2836Crossref PubMed Scopus (69) Google Scholar to study the progression of specific motor symptoms known to be associated with dysfunction and/or degeneration of the dorsolateral sensorimotor putamen of NHP.17Burns LH Pakzaban P Deacon TW Brownell AL Tatter SB Jenkins BG et al.Selective putaminal excitotoxic lesions in non-human primates model the movement disorder of Huntington disease.Neuroscience. 1995; 64: 1007-1017Crossref PubMed Scopus (76) Google ScholarResultsInjection of the Htt171-82Q vector induces HD neuropathology in the primate striatumWe studied the behavioral consequences of selective overexpression of a mutated Htt fragment in the primate striatum. We selected lentiviral vectors encoding the first 171 amino acids of the Htt protein with 19 (wild-type) or 82 (mutated) polyglutamine repeats.18de Almeida LP Ross CA Zala D Aebischer P Déglon N Lentiviral-mediated delivery of mutant huntingtin in the striatum of rats induces a selective neuropathology modulated by polyglutamine repeat size, huntingtin expression levels, and protein length.J Neurosci. 2002; 22: 3473-3483Crossref PubMed Google Scholar In a first series of experiments, four Macaca fascicularis monkeys received intraputaminal injections of 40 μl Htt171-82Q vector into four tracks distributed into the dorsolateral aspect of the commissural and post-commissural putamen, a region known to be involved in dyskinesia in primates with unilateral excitotoxic striatal lesions17Burns LH Pakzaban P Deacon TW Brownell AL Tatter SB Jenkins BG et al.Selective putaminal excitotoxic lesions in non-human primates model the movement disorder of Huntington disease.Neuroscience. 1995; 64: 1007-1017Crossref PubMed Scopus (76) Google Scholar (Figure 1). The Htt171-19Q vector was injected into the contralateral putamen. As a control for behavioral studies, three additional animals were injected bilaterally with either the Htt171-19Q vector (n = 2) or the phosphate-buffered saline–bovine serum albumin solvent (n = 1) (Figure 1). In these experiments, histological analyses were carried out 9 weeks after the injections. In a second series of experiments, three animals received bilateral intraputaminal injections of Htt171-82Q vector and were subsequently studied for up to 30 weeks.Brain sections from animals sacrificed 9 weeks after injection were immunostained with the EM48 antibody. Htt-containing aggregates were detected in all Htt171-82Q animals, but not in the Htt171-19Q control group (Figures 2a and c, 3a and b). At low magnification, coronal brain sections of the Htt171-82Q-infected animals displayed large areas (up to 14 mm2) containing EM48-positive aggregates/inclusions mostly located within the lateral part of the putamen, extending from the pre-commissural to the post-commissural part and largely encompassing the sensorimotor region of this structure (Figure 2a). The volume of putaminal tissue expressing the transgene varied from 50 to 60 mm3, accounting for a mean of 16% of the total putaminal volume at these rostro-caudal levels. EM48-immunoreactivity was generally detected throughout the nuclei, with a densely stained core resembling the large spherical neuronal intranuclear inclusions (NII) typically observed in HD patients or transgenic animals (Figure 3b). EM48-positive objects detected by immunochemistry at this early stage displayed a mean cross-sectional area of 57 μm2, consistent with the size of the nucleus. The EM48-positive objects observed by immunofluorescence were typical of NII and were often immunoreactive for ubiquitin (Figure 3d), a typical hallmark of Htt inclusions in HD.20DiFiglia M Sapp E Chase KO Davies SW Bates GP Vonsattel JP et al.Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain.Science. 1997; 277: 1990-1993Crossref PubMed Scopus (2284) Google Scholar A few neuropil aggregates were also immunostained for EM48 (Figure 3d). Cell counts on these animal brains showed a high density of EM48-positive aggregates within the infected zones (∼700/mm2 i.e., 18,000/mm3). The total number of nuclei with EM48-positive inclusions was estimated at 850,000 in these primates. This number is in agreement with the transduction efficiency of lentiviral vectors in primates.21Kordower JH Bloch J Ma S Chu Y Palfi S Roitberg BZ et al.Lentiviral gene transfer to the nonhuman primate brain.Exp Neurol. 1999; 160: 1-16Crossref PubMed Scopus (179) Google Scholar A loss of NeuN-ir was observed in the striatum of Htt171-82Q-injected animals (Figure 2b and c), and this loss closely paralleled the regional distribution of EM48-positive NII within the 82Q-injected putamen (Figures 2a and c, 3a and c). However, within the EM48-positive area, NeuN-ir loss was greatest in the center of the infected region, close to the injection track. No such loss of NeuN staining was detected in the contralateral putamen infected with the Htt171-19Q control vector (Figure 3c). A robust glial fibrillary acidic protein immunoreaction was also observed in the putaminal area displaying NII on the Htt171-82Q-infected side (Figure 3e and f), contrasting with the mild glial fibrillary acidic protein immunostaining observed only in the vicinity of the needle track in the contralateral hemisphere receiving the Htt171-19Q control vector (Figure 3e).Figure 2Formation of huntingtin (Htt) aggregates and loss of a neuronal marker 9 weeks after the injection of Htt171-19Q/82Q. (a) Schematic representation of the accumulation of Htt aggregates, illustrating the distribution of pathogenic transgene (EM48 staining) expression in the sensorimotor parts of the putamen (AC: anterior commissure; the coordinates from AC are indicated). (b) NeuN immunocytochemistry in one representative Htt171-82Q animal (0.8 mm posterior to AC), showing striatal pathology 9 weeks after infection. (c) Distribution of EM48-positive Htt aggregates in one adjacent section, showing the overlap between loss of the neuronal marker NeuN and a high density of neuronal intranuclear inclusions containing mutated Htt. Scale bars: 1 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Neuropathology 9 weeks after injection of lentiviral vectors encoding Htt171-19/82Q. (a) Low and (b) high power photomicrographs through the putamen of a monkey illustrating the area with transduced cells and accumulation of huntingtin (Htt)-containing aggregates (EM48 staining). As expected, Htt171-19Q-injected striata contained no EM48-positive objects. (c) The overexpression of mutated Htt was associated with a neuronal dysfunction (NeuN staining). (d) Laser confocal microscopy of the putamen showing the co-localization (yellow nuclei) of ubiquitin-positive Htt neuronal intranuclear inclusions (red) and EM48-positive aggregates (green). (e) The overexpression of mutated Htt was associated with a reactive astrogliosis (glial fibrillary acidic protein staining) which is absent in striata injected with the wild-type Htt fragment (panels c, e). Scale bars: panel (a), 5 mm; panels (c), (d), and (e), 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Interestingly, the EM48 staining from macaques sacrificed at 30 weeks clearly differs from that obtained from macaques sacrificed at the 9 weeks time point (Figure 4). At low magnification, the areas containing EM48-positive objects in the animals examined at 30 weeks did not exceed 10 mm2, an area smaller than that observed in animals sacrificed after 9 weeks. At higher magnification, very pale staining was detected throughout the nucleus, with a more strongly stained circular inclusion (NII; Figure 4a). These NII had a mean apparent size of about 24 μm2. The estimated number of EM48-positive objects in animals analyzed after 30 weeks was only about a quarter that in animals analyzed after 9 weeks. The apparent atrophy of the putamen in primates analyzed 30 weeks after injection is also indicative of real degeneration (Htt171-19Q short-term 40.1 ± 3.5 mm2 (n = 7); Htt171-82 short-term 40.4 ± 7.6 mm2 (n = 4); Htt171-82Q long-term 29.3 ± 1.5 mm2 (n= 3); P = 0.024 and P = 0.036, respectively). The region in which NeuN immunoreactivity was lost seemed to be compact and was surrounded by nuclei with residual EM48-positive staining (Figure 4b and c).Figure 4Long-term transgene expression and neuropathology in Htt171-82Q/82Q monkeys. (a) The size of EM48 huntingtin (Htt) inclusions were smaller 30 weeks after injection than 9 weeks after injection. In addition, apparent atrophy of the striatum was observed, with shrinkage of the NeuN-negative area (b) consistent with the neuronal degeneration induced by Htt171-82Q. (c) Finally, the number of Htt aggregates was decreased at 30 weeks. Scale bars: 1 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Unilateral overexpression of mutated Htt induces an HD-like motor syndrome under dopaminergic stimulationWhen studied under spontaneous conditions, neither the Htt171-82Q/19Q nor the Htt171-19Q/19Q macaques showed any change in behavior over the 9-week study period. Following apomorphine injection, the Htt171-82Q/19Q-injected animals displayed abnormal movements beginning 3 weeks after infection and persisting thereafter. These involuntary movements, typical of striatal dysfunction, were characterized by a series of abrupt abnormal limb movements. Some of these involuntary movements were ballisticin nature and were accompanied by twisting movements of a forearm or hind limb, with occasional foot dystonia. A quantitative analysis of the rotations showed that Htt171-82Q/19Q-infected animals had a significant (n = 7, Kolmogorov–Smirnov test P < 0.05) ipsilateral turning behavior, when compared with control animals (Htt171-19Q/19Q) (Figure 5a). A qualitative assessment of these motor symptoms, based on clinical rating scales, demonstrated a significant increase in the incidence of dyskinetic movements over time in the Htt171-82Q/19Q group, in comparison with both the pre-injection state animals and the Htt171-19Q/19Q animals (Kruskal–Wallis test P < 0.05) (Figure 5b). Consistent with these qualitative observations, quantitative kinematic analysis based on video recordings also showed a significantly greater total distance moved (TDM) for the Htt171-82Q/19Q group than for the pre-injection state and Htt171-19Q/19Q control groups (TDM Kruskal–Wallis test P < 0.05) (Figure 5c). All these symptoms were observed only under dopamine-agonist stimulation, and persisted for up to 60 minutes after apomorphine injection.Figure 5Behavioral deficits in Htt171-82Q-infected primates. (a) Apomorphine-induced turning behavior in Htt171-19Q/82Q and control monkeys (Htt171-19Q/19Q or phosphate-buffered saline) revealing the selective nature of striatal symptoms in Htt171-82Q animals. Kolmogorov–Smirnov test P< 0.05. (b) Dyskinesia index. Kruskal–Wallis test (K–W) P< 0.05. (c) locomotor activity (meters/40 minutes) measurements 9–12 weeks after unilateral and bilateral injections of Htt171-82Q. K–W P< 0.05. (a: Mann–Whitney U-test (M–W) P< 0.05 82/19 versus PRE; b: M–W P< 0.05 82/19 versus 19/19; c: M–W P< 0.05; 82/82 versus PRE; d: M–W P< 0.05 82/82 versus 82/19). PRE, pre-infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Bilateral overexpression of mutated Htt induces a progressive, spontaneous, HD-like phenotypeWe occasionally observed brief, unilateral, spontaneous dyskinetic movements under stress conditions in one of the Htt171-82Q/19Q-injected animals 9 weeks after injection. We therefore investigated whether the bilateral injection of Htt171-82Q/82Q into the sensorimotor part of the putamen would elicit abnormal movements under spontaneous conditions. Three Macaca fascicularis monkeys (#6682, 6835, and 6633) received bilateral intraputaminal injections of the Htt171-82Q vector into the dorsal and lateral aspects of the commissural and post-commissural putamen (Figure 1). However, as shown in Figure 7, the viral vector injections were not made at exactly the same coordinates, with the primate #6682 receiving injections in the most lateral part of the putamen, the primate #6835 in the dorso-lateral putamen and the third macaque (#6633) receiving the viral vector injections more medially in the structure.Figure 7Analysis of spontaneous behavior in Htt171-82Q/82Q animals. Graphs showing the mean (bars) and individual curves for (a) dyskinesia index and (b) locomotor activity (total distance moved; meters/30 minutes) in Htt171-82Q/82Q-injected primates. (c) Schematic representation of coronal sections of macaque (Mac) brains showing the distribution of Htt aggregates in Htt171-82Q/82Q animals. Mann–Whitney U-test *P < 0.05, PRE, pre-infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Following apomorphine administration, the incidence of dyskinetic movements significantly increased in Htt171-82Q/82Q animals at 12 weeks after infection, and was significantly higher than that in Htt171-19Q/19Q animals (Kruskal–Wallis test P < 0.05) (Figure 5b and 5c). Following apomorphine injection, all Htt171-82Q/82Q animals displayed choreiform movements and dystonic postures. These motor symptoms were characterized by abrupt, bilateral and random involuntary leg, arm, and trunk twisting movements, the incidence of which increased significantly over the 30-week test period (dystonia Mann–Whitney U-test (M–W) P < 0.05 at 16 and 27–30 weeks; chorea M–W P < 0.05 at 6.9–12.16 and 27–30 weeks) (Figure 6c and c). Quantitative kinematic analysis based on video recordings showed that TDM was significantly increased at 9–12.16 and 27–30 weeks post-injection in the Htt171-82Q/82Q group compared to pre-injection values (TDM M–W P < 0.05) (Figure 6d). Unlike animals unilaterally infected with the Htt171-82Q construct, animals infected bilaterally with Htt171-82Q/82Q displayed no significant turning behavior (data not shown).Figure 6Apomorphine-induced abnormal movements in Htt171-82Q/82Q-injected primates. Motor deficits were measured before the injection of lentiviral vectors into the putamen and up to 30 weeks after injection. (a) Dyskinesia, (b) dystonia (c) chorea, and (d) total distance moved (meters/40 minutes) were assessed. Mann–Whitney U-test *P < 0.05, PRE, pre-infection.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In spontaneous conditions, two of the three Htt171-82Q/82Q (#6682 and 6633) macaques displayed choreiform movements, beginning as early as 16 weeks after injection and persisting for up to the 30 weeks after injection, the last time-point studied (M–W P < 0.05) (Figure 7a). These spontaneous abnormal movements consisted of sudden, random, and abnormal motion of the leg, arm and trunk, resembling the choreiform movements observed in HD patients. Assessment of these motor symptoms using a clinical rating scale indicated a progressive increase in the incidence of spontaneous dyskinetic movements, beginning 16 weeks after injection and persisting thereafter (Figure 7a). Interestingly, one monkey (#6682) displayed additional sudden and rapid stereotyped behavior resembling motor tics, involving the hands, legs and head. Based on the modified rating scale for tics, the severity of tics peaked 9 weeks after injection (304 tics/30 minutes), and remained high until the end of the experiment (75 tics/30 minutes at 27–30 weeks). Quantitative analysis of locomotor activity in the two dyskinetic animals (Figure 7b) also showed a more than 50% increase in locomotor activity at 30 weeks compared to their control condition whereas monkey #6835 displayed no increase in locomotor activity (TDM #6682 = +52%; #6633 = +50%; #6835 = −57%).DiscussionOur main finding is the demonstration that intraputaminal injections of a lentiviral vector encoding a mutated Htt fragment generate spontaneous and apomorphine-induced choreiform movements that are associated with the presence of neuronal Htt aggregates and a significant loss of neuronal markers in the striata of primates. This is the first series of experiments to describe the occurrence of spontaneous choreic movements in primates, involving a pathological mechanism similar to that operating in the human disorder—local production of a mutated form of Htt. In this genetic primate model of HD, local overexpression of Htt171-82Q in the dorsolateral putamen reproduces a spectrum of basal ganglia motor deficits typical of HD, including hand, leg, and head dyskinesia, leg dystonia, and even tics in one of the animals. Abnormal movements were observed both under spontaneous conditions and after the systemic administration of a mixed D1/D2 dopamine receptor-agonist, which is known to elicit abnormal movements in patients with pre-symptomatic HD.Detailed behavioral analysis showed a succession of events following viral injections. The first indicator of striatal neuronal dysfunction was an ipsilateral turning behavior detected as early as 3 weeks after injection in the Htt171-82Q/19Q group. After 9 weeks, apomorphine-induced dyskinetic movements were observed in both the Htt171-82Q/19Q and Htt171-82Q/82Q groups, whereas the spontaneous dyskinetic movements and tics that were clearly evident 6 weeks after injection occurred only in the Htt171-82Q/82Q group of animals. This progression of neurological deficits resembles that in HD patients, in whom a pre-symptomatic state can be revealed with the administration of dopaminergic agonists.22Klawans HL Goetz CG Perlik S Presymptomatic and early detection in Huntington's