Introduction

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion in huntingtin (Htt), a ubiquitously distributed protein.¹ 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.² 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.³ 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.³

Various 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.⁴ 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,⁵ 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 disease^6,7,8,9 and HD,^10,11 and for the assessment of new treatments.^12,13,14,15 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.^10,11 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,^16,17 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,^18,19 to study the progression of specific motor symptoms known to be associated with dysfunction and/or degeneration of the dorsolateral sensorimotor putamen of NHP.¹⁷

Results

Injection of the Htt171-82Q vector induces HD neuropathology in the primate striatum

We 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.¹⁸ In a first series of experiments, four Macaca fascicularis monkeys received intraputaminal injections of 40 l 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 lesions¹⁷ (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.

Figure 1.

Experimental paradigm: lentiviral vectors encoding wild-type (Htt171-19 CAG repeats) or mutated Htt fragments (Htt171-82 CAG repeats) were injected into the dorsolateral putamen of adult macaques.

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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 m², 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.²⁰ 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.²¹ 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 2.

Formation 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.

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Figure 3.

Neuropathology 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 m.

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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 m². 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 4.

Long-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.

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Unilateral overexpression of mutated Htt induces an HD-like motor syndrome under dopaminergic stimulation

When 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 5.

Behavioral 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.

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Bilateral overexpression of mutated Htt induces a progressive, spontaneous, HD-like phenotype

We 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 7.

Analysis 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.

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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 6.

Apomorphine-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.

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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%).

Discussion

Our 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.²² This early stage of the disease is followed by a symptomatic phase characterized by spontaneous choreiform movements.²² Motor disabilities progress over a period of 10–15 years, from a hyperkinetic to an akinetic-rigid syndrome. Typically, the earliest motor signs are eye movement abnormalities, followed by the progressive occurrence of isolated dyskinesia and finally, chorea. At very early stages of the disease, choreic movements are usually considered to be involuntary, but they may appear purposeful and resemble tics.^23,24 In the experiments described here, very similar early hyperkinetic symptoms were observed in Htt171-82Q/82Q primates, consisting of a combination of tics and choreiform movements in one macaque (#6682). In contrast, although some isolated dystonic movements of the trunk and feet were observed in animals receiving bilateral injections, severe progressive dystonia with rigidity was not observed. This suggests that a local striatal injection of a lentiviral vector encoding a mutated Htt fragment is sufficient to reproduce the early and moderate stages of HD motor disability. Indeed, neuropathological examination also revealed features of the early and moderate stages of HD, with the presence of NII²⁵ in both short-term Htt171-82Q/19Q (9 weeks after injection) and long-term Htt171-82Q/82Q (30 weeks after injection) experiments. Aggregates appeared to be smaller and less abundant in the long term than in the short term. This observation probably reflects the progressive degeneration of infected neurons, leading to a shrinkage of the area of lesion, as demonstrated in the rodent model.^18,19 NII were also observed in the striatum of transgenic and knock-in HD models.^26,27,28,29 The striatal neuropathological changes observed in all the animals in our experiment appeared to be consistent with behavioral deficits typical of early and moderate HD.

The organization of the basal ganglia postulates the existence of functional partitioning into sensorimotor, associative, and limbic territories, and these are involved in motor, cognitive and emotional aspects of behavior, respectively. Studies involving non-human primates have described GABAergic striato-pallidal neuronal connections, the inactivation of which (by intrapallidal injections of a GABAergic antagonist) is associated with abnormal behavior, such as leg dyskinesia, attention deficits, hyperactivity, or stereotypies.^30,31,32,33 These reports indicate that the inactivation of neuronal pathways originating in the posterior part of the putamen is related to leg dyskinesia, the inhibition of neural circuits originating in the dorsal part of the pre-commissural putamen and caudate nucleus is related to attention deficits, the ventral part of the pre-commissural putamen is associated with hyperactivity and attention deficits, and the ventromedial striatum is associated with stereotyped behavior. Taking this anatomical and functional organization into account, it is interesting to analyze the relationship between the abnormal movements observed in Htt171-82Q/82Q monkeys and the location of the infected areas within the dorsolateral post-commissural putamen. In animals displaying spontaneous abnormal movements (#6682 and 6633), EM48-ir aggregates were found in both the ventral and lateral parts of the putamen, which receive afferences mostly from the primary motor cortex (M1) and premotor cortex. In contrast, animal #6835, which displayed limited spontaneous choreiform movements, had EM48-ir aggregates only in the dorsal and central parts of the putamen, corresponding to the pre-motor area of the putamen, as described by Nambu et al. (Figure 7).³⁴ It remains unclear whether neuronal dysfunction and/or neuronal degeneration of the striatum are necessary in order to generate dyskinesia and motor tics. Nevertheless, the apomorphine-induced turning behavior in the Htt171-82Q/19Q group and the motor tics in the Htt171-82Q/82Q group were observed soon after viral injections, thereby suggesting that neuronal dysfunction, rather than actual neuronal cell loss, may be the predominant mechanism, as reported in other primate models of tics or abnormal movements.³¹

Various primate models of HD mimic different clinical and neuropathological aspects of the disease. Each of these primate models may address specific questions in studies of the potential mechanisms involved in the degenerative process observed in HD, or may help in the evaluation of restorative or protective therapeutic strategies. The intrastriatal injection of glutamatergic agonists (quinolinic and ibotenic acid) was one of the first primate models of HD to be developed.^13,35 These toxins induce acute and extensive striatal degeneration but do not produce the progressive and sustained spontaneous behavioral abnormalities reminiscent of HD.³⁶ In contrast, prolonged systemic treatment with a daily dose of mitochondrial toxin, 3-nitropropionic acid, leads to the progressive appearance of abnormal movements reminiscent of early to severe HD associated with selective striatal degeneration.^11,37 Although the chronic 3-nitropropionic acid model reproduces many phenotypic features of HD, including a frontal-type syndrome resembling the cognitive deficit associated with HD,^11,12,15 it does not replicate the early events of HD pathogenesis that are specifically triggered by mutated Htt, and eventually lead to striatal cell death.

The intraputaminal injection of lentiviruses expressing mutated Htt into non-human primates therefore provides a unique opportunity to study, in the NHP brain, the possible relationships between mechanisms that are specifically triggered by mutated Htt, and behavioral deficits. The use of this genetic model may also facilitate studies of higher-order cognitive dysfunction, a devastating feature of HD. Lentiviral vector injection into the putamen and caudate nucleus could be used for reproducing both motor and cognitive deficits, better representing the spectrum of symptoms seen in patients with HD. However, till such time as it is demonstrated that striatal lentiviruses expressing mutated Htt can reproduce the whole spectrum of HD symptoms, the 3-nitropropionic acid NHP model of HD remains the gold standard for preclinical therapeutic research in HD.Finally, these experiments provide a proof-of-principle for the development of genetic models of HD in NHPs. These models could be based on the direct injection of viral vectors into the brain or on new transgenic techniques recently used for producing animal models in various species, including NHPs.^38,40

Materials and Methods

Animals. Ten adult male Macaca fascicularis monkeys, each weighing 5–7 kg, were included in this study (seven in experiment 1 and three in experiment 2). Animals were housed individually, under conditions of a 12 hours/12 hours light/dark cycle. Because the study used lentiviral vectors expressing a mutated form of a human protein, it was necessary to carry out all experiments, including behavioral tests, under biosafety level 3conditions. Studies were conducted in accordance with the European convention for animal care (86/609/EEC) and the guide for the care and use of laboratory animals adopted by the National Institutes of Health.

Lentiviral production. Previous studies aimed at assessing the relative contributions of polyglutamine repeat size, htt expression levels, protein length, and promoter choice (either cytomegalovirus or phosphoglycerate kinase)to the onset and specificity of the pathology in rats¹⁸ have shown that earlier onset and more severe disease are associated with shorter fragments, longer CAG repeats, and higher levels of expression. We aimed to induce a severe HD-like pathology in primates. We therefore selected the SIN-W-PGK transfer vector^18,41 encoding the first 171 amino acids of the human Htt with 19 or 82 CAG repeats (Htt171-19Q/82Q).

Vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vectors were produced with a four-plasmid system, as previously described,⁴² by transient transfection of 293T cells.⁴³ The particle content was determined by enzyme-linked immunosorbent assay (Perkin Elmer Life Sciences, Courtaboeuf, France). The batches of viruses were matched for particle content (200,000 ng p24 antigen/ml).

Surgical procedure and lentiviral injection. The 10 male macaques received 4 stereotactic injections in 10 operative sessions conducted under strict biosafety level 3 conditions. The stereotactic coordinates were determined for each monkey on the basis of a preoperative magnetic resonance imaging study, as previously described in humans.⁴⁴ Macaques were placed under general anesthesia with a mixture of ketamine and xylazine (Coveto, Montaigu, France; 15 mg/kg + 1.5 mg/kg, renewed every hour). They were then placed in a prone position in a stereotaxic magnetic resonance imaging-compatible frame and received four stereotaxic injections (10 l/injection i.e., 40 l/putamen) of lentiviral vectors, expressing Htt171-19Q or Htt171-82Q. The injections were administered to target the sensorimotor region of the putamen, one injection into the commissural putamen (10 l) and three into the post-commissural putamen (3 10 l). Target coordinates were determined by magnetic resonance imaging guidance, using a 1.5-T MR magnet (General Electric Medical Systems, Waukesha, WI), as described elsewhere.¹² Briefly, the first injection was aimed at the commissural level of the putamen, followed by a group of two injections (second and third injection, 1 mm apart) 2 mm caudal to the anterior commissure, and a fourth injection 5 mm caudal to the anterior commissure. Among the four animals belonging to the unilateral group, two were injected in the right putamen with the lentiviral vectors expressing Htt171-19Q, and in the left with those expressing Htt171-82Q, whereas the two others were injected in the left putamen with the lentiviral vectors expressing Htt171-19Q and in the right with those expressing Htt171-82Q.

All injections were administered into the dorsolateral part of the putamen—a region previously identified as being critically involved in the expression of abnormal movements following excitotoxic striatal injections.^16,17 The lentiviral suspension was injected manually, at the rate of 1 l/min, in each of the tracks, using a 10-l Hamilton syringe fitted with a 45° beveled 22-gauge needle. After the first 5 l had been injected, the needle was partially withdrawnby 1 mm to optimize the dispersal of the lentiviral vector along the dorso-ventral axis of the intended target. Once all 10 l had been injected, the needle was left in situ for an additional 2 minutes to facilitate the complete diffusion of the lentiviral vector from the needle tip and to limit the spreading of viral particles into the underlying cerebral structures during needle withdrawal.

Behavioral analysis. We repeatedly assessed the motor behavior of the animals, by both quantitative and qualitative analyses.

Video-based recording and analysis. Primates were placed in a transparent Plexiglas cage and video recordings were taken from the front, with no observers present in the room. Videotapes were analyzed off-line, using motion tracking and analysis software (Ethovision 2.3; Noldus, Wageningen, The Netherlands), by an examiner who was not in the know of the experimental conditions, thereby providing an objective measurement of kinematic parameters, such as the TDM as an indicator of general locomotor activity.

Dyskinesia rating scale. In addition to quantitative assessment of the behavioral syndrome, time-sampled neurological observations were used in order to characterize qualitatively the incidence of abnormal movements following viral injection, as previously described.⁴⁵ Briefly, orofacial dyskinesia, dystonia, and choreiform movements were identified axially, and for each limb. Each of these abnormal movements was rated as present (=1) or absent (=0) during each 5-minute period of each test session: a 30-minute test session for spontaneous behavioral analysis, and a 40-minute session for an apomorphine-induced test. A dyskinesia index (sum of incidences) was calculated by adding the individual incidences of each symptom during the total duration of the test period.

Tic rating scale. Each videotape segment was also rated for the presence or absence of tics under spontaneous/no drug condition, using a modified Tics rating scale adapted from the Rush Videotape Rating Scale.⁴⁶ In contrast to choreiform movements, which were defined as abnormal if they were involuntary, brief, and random, motor tics were defined as repetitive and stereotyped sudden involuntary movements. In the modified Rush rating scale we quantified two tic variables: the anatomic distribution of the tics, defined as the number of body areas affected (eyes, nose, mouth, neck, shoulders, arms, hands, trunk, pelvis, legs, and feet) and the frequency of motor tics (expressed as the total number of discrete motor tics observed during each 5-minute video segment during the 30-minute test period).

All animals were analyzed in two consecutive sessions, performed at different time points during the experiment. The first session was a 30-minute one recorded under normal conditions (i.e., no drug injected into the animals). This session was immediately followed by a 40-minute video recording session beginning with an intramuscular injection of 0.3 mg/kg apomorphine. The animals given unilateral injections were studied repeatedly before injection and at 3, 6, and 9 weeks after injection. Animals given bilateral injections were studied repeatedly before injection and at 1, 3, 6, 9–12, and 27–30 weeks after injection.

Immunohistochemical analysis. Once the behavioral study had been completed, 9 weeks (unilaterally injected) and 30 weeks (bilaterally injected) after injection, respectively, the macaques were deeply anesthetized with ketamine (15 mg/kg) and killed by the administration of an overdose of pentobarbital (100 mg/kg, intravenously; Sanofi, France). The monkeys were perfused transcardially with cold saline and their brains were immediately removed and cut in half along a midline sagittal plane. Each hemisphere was then cut into slabs on a monkey brain slicer. The tissue slabs were immersed in cold 4% paraformaldehyde fixative solution for 6 days, washed in a series of cold graded sucrose solutions for 4 days and sectioned in the coronal plane on a freezing microtome (40 m sections).

The presence of aggregates, which are a hallmark of the disease,^20,26,47 and possible changes in various striatal markers, were assessed qualitatively and quantitatively in order to characterize the progressive appearance of an HD-related pathology. We used the well-characterized EM48 antibody⁴⁷ for the detection of neuropil aggregates and forNII containing mutated Htt. For immunohistochemical labeling, sections were first incubated for 48 hours at room temperature or 72 hours at 4°C in phosphate-buffered saline containing 0.5% Triton-X100, 2% bovine serum albumin, 3.5% normal serum, and an appropriate dilution of the primary antibody: anti-Htt (EM48), 1:5,000 dilution;⁴⁷ anti-NeuN, 1:1,000 dilution (Chemicon International, Temecula, CA) or anti-glial fibrillary acidic protein, 1:50,000 dilution (DakoCytomation, Glostrup, Denmark). Sections were then processed using the avidin-biotin peroxidase method,⁴⁸ with tyramine amplification.⁴⁹

See Supplementary Materials and Methods for details on confocal microscopy analysis, cell counts and localization of EM48-positive aggregates.

Statistical analysis. The values indicated are mean values SEM. The M–W was used for comparing independent groups for single measures. The Kruskal–Wallis test was employed as a non-parametric alternative to the one-way independent-samples analysis of variance. The Kolmogorov–Smirnov test was used in comparing two independent groups for consecutive measures (Statview 4.0; Abbacus Concepts, Berkeley, CA).

References

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Acknowledgments

This work was supported by the Swiss National Science Foundation, the CEA and Association pour la Recherche sur la Stimulation Cérébrale. We also thank Fabienne Pidoux, Vivianne Padrun and Maria Rey from the Ecole Polytechnique Fédérale de Lausanne for technical assistance.